Escherichia coli

J Bacteriol, 1986 Jun, 166(3), 733 - 8
Reversibility of SOS-associated division inhibition in Escherichia coli; Maguin E et al.; In Escherichia coli the SOS response, induced by DNA-damaging treatments, includes two systems of cell division inhibition, SfiA and SfiC, which are thought to prevent cell division by interacting with the division protein FtsZ . It is shown here that SfiA-mediated division inhibition is readily reversible, even in the absence of de novo protein synthesis, suggesting that functional FtsZ molecules can be recovered from SfiA-FtsZ complexes . The action of SfiC, on the other hand, is essentially irreversible; induction by expression of the recA (Tif) mutation for 60 min results in division inhibition that continues for at least 180 min after the end of the induction period . An excess of the presumed target molecule FtsZ, furnished by a multicopy plasmid, suppresses the action of SfiA but not SfiC . Simultaneous induction of SfiA and SfiC results in irreversible division inhibition, showing that SfiC is epistatic to SfiA . The irreversibility of SfiC action is most readily accounted for by assuming that the SfiC product, unlike SfiA, is stable . The reversibility of SfiA action is slower in a lon mutant, in which the SfiA protein is partially stabilized . From the kinetics of division resumption in the absence of protein synthesis, we estimated the in vivo half-life of the SfiA protein to be 10 min in a lon+ strain and 170 min in a lon mutant.

J Bacteriol, 1986 Jun, 166(3), 713 - 21
Nucleotide sequence of the surface exclusion genes traS and traT from the IncF0 lac plasmid pED208; Finlay BB et al.; pED208 is a 90-kilobase conjugative plasmid belonging to the incompatibility group IncF0 lac . The surface exclusion system from this plasmid was cloned and sequenced, and two genes demonstrated exclusion ability . traS encoded a 186-amino-acid hydrophobic protein which, when transcribed from a vector promoter, caused exclusion of pED208 . The product of traT (TraTp) was a 245-residue protein which was highly expressed independently of a vector promoter in Escherichia coli minicells . The TraTp from pED208 was homologous with traT products from the IncF plasmids R-100 and F (80% homology), but recombinants containing the pED208 surface exclusion system excluded F poorly.

J Bacteriol, 1986 Jun, 166(3), 706 - 12
Contrasting mechanisms of envZ control of mal and pho regulon genes in Escherichia coli; Case CC et al.; The envZ11 missense mutation in the regulatory gene envZ pleiotropically repressed synthesis of OmpF, alkaline phosphatase, and several proteins of the maltose regulon . Procaine treatment of wild-type cells resulted in the same phenotype through an envZ+-mediated mechanism . Here we show that envZ11-procaine act differently on the mal and pho regulons . In the mal system, the expression of the positive regulator gene malT, measured as beta-galactosidase activity of a malT-lac+ operon fusion, was drastically reduced by procaine treatment or by the envZ11 mutation . In contrast, expression of the positive regulator of the pho regulon phoB was not reduced by procaine treatment . The products of the regulatory genes phoM, phoR, and phoU were also not required for procaine action . Procaine and envZ11 inhibited expression of only two products of the pho regulon, alkaline phosphatase and the PhoE porin . The conclusion that envZ11-procaine act differently on the mal and the pho regulons is supported by our ability to isolate second-site mutations with a Mal+ PhoA- phenotype in an envZ11 strain.

Exp Parasitol, 1986 Jun, 61(3), 369 - 72
Caenorhabditis elegans: comparisons of chemotactic behavior from monoxenic and axenic culture; Jansson HB et al.; Significant differences in chemotactic response of Caenorhabditis elegans were demonstrated for nematodes from monoxenic culture as compared to nematodes from axenic culture . These results support those of a previous study in which large differences in growth, development, behavior, and longevity were shown for C . elegans in comparative assays of the monoxenic and axenic regimes.

Virology, 1986 Jun, 151(2), 390 - 3
The spiroplasma virus 4 replicative form cloned in Escherichia coli transfects spiroplasmas; Pascarel-Devilder MC et al.; The replicative form (RF) of spiroplasma virus 4 (SpV4) has been purified from infected cells of Spiroplasma melliferum strain G1 by alkaline lysis followed by low melting point agarose gel electrophoresis . A partial restriction map has been established . The circular RF was linearized by cutting at the unique ClaI restriction site and has been cloned in Escherichia coli HB101 using the plasmid pBR328 as a vector . The recombinant plasmid was purified by equilibrium centrifugation in ethidium bromide-cesium chloride gradient . After ClaI endonuclease digestion, the inserted SpV4 RF DNA was recovered by low melting point agarose gel electrophoresis and was recircularized by ligation . The cloned SpV4 RF DNA was demonstrated to be infectious by transfection.

J Virol, 1986 Jun, 58(3), 893 - 9
A second protease of foot-and-mouth disease virus; Strebel K et al.; Foot-and-mouth disease virus (FMDV) genes are expressed as a polyprotein which is rapidly processed into the four primary cleavage products L, P1, P2, and P3 . In secondary cleavage reactions, these are further processed into the mature proteins . The FMDV L protein is located at the N terminus of the polyprotein and is the first gene product released from the nascent polyprotein . For analysis of its biological function, the L gene was mutated by site-directed mutagenesis of cloned cDNA . In vitro translation of in vitro transcripts of these DNAs and expression studies in Escherichia coli showed that the L mutants affect the processing of the viral polyprotein . The mutants isolated were partially or totally defective in processing the polyprotein at the L/P1 junction . These mutants could, however, be processed in the presence of the wild-type L protein . Furthermore, an antiserum directed against the L protein inhibited processing at the L/P1 cleavage site, so that the release of the L protein from the polyprotein was blocked . These data reveal that the L gene product represents a viral protease which catalyzes its own release from the nascent polyprotein.

J Biochem (Tokyo), 1986 Jun, 99(6), 1579 - 90
Determination of the initiation sites of transcription and translation of the uvrD gene of Escherichia coli; Yamamoto Y et al.; Prior to the analysis of transcription and translation, the nucleotide sequence of the uvrD gene and its neighboring regions was determined by the method of Maxam and Gilbert (Maxam & Gilbert (1980) Methods Enzymol . 65, 499-560) . Disagreement in 14 positions between the nucleotide sequence determined by us and that reported previously (Finch & Emmerson (1984) Nucl . Acids Res . 11, 5789-5799) was found . We reexamined these disputed regions . The initiation site of transcription of the uvrD gene was determined by analyzing the transcripts synthesized in vitro . It was found that transcription of the uvrD gene starts from the A nucleotide, which is the first one of the SOS box of the uvrD . The amino terminal sequence and the amino acid composition of the purified UvrD protein (helicase II) were determined . It was found that translation starts from the first ATG codon, which lies 77 nucleotides downstream from the initiation site of transcription . The amino acid composition of the purified UvrD protein agreed well with that deduced from the nucleotide sequence.

J Bacteriol, 1986 Jun, 166(3), 746 - 50
Deletion of the spf (spot 42 RNA) gene of Escherichia coli; Hatfull GF et al.; To investigate the function of spot 42 RNA, a small RNA of Escherichia coli, we constructed a strain in which spf, the structural gene for this RNA, is deleted . We achieved this by using a delta att phage lambda carrying a DNA fragment spanning the spf region but with a precise deletion of spf . By integration of this phage at the spf locus and by its subsequent excision, we were able to cross the spf deletion onto the bacterial chromosome . The fact that such a deletion could be obtained indicated that spf is not an essential gene . We did not observe any major defect in delta spf cells, although in one strain background the deletion caused a slight growth impairment.

EMBO J, 1986 Jun, 5(6), 1389 - 93
Two regulatory fim genes, fimB and fimE, control the phase variation of type 1 fimbriae in Escherichia coli; Klemm P; The expression of type 1 fimbriae in Escherichia coli is phase dependent, i.e . a cell is either completely fimbriated or bald . This phenomenon is due to the periodic inversion of a specific 300-bp DNA segment containing the promoter for the fimbrial subunit gene, fimA . The phase switch is controlled by the products of two regulatory genes, fimB and fimE, located upstream of fimA . The fimB and fimE proteins direct the phase switch into the 'on' and 'off' position, respectively . The DNA sequence of a 3000-bp region containing the two genes has been determined . The fimB and fimE proteins exhibit strong homology and have most likely originated by duplication of an ancestral gene . They are highly basic implying that they control the phase switch through interaction at the DNA level.

Proc Natl Acad Sci U S A, 1986 Jun, 83(12), 4219 - 22
Both ATP and the electrochemical potential are required for optimal assembly of pro-OmpA into Escherichia coli inner membrane vesicles; Geller BL et al.; Pro-OmpA is processed to OmpA by isolated inverted plasma membrane vesicles from Escherichia coli . In the presence of ATP and a membrane potential, 58% (+/- 13%) of the OmpA is sequestered in the vesicles . We sought to determine which of these two metabolic energy sources is used for protein translocation . The plasma membrane F1F0-ATPase is the central enzyme that interconverts the energy of membrane electrochemical potential and ATP . To separate the effects of these two forms of energy in vitro, the ATPase was inactivated, either by "stripping" the F1 from the membranes with low salt and EDTA or by using membrane vesicles derived from a strain without the atp operon . In each case, optimal translocation and processing of pro-OmpA required both a membrane potential and ATP . We conclude that ATP and membrane potential are separate requirements for bacterial protein export.

Pediatr Res, 1986 Jun, 20(6), 555 - 60
The immature rat small intestine exhibits an increased sensitivity and response to Escherichia coli heat-stable enterotoxin; Cohen MB et al.; Escherichia coli which elaborate heat stable enterotoxin (ST) are a major cause of endemic diarrhea in infants . The reason(s) for this increased susceptibility of infants to ST-mediated diarrhea is unknown . We investigated the possibility that the immature (14 and 21 day old) rat small intestine is more sensitive to ST than is the adult . Initially we found there was a 600-fold increased jejunal sensitivity to ST in the immature animals as measured by dose required for half maximal secretion . Also there was a greater jejunal secretory response in the immature animals (14 greater than or equal to 21 days old greater than adult) . To determine the cause for this increased sensitivity and secretory response to ST, we examined: 1) binding characteristics of 125I-ST to brush border membrane (BBM) receptors and 2) membrane bound guanylate cyclase activation by ST in both immature and adult rats . Our findings demonstrate that more ST receptors are present in jejunal BBM from 14- and 21-day-old rats than in jejunal BBM from adult rats (2.34 +/- 0.18, 2.85 +/- 0.82, and 0.79 +/- 0.13 X 10(12) receptors/mg BBM protein, respectively), while the affinity of the BBM receptor for ST is similar at all three ages in both jejunum and ileum . Furthermore, both the jejunum and ileum of the rats of all three ages revealed an equal sensitivity of guanylate cyclase to activation by ST . These findings suggest that the increased number of jejunal receptors in the immature rat may, in part, explain the increased sensitivity and secretory response observed in vivo.

J Biochem (Tokyo), 1986 Jun, 99(6), 1719 - 24
High temperature induction of a stringent response in the dnaK(Ts) and dnaJ(Ts) mutants of Escherichia coli; Itikawa H et al.; The cellular concentrations of ppGpp in the dnaK(Ts) and dnaJ(Ts) mutants of Escherichia coli were examined, since the thermosensitive RNA synthesis of these mutants is relaxed by an additional mutation in the relA gene . The results showed that ppGpp accumulated extensively in the dnaK(Ts) and dnaJ(Ts) mutants after a temperature shift up, reaching levels of 5 mM and 0.5 mM, respectively . This unusual accumulation of ppGpp was suppressed by the relA1 mutation, implying that it results from induction of a stringent response in these mutants at a nonpermissive temperature.

EMBO J, 1986 Jun, 5(6), 1383 - 8
Mutations affecting antigenic determinants of an outer membrane protein of Escherichia coli; Desaymard C et al.; The Escherichia coli LamB protein is located in the outer membrane . It is both a component of the maltose and maltodextrin transport system, and the receptor for phages lambda and K10 . It is a trimer composed of three identical polypeptide chains, each containing 421 residues . Six independent mutants have been isolated, in which the LamB protein is altered in its interaction with one or more monoclonal antibodies specific for regions of the protein that are exposed at the cell surface . Some of the mutations also altered the binding site for phage lambda . All of the mutations were clustered in the same region of the lamB gene, corresponding to residues 333-394 in the polypeptide . This and previous results strongly suggest that a rather large segment of the LamB polypeptide, extending from residue 315 to 401, is exposed at the outer face of the outer membrane . This segment would bear the epitopes for the four available anti-LamB monoclonal antibodies that react with the cell surface, and part of the binding site for phage lambda.

Vaccine, 1986 Jun, 4(2), 119 - 24
Synthesis of fusion proteins containing antigenic determinants of foot-and-mouth disease virus; Broekhuijsen MP et al.; Part of the genome of foot-and-mouth disease virus (FMDV) type 01,BFS, including the sequence encoding the capsid polypeptide VP1, was cloned in Escherichia coli following a new cloning strategy . The clone containing the VP1 sequence was used for the construction of two expression plasmids encoding VP1 fusion proteins . Subsequently, substantial amounts of the two VP1-beta-galactosidase fusion proteins, containing either one (amino acid region 140-160) or two (amino acid regions 140-160 and 200-213) antigenic determinants of the virus, were synthesized by E . coli bacteria . The protein containing the amino acid region 140-160 of VP1 fused to beta-galactosidase efficiently induced antibodies in rabbits specifically reacting with FMDV type 01,BFS . The same protein was also capable of eliciting neutralizing antibodies . The fusion protein containing both antigenic determinants did not efficiently induce antibodies reacting with FMDV.

J Med Virol, 1986 Jun, 19(2), 193 - 203
Human and monoclonal antibodies to hepatitis B core antigen recognise a single immunodominant epitope; Ferns RB et al.; Monoclonal antibodies were raised against hepatitis B core antigen (HBcAg) synthesized in Escherichia coli . They identified a single immunodominant determinant on both liver-derived and E . coli-derived HBcAg . Cross-inhibition studies demonstrated that HBsAg-containing human sera which contained antibodies to HBcAg (anti-HBc) together with either hepatitis B e antigen (HBeAg) or its antibody (anti-HBe) essentially only recognised the same single determinant as the murine antibodies . The identification of a single dominant HBcAg determinant may be important if future hepatitis B virus vaccines contain HBcAg.

J Bacteriol, 1986 Jun, 166(3), 945 - 58
Autogenous regulation of the gene for transcription termination factor rho in Escherichia coli: localization and function of its attenuators; Matsumoto Y et al.; We present evidence that the expression of rho is regulated by rho-dependent attenuation of transcription . Gene fusion analysis with nested series of deletions of rho indicated that the transcription of rho is attenuated in a rho-dependent manner in the leader region and that neither a read-through transcription from the upstream gene, trxA, nor a modulation of transcription initiation of the rho promoter is involved in the self-control of rho . S1 mapping and Northern hybridization analyses localized at least six transcription attenuation or termination sites in the region ranging from the 3' end of the trxA structural gene to the middle of the rho structural gene . Among them, the most upstream site overlapping the rho promoter sequence was assigned to the terminator for the trxA gene, and the second and third sites, mapping about 80 and 50 nucleotides upstream from the start codon of rho, were suggested to function as the major attenuation sites for regulation of the rho expression . Further, the start points of the trxA and rho RNAs were determined in an in vitro transcription system to be located 111 nucleotides (U) and 255 nucleotides (G) upstream from their respective start codons . These results necessitate revisions of previous predictions on the sites of transcriptional signals in the trxA and rho genes (S . Brown, B . Albrechtsen, S . Pedersen, and P . Klemm, J . Mol . Biol . 162:283-298, 1982; C.-J . Lim, D . Geraghty, and J . A . Fuchs, J . Bacteriol . 163:311-316, 1985; B.J . Wallace and S.R . Kushner, Gene 32:399-408, 1984).

J Bacteriol, 1986 Jun, 166(3), 1106 - 12
Incompatibility repressor in a RepA-like replicon of the IncFI plasmid ColV2-K94; Weber PC et al.; The replication region Rep1 of the IncFI plasmid ColV2-K94 was cloned on self-replicating restriction fragments . Rep1 was structurally and functionally homologous to the RepA replicon of IncFII R plasmids . Despite this close relationship, these two replication systems were compatible with each other . The nucleotide sequence of the copA incompatibility-replication control gene of Rep1 was determined and compared with the copA sequence of RepA . Six base changes were found in a 24-base-pair span of the copA gene; these may result in the formation of a new, more stable, 49-base stem-loop structure in the potential CopA RNA repressor molecule . We postulate that these alterations weaken the interaction between RNA transcripts of the Rep1 and RepA replicons.

Science, 1986 May 30, 232(4754), 1138 - 40
Brain glutamate decarboxylase cloned in lambda gt-11: fusion protein produces gamma-aminobutyric acid; Kaufman DL et al.; Glutamate decarboxylase (GAD; E.C . 4.1.1.15) converts glutamate to gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the vertebrate central nervous system . This report describes the isolation of a GAD complementary DNA clone by immunological screening of a lambda gt-11 brain complementary DNA expression library . The fusion protein produced by this clone catalyzes the conversion of glutamate to GABA and carbon dioxide, confirming its identity as GAD . Antibodies to beta-galactosidase remove GAD enzymatic activity from solution, showing that this activity is associated with the fusion protein . In immunoblotting experiments all three available antisera to GAD reacted with the fusion polypeptide and with two major polypeptides (molecular size, 60,000 and 66,000 daltons) in brain extracts.

Biochem Biophys Res Commun, 1986 May 29, 137(1), 468 - 73
An antibody-binding site in the native enzyme between amino acid residues 205-287 of the gamma-subunit of F1 from Escherichia coli; Ehrig K et al.; A monoclonal antibody was isolated specific for the isolated denatured gamma-subunit of F1 from Escherichia coli and binding to native F1 . The binding site of this antibody was identified between amino acid residues 205-287 of the polypeptide chain thus being located at the surface of the F1 complex.

Nucleic Acids Res, 1986 May 27, 14(10), 4095 - 111
Studies on the kinetic sequence of in vitro ribosome assembly using cibacron blue F3GA as a general assembly inhibitor; Datta D et al.; We have found that all E . coli ribosomal proteins strongly bind to an agarose affinity column derivatized with the dye Cibacron Blue F3GA . We have also shown that the capacity to bind the dye is lost when the proteins are organized within the structure of the ribosome or are members of pre-formed protein-RNA complexes . We conclude that the binding of ribosomal proteins to this dye involves specific protein-RNA recognition sites . These observations led us to discover that Cibacron Blue can be used to inhibit in vitro ribosome assembly at any stage of the assembly process . This has allowed us to determine a kinetic order of ribosome assembly.

Nucleic Acids Res, 1986 May 27, 14(10), 4253 - 66
Tn10 tet operator mutations affecting Tet repressor recognition; Wissmann A et al.; The effect of single base pair alterations of the Tn10 encoded tet operator on recognition of Tet repressor was studied in vivo using a repressor titration system and in vitro by dissociation rate determinations of the respective complexes . Both methods reveal that the two operators, O1 and O2, which are in a tandem arrangement in the wild type, are recognized with a two-fold different affinity when separated . Studies on synthetic operator sequences indicate that the Tet repressor binds with higher affinity to the non-palindromic O2 wildtype than to the respective palindromic sequences . The in vivo repressor titration system links the expression of lacZ to the affinity of tet operator to Tet repressor . It was used to isolate tet operator mutations with reduced affinity to the repressor . The in vivo and in vitro obtained results with these mutants agree quantitatively and indicate, that the GC base pairs at positions 2, 6, and 8 are involved in interaction with the Tet repressor . Their importance for recognition decreases in that order . Transitions at position 7 of the tet operator show smaller effects on recognition than transversions.

Nucleic Acids Res, 1986 May 27, 14(10), 4207 - 27
Activation of the Bradyrhizobium japonicum nifH and nifDK operons is dependent on promoter-upstream DNA sequences; Alvarez-Morales A et al.; Previous analysis of B . japonicum nifH'- and nifD'-'lacZ translational fusions showed that these promoters could be activated by the K . pneumoniae nifA plus the E . coli ntrA gene products . To study the functions of the DNA 5' to these promoters, plasmids carrying deletions in this region were constructed and analyzed in vivo in a heterologous system consisting of an E . coli (NtrA+) background with a plasmid that constitutively expresses the K . pneumoniae nifA gene . Activation of the B . japonicum promoters was completely dependent on sequences located between positions -165 and -100, relative to the start of transcription . Some of the nifD deletion-fusions were mobilized to the wild-type B . japonicum and the exconjugants tested in an ex planta micro-aerobic system, and also used to infect soybean seedlings . The time course of derepression was followed by assaying beta-galactosidase activity from samples withdrawn from the microaerobic cultures or from root-nodule extracts . The results conclusively show that in the homologous system the sequences upstream of the promoter are required to achieve wild-type activity.

Nucleic Acids Res, 1986 May 27, 14(10), 3995 - 4007
Highly selective chemical modification of cruciform loops by diethyl pyrocarbonate; Furlong JC et al.; Diethyl pyrocarbonate reacts with the single-stranded loops of cruciform structures with great selectivity . Adenine bases are carbethoxylated, as a result of which the backbone may be cleaved with piperidine, and the level of chemical modification at each base may be determined . We have studied the ColE1 and (A-T)34 cruciforms of pColIR315 and pXG540 . In each case we observe maximal modification at the most central adenosine of the loop, and an overall pattern of modification corresponding to a total loop size of about six bases . The results may be interpreted in terms of a model in which the loop has a defined tertiary structure . No modification was detected at either cruciform four-way junction, suggesting that this region is fully base-paired.

Biochim Biophys Acta, 1986 May 27, 867(1-2), 45 - 56
The effect of the trp repressor from Escherichia coli on the structure and dynamics of dA20dT20; Lane AN et al.; We have determined the effect of the tryptophan (trp) repressor from Escherichia coli on the structure and dynamics of dA20dT20 . The structure was determined using time-dependent nuclear Overhauser effects and spin-lattice relaxation times . The deoxyribose conformation is near C3' endo for the thymine residues, and a mixture of about 30% C3' endo and 70% C2' endo for the adenine residues . The glycosidic torsion angles are -50 degrees for T and -60 degrees for A . The roll is 20 degrees and the propellor twist is about 29 degrees . The conformation is consistent with recent calculations (Rao, K . and Kollman, P.A . (1985) J . Am . Chem . Soc . 107, 1507-1511) . The rate constant for exchange of the imino protons is similar to that usually found for AT base-pairs, with an activation energy of 20 +/- 2 kcal/mol, and an activation entropy of 17 +/- 7 cal/mol per K . The repressor greatly retards the exchange of imino protons, and the activation energy increases to 38 kcal/mol . There are small changes in the structure of the DNA on forming the complex, with the adenine and thymidine residues becoming more similar in conformation.

Biochim Biophys Acta, 1986 May 27, 867(1-2), 24 - 30
Relationship between DNA acid-solubility and frequency of single-strand breaks near apurinic sites; Bricteux-Gregoire S et al.; Using {32P}DNA alkylated with {3H}methyl methanesulfonate, depurinated by heating at 50 degrees C for various periods, then treated with sodium hydroxide, a table was constructed giving the DNA fraction soluble in 5% perchloric acid at 0 degree C as a function of the frequency of strand breaks . The alkaline treatment placed a break near each apurinic site; the apurinic sites were counted in two ways which gave consonant results: by the loss of {3H}methyl groups and by reaction with {14C}methoxyamine . The 32P label of DNA was used to measure the acid-solubility.

Nucleic Acids Res, 1986 May 27, 14(10), 4009 - 23
Multiple crosslinks of proteins S7, S9, S13 to domains 3 and 4 of 16S RNA in the 30S particle; Hajnsdorf E et al.; Functionally active 70S ribosomes containing 4-thiouracil in place of uracil (substitution level 2%) were prepared by an in vivo substitution method . RNA-protein crosslinks were introduced by 366 nm photoactivation of 4-thiouracil in the purified 30S subunits . Seven single stranded M13 probes containing rDNA inserts complementary to domains 3 and 4 of 16S RNA were constructed . These inserts approximately 100 nucleotides long starting at nucleotide 868 and ending at the 3' OH terminus were used to select contiguous RNA sections . The proteins covalently linked to each selected RNA section were identified by 2D gel electrophoresis . Proteins S7, S9, S13 were shown to be efficiently crosslinked to multiple sites belonging to both domains.

J Biol Chem, 1986 May 25, 261(15), 6893 - 9
Sugar binding properties of the melibiose permease in Escherichia coli membrane vesicles . Effects of Na+ and H+ concentrations; Damiano-Forano E et al.; The substrate binding reaction of the melibiose carrier was analyzed by studying {3H}p-nitrophenyl-alpha-D-galactopyranoside (Np alpha Gal) binding to de-energized membrane vesicles from Escherichia coli RA11 as a function of H+ and Na+ (or Li+) concentrations . The data indicate first that Na+ (or Li+) activates Np alpha Gal binding at all pH values tested between 5.5 and 7.5 and second that H+ inhibits the Na+ (or Li+)-dependent activating effect on Np alpha Gal binding . Similar conclusions were drawn for melibiose and methyl-1-thio-beta-D-galactoside binding activities . Unexpectedly, Np alpha Gal, melibiose, and methyl-1-thio-beta-D-galactoside binding activities are insensitive to a variety of SH reagents which completely block transport activity . Quantitative analysis of the effects of H+ and Na+ ions on the parameters of Np alpha Gal binding show that 1) the maximal number of binding sites is constant irrespective of the concentration of Na+ or Li+ in the range of pH between 6 and 7.5 and 2) the apparent dissociation constant for Np alpha Gal binding varies with both Na+ and H+ according to a relation described by a linear combination of the concentration of H+ and the reciprocal of Na+ concentration . These results can be accounted for by a model which assumes sequential binding of the cation and substrate in this order and competition between Na+ and H+ for a common cationic binding site on the porter . Predictions of the proposed binding model for a carrier mechanism catalyzing sugar transport according to a Na+ symport mode or a H+ symport mode are discussed.

J Biol Chem, 1986 May 25, 261(15), 6765 - 71
Phosphoribosylpyrophosphate synthetase of Escherichia coli . Properties of the purified enzyme and primary structure of the prs gene; Hove-Jensen B et al.; Phosphoribosylpyrophosphate (P-Rib-PP) synthetase of Escherichia coli has been purified to near homogeneity from a strain harboring the prs gene, encoding P-Rib-PP synthetase, on a multicopy plasmid . Analysis of the enzyme showed that it required inorganic phosphate for activity and for stability . Magnesium ions were required both as a complex with the substrate ATP and as a free cation . P-Rib-PP synthetase activity was inhibited strongly by ADP . Kinetic analysis indicated multiple sites of action of ADP . In addition apparent substrate inhibition was exerted by ribose 5-phosphate in the presence of ADP . The nucleotide sequence of the E . coli prs gene has been determined and the coding segment established . The deduced amino acid sequence of P-Rib-PP synthetase contained 314 amino acid residues and the molecular weight was calculated as 34,060 . The initiation site of transcription was determined . This site was preceded by well conserved -10 and -35 consensus sequences (pdT-dA-dG-dA-dA-dT and pdT-dT-dG-dA-dT-dG, respectively) . The transcription initiation site preceded the potential translation initiation site by 302 nucleotides . Transcription terminated approximately 35 nucleotides downstream from the UAA translation stop codon, within a Thy-rich region following an inverted repeat sequence, indicative of an rho-independent transcription terminator.

J Biol Chem, 1986 May 25, 261(15), 7070 - 5
Mutational replacements of conserved amino acid residues in the beta subunit resulted in defective assembly of H+-translocating ATPase (F0F1) in Escherichia coli; Noumi T et al.; Mutant genes for the beta subunit of H+-translocating ATPase (F0F1) were cloned from Escherichia coli strains isolated in this laboratory . Determination of their nucleotide sequence revealed four missense mutations (strain KF39, Glu-41----Lys; strain KF16 and KF42, Glu-185----Lys; strain KF48, Gly-223----Asp; KF26 and 4 other strains, Ser-292----Phe) . Two nonsense mutants (strain KF40, Gln-361----end; strain KF20, Gln-397----end) were also identified . Glu-41, Glu-185, and Ser-292 are conserved in the amino acid sequences of the beta subunits so far studied, and Gly-223, Gln-361, and Gln-397 are conserved in beta subunits from bacteria and mitochondria, but not in those from chloroplasts . The amounts of F1 subunits in the membranes of these strains were studied by immunochemical assay and two-dimensional gel electrophoresis . In the mutants studied, the amounts of alpha and beta subunits in the membranes were 69-21 and 46-2%, respectively, of the amounts in wild-type membranes, the amount depending on the strain . No delta and epsilon subunits were detected in membranes of a missense mutant KF16, although reduced amounts of alpha and beta subunits could be detected, suggesting that the F1 portion may not be connected to F0 through the delta and epsilon subunits . The altered residues in missense mutants or missing domains in nonsense mutants may be important for the subunit-subunit interactions or assembly of the entire complex . Genetic experiments on introduction of suppressor tRNA into strains KF40 and KF20 suggested that F1 could be active even when residue 361 or 397 was replaced by a Ser, Leu, or Tyr residue.

J Biol Chem, 1986 May 25, 261(15), 6924 - 32
Localization of two epitopes of protein L7/L12 to both the body and stalk of the large ribosomal subunit . Immune electron microscopy using monoclonal antibodies; Olson HM et al.; Four molecules of ribosomal protein L7/L12 are found as two dimers on the Escherichia coli 50 S ribosomal subunit . Immune electron microscopy using monoclonal antibodies directed against two epitopes of protein L7/L12 has allowed placement of elements of each dimer . One monoclonal antibody, directed against a determinant in the COOH-terminal domain, allows localization of two identical determinants at or near the end of the subunit stalk . The same antibody was used to place two additional determinants at the periphery of stalkless subunits, in an area from which a stalk might be expected to project . A second antibody, directed against an epitope in the amino-terminal portion of L7/L12, caused loss of stalks from the 50 S subunits . The micrographs showed symmetrical oligometric complexes of the dissociated dimeric protein with bivalent antibody . Antibodies were also seen to bind to the body of stalkless subunits, in a region near the COOH-terminal sites . The results are explained by a model in which one dimer of protein L7/L12 exists in a folded conformation on the subunit body and the second dimer occurs in an extended conformation in the subunit stalk.

J Biol Chem, 1986 May 25, 261(15), 6919 - 23
The selective release of one of the two L7/L12 dimers from the Escherichia coli ribosome induced by a monoclonal antibody to the NH2-terminal region; Tewari DS et al.; Two monoclonal antibodies against different epitopes in Escherichia coli ribosomal protein L7/L12 were prepared and characterized as reported previously (Sommer, A., Etchison, J.R., Gavino, G., Zecherle, N., Casiano, C., and Traud, R.R . (1985) J . Biol . Chem . 260, 6522-6527) . Both antibodies strongly inhibited polyuridylic acid-directed polyphenylalanine synthesis, ribosome-dependent GTPase activity, and the binding of elongation factor G to the ribosome at mole ratios over ribosomes of 4:1 or less . One epitope was shown to be within residues 1-73 (Ab 1-73) and the other within 74-120 (Ab 74-120) . Incubation of 50 S ribosomal subunits or 70 S ribosomes with Ab 1-73, but not with Ab 74-120, leads to a partial loss of L7/L12 from the particle with no loss of any other protein . The experiment was repeated with ribosomes reconstituted with pure radioactive L7/L12 of determined specific activity in order to quantify the L7/L12 in the antibody-treated particle . The protein-deficient core particles isolated by sucrose gradient centrifugation after incubation with Ab 1-73 were found to contain, on average, two copies of L7/L12 and one Ab 1-73 . The constancy of this stoichiometry in many experiments and the demonstration of Ab 1-73 on all particles indicate the presence of a homogeneous population of ribosomes, each with only one of the two L7/L12 dimers originally present . The results show a difference in the interactions of the two dimers with the ribosome and present a means of preparing ribosomes with one dimer in a specific binding site . The accompanying paper (Olson, H.M., Sommer, A., Tewari, D . S., Traut, R.R., and Glitz, D.G . (1986) J . Biol . Chem . 261, 6924-6932) shows by immune electron microscopy the location of the two antibody-binding sites and the effect of Ab 1-73 on structure.

Nature, 1986 May 22-28, 321(6068), 429 - 31
Amino-acid sequence of the beta-subunit of the (Na+ + K+)ATPase deduced from a cDNA; Shull GE et al.; The sodium/potassium-dependent ATPase {(Na+ + K+)ATPase}, which establishes and maintains the Na+ and K+ gradients across the plasma membrane of animal cells, consists of two subunits, alpha and beta . Complementary DNA clones encoding the catalytic (alpha) subunit of sheep kidney and Torpedo californica electroplax enzymes have previously been isolated and characterized . However, there is little information concerning the primary structure of the beta-subunit, a glycoprotein of unknown function and relative molecular mass (Mr) approximately 55,000 (ref . 3) . Here we describe the isolation and characterization of a cDNA clone containing the entire coding region of the beta-subunit of the sheep kidney (Na+ + K+)ATPase . We also discuss structural aspects of the protein and present evidence for a possible evolutionary relationship with the KdpC subunit of the Escherichia coli K+-ATPase.

Biochemistry, 1986 May 20, 25(10), 3064 - 72
Xenopus nucleoplasmin: egg vs . oocyte; Sealy L et al.; Nucleoplasmin has been purified from either oocytes or unfertilized eggs of the frog, Xenopus laevis . We find that the pentameric form of egg nucleoplasmin exhibits an apparent molecular mass approximately 15 000 daltons larger than its oocyte counterpart upon sodium dodecyl sulfate (SDS)-acrylamide gel electrophoresis . Egg nucleoplasmin monomers are more heterogeneous, substantially more acidic, and overall larger in apparent molecular weight than oocyte nucleoplasmin monomers when analyzed by isoelectric focusing or SDS gel electrophoresis . Protease digestions indicate that the structural differences between egg and oocyte nucleoplasmin are primarily confined to the N-terminal halves of the proteins . The structural diversity observed is accompanied by a difference in the ability of nucleoplasmin from the two sources to act as a nucleosome assembly agent in vitro . Egg nucleoplasmin efficiently promotes the formation of nucleosomes onto circular pBR322 DNA in vitro at physiological ionic strength and at physiological histone:DNA ratios, while oocyte nucleoplasmin is markedly deficient in serving as an in vitro chromatin assembly agent under all conditions which we have tested . Treatment of egg nucleoplasmin in vitro with alkaline phosphatase demonstrates that the structural diversity between egg and oocyte nucleoplasmin results primarily from extensive additional phosphorylation of the egg protein . The relevance of nucleoplasmin phosphorylation in leading to differences in the chromatin assembly activity of this protein both in vitro and in vivo is considered.

Biochemistry, 1986 May 20, 25(10), 2770 - 7
Circular dichroism and 500-MHz proton magnetic resonance studies of the interaction of Escherichia coli translational initiation factor 3 protein with the 16S ribosomal RNA 3' cloacin fragment; Wickstrom E et al.; The RNA helix destabilizing properties of Escherichia coli initiation factor 3 protein (IF3), and its affinity for an evolutionarily conserved sequence at the 3' end of 16S rRNA, led us to examine the details of the protein-nucleic acid interactions upon IF3 binding to the 49-nucleotide 3'-terminal cloacin DF13 fragment of 16S rRNA by studying the circular dichroism (CD) and proton magnetic resonance spectra of the RNA, the protein, and their complex . In a physiological tris(hydroxymethyl)aminomethane buffer, where the interaction is primarily nonionic and sequence specific, addition of IF3 decreases the RNA 268-nm CD peak hyperbolically by 19% to an end point of about one IF3 per RNA strand . The titration curve is best fit by an association constant of (1.80 +/- 0.05) X 10(7) M-1, within the range estimated by a nuclease mapping study of the same system {Wickstrom, E . (1983) Nucleic Acids Res . 11, 2035-2052} . In a low-salt phosphate buffer without Mg2+, where the interaction is primarily ionic and nonspecific, titration with IF3 decreases the peak CD sigmoidally by 35% to an end point of two IF3 per strand . The titration curve is best fit by an intrinsic association constant of (1.7 +/- 0.7) X 10(6) M-1 for each IF3 and a cooperativity constant of 33 +/- 6 . In a physiological phosphate buffer lacking Mg2+, the dispersion of aromatic proton magnetic resonance peaks and upfield-shifted methyl proton resonances indicates a high degree of secondary and tertiary structure in the protein . In an equimolar mixture of IF3 and RNA cloacin fragment, several changes in identifiable IF3 and RNA resonances are observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1986 May 20, 25(10), 2756 - 65
Binding of Escherichia coli ribosomal protein S8 to 16S rRNA: kinetic and thermodynamic characterization; Mougel M et al.; A sensitive membrane filter assay has been used to examine the kinetic and equilibrium properties of the interactions between Escherichia coli ribosomal protein S8 and 16S rRNA . In standard conditions (0 degrees C, pH 7.5, 20 mM Mg2+, 0.35 M KCl) the apparent association constant is 5 +/- 0.5 X 10(-7) M-1 . The interaction is highly specific, and the kinetics of the reaction are consistent with the apparent association constant . Nevertheless, the rate of association is somewhat slower than that expected for a diffusion-controlled reaction, suggesting some steric constraint . The association is only slightly affected by temperature (delta H = -1.8 kcal/mol) . The entropy change {delta S = +29 cal/(mol K)} is clearly the main driving force for the reaction . The salt dependence of Ka reveals that five ions are released upon binding at pH 7.5 and in the presence of 10 mM magnesium . The substitution of various anions for Cl- has an appreciable effect on the magnitude of Ka, following the order CH3COO- greater than Cl- greater than Br-, thus indicating the existence of anion binding site(s) on S8 . An equal number of ions were released when Cl- was replaced by CH3COO-, but the absence of anion release upon binding cannot be excluded . On the other hand, the free energy of binding appears not to be exclusively electrostatic in nature . The effect of pH on both temperature and ionic strength dependence of Ka has been examined . It appears that protonation of residue(s) (with pK congruent to 9) increases the affinity via a generalized charge effect . On the other hand, deprotonation of some residue(s) with a pK congruent to 5-6 seems to be required for binding . Furthermore, the unique cysteine present in S8 was shown to be essential for binding.

Biochemistry, 1986 May 20, 25(10), 2749 - 56
tRNA topography during translocation: steady-state and kinetic fluorescence energy-transfer studies; Paulsen H et al.; The distances between the anticodon loops of fluorescent tRNAPhe bound to the E site and to either the A or the P site of poly(U)-programmed Escherichia coli ribosomes were measured by fluorescence energy transfer . Donor and acceptor molecules were wybutine and proflavin, respectively, both located 3' to the anticodon of tRNAPhe . The anticodon loops were found to be separated by 42 +/- 10 A (A to E site) and 34 +/- 8 A (P to E site) . The latter distance is much larger than the one measured between the anticodon loops of A and P site bound tRNAs {24 +/- 4 A; Paulsen, H., Robertson, J . M., & Wintermeyer, W . (1983) J . Mol . Biol . 167, 411-426}, rendering unlikely simultaneous codon-anticodon interaction in the P and E sites . In kinetic stopped-flow measurements, the energy transfer between the anticodon loops of the tRNA molecules was followed during translocation . The transfer efficiency decreases in three steps with apparent rate constants on the order of 1, 0.1, and 0.01 s-1 . The fast step is ascribed to the simultaneous displacement of the deacylated tRNAPhe out of the P site and of the N-AcPhe-tRNAPhe from the A site to the P site . The distance between the anticodon loops does not change appreciably during this reaction . A significant separation of the two tRNAs occurs during the intermediate and the slow steps . The latter most likely represents a rearrangement of the posttranslocation complex containing both tRNA molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1986 May 20, 25(10), 2941 - 5
Inhibition of Euglena gracilis and wheat germ zinc RNA polymerases II by 1,10-phenanthroline acting as a chelating agent; Mazus B et al.; Copper complexes of 1,10-phenanthroline (OP-Cu) hydrolyze DNA {D'Aurora, V., Stern, A . M., & Sigman, D . S . (1978) Biochem . Biophys . Res . Commun . 80, 1025-1032; Marshall Pope, L., Reich, K . A., Graham, D . R., & Sigman, D . S . (1982) J . Biol . Chem . 257, 12121-12128} . This reaction has been studied to determine whether the 1,10-phenanthroline (OP) inhibition of the activity of RNA and DNA polymerases is the result of template hydrolysis or the chelation of a metal associated with and essential to the function of these enzymes . Addition of 4',6-diamino-2-phenylindole dihydrochloride (DAPI) to DNA generates a fluorescence signal with a linear increase of the intensity over a broad range of DNA concentrations from 0 to 100 micrograms/mL . The progress of hydrolysis of DNA by DNase I or OP (2 mM) is monitored by the time-dependent decrease in DAPI-induced fluorescence . In the presence of OP, the rate of hydrolysis increases as the Cu2+ concentration in the reaction mixture rises from 10(-8) to 10(-5) M . The rate differs for each nucleic acid template used; hydrolysis of poly(dA-dT) greater than denatured DNA greater than double-stranded DNA . However, millimolar amounts of OP do not hydrolyze the template even in the presence of Cu2+ (10(-6) M) when DNA is complexed with either Escherichia coli DNA polymerase I or Euglena gracilis or wheat germ RNA polymerase II . Under the same conditions, OP inhibits the activity of both varieties of RNA polymerase II with pKi's of 3.4 and 3.0, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1986 May 20, 189(2), 293 - 303
Mini-F E protein: the carboxy-terminal end is essential for E gene repression and mini-F copy number control; Bex F et al.; Mini-F is a segment of the conjugative plasmid F consisting of two origins of replication flanked by regulatory regions, which ensure a normal control of replication and partitioning . Adjacent to the ori-2 origin is a complex coding region that consists of the E gene overlapped by three open reading frames with the coding potential for 9000 Mr polypeptides here designated 9 kd-1, 9 kd-2 and 9 kd-3 . In this paper, we show that open reading frame 9 kd-3 is preceded by active promoter and Shine-Dalgarno sequences . The E coding region specifies: an initiator of replication, which acts at the ori-2 site; a function that negatively regulates the expression of the E gene; and a function involved in mini-F copy number control . To assign one of these functions to one of the overlapping coding sequence, we have isolated, characterized and sequenced mutations mapping in the E coding region . In this paper, we analyse two mutations (cop5 and pla25) that abolish the repression of the E gene . As these mutations affect the primary structure of protein E itself but not the 9 kd polypeptides, we conclude that protein E takes part in the negative regulation of its own synthesis . In addition, the localization of the cop5 and pla25 mutations indicates that the carboxy-terminal end of the E protein is involved in the autorepression function . The cop5 mutation causes an eightfold increase of the mini-F copy number . The pla25 mutation leads to the inability of the derived mini-F plasmid to give rise to plasmid-harbouring bacteria . The ways in which the cop5 and pla25 mutations may lead to such phenotypes are discussed in relation to the different functions mapping in the E coding sequence.

J Mol Biol, 1986 May 20, 189(2), 273 - 84
Mechanisms of spontaneous mutagenesis: an analysis of the spectrum of spontaneous mutation in the Escherichia coli lacI gene; Schaaper RM et al.; We have obtained via DNA sequence analysis a spectrum of 174 spontaneous mutations occurring in the lac I gene of Escherichia coli . The spectrum comprised base substitution, frameshift, deletion, duplication and insertion mutations, of which the relative contributions to spontaneous mutation could be estimated . Two thirds of all lacI mutations occurred in the frameshift hotspot site . An analysis of the local DNA sequence suggested that the intensity of this hotspot may depend on structural features of the DNA that extend beyond those permitted by the repeated tetramer at this site . Deletions comprised the largest non-hotspot class (37%) . They could be divided into two subclasses, depending on whether they included the lac operator sequence; the latter was found to be a preferred site for deletion endpoints . Most of the deletions internal to the lacI gene were associated with the presence of directly or invertedly repeated sequences capable of accounting for their endpoints . Base substitutions comprised 34% of the non-hotspot events . Unlike the base substitution spectrum obtained via nonsense mutations, G . C----A . T transitions do not predominate . A new base substitution hotspot was discovered at position +6 in the lac operator; its intensity may reflect specific features of the operator DNA . IS1 insertion mutations contributed 12% of the non-hotspot mutations and occurred dispersed throughout the gene in both orientations . Since the lacI gene is not A + T-rich, the contribution of IS1 insertion to spontaneous mutation in general might be underestimated . Single-base frameshift mutations were found only infrequently . In general, they did not occur in runs of a common base . Instead, their occurrence seemed based on the "perfection" of direct or inverted repeats in the local DNA sequence . Three (tandem) duplication events were recovered . No repeated sequences were found that might have determined their endpoints.

Biochemistry, 1986 May 20, 25(10), 2965 - 74
Effects of the phenylalanine-22----leucine, glutamic acid-49----methionine, glycine-234----aspartic acid, and glycine-234----lysine mutations on the folding and stability of the alpha subunit of tryptophan synthase from Escherichia coli; Beasty AM et al.; The effects of four single amino acid replacements on the stability and folding of the alpha subunit of tryptophan synthase from Escherichia coli have been investigated by ultraviolet differences spectroscopy . In previous studies {Miles, E . W., Yutani, K., & Ogasahara, K . (1982) Biochemistry 21, 2586}, it had been shown that the urea-induced unfolding at pH 7.8, 25 degrees C, proceeds by the initial unfolding of the less stable carboxyl domain (residues 189-268) followed by the unfolding of the more stable amino domain (residues 1-188) . The effects of the Phe-22----Leu, Glu-49----Met, Gly-234----Asp, and Gly-234----Lys mutants on the equilibrium unfolding process can all be understood in terms of the domain unfolding model . With the exception of the Glu-49----Met replacement, the effects on stability are small . In contrast, the effects of three of the four mutations on the kinetics of interconversion of the native form and one of the stable partially folded intermediates are dramatic . The results for the Phe-22----Leu and Gly-234----Asp mutations indicate that these residues play a key role in the rate-limiting step . The Glu-49----Met mutation increases the stability of the native form with respect to that of the intermediate but does not affect the rate-limiting step . The Gly-234----Lys mutation does not affect either the stability or the kinetics of folding for the transition between native and intermediate forms . The changes in stability calculated from the unfolding and refolding rate constants agree quantitatively with those obtained from the equilibrium data . When considered with the results from a previous study on the Gly-211----Glu replacement {Matthews, C . R., Crisanti, M . M., Manz, J . T., & Gepner G . L . (1983) Biochemistry 22, 1445}, it can be concluded that the rate-limiting step in the conversion of the intermediate to the native conformation involves either domain association or some other type of molecule-wide phenomenon.

Biochemistry, 1986 May 20, 25(10), 2778 - 81
Substitution of glutamine-60 with glutamic acid causes the lac permease of Escherichia coli to become temperature sensitive; Sarkar HK et al.; The lac Y gene of Escherichia coli was modified by oligonucleotide-directed, site-specific mutagenesis so that Gln-60 is replaced with Glu . Although the replacement introduces a negative charge into a putative hydrophobic, transmembrane alpha-helical segment of the lac permease, lactose/H+ symport is unimpaired . However, the modified permease is more susceptible to heat inactivation . That is, upon incubation at 45 degrees C, Glu-60 permease loses activity with a t1/2 of 20 min relative to a t1/2 of 50 min with wild-type permease.

Biochemistry, 1986 May 20, 25(10), 3050 - 5
Differential effects of captan on DNA polymerase and ribonuclease H activities of avian myeloblastosis virus reverse transcriptase; Freeman-Wittig MJ et al.; Captan was used as an inhibitor of avian myeloblastosis virus reverse transcriptase to study the polymerase and RNase H catalytic activities . With purified enzyme, RNase H activity was 10-fold more sensitive to captan than was either the DNA-dependent or RNA-dependent DNA polymerase activity . Inhibition of the RNA-dependent polymerase activity could be prevented by dTTP . Conversely, inhibition of this polymerase activity was enhanced by template/primer . The calculated KdTTP of the uninhibited reaction was 5.6 microM . Kinetic studies allow for the proposition of a model for the interaction of captan with the polymerase active center . RNase H activity showed a sigmoidal relationship between activity and substrate concentration . Nuclease activity decreased in Vmax with no change in the Hill coefficient in the presence of captan . Addition of dithiothreitol to the incubation cocktail prevented inhibition by captan of both RNA-dependent polymerase and RNase H activities, suggesting that the (trichloromethyl)thio moiety of captan is involved in the inhibitory action . Captan inhibition suggests the presence of essential amino residues in both polymerase and RNase H active centers.

J Biol Chem, 1986 May 15, 261(14), 6450 - 3
High-level overexpression, rapid purification, and properties of Escherichia coli tRNA nucleotidyltransferase; Cudny H et al.; The cloned Escherichia coli cca gene, described in the accompanying paper (Cudny, H., Lupski, J . R., Godson, G . N., and Deutscher, M . P . (1986) J . Biol . Chem . 261, 6444-6449), has been used to construct strains that overproduce tRNA nucleotidyltransferase, the enzyme that synthesizes the CCA terminus of tRNA . Strain UT481 (pEC4), which contains a 1.9-kilobase cca gene insert in plasmid pUC8, overproduces the enzyme by about 100-150-fold, probably under the control of the cca gene promoter . A second strain, containing a plasmid with a 1.5-kilobase insert, overproduces tRNA nucleotidyltransferase by about 650-fold, to a level of about 3-4% of the soluble cell protein . In this case, overexpression was dependent on the lac promoter of the plasmid . A rapid, two-step procedure was developed to purify large amounts of the enzyme from strain UT481 (pEC4) that was about 40% pure, free of ribonucleases, and suitable for use as a reagent for modification of tRNA molecules . Preparation of milligram quantities of homogeneous tRNA nucleotidyltransferase was accomplished by two further chromatographic steps . The structural and catalytic properties of this purified enzyme were similar to those from partially purified preparations previously described . The availability of large amounts of pure tRNA nucleotidyltransferase will not permit a variety of structural and functional studies of the enzyme that previously were not possible.

J Biol Chem, 1986 May 15, 261(14), 6272 - 8
Pressure dependence of equilibria and kinetics of Escherichia coli ribosomal subunit association; Pande C et al.; Equilibria and rates were observed over the ranges 1-1600 atm, 3-10 mM Mg2+, at 60 mM NH4Cl, pH 7.5, 20 degrees C, by light scattering . The main reaction is accurately represented at all conditions by the following phenomenological equations . 30 S + 50 S = 70 S, KA70 = ka/kd = {70 S}/{30 S}{50 S} The equilibrium constants obey simple rules: the volume of association, delta VA0, has the constant value 242 +/- 9 ml/mol, independent of pressure, at all Mg2+ concentrations; the derived values of log KA70 at 1 atm increase linearly with log {Mg2+} at a slope of 7.5 . In contrast, the rate constants show a clear break at 6 mM Mg2+: below 6 mM, log ka decreases with pressure with a delta Va of 105 +/- 9 ml/mol and increases with log {Mg2+} at a slope of 4.9; above 6 mM, these values are halved; a split can actually be seen at 6 mM Mg2+, near 500 atm . The usual two-step mechanism for second order reactions in solution, which would insert a 70 S' species, either an encounter complex or a true low concentration steady state intermediate, into the above equation can accommodate these results: as {Mg2+} increases, the rate of transformation of 70 S' into 70 S finally predominates over the rate of dissociation of 70 S' into subunits . The bulk of the pressure effects and all of the {Mg2+} dependence arise from the progressive increase in delta GA0 (electrostatic) that occurs when 30, 50, and 70 S particles all lose equivalent fractions of their internal Mg2+ in response to increases in pressure or decreases in {Mg2+}.

J Biol Chem, 1986 May 15, 261(14), 6141 - 4
Production of pea lectin in Escherichia coli; Stubbs ME et al.; In order to explore the molecular basis for the glycopeptide specificity of legume lectins, we have developed an experimental system in which specific amino acid alterations can be introduced into the carbohydrate binding site of pea lectin . This system is based on the production of pea lectin in Escherichia coli . The plasmid coding for the lectin was constructed from two lectin cDNA sequences isolated from Pisum sativum seeds (Higgins, T . J . V., Chandler, P . M., Zurawski, G., Button, S . C., and Spencer, D . (1983) J . Biol . Chem . 258, 9544-9549) and an expression vector based on the gene for the outer membrane lipoprotein of E . coli (Nakamura, K., and Inouye, M . (1982) EMBO J . 1, 771-775) . The lectin is produced as a single polypeptide chain and forms insoluble aggregates in E . coli cells (2-5 mg/liter) . Functional lectin is recovered by solubilization of the aggregates in guanidinium hydrochloride, renaturation in the presence of MnCl2 and CaCl2, and affinity purification on Sephadex . This procedure yields a homogeneous 28,000-dalton protein . Comparison of the recombinant lectin with natural pea lectin in an inhibition of hemagglutination assay demonstrated that there is no detectable difference in the carbohydrate binding properties of the two lectins.

Nature, 1986 May 15-21, 321(6067), 213 - 9
Changing the identity of a transfer RNA; Normanly J et al.; A leucine transfer RNA has been transformed into a serine transfer RNA by changing 12 nucleotides . This result indicates that a limited set of residues determine tRNA identity.

Eur J Biochem, 1986 May 15, 157(1), 107 - 14
Mechanism of the phosphorylase reaction . Utilization of D-gluco-hept-1-enitol in the absence of primer; Klein HW et al.; alpha-Glucan phosphorylases from rabbit skeletal muscle, potato tubers and Escherichia coli catalyze the utilization of 2,6-anhydro-1-deoxy-D-gluco-hept-1-enitol (heptenitol) in the presence of arsenate or phosphate . 1H-NMR analysis in the presence of 2H2O and arsenate indicated formation of 1-{1-2H}deoxy-alpha-D-glucoheptulose with rates comparable to the arsenolysis of poly- or oligosaccharides . The reaction depends on the presence of a dianionic 5'-phosphate group of pyridoxal in the active conformation of the phosphorylases . Heptenitol is the first known substrate of alpha-glucan phosphorylases which does not require a primer . This is explained by the finding that heptenitol is exclusively used as substrate for the degradative pathway of the phosphorylase reaction where it competes with polysaccharide substrates . In the presence of phosphate the reaction product is 1-deoxy-alpha-D-gluco-heptulose 2-phosphate (heptulose-2-P), which subsequently inhibits the reaction . This characterizes heptulose-2-P as an enzyme-derived inhibitor . The Ki = 1.9 X 10(-6) M with potato phosphorylase suggests the formation of a transition-state-like enzyme-ligand complex . These findings, together with the fact that the phosphates of heptulose-2-P and pyridoxal 5'-phosphate are linked by hydrogen bridges {Klein, H . W., Im, M . J., Palm, D . & Helmreich, E . J . M . (1984) Biochemistry 23, 5853-5861}, make it likely that both phosphates are involved in phosphorylase catalysis . A catalytic mechanism of phosphorylase action is proposed in which a 'mobile' phosphate anion plays a versatile role . It serves as proton carrier for the substrate activation, it stabilizes the intermediate and acts as a nucleophile which can accept a glycosyl residue reversibly.

J Immunol, 1986 May 15, 136(10), 3884 - 90
Correlation between enhanced oxidative metabolism and leishmanicidal activity in activated macrophages from healer and nonhealer mouse strains; Buchmuller-Rouiller Y et al.; A test system that allows a precise monitoring of intracellular killing of Leishmania parasites was used to measure the leishmanicidal capacity of activated macrophages from different strains of mice . Activation was obtained by exposure to dilutions of lymphokine (LK)-rich medium in the presence of ng/ml amounts of E . coli lipopolysaccharide (LPS) . Highest leishmanicidal activity was displayed by macrophages from the healer mouse strain CBA/T6, whereas cells from the nonhealer strains DBA/2 and BALB/c were less effective . C3H/HeJ macrophages from a healer but LPS-unresponsive mouse strain failed to destroy leishmanias under these conditions . Experiments were then performed to determine the level of respiratory burst activity in the various macrophage populations . Hexose monophosphate shunt stimulation was higher in CBA/T6 than in BALB/c or DBA/2 macrophages, and only marginal in C3H/HeJ cells, correlating with differing leishmanicidal activities of such macrophages under the present experimental conditions . Measurements of O2- and H2O2 secretion and of chemiluminescence led to similar findings, i.e., CBA/T6 macrophages released higher amounts of oxygen metabolites than BALB/c cells activated under the same conditions, whereas C3H/HeJ cells were the least active . The results obtained by the four assays of oxidative metabolism were consistent in that endotoxin itself (in the 10 to 30 ng/ml range) stimulated the oxidative response of macrophages to a level close to that achieved by using LPS and LK together, however, only in the latter situation were parasites destroyed.

J Biol Chem, 1986 May 15, 261(14), 6239 - 47
sn-1,2-Diacylglycerol kinase of Escherichia coli . Mixed micellar analysis of the phospholipid cofactor requirement and divalent cation dependence; Walsh JP et al.; Efficient delivery of hydrophobic water-insoluble substrates and cofactors to membrane-bound enzymes is a recurring problem which has impeded kinetic analyses . Kinetic analysis of the Escherichia coli sn-1,2-diacylglycerol kinase, an extremely hydrophobic integral membrane protein of 122 residues, was facilitated by the development of a mixed micellar assay . beta-Octyl glucoside micelles quantitatively solubilized diacylglycerol kinase from membranes of strains which overproduced the enzyme up to 250-fold and provided an effective method to disperse and deliver the hydrophobic water-insoluble substrate, sn-1,2-dioleoyglycerol . Diacylglycerol kinase was active in mixed micelles containing octyl glucoside and dioleoyglycerol . Several phospholipids stimulated activity up to 6-fold, suggesting a cofactor function . Activation by phospholipids was not stereospecific and was mimicked partially by fatty acids . Half-maximal activation was observed at 1 mol % cardiolipin, suggesting that a small number of phospholipids are sufficient to activate the enzyme . Activity was dependent on the mole fractions of dioleoylglycerol and phospholipid in the mixed micelles, but independent of micelle number . Several lines of evidence indicated that the transfer of diacylglycerol between micelles was much more rapid than its utilization by the enzyme . Diacylglycerol kinase exhibited Michaelis-Menten kinetics with respect to diacylglycerol and MgATP . A second Mg2+ ion (in addition to MgATP) was required for activity . When Mg2+ was excluded from the assay, Mn2+, Zn2+, Cd2+, and Co2+ supported activity to lesser extents . These data establish a suitable system for in-depth kinetic analysis of the E . coli diacylglycerol kinase and its phospholipid cofactor requirements.

Anal Biochem, 1986 May 15, 155(1), 51 - 5
A single-step isolation of K99 pili from B-44 strain of Escherichia coli; Karkhanis YD et al.; A single-step procedure has been developed to isolate pili from enterotoxigenic Escherichia coli strain B44 of calf origin . Pili, removed from the bacteria by heat shock treatment, were allowed to aggregate at 4 degrees C for 16 h . The precipitated pili, isolated by centrifugation, had typical pili morphology as shown by electron microscopy; ability to bind pig brush border; molecular weight greater than 6 million; and predominance of hydrophobic amino acids . On sodium dodecyl sulfate gel electrophoresis, the subunit pilin migrated as a single polypeptide of molecular weight 17,000.

Biochem Biophys Res Commun, 1986 May 14, 136(3), 1136 - 41
Mycobacteria possess a surprisingly small number of ribosomal RNA genes in relation to the size of their genome; Bercovier H et al.; DNAs from Mycobacterium tuberculosis, M . intracellulare, M . phlei and M . smegmatis were digested by restriction enzymes and hybridized with three probes consisting of the 5' (16S rRNA), the middle (16S and 23S rRNA), and the 3' (23S and 5S rRNA) portions of the Escherichia coli rrnB operon . The resulting hybridization patterns indicate that slow-growing Mycobacteria species (i.e., M . tuberculosis and M . intracellulare), with genome size 3.13 - 4.29 X 10(9) daltons, appear to possess only one rRNA operon, whereas fast-growing species (i.e., M . phlei and M . smegmatis), with genome size 4.30 - 5.20 X 10(9) daltons, appear to possess two rRNA operons.

Biochem Biophys Res Commun, 1986 May 14, 136(3), 955 - 61
Cloning and expression in Escherichia coli of cDNA for arginase of rat liver; Kawamoto S et al.; A cDNA expression library constructed in a plasmid pUC8 from poly(A)+ RNA of rat liver was screened immunologically, using an antibody against arginase of rat liver . A cDNA clone was isolated and identified by hybrid-selected translation . The clone contained an insert approximately 1.35 kilobase pairs in length . In the bacterial clone, we detected a specific protein of Mr = about 43,000 that is slightly larger than the purified arginase (Mr = about 40,000) and a high activity of arginase was expressed . The arginase mRNA species of about 1600 bases long was detected in the liver, but not in the small intestine, kidney, spleen and heart of the rats.

Nucleic Acids Res, 1986 May 12, 14(9), 3827 - 39
Peculiar feature of the organization of rRNA genes of the Chlorella chloroplast DNA; Yamada T et al.; The organization of a cloned rRNA gene cluster from Chlorella ellipsoidea chloroplast DNA (cpDNA) has been analyzed . Southern hybridization experiments with labelled chloroplast rRNAs as probes revealed an extraordinarily large size of the 16S-23S rRNA spacer region, ca . 4.8 kbp, almost twice as large as those of most higher plants . The nucleotide sequence determined on this region has shown that: (1) The tRNAIle gene locating in this region is similar to those of higher plant chloroplasts, blue-green algae and E . coli but does not contain any introns in contrast to higher plant chloroplasts . (2) The tRNAAla gene is absent from this region . (3) There are four open reading frames (ORFs) coding for 55, 102, 107 and 110 amino acids, respectively . (4) A few sets of unique sequence were found repeatedly in this region . (5) The 23S rRNA gene is coded on the opposite strand in the reverse order . This arrangement of the 16S-23S rRNA region of Chlorella cpDNA is quite different from any of those reported so far for various organisms.

FEBS Lett, 1986 May 12, 200(2), 317 - 21
Tick-borne encephalitis virus genome . The nucleotide sequence coding for virion structural proteins; Pletnev AG et al.; RNA of a flavivirus, tick-borne encephalitis virus (TBEV; strain Sofjin), was subjected to reverse transcription and the DNA copy was transformed into double-stranded DNA by the action of E . coli DNA-polymerase I (Klenow fragment) . This DNA was annealed with plasmid pBR322 . The recombinant plasmids were cloned in E . coli K802 . The nucleotide sequence of the inserts of the clones, coding for region structural proteins C, M, E and nonstructural protein NS1, was determined by the Maxam-Gilbert method . The genes of structural proteins form a compact cluster . Homology has been studied of the TBEV sequences found with the structures of proteins and RNAs of other flaviviruses, yellow fever virus and West Nile virus, and a high degree of homology was found.

Nucleic Acids Res, 1986 May 12, 14(9), 3763 - 72
Nucleotide sequence of the tag gene from Escherichia coli; Steinum AL et al.; We have determined the complete nucleotide sequence of the tag gene, encoding 3-methyladenine DNA glycosylase I from Escherichia coli . From the nucleotide sequence it is deduced that the tag enzyme consists of 187 amino-acids and has a calculated molecular weight of 21.1 kdaltons . The tag enzyme is unusually rich in cysteine (8 residues) with a cluster of three consecutive cysteines near the C-terminal end . The tag coded DNA glycosylase does not show significant sequence homology to the alkA coded glycosylase in spite of that both of these enzymes catalyze the release of free 3-methyladenine from alkylated DNA.

J Theor Biol, 1986 May 7, 120(1), 99 - 110
Effect of distribution of unfavourable codons on the maximum rate of gene expression by an heterologous organism; Varenne S et al.; We have analysed theoretically the effect of the relative position of unfavourable codons on the maximum level of synthesis of foreign proteins in E . coli . We predict that the occurrence of such codons scattered in the corresponding genes has little effect . In contrast, clustering (in our terminology indicating directly adjacent codons) of unfavourable codons is predicted to dramatically reduce the maximum level of protein synthesis . The context effect would explain the reduction of expression level for a chloramphenicol acetyl transferase gene modified by Robinson et al . (1984), which contains 4 contiguous unfavourable codons . As an example, we predict that due to the different downstream contexts of unfavourable codons in the alpha 1 and beta interferon genes, the maximum level of synthesis in E . coli for these proteins will be different.

J Theor Biol, 1986 May 7, 120(1), 63 - 71
Energy storage during DNA girase activity; Mizraji E et al.; In this work, we develop a minimal two-cycle model for the action of DNA girase . One of the cycles describes the ATP dependent chemicomechanical transduction performed by the enzyme . The other cycle describes the relaxing activity exhibited by girase on supercoiled DNA in the absence of substrates . Supercoiling of DNA is described as a random walk on a topological index . The mechanical energy storage in DNA is manifested in the fact that in general, the kinetic constants for the cycles do not satisfy Wegscheider relations . Finally, a connection is established between the degree of DNA supercoiling and the hydrolysis of ATP.

Biochemistry, 1986 May 6, 25(9), 2736 - 42
Glutathione reductase from Escherichia coli: cloning and sequence analysis of the gene and relationship to other flavoprotein disulfide oxidoreductases; Greer S et al.; A glutathione reductase negative strain of Escherichia coli K-12 was isolated as a thermoresistant survivor when a gor::MuctsAp lysogen was subjected to elevated temperature . It was found that in addition to being ampicillin sensitive this mutant was hypersensitive to arsenate, which may be connected with the fact that the gor gene maps between 77 and 78 min on the E . coli genome, close to the pit locus encoding the major arsenate transport system of E . coli . A derivative of this mutant was used as the recipient in a screen of the Clarke and Carbon hybrid plasmid bank of E . coli DNA . A plasmid, pGR, was isolated that encodes both an arsenate-resistance element and glutathione reductase . Restriction mapping of this plasmid showed that the insert DNA is approximately 10 kilobase pairs in length, and a fragment of the gor gene was identified that allowed the gor gene to be accurately mapped on pGR by a combination of restriction analysis and Southern blotting . The DNA sequence of the gor gene was determined and found to encode a protein of 450 amino acid residues . The glutathione reductase of E . coli is very homologous to the human enzyme and is also related (though less closely) to other flavoprotein disulfide oxidoreductases whose sequences are available . These enzymes have retained a common mechanism while evolving different specificities.

Biochemistry, 1986 May 6, 25(9), 2502 - 8
Conformational effects of ligand binding on the beta 2 subunit of Escherichia coli tryptophan synthase analyzed with monoclonal antibodies; Djavadi-Ohaniance L et al.; Five monoclonal antibodies recognizing five different epitopes of the native beta 2 subunit of Escherichia coli tryptophan synthase (EC 4.1.2.20) were used to analyze the conformational changes occurring upon ligand binding or chemical modifications of the enzyme . For this purpose, the affinities of each antibody for the different forms of the enzyme were determined by using an enzyme-linked immunosorbent assay which allows measurement of the dissociation constant of antigen-antibody equilibrium in solution . The fixation of the coenzyme pyridoxal 5'-phosphate and the substrate L-serine modifies the affinity constants of most of the antibodies for the enzyme, thus showing the existence of extended conformational rearrangements of the protein . The association of the alpha subunit with the beta 2 subunit, which brings about an increase of the tryptophan synthase activity and abolishes the serine deaminase activity of beta 2, is accompanied by an important conformational change of the N-terminal domain of beta 2 (F1) since none of the anti-F1 monoclonal antibodies can bind to alpha 2 beta 2 . Similarly, chemical modifications of beta 2 which are known to produce significant effects on the enzymatic activities of beta 2 result in changes of the affinities of the monoclonal antibodies which can be interpreted as the acquisition of different conformational states of the enzyme.

Biochemistry, 1986 May 6, 25(9), 2485 - 9
Isolation and properties of N epsilon-hydroxylysine:acetyl coenzyme A N epsilon-transacetylase from Escherichia coli pABN11; Coy M et al.; The enzyme N epsilon-hydroxylysine acetylase has been isolated from Escherichia coli 294 carrying recombinant plasmid ABN11 . Activity of the enzyme was followed by measurement of the rate of appearance of 2-nitro-5-thiobenzoate, the product of cleavage of 5,5'-dithiobis(2-nitrobenzoate) by free coenzyme A released from its acetyl derivative . The enzyme bound firmly to Reactive Blue 2-Sepharose CL-6B and was eluated with 1.5 M KCl . The protein gave a single band, corresponding to a Mr of 33,000, on polyacrylamide gel electrophoresis in sodium dodecyl sulfate . In contrast, gel filtration of the native enzyme gave a Mr of 150,000-200,000 . A sequence analysis of the DNA at the junction of the first and second genes in the aerobactin operon, considered in conjunction with the N-terminal amino acid sequence of the isolated protein, enabled the conclusion that the acetylase is specified by the second gene in the complex . The enzyme transfers the acetyl moiety from acetyl coenzyme A to a variety of hydroxylamines, with N epsilon-hydroxylysine as the preferred substrate . In agreement with the results found by affinity chromatography, Coomassie Blue was observed to act as a potent inhibitor.

Biochemistry, 1986 May 6, 25(9), 2403 - 9
Anti-peptide antibodies and proteases as structural probes for the lactose/H+ transporter of Escherichia coli: a loop around amino acid residue 130 faces the cytoplasmic side of the membrane; Seckler R et al.; From the amino acid sequence of the Escherichia coli lactose/H+ transporter, 7 hydrophilic segments were selected, 8-13 amino acids in length, and chemically synthesized, and anti-peptide antibodies were raised in rabbits . Apart from the antiserum to the synthetic COOH terminus (P408-417), which reacted strongly with the lactose/H+ transporter and has previously been used to localize the COOH terminus on the cytoplasmic face of the membrane, only those antibodies directed against the peptide corresponding to amino acid residues 125-135 (P125-135) exhibited a marked reaction with the transporter, while antibodies to the five other peptides reacted very weakly or not at all, suggesting that most of the hydrophilic segments are conformationally restricted or buried in the interior of the protein . Thermolysin treatment destroys the epitope on the transporter which is recognized by anti-P125-135 antibodies . Comparison of the kinetics and the extent of proteolysis of the transporter in right-side-out or inside-out cytoplasmic membrane vesicles or in reconstituted proteoliposomes suggests that the hydrophilic sequence from amino acid 125 to amino acid 135 is accessible to thermolysin only from one side, corresponding to the cytoplasmic face of the membrane . Furthermore, the experiments demonstrate that the transporter is inserted bimodally in a nonpreferential fashion into the proteoliposomes, confirming earlier results using antibodies to the synthetic COOH terminus of the transporter in conjunction with carboxypeptidase A treatment.

Biochemistry, 1986 May 6, 25(9), 2494 - 501
Characterization of the reaction of L-serine and indole with Escherichia coli tryptophan synthase via rapid-scanning ultraviolet-visible spectroscopy; Drewe WF Jr et al.; The pre-steady-state reaction of indole and L-serine with the alpha 2 beta 2 complex of Escherichia coli tryptophan synthase has been investigated under different premixing conditions with rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy for the spectral range 300-550 nm . When alpha 2 beta 2 was mixed with indole and L-serine, the reaction of alpha 2 beta 2 was found to occur in three detectable relaxations (1/tau 1 greater than 1/tau 2 greater than 1/tau 3) with rate constants identical with the three relaxations seen in the partial reaction with L-serine {Drewe, W.F., Jr., & Dunn, M.F . (1985) Biochemistry 24, 3977-3987} . Kinetic isotope effects due to substitution of 2H for the alpha-1H of serine were found to be similar to the effects observed in the reaction with serine only . The observed spectral changes and isotope effects indicate that the aldimine of L-serine and PLP and the first quinoid derived from this external aldimine are transient species that accumulate during tau 1 . Conversion of these intermediates to the alpha-aminoacrylate Schiff base during tau 2 and tau 3 limits the rate of formation of the second quinoidal species (lambda max 476 nm) generated via C-C bond formation between indole and the alpha-aminoacrylate intermediate . The pre-steady-state reaction of the alpha 2 beta 2-serine mixture with indole is comprised of four relaxations (1/tau 1* greater than 1/tau 2* greater than 1/tau 3* greater than 1/tau 4*).(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1986 May 6, 25(9), 2321 - 7
D-lactate oxidation and generation of the proton electrochemical gradient in membrane vesicles from Escherichia coli GR19N and in proteoliposomes reconstituted with purified D-lactate dehydrogenase and cytochrome o oxidase; Matsushita K et al.; The respiratory chain in the cytochrome d deficient mutant Escherichia coli GR19N is a relatively simple, linear system consisting of primary dehydrogenases, ubiquinone 8, cytochrome b-556, and cytochrome o oxidase . By use of right-side-out and inside-out membrane vesicles from this strain, various oxidase activities and the generation of the H+ electrochemical gradient were studied . Oxidation of ubiquinol 1 or N,N,-N',N'-tetramethyl-p-phenylenediamine, which donate electrons directly to the terminal oxidase, generates a H+ electrochemical gradient comparable to that observed during D-lactate oxidation . In contrast, D-lactate/ubiquinone 1 or D-lactate/ferricyanide oxidoreductase activity does not appear to generate a membrane potential, suggesting that electron flow from D-lactate dehydrogenase to ubiquinone is not electrogenic . Moreover, proteoliposomes reconstituted with purified D-lactate dehydrogenase, ubiquinone 8, and purified cytochrome o catalyze D-lactate and ubiquinol 1 oxidation and generate a H+ electrochemical gradient similar to that observed in membrane vesicles . Strikingly, in inside-out vesicles, NADH oxidation generates a H+ electrochemical gradient that is very significantly greater than that produced by either D-lactate or ubiquinol 1; furthermore, NADH/ubiquinone 1 and NADH/ferricyanide oxidoreductase activities are electrogenic . It is suggested that the only component between D-lactate dehydrogenase or ubiquinol and oxygen in GR19N membranes that is directly involved in the generation of the H+ electrochemical gradient is cytochrome o, which functions as a "half-loop" (i.e., the oxidase catalyzes the scalar release of 2 H+ from ubiquinol on the outer surface of the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1986 May 6, 25(9), 2314 - 21
Coulometric and spectroscopic analysis of the purified cytochrome d complex of Escherichia coli: evidence for the identification of "cytochrome a1" as cytochrome b595; Lorence RM et al.; Coulometric and spectroscopic analyses were performed on the three cytochrome components (cytochrome d, cytochrome b558, and the cytochrome previously described as cytochrome a1) of the purified cytochrome d complex, a terminal oxidase of the Escherichia coli aerobic respiratory chain . On the basis of heme extraction, spectroscopic, and coulometric data, the "cytochrome a1" component was identified as a b-type cytochrome: cytochrome b595 . The pyridine hemochromogen technique revealed the presence of two molecules of protoheme IX per cytochrome d complex . This quantity of protoheme IX fully accounted for the sum of the cytochrome b558 and cytochrome b595 components as determined coulometrically . The renaming of cytochrome a1 as cytochrome b595 was further indicated by the lack of any heme a in the complex and by its resolved reduced-minus-oxidized spectrum . The latter was found to be similar to that of cytochrome c peroxidase, which contains protoheme IX . Coulometric titrations and carbon monoxide binding titrations revealed that there are two molecules of cytochrome d per complex . A convenient measurement of the amount of cytochrome b558 was found to be the beta-band at 531 nm since cytochrome b558 was observed to be the only component of the cytochrome d complex with a peak at this wavelength . By use of this method and the extinction coefficient for the purified cytochrome b558, it was estimated that there is one molecule of cytochrome b595 and one of cytochrome b558 per cytochrome complex.

Biochemistry, 1986 May 6, 25(9), 2309 - 14
Specific overproduction and purification of the cytochrome b558 component of the cytochrome d complex from Escherichia coli; Green GN et al.; In Escherichia coli strain GR84N{pNG10}, the cloned gene for subunit I of the membrane-bound cytochrome d complex resulted in the overproduction of cytochrome b558 and facilitated purification of this cytochrome . Extracting membranes with 1% Triton X-100 followed by two chromatographic steps yielded a single band on sodium dodecyl sulfate-polyacrylamide gels corresponding to subunit I (Mr 57 000) . Purified cytochrome b558 was in its native state as determined by difference absorption spectroscopy and by potentiometric analysis . Both the membranes of strain GR84N{pNG10} and the purified subunit I lacked the other two spectroscopically defined cytochromes, b595 (previously "a1") and d, of the cytochrome d complex . Reconstitution of cytochrome b558 in phospholipid vesicles demonstrated that cytochrome b558 can be reduced by ubiquinol but that it does not reduce molecular oxygen . Heme extraction of cytochrome b558 yielded an extinction coefficient of 22 000 M-1 cm-1 for the wavelength pair of 560 and 580 nm in the reduced-minus-oxidized spectrum . The mutation on pNG10 that eliminates subunit II was mapped to a 250 base pair DNA fragment.

Biochemistry, 1986 May 6, 25(9), 2490 - 3
Limited proteolysis at the carboxy end modifies interactions between the subunits of Escherichia coli phosphofructokinase; Le Bras G et al.; The limited proteolysis of Escherichia coli phosphofructokinase by subtilisin involves the removal of a segment of 40-50 residues at the C-terminal end of each polypeptide chain {Le Bras, G., & Garel, J.R . (1985) J . Biol . Chem . 260, 13450-13453} . The time course of proteolysis has been followed by the appearance of shorter chains, the loss of allosteric inhibition by phosphoenolpyruvate, and the weakening of the tetrameric structure in the absence of fructose 6-phosphate . It is found that with only one shorter chain out of four the stability of the tetramer is altered so that it is no longer stable in the absence of fructose 6-phosphate . Also, the reduction in size of only two chains is sufficient to render the enzyme insensitive to allosteric effectors, albeit the protein still possesses the ability to bind such an effector (at least partially); the cleavage of all four chains is needed to lose all the effector binding ability . The C-terminal segment therefore plays an important role in subunit interactions as seen from the gradual changes in structural and functional properties which follow its removal from one, two, or four chains.

Biochemistry, 1986 May 6, 25(9), 2480 - 5
Purification and properties of acyl coenzyme A thioesterase II from Rhodopseudomonas sphaeroides; Seay T et al.; A high molecular weight acyl coenzyme A (acyl-CoA) thioesterase, designated thioesterase II, has been purified 5300-fold from photoheterotrophically grown cells of Rhodopseudomonas sphaeroides . In contrast to R . sphaeroides acyl-CoA thioesterase I {Boyce, S.G., & Lueking, D.R . (1984) Biochemistry 23, 141-147}, thioesterase II has a native molecular mass (Mr) of 120,000, is capable of hydrolyzing saturated and unsaturated acyl-CoA substrates with acyl chain lengths ranging from C4 to C18, and is completely insensitive to the serine esterase inhibitor diisopropyl fluorophosphate . Palmitoyl-CoA and stearoyl-CoA are the preferred (lowest Km) saturated acyl-CoA substrates and vaccenoyl-CoA is the preferred unsaturated substrate . However, comparable Vmax values were obtained with a variety of acyl-CoA substrates . Unlike a similar thioesterase present in cells of Escherichia coli {Bonner, W.M., & Bloch, K . (1972) J . Biol . Chem . 247, 3123-3133}, R . sphaeroides thioesterase II displays a high ratio of decanoyl-CoA to palmitoyl-CoA activities and exhibits little ability to hydrolyze 3-hydroxyacyl-CoA substrates . Only 3-hydroxydodecanoyl-CoA supported a measurable rate of enzyme activity . With the purification of thioesterase II, the enzymes responsible for greater than 90% of the acyl-CoA thioesterase activity present in cell-free extracts of R . sphaeroides have now been identified.

J Mol Biol, 1986 May 5, 189(1), 251 - 5
Alteration of the growth-rate-dependent regulation of Escherichia coli tyrT expression by promoter mutations; Travers AA et al.; The growth-rate regulation of transcription of the Escherichia coli tyrT gene depends on sequences in at least two distinct regions of the promoter, the upstream element required for optimal activity and the discriminator adjacent to the transcription start-point . Since mutations in the discriminator also alter the response of the promoter to amino acid starvation, we conclude that growth rate and amino acid control mechanisms share a common target molecule, probably RNA polymerase.

J Mol Biol, 1986 May 5, 189(1), 227 - 38
Involvement of tryptophan 209 in the allosteric interactions of Escherichia coli aspartate transcarbamylase using single amino acid substitution mutants; Smith KA et al.; Five mutant versions of aspartate transcarbamylase have been isolated, all with single amino acid substitutions in the catalytic chain of the enzyme . A previously isolated pyrB nonsense mutant was suppressed with supB, supC, supD and supG to create enzymes with glutamine, tyrosine, serine or lysine, respectively, inserted at the position of the nonsense codon . Each of these enzymes was purified to homogeneity and kinetically characterized . The approximate location of the substitution was determined by using tryptic fingerprints of the wild-type enzyme and the enzyme obtained with a tyrosine residue inserted at the position of the nonsense codon . By first cloning the pyrBI operon, from the original pyrB nonsense strain, followed by sequencing of the appropriate portion of the gene, the exact location of the mutation was determined to be at position 209 of the catalytic chain . Site-directed mutagenesis was used to generate versions of aspartate transcarbamylase with tyrosine and glutamic acid at this position . The Tyr209 enzyme is identical with that obtained by suppression of the original nonsense mutation with supC . The two enzymes produced by site-directed mutagenesis were purified using a newly created overproducing strain . Kinetic analysis revealed that each mutant has an altered affinity for aspartate, as judged by variations in the substrate concentration at one-half maximal activity . In addition, the mutants exhibit altered Hill coefficients and maximal activities . In the wild-type enzyme, position 209 is a tryptophan residue that is involved in the stabilization of a bend in the molecule near the subunit interface region . The alteration in homotropic cooperativity seems to be due to changes induced in this bend in the molecule, which stabilizes alternate conformational states of the enzyme.

J Biol Chem, 1986 May 5, 261(13), 6026 - 33
Denaturation of covalently closed circular DNA . Kinetics, comparison of several DNAs, mechanism and ionic effects; Camien MN et al.; The rates of the alkaline denaturation of the covalently closed, circular DNAs (form I) of the replicative forms (RF) of phages G4, phi X174, and fd, and of plasmid pBR322 and phage PM2 have been measured at 0 degrees C and some at higher temperatures . These rates are orders of magnitude slower than the denaturation of linear DNA because of the increased stability of the helix to deprotonation that results from the accumulating positive superhelicity during denaturation . Denaturation reactions were initiated by rapid, infrasonic mixing (Camien, M.N., and Warner, R.C . (1984) Anal . Biochem . 138, 329-334), and their progress was measured by analytical ultracentrifugal analysis for the amounts of form I and denatured (Id) DNA after neutralization of the alkaline reaction . The comparative rates of the five DNAs varied over a wide range; the fastest, G4-RF, denatured at 500-fold the rate of the slowest, fd-RF . The differences are accounted for by the interaction of positive superhelicity with the sequence-dependent regions of relative helix stability in the various DNAs . Renaturation rates of Id DNAs varied similarly for Ids prepared at 0 degrees C, but only a few-fold for Ids prepared at 50 degrees C . The rate of denaturation of G4-RF was determined over a wide range of NaOH and NaCl concentrations at 0 degrees C, and the pHm was determined as a function of ionic strength and temperature . The effects of ionic strength have been analyzed in an application of the Manning ion condensation-screening theory (Manning, G.S . (1978) Q . Rev . Biophys . 11, 179-246) which is shown to account for the large destablizing effect of salts on the helix . The pH region of transition at 50 degrees C from renaturation to denaturation was examined, and it was found that the maximum rate of renaturation occurred at a pH about 0.05 units below the pHm.

J Mol Biol, 1986 May 5, 189(1), 85 - 102
Site-specific and illegitimate recombination in the oriV1 region of the F factor . DNA sequences involved in recombination and resolution; O'Connor MB et al.; We have defined some of the sequences involved in high frequency recA-independent recombination at the oriV1 region of the F factor . Using a mobilization assay, we determined that plasmid pMB080, a pBR322 derivative bearing the PvuII-BamHI (F factor co-ordinates 45.43 to 46.0) fragment from the oriV1 region of F, contained all sequences necessary to undergo efficient site-specific recombination with the F derivative pOX38, which retains the oriV1 region . We constructed a series of pMB080 deletions in vitro using exonucleases S1 and Bal31 . Deletions removing a ten base-pair sequence, which forms part of an inverted repeat segment located 62 base-pairs to the left of the NcoI site (45.87) within the cloned fragment, totally eliminated the recA-independent recombination reaction . Other deletions differentially affected both the frequency and stability of cointegrate molecules formed by the site-specific recombination system . The F factor oriV1 region is involved also in low-frequency recombination with several sites on pBR322 and related plasmids . We have determined the precise location of these recombination sites within oriV1 by DNA sequencing . These studies revealed that recombination always took place within an eight base-pair spacer region between the ten base-pair inverted repeats found to be important for oriV1-oriV1 interactions . We propose that the low-efficiency recombination between pBR322 and pOX38 results from the ability of the F site-specific recombination apparatus to weakly recognize and interact with sequences that bear some resemblance to the normal oriV1 recognition elements . Furthermore, we suggest, by analogy with the lambda paradigm, that the nucleotide sequences at the junctions of secondary site recombinants define at least one crossover site used during the normal site-specific recombination process.

J Mol Biol, 1986 May 5, 189(1), 25 - 36
Molecular organization of the rudimentary gene of Drosophila melanogaster; Freund JN et al.; We have determined the molecular structure of the rudimentary gene of Drosophila melanogaster . The transcription unit, which extends over 14,000 bases, is split into seven exons and six introns . Sequences presenting homologies with a TATA box and a CAT box are located upstream from the transcription initiation site, and there is a consensus polyadenylation signal in the vicinity of the 3' end of the messenger RNA coding sequence . Short intronic sequences fit well with consensus signals found in other eukaryotic genes and involved in the splicing of the precursor messenger RNA . Partial genomic and complementary DNA sequencing has revealed stretches of amino acid sequence homologies with the corresponding enzymes in Saccharomyces cerevisiae and Escherichia coli . This is in good agreement with the genetic map of the locus, which indicates that the enzymic activities encoded by the gene are organized linearly along the DNA.

J Mol Biol, 1986 May 5, 189(1), 103 - 11
Regulatory defects of a conditionally lethal nusAts mutant of Escherichia coli . Positive and negative modulator roles of NusA protein in vivo; Nakamura Y et al.; Previous studies have attributed two activities to the NusA protein of Escherichia coli; namely, termination and antitermination of transcription . To examine these activities, we isolated a temperature-sensitive mutant of the nusA gene (nusAts11) . The mutant cells produce a thermolabile NusA protein and grow at 32 degrees C, but not at 42 degrees C . At 42 degrees C, nusAts11 is recessive to nusA+ and nusA1, indicating the absence of its active gene product at that temperature . In the mutant, the efficiency of termination at the lambda tR1 terminator decreases, resulting in an increased expression of distal gene(s) . On the other hand, the synthesis of the beta-galactosidase and beta beta' subunits of RNA polymerase is reduced in the mutant . This mimics effects seen in vitro when NusA protein is removed from a coupled transcription-translation system . These results suggest that the NusA protein plays both negative and positive modulator roles in vivo . The mutation nusAts11, unlike nusA1, does not block lambda phage growth at non-permissive temperatures, suggesting that NusA protein is not required for N antitermination in the mutant . Besides, the nusAts11 allows lambda Nam7Nam53byp phage growth under sup0 conditions, indicating that the N antitermination function is dispensable (at least partly) in this mutant.

J Biol Chem, 1986 May 5, 261(13), 6090 - 5
Photodynamic guanine modification by hematoporphyrin is specific for single-stranded DNA with singlet oxygen as a mediator; Kawanishi S et al.; Photodynamic modification of DNA by hematoporphyrin (Hp) was characterized by the DNA sequencing technique using 32P-labeled DNA fragments, and the reaction mechanism was investigated by ESR spectroscopy . Mild photodynamic treatment of single-stranded DNA with Hp induced an alteration of guanine residues, and subsequent treatment with piperidine led to chain cleavages at each guanine residue . On the other hand, methylene blue plus light modified the guanine residues in both single-stranded and double-stranded DNA . ESR studies using 2,2,6,6-tetramethylpiperidine and 2,2,6,6-tetramethyl-4-piperidone as singlet oxygen traps demonstrated that Hp plus light produced almost the same amount of singlet oxygen as methylene blue plus light and that the photochemically generated singlet oxygen reacts significantly with guanylate but only slightly with other mononucleotides . An ESR spin destruction method revealed that photoexcited Hp generated porphyrin radical, but guanylate did not react with this radical . These results indicate that photoexcited Hp reacts with oxygen to generate singlet oxygen which oxidizes the guanine residues of single-stranded DNA and that the difference in photoreactivities of DNA with Hp and methylene blue may be explained in terms of the structural difference in their intercalating abilities.

FEBS Lett, 1986 May 5, 200(1), 23 - 6
Expression of the cloned subunits of Escherichia coli transhydrogenase from separate replicons; Clarke DM et al.; The pntA and pntB genes of Escherichia coli, encoding the alpha- and beta-subunits of the pyridine nucleotide transhydrogenase, were cloned individually in two different compatible plasmids into Escherichia coli mutants lacking transhydrogenase activity . Energy-linked and non-energy-linked transhydrogenase activities were produced only in cells carrying both plasmids thus showing that the products of both genes are required for the formation of an active enzyme . ATP-energized transhydrogenase activity was not increased in cells containing amplified levels of the transhydrogenase when the cell membrane ATPase was also amplified . It is suggested that the excess transhydrogenase is effectively uncoupled from the ATPase by compartmentalization in the cell.

FEBS Lett, 1986 May 5, 200(1), 11 - 7
The complete amino acid sequence of 3-dehydroquinate synthase of Escherichia coli K12; Millar G et al.; The complete amino acid sequence of the Escherichia coli 3-dehydroquinate synthase has been determined by a combined nucleotide and direct amino acid sequencing strategy . E . coli 3-dehydroquinate synthase is 362 amino acids long and has a calculated Mr of 38 880 . Analysis of the aroB nucleotide sequence and its 5'- and 3'-flanking regions has identified the aroB promoter elements and a possible 3'-terminator site.

J Biol Chem, 1986 May 5, 261(13), 5920 - 9
Dihydroorotase from Escherichia coli . Sulfhydryl group-metal ion interactions; Washabaugh MW et al.; We have obtained 53 mg of 99% pure dihydroorotase from 10.9 g of frozen Escherichia coli pyrC plasmid-containing E . coli cells using a 4-step 16-fold purification procedure, a yield of 60% . We characterize the enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (a dimer of subunit molecular weight 38,300 +/- 2,900), high performance liquid chromatography gel sieving, amino acid analysis, amino terminus determination (blocked), and specific activity . The isolated enzyme contains 1 tightly bound essential zinc atom/subunit, and readily but loosely binds 2 additional Zn(II) or Co(II) ions/subunit which modulate catalytic activity; treatment of crude extracts with weak chelators suggests that the enzyme contains 3 zinc atoms/subunit in vivo . Two of the 6 thiol groups/subunit react rapidly with 5,5'-dithiobis(2-nitrobenzoate) when 1 Zn/subunit enzyme is used, but slowly when 3 Zn/subunit enzyme is used . The 2 weakly bound Zn(II) ions/subunit protect against the reversible air oxidation which lowers the specific activity of the enzyme and renders it unreactive with 5,5'-dithiobis(2-nitrobenzoate) . The dilution activation observed in the presence of substrate, the dilution inactivation observed in the absence of substrate, and the transient activation by the metal chelator oxalate are interpreted as evidence for an unstable, hyperactive monomer.

J Biol Chem, 1986 May 5, 261(13), 5917 - 9
Dihydroorotase from Escherichia coli . Cloning the pyrC gene and production of tryptic peptide maps; Brown DC et al.; We have inserted a 1.7-kilobase pair Escherichia coli DNA fragment containing the 1-kilobase pair pyrC gene into the high copy number plasmid pKC16 . Dihydroorotase expressed by the pyrC plasmid in E . coli constituted 6.3% of the soluble protein in frozen cell paste . Pure dihydroorotase derived from this frozen cell paste was compared with pure enzyme derived from an E . coli strain lacking the pyrC plasmid: tryptic peptide maps from the two dihydroorotase preparations, produced using reverse-phase high performance liquid chromatography, were indistinguishable . We conclude that the entire pyrC gene is present on the hybrid plasmid and that the dihydroorotase produced from this plasmid is identical to the wild type.

Lancet, 1986 May 3, 1(8488), 1007 - 9
Membrane filter assay for detection of enterotoxigenic Escherichia coli in epidemiological studies; Vadivelu J et al.; A membrane filter assay is described for detection of heat-labile toxin (LT) production by Escherichia coli . Bacterial colonies were isolated on a membrane filter which was then incubated on an agar medium containing anti-cholera toxin . LT produced by bacterial colonies diffused through the membrane filter, complexed with the antiserum, and formed a zone of precipitation in the agar . Requirements for reagents and materials were simple . The membrane filter assay showed good agreement with both the ganglioside-enzyme-linked immunosorbent assay and the DNA-DNA hybridisation assay and may prove to be an important technique for the study of the epidemiology of enterotoxigenic E coli in developing countries.

J Chromatogr, 1986 May 2, 357(2), 283 - 92
Purification of guanosine triphosphate cyclohydrolase I from Escherichia coli . The use of competitive inhibitors versus substrate as ligands in affinity chromatography; Ferre J et al.; Different affinity chromatography ligands have been compared for the purification of guanosine triphosphate (GTP) cyclohydrolase I, an enzyme that catalyses the transformation of GTP into formate and dihydroneopterin triphosphate, the first metabolite in the biosynthetic pathway of the pterins . When this enzyme is purified by affinity chromatography on GTP-Sepharose a major fraction of the activity is lost and the yield of enzyme decreases as the amount of enzyme applied to the column decreases . The use of nucleotide competitive inhibitors (UTP and ATP) as ligands in the affinity column has shown that the extent of inactivation of the enzyme is related to the affinity of the enzyme for the ligand . Further, the extent of inactivation was reduced by reducing the length of the columns when using the same volume of GTP-Sepharose . Dihydrofolate-Sepharose gave consistently higher yields of GTP cyclohydrolase I regardless of the amount of enzyme applied, but several other proteins were also obtained . For a high purification of GTP cyclohydrolase I the best yield may be obtained with UTP as the affinity ligand and with the shortest length possible of the affinity column, and the purity of enzyme is comparable with that obtained with GTP-Sepharose.

Eur J Biochem, 1986 May 2, 156(3), 669 - 75
Primary structures of mutationally altered ribosomal protein L7/L12 and their effects on cellular growth and translational accuracy; Kirsebom LA et al.; The amino acid sequences of mutationally altered ribosomal protein L7/L12 from four different rplL mutants of Escherichia coli were determined and correlated with some features of the mutant ribosomes . Two of the rplL mutations are deletions around position 40, which give rise to a shortened hinge region between the two domains of L7/L12 . The other two mutants harbor point mutations at position 74 (Gly----Asp) or at position 82 (Glu----Lys), which are in or close to an evolutionarily conserved sequence in the C-terminal domain . The two latter mutations are associated with decreased rates of growth and translational elongation . All four mutants show increased nonsense codon read-through in vivo . Ribosomes from one of the deletion mutants show clearly increased missense error rates in vitro.

Eur J Biochem, 1986 May 2, 156(3), 497 - 503
Localization of L11 on the Escherichia coli ribosome by singlet-singlet energy transfer; Deng HY et al.; Isolated Escherichia coli ribosomal protein L11 was labeled with maleimidyl derivatives of coumarin or fluorescein at the thiol group of its single cysteine, then reconstituted singly or in pairs with other fluorescently labeled ribosomal components . The characteristics of fluorescence from the labeled protein were studied and its distance to other components was determined by non-radiative energy transfer . The distance between probes on L11 and cysteine residues on other proteins or the 3' end of the ribosomal RNAs were found to be: S1, 7.4-8.3 nm; S21, 7.6 nm; 23S RNA, 6.9 nm; 5S RNA, 7.6 nm; 16S RNA, greater than 8.5 nm . Considered together with previously published results these distances indicate that the location of L11 in the 50S subunit is below the lateral protuberance characterized by L7/L12.

Mol Cell Biol, 1986 May, 6(5), 1830 - 3
Cell-specific expression of the human parathyroid hormone gene in rat pituitary cells; Igarashi T et al.; The promoter region of the human parathyroid hormone gene was fused to the Escherichia coli neo gene and introduced into GH4C1 rat pituitary and human HeLa cells . Both TATA boxes of the human parathyroid hormone gene accurately directed transcription in GH4C1 cells; the parathyroid hormone promoter was inactive in HeLa cells.

EMBO J, 1986 May, 5(5), 1111 - 3
Higher order structure in ribosomal RNA; Gutell RR et al.; The only reliable general method currently available for determining precise higher order structure in the large ribosomal RNAs is comparative sequence analysis . The method is here applied to reveal 'tertiary' structure in the 16S-like rRNAs, i.e . structure more complex than simple double-helical, secondary structure . From a list of computer-generated potential higher order interactions within 16S rRNA one such interaction considered likely was selected for experimental test . The putative interaction involves a Watson-Crick one to one correspondence between positions 570 and 866 in the molecule (E . coli numbering) . Using existing oligonucleotide catalog information several organisms were selected whose 16S rRNA sequences might test the proposed co-variation . In all of the (phylogenetically independent) cases selected, full sequence evidence confirms the predicted one to one (Watson-Crick) correspondence . An interaction between positions 570 and 866 is, therefore, considered proven phylogenetically.

J Virol, 1986 May, 58(2), 522 - 5
Pre-S1 antigens and antibodies early in the course of acute hepatitis B virus infection; Klinkert MQ et al.; The presence of the two "large" surface proteins of hepatitis B virus (HBV), P39 and GP42 of pre-S1-hepatitis B surface antigen, was assayed in the serum of an experimentally infected chimpanzee by using antibodies to a pre-S1-specific fusion protein synthesized in Escherichia coli . The immune response to pre-S1-hepatitis B surface antigen was monitored by using the pre-S1 fusion protein as an antigen . pre-S1 proteins were detected in the serum early in the course of infection and prevailed as long as hepatitis B surface antigen did, together with hepatitis B e antigen and viral DNA . Thus, the pre-S1 antigen can be considered a novel diagnostic marker for acute HBV infection . Antibodies to pre-S1, both immunoglobulin M and G classes, were also detected early in infection, shortly after the appearance of the pre-S1 antigen, suggesting its strong immunogenicity in vivo . The anti-pre-S1 antibodies therefore also represent an early serological marker for acute HBV infection and, owing to their early appearance and persistence, may play a role in the neutralization of the virus.

Mol Gen Mikrobiol Virusol, 1986 May, (5), 35 - 8
{Effect of mutation crp11 in the 3',5'-cyclic adenosine monophosphate receptor on the expression of the udp gene in Escherichia coli K12 strains deficient for transcription termination factors (rho ts15)}; Astvatsaturian MZ; The suppression of temperature sensitive phenotype caused by mutation rho ts 15, making cells deficient in transcription termination, but mutation crp11 impairing the cAMP-receptor protein (cap) restores transcription termination on IS3 sequence in galactose operon and on the site before the promoter of udp gene in strains crp11 rho ts15 . The obtained data suggest the direct interaction between factor Rho and cAMP-CAP complex in transcription process.

Mol Gen Mikrobiol Virusol, 1986 May, (5), 31 - 4
{Lethal effect of formaldehyde on Escherichia coli strains carrying or lacking the plasmid pKM101}; Kalashnikov NV; The presence of plasmid pKM101 in Escherichia coli cells results in a slight increase in their sensitivity of lethal effect of formaldehyde . Plasmid ability to sensitize bacterial cells to formaldehyde inactivation is controlled by some chromosomal (uvrE, uvrA, recA) and plasmid-borne (mucAB) genes and depends on SOS-DNA repair activity . Plasmid pKM101 is capable of decreasing the level of repair reliability of DNA damaged by formaldehyde thus causing increased bacterial sensitivity to this agent.

Mol Cell Biol, 1986 May, 6(5), 1608 - 14
Extrachromosomal and chromosomal gene conversion in mammalian cells; Rubnitz J et al.; We constructed substrates to study gene conversion in mammalian cells specifically without the complication of reciprocal recombination events . These substrates contain both an insertion mutation of the neomycin resistance gene (neoX) and an internal, homologous fragment of the neo gene (neo-526), such that gene conversion from neo-526 to neoX restores a functional neo gene . Although two reciprocal recombination events can also produce an intact neo gene, these double recombination events occur much less frequently that gene conversion in mammalian cells, We used our substrates to characterize extrachromosomal gene conversion in recombination-deficient bacteria and in monkey COS cells . Chromosomal recombination was also studied after stable integration of these substrates into the genome of mouse 3T6 cells . All extrachromosomal and chromosomal recombination events analyzed in mammalian cells resulted from gene conversion . Chromosomal gene conversion events occurred at frequencies of about 10(-6) per cell generation and restored a functional neo gene without overall effects on sequence organization.

Mol Cell Biol, 1986 May, 6(5), 1440 - 5
DNA methyltransferase induced by PBCV-1 virus infection of a Chlorella-like green alga; Xia YN et al.; A DNA methyltransferase was isolated from a eucaryotic, Chlorella-like green alga infected with the virus PBCV-1 . The enzyme recognized the sequence GATC and methylated deoxyadenosine solely in GATC sequences . Host DNA, which contains GATC sequences, but not PBCV-1 DNA, which contains GmATC sequences, was a good substrate for the enzyme in vitro . The DNA methyltransferase activity was first detected about 1 h after viral infection; PBCV-1 DNA synthesis and host DNA degradation also began at about this time . The appearance of the DNA methyltransferase activity required de novo protein synthesis, and the enzyme was probably virus encoded . Methylation of DNAs with the PBCV-1-induced methyltransferase conferred resistance of the DNAs to a PBCV-1-induced restriction endonuclease enzyme described previously (Y . Xia, D . E . Burbank, L . Uher, D . Rabussay, and J . L . Van Etten, Mol . Cell . Biol . 6:1430-1439) . We propose that the PBCV-1-induced methyltransferase protects viral DNA from the PBCV-1-induced restriction endonuclease and is part of a virus-induced restriction and modification system in PBCV-1-infected Chlorella cells.

Vestn Khir Im I I Grek, 1986 May, 136(5), 28 - 32
{Treatment of peritonitis}; Osipov AP et al.; Different forms of peritonitis were observed in 209 patients . Generalized results of treatment have shown positive effects of the complex treatment including preoperative management, timely radical operation aimed at liquidation of the source of peritonitis, lavage of the abdominal cavity and adequate drainage and finally, intensive therapy in the postoperative period . Lethality in generalized peritonitis was 11.1%.

Mikrobiologiia, 1986 May-Jun, 55(3), 357 - 61
{Succinate dehydrogenase activity of Escherichia coli cells after heat stress and during the reparative process}; Zhdan-Pushkina SM et al.; The resting cells of different E . coli cells remained viable after their heating at 48 degrees C for 30 min . The activity of their succinate dehydrogenase (SDH) (EC 1.3.99.1) was not more than 50% of the control one . When the cells were inoculated after a heat stress into a peptone medium, they started to grow at a high rate . However, their maximal specific growth rate mu and the overall biomass yield were less than in the control . The SDH activity of the cells reached the original level by the end of the logarithmic growth phase . This did not happen when the cells were incubated in 0.14 M NaCl for a time necessary for the culture to reach the end of the logarithmic growth phase . The SDH activity (in absolute values) of cell-free extracts was not greater than 35% of the cell SDH activity . The SDH activity of the cell-free extracts did not change after their heating at 48 degrees C . The SDH activity of E . coli cells is recommended to be used as a parameter indicative of their stress state.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1986 May, 261(3), 266 - 79
Characterization of hemolytic strains of Escherichia coli belonging to classical enteropathogenic O-serogroups; Beutin L et al.; 147 isolates of enteropathogenic E . coli (EPEC) belonging to eleven different O-serogroups were examined for hemolysin-synthesis . Hemolysin-producing (Hly+) strains were found at high frequency in O-serogroups 26 and 114 and at low frequency in O-serogroup 126 . The hemolytic activities of EPEC strains of different serogroups were found to be low when compared to Hly+ non-EPEC E . coli strains of different origins . Similarities between the hly determinants of EPEC strains and the hly region of an uropathogenic strain were detected by DNA-hybridization . By this method hly genes of EPEC strains belonging to O-serogroups 114 and 126 were localized on the bacterial chromosome . The hly determinants of O26 strains were localized on 100 +/- 5 MD size conjugative plasmids . The hly plasmid pEO5 of the O26 strain C4115 was transferred by conjugation into an E . coli K12 recipient and found to be stable inherited . The hemolytic activity of the transconjugant was comparable to the wildtype O26 donor . The plasmid pEO5 was found to be different in size, compatibility and hemolysin expression from a number of described hemolysin-plasmids.

Radiobiologiia, 1986 May-Jun, 26(3), 375 - 7
{Modification of the damaging effect of alpha-particles on Escherichia coli K-12 by low-intensity laser irradiation}; Voskanian KSh et al.; A study was made of the combined effect of laser (helium-neon laser, lambda = 633 nm) and alpha-radiation on survival of Escherichia coli K-12 cells of different genotypes . Pre- and post-irradiation exposures to laser-radiation diminished the damaging effect of alpha-particles . In the latter case, modification of survival was more pronounced.

Radiobiologiia, 1986 May-Jun, 26(3), 372 - 4
{Oxygen effect in the radioresistant Escherichia coli Gamr444 strain}; Korystov IuN et al.; In experiments on E . coli Gamr 444 the oxygen effect has been studied . Cells were exposed to gamma-rays with constant bubbling of oxygen or nitrogen through the suspension or without bubbling . In the latter case the dose-effect curve was distorted due to radiochemical absorption of oxygen . The dose curve parameters were determined in the anoxic and oxygenating conditions, they are: lin n(N2) = 3.6; D0(N2) = 371 Gy; In n(O2) = 3.6; D0(O2) = 112 Gy . The oxygen effect for E . coli Gamr 444 was 3.3 as determined by D0 . In studying the radiosensitivity and its modification in radioresistant strains one should eliminate the influence of radiochemical absorption of oxygen by aeration of the medium during exposure.

Mol Gen Genet, 1986 May, 203(2), 281 - 7
Assembly analysis of ribosomes from a mutant lacking the assembly-initiator protein L24: lack of L24 induces temperature sensitivity; Herold M et al.; Previously, we have shown that the ribosomal protein L24 is one of two assembly-initiator proteins . L24 is essential for early steps of the assembly of the 50S ribosomal subunit but it is not involved in both the late assembly and the ribosomal functions . Surprisingly, an E . coli mutant (TA109-130) exists which lacks L24 . This apparent paradox is analyzed and resolved in this paper . The phenotypic is analyzed and resolved in this paper . The phenotypic features of the mutant lacking L24, are a temperature sensitivity (growth severely reduced beyond 34 degrees C), a very low growth rate already at permissive temperatures (at least six-fold slower than wild type) and an underproduction of 50S subunits (molar ratio of 30S to 50S about 1:0.5) . The S value of the mutant large subunits is 47S, and they are normally active in poly(Phe) synthesis . The total protein of the mutant large subunits show negligible activity in the total reconstitution assay using the standard two-step procedure . Number analysis of the assembly-initiator proteins revealed that only one initiator protein is effective, as expected . The activity is restored upon addition of wild-type L24 . However, when the temperature of the first step is lowered from 44 degrees to 36 degrees C, reconstitution of active particles occurs with a 50% efficiency in the absence of L24 . The recovery of activity is accompanied by the appearance of again two initiator proteins, when the mutant TP50 lacking L24 is used in the reconstitution assay at the 'permissive' temperature of 36 degrees C during the first step.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Genet, 1986 May, 203(2), 208 - 13
Inhibition of F plasmid replication in htpR mutants of Escherichia coli deficient in sigma 32 protein; Wada C et al.; The Escherichia coli htpR (= hin, rpoH) mutants are defective in the induction of heat-shock proteins due to a deficiency in sigma 32 and are unable to grow at high temperature . We found that these mutants are also defective in supporting replication of certain plasmids including F and mini-F . When a htpR mutation is introduced into an F' strain, the F' plasmid is effectively excluded . Similarly, when an F' or mini-F plasmid is introduced into htpR mutant cells, transconjugant or transformant clones are obtained at low frequencies and the plasmid is rapidly lost upon subsequent growth in a non-selective medium . In htpR amber mutants carrying a temperature-sensitive suppressor, mini-F replication occurs normally at 30 degrees C, but is inhibited upon transfer to 40 degrees C where the suppressor tRNA is inactivated . A temperature-resistant "pseudo-revertant" of the htpR6 (amber) mutant, that exhibits apparently normal induction of the major heat-shock proteins in the absence of functional sigma 32, fails to support mini-F replication at 40 degrees C, suggesting that inhibition of mini-F replication is not a secondary consequence of the defective induction of the major heat-shock proteins . It is proposed that the function of the sigma 32 protein is directly required for F plasmid replication.

Jpn J Surg, 1986 May, 16(3), 209 - 17
Enhanced endotoxin clearance in reversed Eck fistula rats during a tolerant stage; Yamaguchi Y et al.; The effects of tolerance to Escherichia coli endotoxin on the clearance activity of the hepatic reticuloendothelial system was examined using the experimental animal model of the reversed Eck fistula (REF) . Compared with clearance rate of 51Cr-labeled endotoxin in the normal REF-rat, in the tolerant reversed Eck fistula animals, there was three fold increase in the clearance of endotoxin in the liver . The enhanced endotoxin uptake of the hepatic reticuloendothelial system was observed when the medium containing serum from tolerant animals was infused into the non-tolerant liver with a Harvard pump before the clearance studies, using 51Cr-labeled endotoxin administered via the femoral vein . Heat treatment of tolerant serum at 56 degrees C for 45 min destroyed the enhancing effect . These results suggest that during endotoxin tolerance, humoral factors, particularly complement, play an important role in the uptake of endotoxin from portal vein blood . A direct stimulation of the hepatic reticuloendothelial system may also occur.

Appl Environ Microbiol, 1986 May, 51(5), 956 - 62
Methodology for enumeration of coliphages in foods; Kennedy JE Jr et al.; The effects of eluent composition, pH, and chaotropic agents on the recovery of T2, MS2, and indigenous coliphages from various foods were investigated . Additionally, methods of sample suspension and clarification were evaluated for coliphage recovery and application to various foods . Clarified sample suspensions were assayed for coliphages with a modified agar layer technique and appropriate Escherichia coli hosts . Centrifugation and polypropylene mesh filtration were more rapid and effective than glass wool filtration for clarification of sample suspensions and subsequent recovery of coliphages . Blending, stomaching, and shaking procedures were generally comparable for sample liquefaction and release of coliphages from foods . Complex basal eluents, EC medium and 1% casein, were generally more effective than a less complex eluent, phosphate buffer, for elution of coliphages from foods . For most foods, incorporation of sodium chloride or chaotropic agents, i.e., sodium trichloroacetate, urea, Tween 80, Triton X-100, and sodium nitrate, into basal eluents did not enhance recovery of coliphages . Indigenous coliphage recovery was not affected by sample suspension pH over a range of 6.0 to 9.0 . With an optimal procedure, i.e., EC medium eluent, blending, and centrifugation, the recovery of T2 and MS2 ranged from 48 to 81% and from 58 to 100%, respectively, depending on the food type.

Anal Biochem, 1986 May 1, 154(2), 485 - 91
Quantitation of single- and double-strand DNA breaks in vitro and in vivo; Kohen R et al.; This communication describes a rapid and convenient procedure for quantitation of strand breaks in bacterial DNA, both in vitro and in vivo, using agarose gel electrophoresis . The electrophoretic determination of single strand breaks is carried out in alkaline medium, followed by renaturation of the gel and intercalation of the fluorescent dye, ethidium bromide . Double-strand breaks are determined by electrophoresis in neutral medium containing the dye . The distribution of DNA fragment sizes, the determination of the number-average molecular weight, the quantitation of the average number of DNA breaks per molecule, and the ratio between the single- and double-strand breaks are evaluated from microdensitometric scanning of the gels . The application of this analysis to damage caused by a combination of ascorbate and copper is demonstrated.

J Dairy Sci, 1986 May, 69(5), 1250 - 8
Effects of endotoxin on mammary secretion of lactating goats; Lengemann FW et al.; The objectives were to describe the magnitude and time course of changes in milk pH, Na, K, lactose, and somatic cells and to determine if paracellular pathways were altered after infusion of Escherichia coli endotoxin (serotype #0128:AB12) to produce inflammation in one-half of the udder of the goat . Intramammary infusion of endotoxin increased pH, number of somatic cells, and Na and decreased K and lactose in milk . Sodium and number of somatic cells were increased by as little as .1 microgram of endotoxin; .25 microgram produced changes in most of the other parameters; maximal effect was elicited by 1 microgram of endotoxin . The gland response peaked from 5 to 7 h after infusion of endotoxin with an increase in milk cellularity as the only significant effect noted in the control gland . Infusion of {14C}lactose into the gland and {99mTc}albumin into the blood demonstrated that large molecules were more able to cross into and out of udder halves after endotoxin treatment . It is suggested that ion interchange rather than bulk flow across paracellular paths is responsible for changes . In addition, endotoxin appeared to reduce lactose secretion and synthesis.

J Assoc Off Anal Chem, 1986 May-Jun, 69(3), 531 - 6
DNA colony hybridization method using synthetic oligonucleotides to detect enterotoxigenic Escherichia coli: collaborative study; Hill WE et al.; The genes that encode several of the enterotoxins produced by Escherichia coli have been cloned by recombinant DNA techniques . When the nucleotide sequence of these genes is determined, defined sequence oligonucleotides that include a part of these genes may be synthesized . A 22-base DNA hybridization probe was produced for each of 2 heat-stable E . coli enterotoxin (ST) genes: STH, from strains originally isolated from humans; and STP, from strains first found in pigs . For this study, 32P end-labeled DNA probes, sonicated calf thymus DNA, and 3 known and 20 unknown (10 ST-positive and 10 ST-negative) strains were sent to each of 23 collaborators . Cultures were spotted onto an agar-based medium and grown into colonies, which were transferred by blotting to cellulose filters, lysed by alkali and steam, and used for DNA colony hybridization with the ST DNA probes . Strains containing an ST gene were recognized as dark spots on an autoradiogram . Of the 460 samples analyzed, 440 (95.7%) were correctly classified by the collaborators . The method has been adopted official first action.

EMBO J, 1986 May, 5(5), 913 - 7
Wounding a fibroblast monolayer results in the rapid induction of the c-fos proto-oncogene; Verrier B et al.; The c-fos gene has previously been shown to be transiently induced within minutes after the stimulation of mouse fibroblasts with growth factors . Induction of c-fos was observed specifically with competence factors (e.g., platelet-derived growth factor), not with progression factors (e.g., platelet-poor plasma), suggesting a role for c-fos in conferring competence on fibroblasts . To test this hypothesis we have analyzed c-fos expression in NIH 3T3 cells that were made competent in a different way, namely by wounding a confluent monolayer of cells . Using antibodies raised against either a synthetic fos peptide or a beta-galactosidase--fos fusion protein, we show in this study that in the majority of cells lining the wound c-fos protein is rapidly and transiently induced to high levels . No induction is observed in cells at a distance from the wound greater than approximately 5 cell layers . Induction is equally efficient in both serum-containing and serum-free medium, and is similar in cells that were deprived of fetal calf serum for 40 h prior to making the wound . Our observations support the hypothesis that c-fos may be involved in inducing the 'competent state' in fibroblasts and suggests an early role for c-fos in wound healing and tissue regeneration.

EMBO J, 1986 May, 5(5), 1105 - 9
Enzymatic 2'-O-methylation of the wobble nucleoside of eukaryotic tRNAPhe: specificity depends on structural elements outside the anticodon loop; Droogmans L et al.; We have investigated the specificity of the enzyme tRNA (wobble guanosine 2'-O-)methyltransferase which catalyses the maturation of guanosine-34 of eukaryotic tRNAPhe to the 2'-O-methyl derivative Gm-34 . This study was done by micro-injection into Xenopus laevis oocytes of restructured yeast tRNAPhe in which the anticodon GmAA and the 3' adjacent nucleotide 'Y' were substituted by various tetranucleotides . The results indicate that the enzyme is cytoplasmic; the chemical nature of the bases of the anticodon and its 3' adjacent nucleotide is not critical for the methylation of G-34; the size of the anticodon loop is however important; structural features beyond the anticodon loop are involved in the specific recognition of the tRNA by the enzyme since Escherichia coli tRNAPhe and four chimeric yeast tRNAs carrying the GAA anticodon are not substrates; unexpectedly, the 2'-O-methylation is not restricted to G-34 since C-34, U-34 and A-34 in restructured yeast tRNAPhe also became methylated . It seems probable that the tRNA (wobble guanosine 2'-O-)methyltransferase is not specific for the type of nucleotide-34 in eukaryotic tRNAPhe; however the existence in the oocyte of several methylation enzymes specific for each nucleotide-34 has not yet been ruled out.

Toxicol Lett, 1986 May, 31(2), 131 - 7
Relationship between oxidative metabolites of hydrazine and hydrazine-induced mutagenicity; Noda A et al.; Hydrazine (Hz) mutagenicity was observed in a test using Escherichia coli B/r strain, WP2 uvrA and was enhanced by the addition of rat liver microsomal fraction containing a generating system, while the enhanced mutagenicity was diminished by the addition of metyrapone to the microsome-free levels . On the other hand, an NADPH-dependent difference spectrum of the metabolic intermediate of Hz-complex, characterized by a maximum level of 448 nm, was also inhibited by metyrapone . The results show that the oxidative intermediates, which are diimide and its precursor, hydrazine free radical {Biochem . Biophys . Res . Commun., 133 (1986) 1086}, are responsible not only for hepatotoxicity but also for the enhancement of genotoxicity or mutagenicity.

Plasmid, 1986 May, 15(3), 191 - 8
Effect of the deletion of a fragment dispensable for the autonomous maintenance of plasmid pT181 on the competition between incompatible plasmids; Iordanescu S; The deletion of the 560-bp HindIII C fragment from pT181 derivatives does not change the stability or copy number of the plasmid but affects its ability to compete with undeleted, incompatible plasmids for maintenance in the host cell . The disadvantage of the deleted plasmids seems to be manifested at the level of replication . It results that for plasmid pT181 a sequence dispensable for autonomous maintenance and replication control could affect the outcome of the competition between autonomous, incompatible plasmids.

Plasmid, 1986 May, 15(3), 163 - 71
Purification and characterization of the CopB replication control protein, and precise mapping of its target site in the R1 plasmid; Riise E et al.; The CopB regulatory loop from plasmid R1 has been analyzed . The CopB protein was partially purified, but proteolytic activity in vitro resulted in the recovery of two molecular forms of the polypeptide . Both of these acted as repressors of the repA promoter and had identical activities . The smaller of the proteins was found to be the result of a specific cleavage in the normal in vivo translation product . The active form of the CopB protein is most likely a tetramer, which binds to a DNA region overlapping the repA promoter that also contains a stretch of dyad symmetry . Footprinting analysis and mutant analysis (including nucleotide sequence determination) identified this binding site within 20-25 base pairs . In agreement with in vivo results the binding between CopB and its target site is moderate compared with other operons like lac and trp.

J Biochem (Tokyo), 1986 May, 99(5), 1327 - 37
Isoprenoid synthesis in Escherichia coli . Separation and partial purification of four enzymes involved in the synthesis; Fujisaki S et al.; Isopentenyl pyrophosphate (IPP) isomerase, farnesyl pyrophosphate (FPP) synthetase, octaprenyl pyrophosphate (OPP) synthetase and undecaprenyl pyrophosphate (UPP) synthetase were partially purified from Escherichia coli by DEAE-Toyopearl chromatography . FPP synthetase catalyzed the condensation of IPP with dimethylallyl pyrophosphate (DPP) as well as with geranyl pyrophosphate (GPP) to yield FPP as final product . OPP synthetase and UPP synthetase catalyzed the condensation of IPP with FPP to yield OPP and cis,trans-polyprenyl pyrophosphates (the C45-, C50, and C55-compound), respectively . Neither DPP nor GPP acted as a priming substrate for either enzyme . These four enzymes required Mg2+ or Mn2+ for their activities . UPP synthetase required also Triton X-100 for its activity . The addition of Triton X-100 enhanced OPP synthetase, but it did not affect IPP isomerase and FPP synthetase . It seems possible that the combination of the four enzymes ensures the in vivo synthesis of long-chain isoprenoids in E . coli.

J Biochem (Tokyo), 1986 May, 99(5), 1299 - 310
Phosphoenolpyruvate carboxylase of Escherichia coli K-12 . N- and C-terminal sequences and tentative assignment of the catalytically essential cysteine residue; Ishijima S et al.; The N- and C-terminal amino acid sequences of phosphoenolpyruvate carboxylase {EC 4.1.1.31} from Escherichia coli K-12 were determined to establish the primary structure deduced from the nucleotide sequence of the cloned gene for the enzyme (Fujita, N., Miwa, T., Ishijima, S., Izui, K., & Katsuki, H . (1984) J . Biochem . 95, 909-916) . As predicted from the nucleotide sequence, two polypeptides were produced upon treatment with hydroxylamine, which specifically cleaves the Asn-Gly bond, and their amino acid compositions were also in accordance with those predicted . The tryptic peptides which contained cysteine residues labeled with a fluorescent reagent, N-{7-(dimethylamino)-4-methylcoumarinyl}maleimide, were isolated by high-performance liquid chromatography and partially sequenced . All of them could be assigned on the deduced primary structure . The modified cysteine residues were Cys-157, Cys-385, Cys-458, Cys-568, Cys-665, and Cys-754 . Furthermore, the essential cysteine residue which is presumably located at or near the active site was tentatively identified as Cys-568, since it was consistently protected against the modification by 2-phospholactate, a substrate analog.

Hepatology, 1986 May-Jun, 6(3), 482 - 9
Bile infection documented as initial event in the pathogenesis of brown pigment biliary stones; Cetta FM; Findings in two patients having brown pigment bile stones, recurrent 18 and 36 months after cholecystectomy, are reported . Present data suggest that bile infection precedes rather than follows the formation of brown stones . The present data are part of a prospective study of 600 consecutive patients who underwent operation for gallstones and in whom clinical and laboratory findings, intra- and postoperative bile culture and bile pH were related to the analysis of stone composition by X-ray diffractometry and infrared spectroscopy . Both patients during the first operation underwent cholecystectomy, sphincterotomy and T-tube drainage . Bile culture was negative both at operation and in the first 3 to 5 samples obtained from the T-tube every 2 days, while Escherichia coli was found, starting from the tenth and fourteenth postoperative days, respectively . At the second operation, typical recurrent "earthy" brown stones, easily crushed with the fingers, with no central nucleus of a different structure were found . Previous sphincterotomy became stenotic, and E . coli was found in the operative bile in a concentration higher than 10(6) per ml . It is suggested that bile infection by E . coli, in addition to bile stasis, plays a crucial role in the pathogenesis of brown pigment stones.

Am Rev Respir Dis, 1986 May, 133(5), 866 - 74
Tracing the leukocyte marker protein in lung fluids and lung-draining lymph nodes during endotoxemia in sheep; Belenko M et al.; Infusion of Escherichia coli endotoxin into sheep produces a form of acute lung injury that resembles the adult respiratory distress syndrome (ARDS) . A large portion of the physiologic derangements produced by E . coli endotoxin in this model is thought to be granulocyte-dependent . We measured the level of L1, a granulocyte and monocyte marker protein, in various tissues and fluids after infusion of E . coli endotoxin into sheep . In an acute study, sheep received saline or 1.25 microgram/kg E . coli endotoxin dissolved in saline, or endotoxin after hydroxyurea-induced granulocytopenia . L1 was measured by radioimmunoassay in efferent lymph from the caudal mediastinal lymph node collected between 5 and 6 h after infusion . In addition, L1 was visualized in both lung-draining and extrapulmonary lymph nodes by indirect immunofluorescence . In a chronic study, sheep were prepared with lung lymph fistulas, and L1 was measured in draining pulmonary lymph, plasma, and bronchoalveolar lavage (BAL) fluid, serially, over a 24-h period after infusion . Mean L1 level in pulmonary lymph in the acute study was 6 times higher by absolute concentration, and 19 times higher when lymph flow rates were taken into account, in the sheep that received endotoxin than in saline-infused sheep or endotoxin-infused, granulocytopenic sheep . Fluorescence was greater in the outer cortical region adjacent to subcapsular, afferent sinuses of lung draining-lymph nodes of endotoxin-treated sheep than in the comparable nodes of saline-infused sheep and endotoxin-infused granulocytopenic sheep . In endotoxin-treated sheep, extrapulmonary lymph nodes were less reactive than lung-draining nodes.(ABSTRACT TRUNCATED AT 250 WORDS)

Proc Natl Acad Sci U S A, 1986 May, 83(9), 2860 - 4
Identification, nucleotide sequence, and control of developmentally regulated promoters in the hook operon region of Caulobacter crescentus; Chen LS et al.; The major flagellar proteins, including the flagellins and the hook protein, are synthesized periodically in the Caulobacter crescentus cell cycle at the time of flagellum assembly . Although fla genes are regulated at the transcriptional level {Ohta, N., Chen, L.-S., Swanson, E . & Newton, A . (1985) J . Mol . Biol . 186, 107-115}, the 5' regulatory regions of C . crescentus genes have not been identified . We describe here the results of nuclease S1 protection assays that map the 5' ends of mRNAs synthesized in vivo from transcription units II (hook operon) and II.1 of the hook gene cluster and locate the corresponding promoter regions PII and PII.1 . The two promoters are regulated with different periodicities in the cell cycle and have different genetic requirements for expression . The failure to detect transcripts from either PI or PII in Escherichia coli suggests that developmentally regulated promoters of C . crescentus have different recognition sequences from those of E . coli . There is little nucleotide sequence homology between PII and PII.1 . There are, however, three regions of homology between PII and the nucleotide sequence 5' to the 29-kDa-flagellin-related gene, and two of these are in regions of dyad symmetry . We discuss the possibility that DNA-protein interactions at homologous nucleotide sequences like those identified in PII are part of a regulatory gene cascade that participates in timing fla gene expression in the C . crescentus cell cycle.

Proc Natl Acad Sci U S A, 1986 May, 83(10), 3223 - 7
Cloning, sequence, and expression of bovine interleukin 2; Cerretti DP et al.; Interleukin 2 (IL-2) cDNA clones have been isolated from both human and murine sources . We report here the isolation of a cDNA clone encoding bovine IL-2 . This was accomplished by screening a cDNA library constructed from lectin-stimulated bovine lymph node cells, using a human IL-2 probe . Bovine IL-2 is composed of 155 amino acids and has a predicted molecular weight of 19,555 . Alignment of the amino acid sequence with human IL-2 indicates that mature bovine IL-2 is composed of 135 amino acids and has a predicted molecular weight of 15,452 . It has an amino acid homology of 65% with human IL-2 and 50% with murine IL-2 . Bovine IL-2 is unique among IL-2 homologs in that it has a single N-linked glycosylation site . Biologically active bovine IL-2 was synthesized in an Escherichia coli expression system.

Proc Natl Acad Sci U S A, 1986 May, 83(10), 3116 - 20
Unique type of plasmid maintenance function: postsegregational killing of plasmid-free cells; Gerdes K et al.; The stability locus parB+ of plasmid R1 has been found to specify a unique type of plasmid maintenance function . Two genes, hok (host killing) and sok (suppressor of killing), are required for the stabilizing activity . The hok gene encodes a highly toxic gene product, whose overexpression causes a rapid killing and a concomitant dramatic change in morphology of the host cell . The other gene, sok, was found to encode a product that counteracts the hok gene-mediated killing . The parB+ region was inserted in a plasmid with a temperature-sensitive replication system . At nonpermissive temperature, the parB+ plasmid was maintained in the population for a significantly longer period than the corresponding parB- plasmid . Coupled to this extended maintenance, a large fraction of the population was shown to be nonviable plasmid-free cells with the characteristic hok-induced change in morphology . Based on these findings, we propose that the parB+ locus mediates plasmid stability by killing cells that have lost the parB+ plasmid during the preceding cell division, thereby ensuring that a growing bacterial culture predominantly consists of plasmid-containing cells.

J Med Chem, 1986 May, 29(5), 698 - 708
Conformational energy calculations and electrostatic potentials of dihydrofolate reductase ligands: relevance to mode of binding and species specificity; Andrews PR et al.; Classical potential energy calculations are reported for a series of 11 structurally diverse substrates, products, and inhibitors of dihydrofolate reductase . In almost every case, the calculations reveal a range of potential biologically active conformations accessible to the molecule, and geometry optimization with molecular mechanics and molecular orbital calculations further expands the range of accessible conformations . The energy calculations are supplemented with electrostatic potential energy surfaces for the heterocyclic components of each molecule . These data are used in conjunction with the energy calculations and the crystallographically determined enzyme structures to compare two alternative proposed binding modes of folates known to bind with their pteridine rings inverted relative to that of methotrexate . It is shown that the conformational flexibility of the connecting chain between the benzoyl glutamate and pteridine moieties in the folates actually allows the pteridine ring to shift between these alternative binding modes, a combination of which may offer the best explanation for the observed activity . The electrostatic potentials and conformational energy data are also used in an attempt to account for the species specificity of inhibitors of mammalian, bacterial, and protozoal dihydrofolate reductases . The results show that while these techniques can be used to explain many of the observed results, others require recourse to the observed crystal structures to provide a satisfactory explanation.

J Bacteriol, 1986 May, 166(2), 519 - 27
Bimodal pattern of killing of DNA-repair-defective or anoxically grown Escherichia coli by hydrogen peroxide; Imlay JA et al.; Two modes of killing of Escherichia coli K-12 by hydrogen peroxide can be distinguished . Mode-one killing was maximal with hydrogen peroxide at a concentration of 1 to 2 mM . At higher concentrations the killing rate was approximately half maximal and was independent of H2O2 concentration but first order with respect to exposure time . Mode-one killing required active metabolism during the H2O2 challenge, and it resulted in sfiA-independent filamentation of both cells which survived and those which were killed by the challenge . This mode of killing was enhanced in xth, polA, recA, and recB strains and was accelerated in all strains by an unidentified, anoxia-induced cell function . A strain carrying both xth and recA mutations appeared to undergo spontaneous mode-one killing only under aerobic conditions . Mode-one killing appeared to result from DNA damage which normally occurs at a low, nonlethal level during aerobic growth . Mode-two killing occurred at higher doses of H2O2 and exhibited a multihit dependence on both H2O2 concentration and exposure time . Mode-two killing did not require active metabolism, and killed cells did not filament, although survivors demonstrated a dose-dependent growth lag . Strains with DNA-repair defects were not especially susceptible to mode-two killing.

J Bacteriol, 1986 May, 166(2), 399 - 403
Molecular cloning and expression of hipA, a gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis; Moyed HS et al.; The hipA gene at 33.8 min on the Escherichia coli chromosome controls the frequency of persistence upon inhibition of murein synthesis; for strains bearing hipA+ the frequency is 10(-6), and for hipA- strains the frequency is 10(-2) . hip+ has been cloned by selection for a kanamycin resistance determinant at 33.9 min . hipA+ is dominant over hipA- in both recA+ and recA- backgrounds . The smallest DNA insert which contains hipA+, as determined by the ability of the plasmids to complement hipA- strains, is 1,885 base pairs . Both orientations of hipA+ are obtained when the cloning site of vector is remote from strong promoters; both orientations complement hipA-, and both encode a unique peptide of 50,000 Mr . The probable direction of transcription has been deduced from the pattern of peptides encoded by plasmids from which either end of the insert and adjacent vector sequences have been deleted . This information and the recovery of only one orientation of hipA+ when the cloning site is close to a strong promoter suggest that a high level of expression of the gene is not tolerated by E . coli.

J Bacteriol, 1986 May, 166(2), 380 - 4
In vitro effect of the Escherichia coli heat shock regulatory protein on expression of heat shock genes; Bloom M et al.; In Escherichia coli, the ability to elicit a heat shock response depends on the htpR gene product . Previous work has shown that the HtpR protein serves as a sigma factor (sigma 32) for RNA polymerase that specifically recognizes heat shock promoters (A.D . Grossman, J.W . Erickson, and C.A . Gross Cell 38:383-390, 1984) . In the present study we showed that sigma 32 synthesized in vitro could stimulate the expression of heat shock genes . The in vitro-synthesized sigma 32 was found to be associated with RNA polymerase . In vivo-synthesized sigma 32 was also associated with RNA polymerase, and this polymerase (E sigma 32) could be isolated free of the standard polymerase (E sigma 70) . E sigma 32 was more active than E sigma 70 with heat shock genes; however, non-heat-shock genes were not transcribed by E sigma 32 . The in vitro expression of the htpR gene required E sigma 70 but did not require E sigma 32.

Infect Immun, 1986 May, 52(2), 613 - 6
Evidence for lipid peroxidation in endotoxin-poisoned mice; Peavy DL et al.; Ethane has been identified and quantitated in air exhaled by mice following intraperitoneal injection of 20, 40, or 200 mg of Escherichia coli O111:B4 lipopolysaccharide (LPS) per kg . Significant increases in ethane concentration occurred within 1 to 5 h after LPS administration . In addition, increased concentrations of malondialdehyde were found in crude homogenates of livers obtained from mice 16 h after administration of 20 mg of LPS per kg . These results suggest that lipid peroxidation may be an important mechanism responsible for LPS toxicity.

Infect Immun, 1986 May, 52(2), 586 - 93
Native surface association of a recombinant 38-kilodalton Treponema pallidum antigen isolated from the Escherichia coli outer membrane; Fehniger TE et al.; A recombinant plasmid designated pAW305, containing a 6-kilobase insert of Treponema pallidum DNA, directed the expression of a 38-kilodalton (kDa) treponemal antigen in Escherichia coli . The 38-kDa antigen copurified with the outer membrane fraction of the E . coli cell envelope after treatment with nonionic detergents or sucrose density gradient centrifugation . Rabbits immunized with the recombinant 38-kDa antigen developed antibodies which reacted specifically with a 38-kDa T . pallidum antigen on immunoblots, and 38-kDa antisera specifically immobilized T . pallidum in a complement-dependent manner in the T . pallidum immobilization test . Antisera to the 38-kDa recombinant antigen were also used to demonstrate its native surface association on T . pallidum by immunoelectron microscopy.

Infect Immun, 1986 May, 52(2), 468 - 75
Identification of a nonfimbrial adhesive factor of an enterotoxigenic Escherichia coli strain; Darfeuille-Michaud A et al.; An enterotoxigenic Escherichia coli strain (strain 2230), isolated from a patient with acute infantile diarrhea, was found to adhere only to the brush border of human intestinal epithelial cells . This strain does not hemagglutinate human, bovine, chicken, or guinea pig erythrocytes . The adhesion of E . coli 2230 appears to be mediated by a nonfimbrial bacterial surface protein of 16,000 daltons which can be extracted by heating the bacteria at 60 degrees C for 20 min . This surface protein is implicated as an adhesive factor because pretreatment of enterocytes with this protein extract completely inhibits the adhesion of E . coli 2230 . This adhesive factor is serologically distinct from other adhesive factors found in enterotoxigenic E . coli strains . A plasmid DNA of 66 megadaltons is involved in the synthesis of this nonfimbrial adhesive factor.

J Clin Chem Clin Biochem, 1986 May, 24(5), 299 - 308
Evaluation of elastase and alpha 1-proteinase inhibitor-elastase uptake by polymorphonuclear leukocytes and evidence of an elastase-specific receptor; Dwenger A et al.; Neither resting nor stimulated isolated human polymorphonuclear leukocytes did bind or ingest preformed complexes of alpha 1-proteinase inhibitor and unlabeled/125I-labeled human leukocyte elastase . In contrast, granulocytes bound unlabeled/125I-labeled elastase and the extent of binding was reduced in the presence of respiratory burst stimulators, such as 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, E . coli endotoxin, and N-formyl-L-methionyl-L-leucyl-L-phenylalanine . In association/dissociation and competition inhibition experiments it was demonstrated that granulocyte-elastase binding was specific and saturable . From Scatchard and non-linear regression analysis there was evidence of a two-class receptor model with independent binding sites . Calculated by the non-linear regression method assuming a two-class receptor model the characteristics of the high affinity/low capacity binding site were K1 = 216 +/- 129 X 10(6) l X mol-1 (means +/- s; n = 3) and R1 = 1.38 +/- 0.95 nmol X l-1 corresponding to 0.083 X 10(6) receptors per cell, whereas the low affinity/high capacity binding site had the characteristics K2 = 0.50 +/- 0.09 X 10(6) l X mol-1 and R2 = 237 +/- 103 nmol X l-1 corresponding to 14.3 +/- 6.2 X 10(6) receptors per cell.

Jpn J Antibiot, 1986 May, 39(5), 1297 - 301
{Transfer of cefotetan into exudates from excoriated skin wounds}; Sugiyama H et al.; Transfer of cefotetan (CTT) into exudates from excoriated skin wounds was studied in 9 adult patients . Each subject was given an intravenous bolus injection of 50 mg/kg of body weight . Concentrations of CTT in serum and in exudate fluid were determined by bioassay using Escherichia coli NIHJ as the test organism . The mean (+/- S.D.) CTT concentration in serum reached 274.3 +/- 78.3 micrograms/ml at 30 minutes after the injection and decreased to 30.0 +/- 12.0 micrograms/ml at 8 hours after the injection . The peak value of CTT in exudate fluid was 143.1 +/- 22.3 micrograms/ml at 1 hour . Eight hours after the injection, the mean concentration in the exudate was 25.7 +/- 21.1 micrograms/ml . The data obtained were analysed pharmacokinetically: CTT concentrations in serum were analysed by two-compartment model, and those in exudate fluid from excoriated skin wounds were analysed by the model in which skin was considered as a small part of the peripheral compartment . Thus T1/2(beta) of CTT levels in serum was calculated as 2.38 hours, AUC0----infinity was 1,000.2 micrograms X hr/ml and Vd was 164.5 ml/kg . Tmax and Cmax of CTT levels in exudates were calculated as 1.05 hours and 131.2 micrograms/ml, respectively.

Am J Reprod Immunol Microbiol, 1986 May, 11(1), 6 - 10
Prostaglandin E2 and plasminogen activators in human milk and their secretion by milk macrophages; Le Deist F et al.; Human milk was shown to contain prostaglandin E2 (PGE2) and plasminogen activator (PA) at variable concentrations depending on the time of lactation after delivery . Milk PA was functionally and immunologically identical to urokinase . A follow-up study showed that the maximum PGE2 concentrations occurred during the second week while the maximum PA concentration was observed at the end of the first week of lactation . Milk macrophages cultured in vitro were able to secrete both PGE2 and PA . When cells were activated by concanavalin A (ConA) or E . coli lipopolysaccharide (LPS), PGE2 secretion increased dramatically while PA secretion did not . The ability of activated macrophages to secrete PGE2 was at its highest shortly after delivery . It then progressively decreased during lactation . The possible physiological role of PGE2 and PA on the gastrointestinal tract of breast fed infants is discussed.

Proc Natl Acad Sci U S A, 1986 May, 83(10), 3356 - 60
Efficient recovery and sequencing of mutant genes from mammalian chromosomal DNA; Ashman CR et al.; A retroviral shuttle vector was constructed by introducing the Escherichia coli xanthine (guanine) phosphoribosyltransferase gene (gpt) into the pZip-NeoSV(X)1 vector {Cepko, C . L., Roberts, B . E . & Mulligan, R . C . (1984) Cell 37, 1053-1062} . This vector was packaged into infectious virus which then was used to infect a hypoxanthine (guanine) phosphoribosyltransferase-deficient mouse cell line . Cell lines that expressed the gpt gene were isolated, and it was found that these cells contained a single integrated copy of the vector in a proviral form . Treatment of these cell lines with either ethyl methanesulfonate or BrdUrd produced a greater than 10-fold increase in the frequency of 6-thioguanine-resistant (Sgur) mutants . Intact gpt genes have been recovered from a number of Sgur cell lines after COS cell fusion and introduced into E . coli as part of a plasmid . The complete DNA sequences of three mutant genes have been determined . Two of the mutant genes have a single base substitution, whereas the third has a 34-base-pair deletion . This system should be valuable for analyzing mutagenic specificity and the molecular mechanisms of chemical mutagenesis in mammalian cells . A potentially important feature of the system relative to other shuttle-vector systems is that the mutations are induced in genes integrated into mammalian chromosomes rather than in genes existing as part of autonomously replicating plasmids.

Ann Inst Pasteur Microbiol, 1986 May-Jun, 137A(3), 295 - 9
{Characterization of Escherichia hermannii by electrophoresis of esterases, acid phosphatase and glutamate and malate dehydrogenases}; Goullet P et al.; Esterases, acid phosphatase and glutamate and malate dehydrogenases of 11 strains of Escherichia hermannii were analysed by horizontal electrophoresis in polyacrylamide agarose gel . Seven esterase bands were defined by their range of activity on synthetic substrates and their sensitivity or resistance to di-isopropyl fluorophosphate . These bands were different in activity and in mobility from those produced by E . coli strains . On the basis of variations in mobility of glutamate and malate dehydrogenases and in the number and mobility of esterases, the strains were divided into 3 zymotypes.

Ann Inst Pasteur Microbiol, 1986 May-Jun, 137A(3), 223 - 37
A motility lesion in ColV+ Escherichia coli strains and its possible clinical significance; Tewari R et al.; The introduction of the ColV-K30 or ColV,I-K94 plasmid into Escherichia coli strains produced derivatives which had a motility lesion if grown without shaking at 37 degrees C . Although most ColV+ organisms from shaken cultures were motile, 80-90% of free unclumped organisms from static cultures were flagellate but non-motile . This plasmid effect was temperature-dependent with only those ColV+ organisms grown at 37 degrees C being affected; ColV+ organisms grown at 30 degrees C or below were predominantly motile . The motility lesion depended on the presence, in the ColV+ organisms, of transfer and colicin components together but not of the VmpA protein . Aside from the changed motility, there was extensive autoagglutination (clumping) of ColV+ organisms in static cultures, and the two phenomena (clumping and motility lesion) appeared to be governed by the same factors . The Flac plasmid of FI incompatibility group had a slight inhibitory effect on motility of strain 1829 and caused slight clumping, but representative plasmids of groups FII, FIII, FIV, C, H, I, K, M, N, P, W and X had no appreciable effect on either parameter . Non-motile ColV+ organisms regained motility on incubation with buffered detergent solutions, suggesting that an envelope change might be responsible for the altered motility . It can be hypothesized that ColV+ organisms in the intestine would be motile and hence able to reach the intestinal epithelium for invasion but that, once such organisms had reached the tissues and bloodstream, they would be predominantly non-motile and hence might be less susceptible to phagocytosis.

Clin Exp Immunol, 1986 May, 64(2), 334 - 41
Modulation of gingival Langerhans cell T6 antigen expression in vitro by interleukin 1 and an interleukin 1 inhibitor; Walsh LJ et al.; A human gingival organ culture system was used to test the hypothesis that interleukin 1 (IL-1) modulates the expression of the cortical thymocyte antigen T6 on Langerhans cells (LC) . In cultures enriched with E . coli lipopolysaccharide, T6 expression peaked concurrently with supernatant IL-1 activity . Addition of exogenous IL-1 produced a dose dependent increase in LC T6 expression while not affecting the expression of the Class II antigens DR and DQ by LC . An IL-1 suppressor factor (ILS) was associated with the loss of T6 antigens which occurred in conventional organ cultures . ILS inhibited the thymocyte response to IL-1 and neutralized the effects of IL-1 on both thymocytes and LC . In isolation ILS depressed T6 expression by eliminating resting DR negative LC . This factor may act to regulative negatively, the action of IL-1 as has been suggested for IL-1 inhibitors of similar molecular weight.

Arch Toxicol, 1986 May, 59(1), 4 - 6
Studies on the genotoxicity of the anabolic drugs trenbolone and zeranol; Scheutwinkel M et al.; The androgen trenbolone, and the mycoestrogen zeranol, both anabolic drugs, were tested for their genotoxic potential . Test systems were the SOS-chromotest, the rec-assay and the V79 sister chromatid exchange test without and with metabolic activation using rat liver homogenates and primary rat hepatocytes . It is still a matter of debate if trenbolone has carcinogenic properties, because of its cell transforming activity in vitro . Trenbolone, however, did not demonstrate any genotoxic effect in the assays performed . The results obtained for zeranol were also negative in the SOS-chromotest and V79 sister chromatid exchange test but positive in the rec-assay.

Ann Inst Pasteur Immunol, 1986 May-Jun, 137C(3), 259 - 72
Qualitative differences in effects of recombinant alpha-, beta- and gamma-interferons on human peripheral blood leukocytes in vitro; Billard C et al.; The in vitro effects of bacteria-produced human interferons alpha 2, beta and gamma on several properties of peripheral blood leukocytes from different healthy donors were compared . Treatment with HuIFN-alpha 2 or HuIFN-beta resulted in inhibition of the proliferative response to phytohaemagglutinin and in closely parallel induction of 2'-5'-oligoadenylate synthetase activity . In contrast, HuIFN-gamma had no significant effect on these two activities . However, all three HuIFN were able to enhance natural killer cell cytotoxicity and the expression of HLA-DR surface antigens, with only quantitative variations from donor to donor . Similar results were observed with glycosylated recombinant hamster-cell-derived HuIFN-gamma and with natural HuIFN-gamma . These data demonstrate qualitative differences in the effects of HuIFN-gamma compared to those of HuIFN-alpha 2 or -beta on cells of the immune system.

Res Vet Sci, 1986 May, 40(3), 313 - 7
Attempts to modify changes in the piglet small intestine after weaning; Hampson DJ; The possible causes of changes in the piglet small intestine after weaning were investigated . Groups of piglets weaned at three weeks old were either given oral oxytetracycline daily, weaned on to a regularly fed sow-milk replacer or weaned on to specially formulated diets based on casein or hydrolysed casein as the protein source . After five days, changes in small intestinal structure and brush-border enzyme activities were compared with previous findings in weaned and unweaned animals of the same age . The presence of enterotoxigenic Escherichia coli and rota-viruses in the intestines and the occurrence of diarrhoea were also noted . Consumption of the diet based on hydrolysed casein resulted in significant modifications to crypt depth and brush border enzyme activity at sites between 50 and 75 per cent along the length of the small intestine . Possible reasons for this protective effect, which include the low level of antigenicity of the diet, are discussed in relation to normal intestinal changes after weaning.

Genetika, 1986 May, 22(5), 733 - 40
{Organization and gene expression of plasmid ColD-CA23 connected with colicin biosynthesis}; Pshennikova ES et al.; Organization of genes for colicin, immunity and lysis and regulation of their expression in colicinogenic plasmid ColD-CA23 were studied . The polypeptides synthesised in minicells carrying the ColD plasmid, its Tn5 mutants and the recombinant plasmids constructed by cloning the ColD EcoRV fragments on pBR325 were analysed . The position of the colicin gene promotor is established and direction of the gene transcription determined . The immunity gene was shown to be expressed independently of the gene coding for colicin and believed to have its own SOS-independent promotor . Treatment with mitomycin C of the ColD-carrying cells leads to induction of the synthesis both of colicin and of a 10 KD protein responsible for cell killing and lysis . This protein can be detected in a cultural medium under lysis . Plasmid mutations abolishing synthesis of the lysis protein prevent release of colicin . The colicin and lysis genes are arranged in an operon, the lysis gene being situated next to that of colicin . The ColD genetic map is suggested.

Biochimie, 1986 May, 68(5), 731 - 7
Kinetic properties of 5-carboxymethyl-2-hydroxymuconate semialdehyde dehydrogenase from Escherichia coli; Alonso JM et al.; Kinetic properties of purified 5-carboxymethyl-2-hydroxymuconate semialdehyde (CHMSA) dehydrogenase (EC 1.2.1.-) in the 4-hydroxyphenylacetate meta-cleavage pathway from Escherichia coli have been studied . The temperature--activity relationship for the enzyme from 27 to 45 degrees C showed an Arrhenius plot with an inflexion at 36 degrees C . When 5-carboxymethyl-2-hydroxymuconic semialdehyde and NAD were used as variable substrates, the double reciprocal plots were all linear and the lines intersected at one point below the horizontal axis, suggesting that a sequential mechanism is operating . From the replots of intercepts and slopes against reciprocal substrate concentrations were calculated Km (CHMSA) = 9.0 +/- 1.02 microM, Km (NAD) = 29.1 +/- 4.65 microM and the value for the dissociation constant of enzyme--NAD complex = 6.3 +/- 1.21 microM . ATP and the product of the reaction (NADH) acted as competitive inhibitors of the enzyme with respect to NAD . Apparent Ki values, estimated from Dixon plots, were 25.0 +/- 3.5 and 88.0 +/- 22.1 microM for NADH and ATP, respectively.

J Exp Med, 1986 May 1, 163(5), 1260 - 6
Interleukin 1 and lipopolysaccharide induce an inhibitor of tissue-type plasminogen activator in vivo and in cultured endothelial cells; Emeis JJ et al.; Human IL-1, recombinant murine IL-1 and E . coli LPS were found to be potent inducers of plasminogen activator (PA)-inhibitor activity, both in vivo, in rats, as well as in cultured human endothelial cells . In vivo, LPS rapidly and dose-dependently (0.01-1,000 micrograms/kg) increased plasma PA-inhibitor activity . Infusion of IL-1 into rats resulted in a small but significant increase in PA-inhibitor activity in rat plasma . Likewise, in cultured human umbilical vein endothelial cells, LPS and IL-1 induced increased synthesis of PA-inhibitor . We suggest that the induced rat plasma inhibitor might be of endothelial origin.

Mol Cell Biol, 1986 May, 6(5), 1520 - 8
Molecular cloning of suppressor of sable, a Drosophila melanogaster transposon-mediated suppressor; Chang DY et al.; A hybrid dysgenesis-induced allele {su(s)w20} associated with a P-element insertion was used to clone sequences from the su(s) region of Drosophila melanogaster by means of the transposon-tagging technique . Cloned sequences were used to probe restriction enzyme-digested DNAs from 22 other su(s) mutations . None of three X-ray-induced or six ethyl methanesulfonate-induced su(s) mutations possessed detectable variation . Seven spontaneous, four hybrid dysgenesis-induced, and two DNA transformation-induced mutations were associated with insertions within 2.0 kilobases (kb) of the su(s)w20 P-element insertion site . When the region of DNA that included the mutational insertions was used to probe poly(A)+ RNAs, a 5-kb message was detected in wild-type RNA that was present in greatly reduced amounts in two su(s) mutations . By using strand-specific probes, the direction of transcription of the 5-kb message was determined . The mutational insertions lie in DNA sequences near the 5' end of the 5-kb message . Three of the seven spontaneous su(s) mutations are associated with gypsy insertions, but they are not suppressible by su(Hw).

Mol Gen Genet, 1986 May, 203(2), 305 - 11
Rearrangements of microinjected recombinant DNA in the genome of transgenic mice; Tarantul VZ et al.; Previously, mouse zygotes were microinjected with the recombinant plasmid pMA3, which contains the Herpes simplex virus thymidine kinase gene attached to the promoter region of the Rous sarcoma virus (Gazaryan et al . 1984a) . In the present work the pMA3 fragment with the flanking genomic sequences was isolated from the DNA of one transgenic Fo mouse by the "plasmid rescue" technique . The rescued plasmid (pMAR1) lacked all virus-specific sequences and retained only some pBR322 sequences . The flanking region at one end of the integrated pBR322-specific fragment contained a highly conserved mouse repetitive sequence . The possible mechanisms of rearrangement of foreign DNA in germ line cells are discussed.

Mol Gen Genet, 1986 May, 203(2), 265 - 8
Genetic studies on the beta subunit of Escherichia coli RNA polymerase . VII . RNA polymerase is a target for ppGpp; Glass RE et al.; RNA polymerase was isolated from two suppressed rpoB amber strains exhibiting relaxed control over RNA synthesis in vivo . In vitro transcription analysis of these mutant holoenzymes carrying defined substitutions at known sites in the beta subunit clearly shows that single amino acid changes in beta render RNA polymerase resistant to ppGpp . This unambiguously demonstrates that RNA polymerase is indeed the target for ppGpp.

Mol Gen Genet, 1986 May, 203(2), 256 - 64
Characterization of a collection of deletion mutants at the 3'-end of 16S ribosomal RNA of Escherichia coli; Zwieb C et al.; Deletions were constructed in plasmid pKK3535 in the coding region for the 3'-end of E . coli 16S rRNA . The plasmid was cleaved with restriction endonuclease Hae2 under conditions favoring the production of single cut linear plasmid DNA and deletions were produced by digestion with exonuclease Bal31 . Seven different deletions were isolated ranging in size from 90 to about 200 base pairs . Transcription of ribosomal DNA, processing of ribosomal RNA and incorporation of mutant rRNA into mutant particles was studied in UV-sensitive cells using a modified maxicell labeling procedure . The different mutants were missing defined features in the secondary structure of 16S rRNA and were characterized according to their stability, ability to be processed, sensitivity to colicin E3, and ability to bind ribosomal protein S1 and to interact with 50S subunits . These analyses show that the small stem and loop structure at positions 1350 to 1372 is necessary for the stability of rRNA . The deletion of the long terminal stem structure (1409-1491) in all mutant rRNAs does not block processing of the mutant rRNAs or S1 binding, although processing of the mutant rRNAs or S1 binding, although it does prevent the association of particles containing the mutant rRNA with 50S subunits.

Genetika, 1986 May, 22(5), 777 - 86
{Characteristics of the het mutations in the Escherichia coli chromosome causing an increase in Tn1 transposition frequency}; Ismailov ZF et al.; Four mutations were studied which lead to increasing the frequency of transposon Tn1 translocation into different replicons . These mutations (het1, het2, het3 and het4) increase the frequency of Tn1 translocation 10-20-fold . The het1 mutation is recessive and has been localized in the 90-94.5 min region of the bacterial chromosome . The mutation effects Tn1 transposition in the presence of F plasmid only . As we have demonstrated recently, F-plasmid inhibits Tn1 transposition in Escherichia coli cells . The het1 mutation eliminates this inhibition . Unlike het2, het3 and het4 mutations, het1 is responsible for resistance to male phages f1, f2, MS2 and inhibition of conjugative transfer in F+ bacteria.

Biochimie, 1986 May, 68(5), 715 - 22
The physiology of stringent factor (ATP:GTP 3'-diphosphotransferase) in Escherichia coli; Justesen J et al.; The enzyme ATP:GTP 3'-diphosphotransferase catalyzes the transfer of the beta, gamma-pyrophosphate of ATP to the 3' position of GTP or GDP . The amounts of enzyme were measured in cell extracts of a relA+ strain of E . coli grown at different growth rates between 0.4 and 1.9 generations per hour, using precipitation with specific antibodies to purify the enzyme . The amount of enzyme was found to be a constant fraction of total protein at all growth rates corresponding to about 45 molecules of enzyme per genome equivalent of DNA . The purified enzyme has little catalytic activity by itself but has to be activated either by a complex of 70S ribosomes, mRNA and uncharged tRNA or by a solvent like ethanol at a concentration of about 20% . The kinetic constants of the enzyme for the transfer pyrophosphate from ATP to GTP in the ribosome-activated state were determined . The Vmax was estimated to be 140 mumol/min X mg at 37 degrees C and the S0.5 values for GTP and ATP were 0.35 and 0.53 mM, respectively . The reaction was estimated to have an equilibrium constant of about 300 . In the pyrophosphate transfer from ATP to GDP the Vmax was estimated to be 90 mumol/min X mg at 37 degrees C and the S0.5 for GDP as 0.3 mM . During amino acid starvation of a relA+ strain of E . coli the amounts of enzyme and the catalytic capacity of the enzyme are sufficient to maintain the observed ppGpp levels in the cells at all growth rates.

Antimicrob Agents Chemother, 1986 May, 29(5), 807 - 13
Evolution and spread of IncFIV plasmids conferring resistance to trimethoprim; Campbell IG et al.; Twenty-one IncFIV-group plasmids conferring trimethoprim resistance in Escherichia coli isolates from humans and pigs were examined . Three evolutionary lines of plasmids were identified on the basis of restriction enzyme analysis . One was found exclusively in human isolates and another was found in pig isolates, while the third line consisted of plasmids from both sources . All R plasmids readily transferred to laboratory strains, and evidence was found for transfer to other biotypes of E . coli in the environment . The Tpr genes from representatives of the plasmid lines were cloned and compared by restriction analysis and by hybridization with two characterized Tpr dihydrofolate reductase genes . The sequences flanking the Tpr genes were different for each line, but all showed homology with the type 2 dihydrofolate reductase gene, irrespective of whether they were of human or animal origin . There was no hybridization to the type 1 gene . The remarkable degree of similarity among plasmids of the third line provided clear evidence of the exchange of plasmid-bearing E . coli between humans and pigs.

Anal Biochem, 1986 May 1, 154(2), 671 - 5
Preparation of high-specific-activity D-{3-3H}pantothenic acid; Vallari DS et al.; High-specific-activity D-{3-3H}pantothenic acid (5 Ci/mmol) was prepared from commercially available beta-{3-3H}alanine employing Escherichia coli strain DV1 (panD2 pan F1) . This strain is defective in beta-alanine synthesis and pantothenate uptake, and under appropriate growth conditions converted 85 to 90% of the input beta-{3-3H}alanine to extracellular D-{3-3H}pantothenate . The radiolabeled vitamin was purified from the medium by thin-layer chromatography followed by reverse-phase high-performance liquid chromatography . The overall yield of D-{3-3H}pantothenic acid was 30% and radiochemical purity was greater than 99%.

Mol Biol (Mosk), 1986 May-Jun, 20(3), 745 - 50
{Effect of superhelical DNA on the transcription of cloned genes of the T4 phage}; Ogryz'ko VV et al.; The effect of DNA superhelicity on the transcription of T4 DNA fragments containing early genes uvs W, Y and late genes 25-29 was studied . RNA polymerase transcribes both early and late phage genes within the supercoiled recombinant plasmid . Late genes relative transcription increases essentially when T4-modified RNA polymerase is used . DNA relaxation causes a sharp decrease of modified RNA polymerase activity, especially on the late genes . The same effect is obtained in intact cells with recombinant plasmid, which superhelicity is lowered by temperature-sensitive mutation in DNA gyrase . The data obtained prove that DNA superhelicity is required for the T4 late transcription by phage-modified RNA polymerase . It is suggested that the dependence of late transcription on the phage DNA replication in infected cells is connected with phage DNA superspiralization throughout its replication.

Mol Biol (Mosk), 1986 May-Jun, 20(3), 683 - 96
{Structural instability of co-integrates formed during interaction of plasmid R57 with pB322 and RP1 . Possible role of IS1 element in the degradation of co-integrates}; Danilevich VN et al.; The conjugative plasmid R57 determines resistance to ampicillin and chloramphenicol . Earlier it was shown that R57 encodes site-specific recA-independent recombinase, which acts in cis and resolves IS1-mediated cointegrates arising in the Escherichia coli recA cells between R57 and pBR322 . In the present work the properties of the cointegrates between R57 and pBR322 or RP1 arising in the E . coli rec+ strains were studied . It was found that the cointegrates between R57 and pBR322, obtained by mating of the respective biplasmid donors of E . coli rec+ and the rec+ recipients, lost as a result of deletion a large DNA segment of R57 containing determinant Cmr . The resulting hybrid replicons preserved determinants Apr and Tcr of pBR322 and the R57 conjugative properties and were structurally identical . By using plasmid RP1ts12, which is temperature-sensitive in replication, it was demonstrated that in cells rec+ the cointegrates between R57 and RP1 are extremely unstable . On storage they undergo structural degradation mainly affecting the RP1 replicon . The degradation products of the hydrid complex had lost their RP1 genes but preserved the R57 functional determinants . For elucidation of the observed phenomena the properties of the IS1-mediated cointegrates between pBR322:Tn9 and plasmid pBR3.1--deletion derivative of RP1 were studied . It was found that insertion of IS1 sometimes resulted in formation of unstable cointegrates capable of resolving and loosing determinant Cmr with a high frequency . It was suggested that IS1 encodes the site-specific recombinase responsible for resolution of the IS1-mediated cointegrates and deletion generation . Expression of this recombinase appears to be dependent on structure of the insertion sites . The possible role of IS1 and recombinase encoded by it in resolution and structural instability of the cointegrates between R57 and pBR322 or RP1 is discussed.

EMBO J, 1986 May, 5(5), 1099 - 103
T7 RNA polymerase directed expression of the Escherichia coli rrnB operon; Steen R et al.; Plasmids having Escherichia coli ribosomal DNA sequences under control of a promoter for T7 RNA polymerase have been constructed . Transcription of the rDNA sequences is dependent on T7 RNA polymerase because the tandem promoters for E . coli RNA polymerase, normally used to direct transcription of these sequences, have been removed . The entire 16S, 23S and 5S coding sequences from the rrnB operon can be efficiently transcribed by T7 RNA polymerase in vitro to yield full-length 30S precursor RNA . When such plasmids are placed into an E . coli strain containing a chromosomal copy of the gene for T7 RNA polymerase under control of the lac UV5 promoter, high-level synthesis of rRNAs from the plasmid can be induced by adding IPTG to exponentially growing cells . Subsequent addition of rifampicin to inhibit further initiation of transcription by E . coli RNA polymerase provides a simple method to study the fate of plasmid-coded rRNAs in the complete absence of host-coded rRNA synthesis . Gel electrophoretic analysis demonstrated that the rRNAs synthesized by T7 RNA polymerase in the presence of rifampicin are processed to their mature forms and assembled into ribosomal particles for at least 35 min after rifampicin addition . T7 RNA polymerase is also capable of efficient transcription of the entire rrnB operon in the reverse direction.

EMBO J, 1986 May, 5(5), 1077 - 85
Regulation of the operon encoding ribonucleotide reductase in Escherichia coli: evidence for both positive and negative control; Tuggle CK et al.; The ribonucleotide reductase genes (nrd) are induced by thymine starvation . Deletion analysis of the sequences upstream of the cloned nrd genes was used to identify several regulatory regions . The start of transcription (nrdP) was mapped 110 bp upstream of nrdA, the first structural gene . A site required for positive regulation of nrd was mapped 135 bp upstream of nrdP in a region with two direct repeat sequences as well as potential secondary structure . Two other sites (one upstream of nrdP, the other downstream) were identified as sequences whose deletion markedly increase expression . These latter sites show sequence homology and probably interact since the effects of their individual deletion are not additive when combined.

EMBO J, 1986 May, 5(5), 1037 - 40
Conserved sequence elements upstream of the gene encoding yeast ribosomal protein L25 are involved in transcription activation; Woudt LP et al.; Previous studies have revealed the occurrence of two closely linked conserved sequence elements, designated as HOMOL 1 and RPG box, in front of most yeast ribosomal protein genes examined . To investigate whether these conserved nucleotide elements play a role in the regulation of ribosomal protein gene expression, we performed deletion analysis of the DNA region upstream of the gene encoding ribosomal protein L25 . To that end we constructed a hybrid gene consisting of the pertinent 5'-flanking sequence and the Escherichia coli galK marker gene . The effects on the transcription of this fusion gene of Bal31-generated deletions were measured by Northern analysis of RNA isolated from the respective transformed yeast cells . The results demonstrate that removal of one box has a detrimental effect on the level of transcription, whereas after the deletion of both boxes hardly any transcription can be observed . Subsequently we inserted synthetic oligonucleotides in the upstream region of an L25 gene from which the original boxes had been removed . Expression of the inactivated hybrid gene turned out to be restored even by insertion of one RPG element . Moreover, the RPG box functions in both orientations, though not with equal efficiency.

EMBO J, 1986 May, 5(5), 1023 - 30
Yeast pre-messenger RNA splicing efficiency depends on critical spacing requirements between the branch point and 3' splice site; Cellini A et al.; In the yeast Saccharomyces cerevisiae the 5' and 3' splice junctions and the internal branch acceptor site (TACTAAC box) are highly conserved intron elements . Analyses of mutants have demonstrated the importance of each of these elements in the splicing process . In the present report we show by three different analytical approaches (splicing-dependent beta-galactosidase expression, in vitro splicing assays and in vivo RNA analyses) that at least two of these elements (the TACTAAC and 3' splice signals) also have to fulfill certain spacing requirements to allow efficient splicing to occur . In particular, the spacing of the 3' splice site from the 2'-5' branch site is a critical factor in determining the efficiency for completion of the final reactions of splicing, intron release and exon-exon joining . Whereas insertions within this region have little or no effect on the first reactions in splicing (the 5' cleavage and 2'-5' branch formation), they dramatically affect the efficiency of the final reactions . In contrast, a 15-base deletion between these two sites has no detectable effect on splicing efficiency . We also show that the 5' cleavage and branch formation can take place, albeit inefficiently, in transcripts in which all of the yeast sequences downstream of the branch site have been replaced by Escherichia coli sequences . We conclude from these studies that, in yeast, the 5' and 3' splice sites are recognized independently from one another, but always in conjunction with the TACTAAC signal.

Plasmid, 1986 May, 15(3), 172 - 81
pHG165: a pBR322 copy number derivative of pUC8 for cloning and expression; Stewart GS et al.; During the construction of the Messing pUC plasmid series, the rop(rom) gene of pBR322 which mediates the activity of RNAI was deleted . This has resulted in an elevated copy number for the pUC plasmids which makes the expression of beta-galactosidase activity constitutive in a host containing the Iqtss lac repressor . We describe the construction of a new series of vectors which retain the pUC multiple cloning site (MCS) but in which copy number control has been recovered . In addition, the lac alpha/lac promoter expression region has been inserted into a HpaI cassette . This facilitates the movement of recombinant DNA clones within the MCS . It also increases the complementation activity of the lac alpha peptide by an order of magnitude, allowing selection of recombinants by their Lac- phenotype on MacConkey agar.

J Inorg Biochem, 1986 May, 27(1), 1 - 15
Reactions of triethylphosphine gold(I) complexes with heme proteins: novel spin-state changes in cytochrome b562, myoglobin, and hemoglobin; Grootveld MC et al.; Reactions of bacterial Fe(III) cyt b562, HbO2, met Hb and met Mb with Et3PAuCl and Et3PAuNO3 (and some related complexes) have been investigated by electronic absorption and EPR and NMR spectroscopy . Except for met Hb, which denatured, the products were novel high-spin Fe(III) heme proteins . The reactions of cyt b562 and Mb were reversible . Two distinct kinetic steps were observed in the autoxidation of HbO2 and MbO2 . These may involve the liberation of superoxide . Autoxidation of HbO2 occurred more rapidly than that of MbO2 . The kinetics of the spin-state change of cyt b562 were too fast to measure by conventional (spectrophotometric) methods . The reaction of Et3PAuCl with HbO2 was not blocked by N-ethylmaleimide . The reactions are discussed in terms of attack by Et3PAu+ on histidine residues in the hydrophobic haem pockets of the proteins.

J Biochem (Tokyo), 1986 May, 99(5), 1393 - 400
Membrane phospholipid synthesis in Escherichia coli: alteration by glycerol and physiological consequences in a pss mutant; Kobayashi T et al.; Escherichia coli mutants harboring the pss-1 allele (coding for a temperature-sensitive phosphatidylserine synthase) are temperature sensitive for growth and synthesize less phosphatidylethanolamine at higher temperatures, giving rise to abnormal membrane phospholipid compositions . To obtain information concerning the determinant for the phospholipid polar headgroup composition and the lethal factor in the defective membranes, we have examined the effect of increased supply of sn-glycerol 3-phosphate on the phospholipid synthesis and the growth ability of a pss-1 mutant . For this purpose, a pair of E . coli K-12 derivatives isogenic except for the pss-1 allele was constructed from strain BB26-36 to harbor the mutations related to glycerol metabolism (glpD3, glpR2, glpKi, and phoA8) . Pulse- and uniform-labeling of phospholipids with 32P at 42 degrees C in a synthetic medium with (0.2%) or without glycerol showed that glycerol further lowered the temperature-sensitive formation of phosphatidylethanolamine, removed the phosphatidate and CDP-diacylglycerol accumulated in the absence of glycerol, and resulted in an increase in cardiolipin content in the pss-1 mutant . The phospholipid synthesis and contents in the pss+ strain were not significantly affected by glycerol . Glycerol in the medium markedly enhanced the growth defect of the pss-1 mutant, which was remediable by sucrose . The results indicate that the intracellular pool of sn-glycerol 3-phosphate is the limiting factor for acidic phospholipid synthesis in the pss-1 mutant, and cardiolipin unusually accumulated is injurious to the functional E . coli membranes . Possible determinants for the phospholipid composition of the wild-type E . coli cells are also discussed on the basis of the present observations.

Biull Eksp Biol Med, 1986 May, 101(5), 600 - 2
{Mutagenesis and cloning of hly determinants of the paP20 plasmid}; Sharova IN et al.; The data obtained indicate that pAP20 plasmid determinants for the synthesis and excretion of Escherichia coli haemolysin are located on the plasmid SalI-fragment f3 . The size of f3 fragment is 12 X 10(6) dalton.

Arch Biochem Biophys, 1986 May 1, 246(2), 515 - 24
Energetic studies of lactose active transport in Escherichia coli membrane vesicles; Chen LI et al.; The energetics of D-lactate-driven active transport of lactose in right-side-out Escherichia coli membrane vesicles has been investigated with a microcalorimetric method . Changes of enthalpy (delta Hox), free energy (delta Gox), and entropy (delta Sox) during the D-lactate oxidation reaction in the presence of membrane vesicles are -39.9 kcal, -46.4 kcal, and 22 cal/deg per mole of D-lactate, respectively . The free energy released by this reaction is utilized to form a proton electrochemical potential (delta-microH+) across the membrane . The higher observed heat in the D-lactate oxidation reaction in the presence of carbonylcyanide m-chlorophenylhydrazone (a proton ionophore) supports the postulate that delta-microH+ is formed across the membrane vesicles . Thermodynamic quantities for the formation of delta-microH+ are delta Hm = 14.1 kcal, delta Gm = 0.6 kcal, and delta Sm = 45 cal/deg per mole of D-lactate . The efficiency in the free energy transfer from the oxidation reaction to the formation of delta-microH+ (defined by delta Gm/delta Gox) was 2%, as compared to that in the heat transfer (defined by delta Hm/delta Hox) of 35% . The energetics of the movement of lactose in symport with proton across the membrane as a consequence of the formation of delta-microH+ are delta H1 = -19 kcal, delta G1 = -0.5 kcal, and delta S1 = -62 cal/deg per mole of lactose . No heat of reaction is contributed by lactose movement across the membrane without symport with H+.

Proc Natl Acad Sci U S A, 1986 May, 83(9), 2807 - 11
Overlapping promoters and their control in Escherichia coli: the gal case; Herbert M et al.; Two overlapping promoters compete for RNA polymerase in the region that controls the expression of the galactose operon in Escherichia coli . Kinetics of open complex formation at P1 and P2 can be followed through the rate of formation of two specific abortive transcripts . The corresponding forward kinetic constants appear to be identical over a wide range of enzyme concentrations and temperatures, indicating that the two processes are strongly coupled . We propose a scheme accounting for our observations . In a first step, the competition between the two sites is a simple kinetic process, involving the "on" rate constants . In a second step, a slow reequilibration occurs, implicating the "off" rate constants and the conversion of one open complex to the other through a set of closed complexes . The first step is clearly affected when the complex between cyclic AMP and its receptor is bound at the activator site . An estimate of the various rate constants describing open complex formation at P1 and P2 is provided, as well as a qualitative description of the effect of the activator complex on these two pathways.

Proc Natl Acad Sci U S A, 1986 May, 83(10), 3402 - 6
Chemically induced mutagenesis in a shuttle vector with a low-background mutant frequency; Drinkwater NR et al.; We have developed a recombinant DNA shuttle vector that permits the molecular analysis of mutations induced in human cells by chemical or physical mutagens . The vector is able to replicate as a plasmid in Escherichia coli and in Epstein-Barr virus (EBV)-transformed human lymphoblastoid cell lines and contains the herpes simplex virus type 1 thymidine kinase gene (HSV tk) as the target for mutagenesis studies . After introduction of the vector into an EBV-transformed lymphoblastoid cell line (LCL-721) by electroporation, approximately equal to 2% of the transfected cells expressed the vector-encoded gene for hygromycin resistance . Plasmid DNA isolated from cells immediately after selection for hygromycin resistance (10 population doublings posttransfection) contained mutations in the HSV tk gene at a frequency of 6 X 10(-5) . Treatment of plasmid-bearing LCL-721 cells with N-ethyl-N-nitrosourea resulted in a dose-dependent increase of up to 15-fold in the frequency of mutations in the HSV tk gene . The dose-response for the induction of mutations in the plasmid-encoded gene closely paralleled that for the induction of mutations in the cellular gene for hypoxanthine (guanine) phosphoribosyltransferase.

J Virol, 1986 May, 58(2), 513 - 21
Cloning and characterization of oriL2, a large palindromic DNA replication origin of herpes simplex virus type 2; Lockshon D et al.; An origin of replication within the long unique sequence of herpes simplex virus type 2 designated oriL2 has been identified in a position homologous to its type 1 counterpart, oriL1, between map coordinates 0.398 and 0.413 . The difficulties encountered in previous attempts to clone both oriL2 and oriL1 in an undeleted form were surmounted by minimizing the growth of the host Escherichia coli, using a recBC sbcB E . coli host, and purifying the full-length plasmid from delected forms by using a novel method which exploits the ability of a palindrome-containing plasmid to adopt a cruciform conformation, thereby decreasing its supercoiling . In a previously developed assay for functional origin activity, oriL2 was localized to a 241-base-pair ApaI-SstII fragment . DNA sequence analysis revealed a 136-base pair, almost perfect palindrome . Comparison with oriL1 showed a very high degree of conservation: the two origins differ in only 16 of the 144-base-pair oriL1 palindromic region . Most significantly, the differences between oriL1 and oriL2 mainly occur in pairs so as to generally preserve the potential for intrastrand base pairing . The central region of oriL2 is homologous with the shorter palindromic structures detected in origins located within the repetitive sequences of the short component of herpes simplex virus type 1 or 2.

J Virol, 1986 May, 58(2), 339 - 47
Characterization of a pseudorabies virus glycoprotein gene with homology to herpes simplex virus type 1 and type 2 glycoprotein C; Robbins AK et al.; A pseudorabies virus (Becker strain) glycoprotein gene was located in the UL region at map position 0.40 . The gene was identified by using open reading frame Escherichia coli plasmid expression vectors and specific antibody reagents . A 1.55-kilobase unspliced transcript from the gene was detected in pseudorabies virus-infected tissue culture cells . The DNA sequence revealed a single open reading frame of 1,437 base pairs encoding 479 amino acids . The predicted primary translation product has a molecular weight of 50,860 and contains features of a typical herpesvirus glycoprotein . An E . coli expression plasmid was constructed that contained essentially all of the open reading frame for this gene . Antibodies raised in rabbits against the protein expressed in bacteria by this plasmid immunoprecipitated pseudorabies virus-specific glycoproteins of 92,000 and 74,000 daltons from infected cell extracts . It is likely that these two forms represent different glycosylation states of the protein.

J Bacteriol, 1986 May, 166(2), 565 - 73
Cloning and characterization of livH, the structural gene encoding a component of the leucine transport system in Escherichia coli; Nazos PM et al.; The physical location of the genetically defined livH gene was mapped in the 17-kilobase plasmid pOX1 by using transposon Tn5 inactivation mapping and further confirmed by subcloning and complementation analysis . These results indicated that the livH gene maps 3' to livK, the gene encoding the leucine-specific binding protein . Moreover, the nucleotide sequence of the livH gene and its flanking regions was determined . The livH gene is encoded starting 47 base pairs downstream from the livK gene, and it is transcribed in the same direction as the livK gene . The livK-livH intergenic region lacks promoter sequences and contains a GC-rich sequence that could lead to the formation of a stable stem loop structure . The coding sequence of the livH gene, which is 924 base pairs, specifies a very hydrophobic protein of 308 amino acid residues . Expression of livH-containing plasmids in minicells suggested that a poorly expressed protein with an Mr of 30,000 could be the livH gene product.

J Bacteriol, 1986 May, 166(2), 541 - 4
Isolation of ntrA-like mutants of Azotobacter vinelandii; Santero E et al.; A number of chlorate-resistant mutants of Azotobacter vinelandii affected in a general control of nitrogen metabolism were isolated . These mutants could not utilize dinitrogen, nitrate, or nitrite as a nitrogen source . The reason for this inability is that they were simultaneously deficient in nitrogenase and nitrate and nitrite reductase activities . They were complemented by a cosmid carrying a DNA fragment of A . vinelandii able to complement ntrA mutants of Escherichia coli, so they seemed to be ntrA-like mutants.

J Bacteriol, 1986 May, 166(2), 533 - 40
Cloning and molecular characterization of csm mutations allowing expression of catabolite-repressible operons in the absence of exogenous cyclic AMP; George SE et al.; The cyclic AMP (cAMP) suppressor mutation (csm) of Escherichia coli has been cloned from strain NCR30 in the HindIII-EcoRI site of pBR322 . This mutation has been mapped in or near the crp gene . Wild-type crp DNA hybridized to recombinant plasmids pGM5 and pGM25 containing the cloned csm mutation . These recombinant plasmids encoded a protein product of identical molecular weight and charge as that of the wild-type cAMP receptor protein . Transformants of cya crp deletion strains harboring pBM5 or pGM25 exhibited phenotypic characteristics common to strain NCR30 . These included the expression of catabolite-repressible enzymes, such as arabinose isomerase, tryptophanase, beta-galactosidase, and threonine deaminase; the expression of chemotactic and motility genes; cAMP sensitivity; and the accumulation of toxic levels of methylglyoxal . DNA sequence analysis indicated that the Csm suppressor phenotype was attributable to the insertion of a guanosine residue 17 base pairs downstream from the termination codon of the crp structural gene . The guanosine insertion is located in the stem region of the presumed transcriptional termination loop . This stem region contained a unique BssHII restriction site which was used to construct an in vitro deletion in the wild-type crp insert in plasmid pHA7 . The resulting plasmid, pGM459, renders transformants having a phenotype common to that conferred by the chromosomal or cloned csm mutation . Our results indicate a novel role for the 3' flanking region of the crp structural gene in the expression of the cAMP receptor protein.

J Bacteriol, 1986 May, 166(2), 505 - 12
Does secA mediate coupling between secretion and translation in Escherichia coli?
Strauch KL, Kumamoto CA, Beckwith J.
An amber mutation in the secA gene of Escherichia coli causes a pleiotropic decrease in the synthesis of secreted proteins, including maltose-binding protein (MBP) and alkaline phosphatase . Reversal of the inhibition of MBP synthesis in secA(Am) strains by signal sequence mutations in the malE gene has been reported . These results suggest a coupling between secretion and translation which involves an interaction between the signal sequence of nascent polypeptides and a cellular secretion machinery . Further analysis reported here indicated that signal sequence mutations of MBP or alkaline phosphatase did not selectively overcome the inhibition of MBP or alkaline phosphatase synthesis in secA(Am) strains . Rather, at a given time in parallel experiments there was substantial variability among closely isogenic secA(Am) strains in the magnitude of the synthesis block; this variability could account for the earlier results . Further experiments suggested that the inhibition of MBP synthesis in secA(Am) strains was caused by depletion of cyclic AMP, leading to decreased transcription of the malE gene . However, the secretion defects in secA(Am) strains were not affected by cyclic AMP levels . Therefore, we conclude that the reduction in MBP synthesis was a secondary consequence of the primary export defect in the secA(Am) strains.

J Bacteriol, 1986 May, 166(2), 446 - 52
Tn5-induced mutations affecting sulfur-oxidizing ability (Sox) of Thiosphaera pantotropha; Chandra TS et al.; Mutants of Thiosphaera pantotropha defective in chemolithoautotrophic growth were obtained by transpositional mutagenesis with Tn5 coding for kanamycin resistance . The suicide vehicle for introducing Tn5 to T . pantotropha was pSUP5011 harbored by Escherichia coli . Kanamycin-resistant isolates were screened for the inability to grow with reduced sulfur compounds (Sox-) . Four classes of Sox- mutants were obtained . Three were of different pleiotropic phenotypes: (i) unable to grow with formate, nitrate, and xanthine; (this class strongly suggested the involvement of a molybdenum cofactor in inorganic sulfur-oxidizing ability); (ii) no growth with hydrogen; (iii) slight growth with hydrogen and formate . Two plasmids, pHG41 (about 450 kilobase pairs) and pHG42 (110 kilobases), were identified in lysates of T . pantotropha . In one Sox- mutant pHG41 could not be detected . Revertant analysis suggested that pHG41 and pHG42 were not involved in the Sox character.

J Bacteriol, 1986 May, 166(2), 404 - 11
Preparation of the FhuA (TonA) receptor protein from cell envelopes of an overproducing strain of Escherichia coli K-12; Hoffmann H et al.; A rapid and simple method for purification of the FhuA receptor protein from cell envelopes of a FhuA-overproducing strain of Escherichia coli K-12 was developed . The overproduction of FhuA was programmed by the thermoamplifiable plasmid pHK232, which carried the fhuACD genes of pLC19-19 of the Clarke and Carbon collection . At low temperature (27 degrees C), pHK232 specified the overproduction of FhuA to levels comparable to those of major outer membrane proteins OmpF, OmpC, and OmpA . The amount of these proteins in the outer membrane was reduced along with overproduction of FhuA . Upon runaway replication of pHK232 at 37 degrees C, the precursor of the FhuA protein, proFhuA, was also accumulated in the cell envelope in amounts similar to FhuA . For extraction of the FhuA protein, crude cell envelopes were washed with 2% Triton X-100-6 M urea to remove less tightly bound proteins . Then FhuA but not proFhuA was solubilized by treating Triton X-100-urea-washed membranes with 1% octylglucoside-1 mM EDTA . This procedure yielded FhuA protein free from other membrane proteins . The amount of lipopolysaccharide and phospholipids was low and ranged from 5 to 15% and 10 to 25% of the weight of the FhuA protein, respectively . As shown by direct inactivation and by competition assays, the isolated FhuA protein retained receptor activity for ferrichrome, albomycin, colicin M, and phages T5 and T1.

Cancer Res, 1986 May, 46(5), 2588 - 95
Anti-neurofilament antibodies in the sera of patients with small cell carcinoma of the lung and with visual paraneoplastic syndrome; Kornguth SE et al.; The sera of patients with small cell carcinoma of the lung (SCCL) and an associated visual paraneoplastic syndrome (VPNS) have high titer immunoglobulins that react with retinal ganglion cells and with cloned lines of the SCCL . The immunoglobulins in the sera of two patients with SCCL and VPNS reacted with at least one common antigen shared by neural cells and cloned lines of the SCCL . The molecular weights of the predominant neural and tumor antigens were 205,000, 145,000, 65,000, and 20,000-24,000 as determined by Western blots . Three of the antigens from neural tissue copurify and comigrate electrophoretically with neurofilament proteins . Polyclonal antibodies prepared against authentic neurofilament proteins react with antigens having molecular weights identical to those of proteins that react with immunoglobulins from the SCCL-VPNS patients . Polyclonal antibodies that were prepared against isolated retinal ganglion cells and that were shown previously to cause the immunoablation of the ganglion cells in vivo reacted most intensely with the Mr 205,000 antigen and weakly with the Mr 145,000 and Mr 70,000 antigens . Treatment of the Western blots with alkaline phosphatase from Escherichia coli did not affect the immunoreactivity between the immunoglobulins and the purified neurofilament proteins . It is proposed that the immunoglobulins in the sera of patients with SCCL-VPNS may be involved etiologically in the development of the VPNS.

Mutat Res, 1986 May, 160(3), 171 - 8
Lack of UV-induced respiration shutoff in a recF strain of Escherichia coli: temperature conditional suppression at 30 degrees C by the sfrA mutation; Swenson PA et al.; A mutation in the recF gene of Escherichia coli results in a radiation-sensitive strain . The RecF pathway and the RecBC pathway account for nearly all of the conjugative recombination occurring in E . coli . recBC cells are radiation-sensitive and carry only out a small amount of recombination but these deficiencies are suppressed by an sbcB as recombination is shunted to the RecF pathway . A recBC sbcB recF strain is very radiation-sensitive and is devoid of recombination ability . These deficiencies are suppressed by the srfA mutation; srfA is a recA allele . UV-induced respiration shutoff is a recA+, lexA+ and recBC+ dependent . We report in this paper that respiration does not shutoff in a recF strain at 37 and 30 degrees C . an srfA mutation suppresses this lack of respiration shutoff effect in a recF srfA mutant at 30 degrees C but not at 37 degrees C; no suppression by this mutation occurs at either temperature in a recF recBC sbcB strain . An srfA strain also does not shut off its respiration at 37 degrees C and shows a temperature conditional UV-induced respiration shutoff response at 30 degrees C . The srfA mutation is thought to cause an altered RecA protein to be produced and we suggest that at 37 degrees This altered protein is temperature sensitive . We conclude from the results in this paper that the recF gene product is required for UV-induced respiration shutoff and that the RecA protein plays a special role in the induction process.

Genetics, 1986 May, 113(1), 1 - 12
DNA synthesis at the site of a Red-mediated exchange in phage lambda; Stahl FW et al.; In phage lambda, progeny particles bearing unreplicated chromosomes are recombinant by action of lambda's Red system only near the right end of the chromosome . These recombinants are frequently heterozygous (heteroduplex) for markers located there . In replication-blocked crosses involving two heavy-labeled parents we find that particles in the solitary peak, containing progeny with fully conserved DNA, vary in density . Those on the heavy side of this peak are more apt to be heterozygous than are those on the light side . The data fit a model in which a double chain cut at cos, lambda's packaging origin, is followed by partial exonucleolytic degradation of lambda's r chain from the right end leftward . The exposed l chain, which thereby constitutes a 3' overhang, invades an intact, circular homologue after itself suffering some degradation . Completion of the recombinant chromosome sometimes involves DNA synthesis primed by the invading chain.

J Virol, 1986 May, 58(2), 592 - 9
Posttranslational modification at the N terminus of the human adenovirus type 12 E1A 235R tumor antigen; Lucher LA et al.; The adenovirus E1A transforming region, which encodes immortalization, partial cell transformation, and gene activation functions, expresses two early mRNAs, 13S and 12S . Multiple-T antigen species with different electrophoretic mobilities are formed from each mRNA, presumably by unknown posttranslational modifications . The adenovirus type 12 (Ad12) 13S and 12S mRNAs encode E1A T antigens of 266 and 235 amino acid residues (266R and 235R), respectively . To study possible posttranslational processing at the N and C termini and to distinguish between the Ad12 266R and 235R T antigens, we prepared antibodies targeted to synthetic peptides encoded at the common C (peptide 204) and N (peptide 202) termini of the 266R and 235R T antigens and at the unique internal domain of the 266R T antigen (peptide 206) . The specificity of each anti-peptide antibody was confirmed by immunoprecipitation of the 266R and 235R T antigens produced in Escherichia coli . Immunoprecipitation analysis of the E1A T antigens synthesized in Ad12-infected KB cells revealed the following . Antibody to the common C terminus recognized three T antigens with apparent Mrs of 43,000, 42,000, and 39,000 (43K, 42K, and 39K) . All three forms were phosphorylated and were present in both the nucleus and the cytoplasm . The 43K and 42K T antigens were rapidly synthesized during a 10-min pulse with {35S}methionine in Ad12-infected cells . The 43K T antigen had a half-life of 20 min, the 42K T antigen had a longer half-life of about 40 min, and the 39K T antigen became the predominant E1A T antigen . Antibodies to the unique region immunoprecipitated the 43K T antigen but not the 42K and 39K T antigens . Antibody to the N terminus immunoprecipitated the 43K and 42K T antigens but not the 39K T antigen, suggesting that the 39K T antigen possessed a modified N terminus . Partial N-terminal amino acid sequence analysis showed that the 43K and 42K T antigens contain methionine at residues 1 and 5, as predicted from the DNA sequence, whereas no methionine was released from the 39K T antigen during the first six cycles of Edman degradation . We propose that the short-lived 43K T antigen is the primary product of the 13S mRNA, the 266R T antigen; the somewhat more stable 42K T antigen is the primary product of the 12S mRNA, the 235R T antigen.(ABSTRACT TRUNCATED AT 400 WORDS)

Mutat Res, 1986 May, 160(3), 179 - 93
Relationship between cellular RecA protein concentration and untargeted mutagenesis in Escherichia coli; Chakravarti S et al.; We measured the production of untargeted mutations in the cI and cII genes of untreated lambda phage undergoing a lytic cycle in UV-irradiated bacterial hosts . As previously shown, treatment with 4 micrograms/ml of rifampicin during post-irradiation incubation inhibited amplification of the RecA protein in these cells . In addition, we observed a decreased mutation rate compared to the untreated, irradiated bacteria . Treatment with 4 micrograms/ml or 8 micrograms/ml rifampicin did not prevent the UV induction of the umuDC operon, as judged by assay of beta-galactosidase activity in a umuC-lacZ fusion strain . In contrast, the UV-induction of beta-galactosidase in the sulA-lacZ fusion strain was decreased by 4 micrograms/ml rifampicin . The inhibition of untargeted mutagenesis by this drug treatment was also observed in a strain constitutive for SOS functions (lexA (Def)) as well as in a RecA-overproducing plasmid strain, suggesting the requirement of other factor(s) in wild-type recA+ cells . An htpR165-carrying strain, that blocks induction of heat-shock proteins, exhibited normal UV-promoted mutagenesis . A correlation was observed between the cellular concentration of RecA protein, increased spontaneously by a temperature shift in a lexA(Ts) strain, and the extent of UV-promoted untargeted mutagenesis . These results suggest a mechanistic role of RecA protein in this process.

Am J Vet Res, 1986 May, 47(5), 1145 - 8
Hybridization of bovine Escherichia coli isolates with gene probes for four enterotoxins (STaP, STaH, STb, LT) and one adhesion factor (K99); Mainil JG et al.; Colony hybridizations with 4 enterotoxins (STaP, STaH, STb, and LT) and 1 adherence factor (K99) gene probes were done on Escherichia coli isolated from calves . Agreement between the K99 probing and a serologic assay to detect the K99 antigen was 99% for the identification of K99+ and K99- isolates . Ninety-five of the isolates (22%) hybridized with at least 1 enterotoxin gene probe (Ent+ isolates), and 82 (19%), with the K99 gene probe . The majority of Ent+ isolates (85%) reacted with probes for STaP and K99 genes . The STaP gene was present by itself in 4 of the Ent+ isolates (4%) and with the STb gene in 6 of the Ent+ isolates (6%) . Five of the Ent+ isolates (5%) carried the STb and LT genes, and none (0%) of the isolates carried the STaH gene . All but 2 of the isolates with the K99 gene also had the STaP gene . Twenty-eight isolates shown to produce STa enterotoxin in previous studies failed to hybridize with any of the enterotoxin gene probes . These 28 isolates were also phenotypically negative when the tests for enterotoxin production were repeated . These isolates probably lost their genes for enterotoxin production during storage in the laboratory.

Infect Immun, 1986 May, 52(2), 428 - 36
Oligomannoside-type glycopeptides inhibiting adhesion of Escherichia coli strains mediated by type 1 pili: preparation of potent inhibitors from plant glycoproteins; Neeser JR et al.; Various structurally defined glycopeptides of natural origin were tested as inhibitors of guinea pig erythrocyte agglutination by enteropathogenic Escherichia coli strains expressing type 1 pili . Besides hybrid-type glycoasparagines from ovalbumin which were not active, large oligomannoside-type carbohydrate chains from legume storage glycoproteins moderately inhibited hemagglutinations, whereas the short oligomannoside-type glycoasparagine from ovalbumin Man alpha(1----6) {Man alpha(1----3)}Man alpha(1----6){Man alpha(1----3)} Man beta(1----4)GlcNAc beta(1----4)GlcNAc beta(1----N)Asn exhibited a potent activity . These results strongly suggested that the nonsubstitution of the alpha(1----3)-linked mannosyl residue from the N-linked glycopeptide core structure is the key determinant in the minimal structural requirement specific to this fimbrial lectin . Such Man5GlcNAc2-containing glycopeptides were obtained from larger N-linked carbohydrate chains, occurring abundantly in natural sources . The ability of jack bean alpha-mannosidase to cleave the alpha(1----2)-linked mannoses more rapidly than the others allowed the controlled digestion of large oligomannoside-type glycopeptides from legume storage glycoproteins . Such shortened glycopeptides of plant origin were prepared which strongly inhibited guinea pig erythrocyte agglutinations as well as bacterial adhesion on human buccal cells, thus confirming their similarity (if not identity) with the receptor of type 1 pili on mammalian cells . The importance of this preparation of a receptorlike compound that inhibits bacterial adhesion with regard to the research on the role of type 1 pili in E . coli pathogenicity is discussed.

Mutagenesis, 1986 May, 1(3), 191 - 4
RecA-independent mutagenesis in Escherichia coli: effects of umuC and mucB mutations; Thomas SM et al.; We have studied the effects of a umuC mutation on reversion of the lacZ19124 and lacZ19136 frameshift markers of Escherichia coli . Introduction of a umuC::Tn5 mutation into a strain carrying the lacZ19136 marker resulted in enhanced reversion by 9-aminoacridine and the acridine half-mustard ICR191, whereas reversion of the lacZ19124 marker was decreased (but not abolished) in a umuC strain . The reversion frequency of the lacZ19136 marker was decreased by the presence of plasmid pKM101, and further decreased by a derivative of pKM101 in which the mucB gene was inactivated by a Tn5 insertion . The reversion frequency of the lacZ19124 marker was relatively unchanged by either plasmid . Since both 9-aminoacridine and ICR191 mutagenesis of these strains is independent of the recA+ and lexA+ gene products, these results may suggest a broader role for the UmuC protein in regulating induced mutation frequencies than has previously been suspected . The contrasting effects of the umuC and mucB mutations on reversion of the lacZ19136 marker may suggest a copy number effect, or perhaps more likely that there are inherent (functional) differences between the UmuC and MucB proteins.

Mol Cell Biol, 1986 May, 6(5), 1843 - 6
A transforming ras gene can provide an essential function ordinarily supplied by an endogenous ras gene; Papageorge AG et al.; Microinjection of monoclonal antibody Y13-259, which reacts with all known mammalian and yeast ras-encoded proteins, has previously been shown to prevent NIH 3T3 cells from entering the S phase (L . S . Mulcahy, M . R . Smith, and D . W . Stacey, Nature {London} 313:241-243, 1985) . We have now found several transformation-competent mutant v-rasH genes whose protein products in transformed NIH 3T3 cells are not immunoprecipitated by this monoclonal antibody . These mutant proteins are, however, precipitated by a different anti-ras antibody . Each of these mutants lacks Met-72 of v-rasH . In contrast to the result for cells transformed by wild-type v-rasH, Y13-259 microinjection of NIH 3T3 cells transformed by these mutant ras genes did not prevent the cells from entering the S phase . These results imply that a transformation-competent ras gene can supply a normal essential function for NIH 3T3 cells . When the proteins encoded by the mutant ras genes were overproduced in Escherichia coli, several mutant proteins that lacked Met-72 failed to bind Y13-259 in a Western blot . However, a ras protein from a mutant lacking amino antibody, but a ras protein from a mutant lacking amino acids 72 to 84 did not . These results suggest that Y13-259 may bind to a higher ordered structure that has been restored in the mutant lacking amino acids 72 to 82.

Biochem J, 1986 May 1, 235(3), 651 - 61
Topographic labelling of pore-forming proteins from the outer membrane of Escherichia coli; Page MG et al.; The topography of three pore-forming proteins from the outer membrane of Escherichia coli has been explored by using two labelling techniques . Firstly, the distribution of nucleophilic residues has been investigated by selective chemical modification using arylglyoxals (for arginine residues), isothiocyanates (for lysine residues), carbodi-imides (for carboxy residues) and diazonium salts . Secondly, the membrane-embedded domains have been investigated by labelling with photoactivatable phospholipid analogues and a reagent that partitions into the membrane . Few nucleophilic groups are found to be freely accessible to pore-impermeant probes reacting in the aqueous medium . More groups are accessible to small, pore-permeant probes, suggesting that several groups of each sort are contained within the pore . In addition, there appear to be a number of arginine, lysine, carboxyl and many tyrosine residues that are rather inaccessible and that react only with small, hydrophobic probes, if at all . Amongst these more deeply buried residues there are four arginine residues and an as-yet-undetermined number of carboxy residues that appear to be essential to the structural integrity of the oligomeric molecule.

Virus Res, 1986 May, 4(3), 289 - 96
Identification and genomic localization of a pseudorabies glycoprotein via expression in Escherichia coli; Saman E et al.; A glycoprotein (gp-88) secreted by pseudorabies virus (PRV) infected cells was isolated and purified . Anti gp-88 antibodies were used to screen an expression library constructed in Escherichia coli using genomic PRV DNA . Two positive clones were identified and the cloned genetic information was used to localize the corresponding gene in the unique short region of the PRV genome . Antibodies to the glycoprotein were also used to identify the unglycosylated precursor as synthesized in an in vitro translation system . A major precursor of 65 kDa was detected . Although the glycoprotein described here was not found as a major structural glycoprotein of the virion, antibodies to gp-88 are able to neutralise viral activity in vitro . The possible relationship with known glycoproteins of PRV is discussed.

Zh Mikrobiol Epidemiol Immunobiol, 1986 May, (5), 73 - 7
{New data on the biological action of normal immunoglobulins}; Ulanova AM et al.; Experiments on 2,520 CBA mice (CBA X X C57BL) F1 nice have shown that the injection of homologous serum immunoglobulins (obtained from intact and blood-stimulated animals), made 2 hours after gamma irradiation from a 60Co source, prevents the development of intestinal dysbacteriosis and endogenous infection . The injection of mouse and human immunoglobulins to nonirradiated mice improved their resistance to experimental infection with Escherichia coli live culture, increased the expression of receptors to the Fc-fragments of IgG in peritoneal macrophages and enhanced the physical working capacity of the animals . The preparations containing normal antitissular antibodies have proved to be particularly effective . In mice, rabbits and dogs the preparations under test have produced no changes in the general state of the animals, no local reactions and no disturbances in the cardiovascular activity.

Mol Biochem Parasitol, 1986 May, 19(2), 117 - 23
A DNA probe cloned in Escherichia coli for the identification of Brugia malayi; Sim BK et al.; This report describes a specific and sensitive DNA probe for the identification of Brugia malayi . A genomic DNA library produced from subperiodic B . malayi microfilariae was screened to detect clones containing DNA sequences which are highly repeated within the parasite genome . Several clones were further analyzed to identify those which hybridize specifically with B . malayi DNA but not with DNA from B . pahangi and Dirofilaria immitis . From these, clone pBm15 was selected because it hybridized with high sensitivity to B . malayi DNA as detected by autoradiography . Clone pBm15 was sensitive enough to detect two infective larvae or five microfilariae or 300 pg of purified B . malayi microfilarial DNA . This study forms the basis for the development of a specific and sensitive DNA probe for the identification of B . malayi in field specimens.

Arch Biochem Biophys, 1986 May 1, 246(2), 564 - 71
Synthesis and properties of adenosine-5'-triphosphoro-gamma-1-(5-sulfonic acid)naphthyl ethylamidate: a fluorescent nucleotide substrate for DNA-dependent RNA polymerase from Escherichia coli; Wu FY et al.; A new fluorescent ATP analog, adenosine-5'-triphosphoro-gamma-1-(5-sulfonic acid)naphthyl ethylamidate (gamma-1,5-EDANS)ATP, containing the fluorophore N-(aminoethyl)-5-naphthylamine-1-sulfonic acid attached via a gamma-phosphoamidate bond was synthesized in good yield . It has absorption maxima at 255 and 344 nm and a fluorescence emission maximum at 490 nm . These spectral characteristics permit its uses as an energy acceptor for energy transfer from the intrinsic protein fluorophores and as an energy donor for the energy transfer to the intrinsic Co of Co-substituted RNA polymerases . This analog is a good substrate for Escherichia coli RNA polymerase and can be used to initiate the RNA chain . Incorporation of this analog into the total RNA synthesized was about 60% of that observed for ATP, independent of the templates used . Its Km values (22 and 118 microM) are twofold higher and its Vmax values (45 and 59 nmol/min/mg of enzyme) are 40% lower than those for ATP using calf thymus DNA and poly{d(A-T)}, respectively, as template . For abortive initiation reaction using pAR1435 plasmid DNA as template, the Km and Vmax values of this analog are 2.7 times higher and 7 times lower, respectively, than those of ATP . With its desirable spectroscopic properties, (gamma-1,5-EDANS)ATP is a good probe for the studies of nucleotide-protein interactions, active site mapping of RNA polymerase, and other ATP-utilizing biological systems.

J Infect Dis, 1986 May, 153(5), 893 - 901
In vivo and in vitro effects of a novel enterotoxin, STb, produced by Escherichia coli; Weikel CS et al.; Escherichia coli may produce a heat-labile enterotoxin (LT) or two heat-stable enterotoxins (STa, STb) . STb consistently causes secretion in vivo in 5-hr weaned-pig intestinal loops (P less than .0001) . In Ussing chambers in vitro, crude culture filtrates of STb initiate a prompt increase in short circuit current (SCC; P less than or equal to .0001) and potential difference (P less than or equal to .0001) when compared with nontoxigenic culture filtrates . In bidirectional in vitro studies of ion flux (22Na and 36Cl), STb did not alter 22Na or 36Cl unidirectional or net fluxes . The calculated residual ion flux increased significantly (P less than .03), however, in tissues treated with STb and fully accounted for the STb-induced increase in SCC . Measurement of the electrolyte content of ligated intestinal segments in vivo further suggested that STb stimulated bicarbonate secretion . Relative to controls, significant accumulation of Na and Cl also occurred intraluminally in vivo . These data indicate that STb is a unique enterotoxin that causes net secretion in pig jejunum in vivo . In vitro and in vivo studies show that STb stimulates active secretion of nonchloride anion . We postulate that STb causes active bicarbonate secretion in weaned-piglet jejunum.

J Immunol Methods, 1986 May 1, 89(1), 123 - 30
Characterization of monoclonal antibody to DNA.RNA and its application to immunodetection of hybrids; Boguslawski SJ et al.; Monoclonal antibodies were raised to a DNA.RNA heteropolymer duplex prepared by transcription of phi X174 single-stranded DNA with DNA-dependent RNA polymerase . A monoclonal antibody with the highest affinity and specificity was selected . This antibody bound the DNA.RNA heteropolymer and poly(I).poly(dC) equally but 100-fold higher levels of poly(A).poly(dT) were required to achieve a similar degree of binding in competitive binding assays using DNA.{3H}RNA . Single-stranded DNA, double-stranded DNA and RNA, and ribosomal RNA were not bound by the antibody . The observed association constant for the antibody and DNA.{3H}RNA, determined by Scatchard analysis, was 8.5 X 10(10) l/mol assuming independent antibody binding sites . The antibody and an alkaline phosphatase-labeled second antibody were used in an immunodetection method for measurement of hybrids formed between immobilized DNA probes of various lengths and 23 S ribosomal RNA . The colorimetric response of this assay increased linearly with the amount of hybrid formed.

J Bacteriol, 1986 May, 166(2), 439 - 45
Role of ribosome degradation in the death of starved Escherichia coli cells; Davis BD et al.; In Escherichia coli cultures limited for phosphate, the number of ribosomal particles was reduced to a small percentage of its earlier peak value by the time the viable cell count began to drop; the 30S subunits decreased more than the 50S subunits . Moreover, the ribosomal activity was reduced even more: these cells no longer synthesized protein, and their extracts could not translate phage RNA unless ribosomes were added . The translation initiation factors also disappeared, suggesting that they become less stable when released from their normal attachment to 30S subunits . In contrast, elongation factors, aminoacyl-tRNA synthetases, and tRNA persisted . During further incubation, until viability was reduced to 10(-5), the ribosomal particles disappeared altogether, while tRNA continued to be preserved . These results suggest that an excessive loss of ribosomes (and of initiation factors) may be a major cause of cell death during prolonged phosphate starvation.

Virology, 1986 Apr 30, 150(2), 373 - 80
T1 pip: a mutant which affects packaging initiation and processive packaging of T1 DNA; Drexler H et al.; The pip mutation of phage T1 is located between the tar (gene 2.5) and am6 (gene 3) mutations in the region of the T1 genome which codes for early functions . The tar and pip mutations are additive in increasing the efficiency of transduction by T1 . When T1 carries the pip mutation the initiation of DNA packaging by the phage at the non-T1, esp-lambda site is more efficient than when the phage is pip+; the small average burst size of 8 to 10 by T1pip suggests that pip causes a reduction in the efficiency with which T1 utilizes pac, the normal packaging initiation site of the phage . The presence of the BglII-D fragment (cut at one end at pac and the other by BglII) after digestion of T1pip DNA by BglII shows that T1pip continues to initiate DNA packaging at pac . The increased molarity of BglII-D coupled with the absence of the BglII-C fragment (which contains DNA on both sides of pac and can only be cut from processively packaged genomes) shows that T1pip packages only genomes which are initiated at pac and is defective in processive packaging.

Biochem Biophys Res Commun, 1986 Apr 29, 136(2), 822 - 6
The binding of N-(phosphonacetyl)-L-aspartate to aspartate carbamoyltransferase of Escherichia coli; Volz KW et al.; A more precise description of the binding of N-(phosphonacetyl)-L- aspartate to the catalytic chains of aspartate carbamoyltransferase clarifies aspects of the specificity of this enzyme toward its substrates, carbamoylphosphate and L-aspartate, and suggests a catalytic role for His-134.

Biochem Biophys Res Commun, 1986 Apr 29, 136(2), 529 - 34
The kanamycin resistance gene expression in Escherichia coli as affected by specific yeast sequences; Polumienko AL et al.; It was shown that the transcription initiation of the kanamycin resistance gene (the Km gene) from transposon Tn5 in E.coli HB101 strain can be provided by specific yeast sequences . To localize a region of the yeast DNA involved in transcription initiation, a number of hybrid plasmids containing promoterless part of the Km gene fused with DNA sequences from chromosome III of the yeast Saccharomyces cerevisiae was constructed . Comparison of nucleotide sequences lying in the LTR of the yeast transposon Tyl-17 with the consensus bacterial promoter revealed strong similarities.

Nucleic Acids Res, 1986 Apr 25, 14(8), 3343 - 62
Persistence of DNA synthesis arrest sites in the presence of T4 DNA polymerase and T4 gene 32, 44, 45 and 62 DNA polymerase accessory proteins; Charette MF et al.; DNA synthesis by phage T4 DNA polymerase is arrested at specific sequences in single-stranded DNA templates . To determine whether or not T4 DNA polymerase accessory proteins 32, 44, 45 and 62 eliminated recognition of these arrest sites, unique primer-templates were constructed in which DNA synthesis began at a DNA primer located at different distances from palindromic and nonpalindromic arrest sites . Nucleotide positions that caused polymerase to pause or leave the template were identified by sequence analysis of 5'-end labeled nascent DNA chains . Stable hairpin structures at palindromic sequences were confirmed by acetylation of single-stranded sequences with bromoacetaldehyde . Our results confirmed that these T4 DNA polymerase accessory proteins stimulated T4 DNA polymerase activity and processivity on natural as well as homopolymer primer-templates . However, they did not alter recognition of DNA synthesis arrest sites by T4 DNA polymerase . Extensive DNA synthesis resulted from an increased rate of translocation and/or processivity to the same extent over all DNA sequences.

J Biol Chem, 1986 Apr 25, 261(12), 5616 - 24
Complete enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome; Funnell BE et al.; During enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome, oriC, formation of an active initiation complex consisting of dnaA, dnaB, dnaC, and HU proteins, requires a supercoiled DNA template . Relaxed covalently closed plasmids are active only if supercoiled by gyrase prior to initiation; nicked and linear DNAs are inactive . Semi-conservative replication proceeds via delta structure as intermediates . Daughter molecules include nicked intermediates . Daughter molecules include nicked monomers and catenated pairs . Elongation is rapid, but late replicative intermediates accumulate because the final elongation and termination steps are slow . Production of covalently closed circular daughter DNA molecules requires removal of ribonucleotide residues (primers) by DNA polymerase I, assisted by ribonuclease H, gap filling, and ligation of nascent strands by ligase . Reconstitution of a complete cycle of oriC plasmid replication, beginning and ending with supercoiled molecules, has been achieved with purified proteins.

J Biol Chem, 1986 Apr 25, 261(12), 5568 - 74
Nucleotide sequence of Escherichia coli pyrG encoding CTP synthetase; Weng M et al.; The amino acid sequence of Escherichia coli CTP synthetase was derived from the nucleotide sequence of pyrG . The derived amino acid sequence, confirmed at the N terminus by protein sequencing, predicts a subunit of 544 amino acids having a calculated Mr of 60,300 after removal of the initiator methionine . A glutamine amide transfer domain was identified which extends from approximately amino acid residue 300 to the C terminus of the molecule . The CTP synthetase glutamine amide transfer domain contains three conserved regions similar to those in GMP synthetase, anthranilate synthase, p-aminobenzoate synthase, and carbamoyl-P synthetase . The CTP synthetase structure supports a model for gene fusion of a trpG-related glutamine amide transfer domain to a primitive NH3-dependent CTP synthetase . The major 5' end of pyrG mRNA was localized to a position approximately 48 base pairs upstream of the translation initiation codon . Translation of the gene eno, encoding enolase, is initiated 89 base pairs downstream of pyrG . The pyrG-eno junction is characterized by multiple mRNA species which are ascribed to monocistronic pyrG and/or eno mRNAs and a pyrG eno polycistronic mRNA.

Science, 1986 Apr 25, 232(4749), 522 - 4
Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes; Ananthan J et al.; Heat shock protein (hsp) genes, a group of ubiquitous genes, are activated by various metabolic stresses . The suggestion that denaturation of intracellular proteins may be produced by the metabolic stresses and then signal the activation of the hsp genes was examined by co-injection of purified proteins and hsp genes into frog oocytes . Activation of hsp genes was observed if the proteins were denatured prior to injection but not if they were introduced in their native form . Furthermore, the activation of hsp genes by abnormal proteins and by heat shock appears to occur by a common mechanism . A model for the transcriptional regulation of the genes is based on competition for degradation between abnormal intracellular proteins and a labile regulatory factor.

Nucleic Acids Res, 1986 Apr 25, 14(8), 3463 - 74
Role of thymidine residues in DNA recognition by the EcoRI and EcoRV restriction endonucleases; Fliess A et al.; We have synthesized a series of oligonucleotides containing the EcoRI (GAATTC) or EcoRV (GATATC) recognition site within which or adjacent to which thymidine was substituted by uridine or derivatives of uridine . The effects of these substitutions on the rate of the EcoRI and EcoRV catalyzed cleavage reaction were investigated . Our results show that most of the substitutions within the site are quite well tolerated by EcoRI, not, however, by EcoRV . We conclude that the thymin residues most likely are not directly involved in the recognition process of the EcoRI reaction . In contrast, they are major points of contact, between substrate and enzyme in the EcoRV reaction . The effects of substitutions in the position adjacent to the recognition site is also markedly different for EcoRI and EcoRV . Here, EcoRI seems to be considerably more selective than EcoRV.

J Biol Chem, 1986 Apr 25, 261(12), 5658 - 62
Overproduction and purification of the B2 subunit of ribonucleotide reductase from Escherichia coli; Sjoberg BM et al.; The nrdB gene of Escherichia coli, coding for the B2 protein of ribonucleotide reductase, has been cloned in a runaway-replication vector . The runaway derivative pBEU17 carries the promoter-proximal portion of the E . coli alanyl-tRNA synthetase gene and proved useful for expressing cloned genes lacking their native transcription initiation signals . The alaS promoter is located approximately 500 base pairs upstream of a single BamHI restriction endonuclease cleavage site utilized in the construction of an expression recombinant plasmid, pBS1, for the nrdB product . After 5-h thermal induction of cells carrying the runaway recombinant pBS1, protein B2 constituted 40% of the soluble protein fraction of the cells . The high concentration of protein B2 in crude extracts of induced cells has enabled a simplified purification scheme to be developed for production of homogeneous and concentrated B2 preparations . Protein B2 produced from pBS1 is identical to the chromosomally encoded nrdB product of E . coli as regards molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme activity, tyrosine radical content, and structure of the binuclear iron center . Amino acid sequence analysis showed that the two polypeptide chains of protein B2 are identical . They start with an alanine residue, and the first 30 residues confirmed the amino acid sequence predicted from the nucleotide sequence of the nrdB gene, apart from an NH2-terminal processing removal of the initiator methionine.

Science, 1986 Apr 25, 232(4749), 500 - 3
Selectivity of intracellular proteolysis: protein substrates activate the ATP-dependent protease (La); Waxman L et al.; A critical enzyme in protein breakdown in Escherichia coli is protease La (the lon gene product), which hydrolyzes proteins and adenosine triphosphate (ATP) in a coupled process . The mechanism of this process was studied with fluorogenic tripeptides . Although proteins and peptides are degraded at the same active site, protein substrates enhance the ability of the enzyme to degrade these peptides two- to tenfold . Proteins that are not substrates had little or no effect . Thus, protein substrates must bind to protease La at two sites, the active site and an allosteric site whose occupancy enhances proteolytic activity . This effect did not require that the proteins themselves be degraded . Proteins could induce peptide breakdown even in the absence of ATP, and proteins and ATP had additive effects in stimulating peptidase activity . A multistep cyclical mechanism is proposed in which the binding of the substrate and ATP activates the protease . The enzyme can then cleave a peptide bond, but is inactivated through ATP hydrolysis . Such a mechanism may help account for the selectivity of protein breakdown and prevent inappropriate or excessive proteolysis in vivo.

Cell, 1986 Apr 25, 45(2), 177 - 83
M1 RNA with large terminal deletions retains its catalytic activity; Guerrier-Takada C et al.; Truncated transcripts of the rnpB gene from E . coli, coding for M1 RNA, the catalytic subunit of RNAase P, and fragments of M1 RNA generated by nuclease treatment have been prepared, and their ability to function catalytically in vitro has been determined . Molecules missing as many as 122 nucleotides at the 3' terminus retain catalytic activity, although at a much lower level than M1 RNA itself . No activity is observed with an RNA that is missing 70 nucleotides at the 5' terminus . The removal of even a small number of nucleotides from both termini eliminates all catalytic function . The preservation of one intact terminus may be essential for the tertiary and quaternary interactions required to generate the conformation of an active RNA species.

Science, 1986 Apr 25, 232(4749), 485 - 7
Potential metal-binding domains in nucleic acid binding proteins; Berg JM; A systematic search for sequences that potentially could form metal-binding domains in proteins has been performed . Five classes of proteins involved in nucleic acid binding or gene regulation were found to contain such sequences . These observations suggest numerous experiments aimed at determining whether metal-binding domains are present in these proteins and, if present, what roles such domains play in the processes of nucleic acid binding and gene regulation.

J Biol Chem, 1986 Apr 25, 261(12), 5467 - 72
Autoantibodies specific for U1 RNA and initiator methionine tRNA; Wilusz J et al.; Autoantibodies reactive with specific nuclear and cytoplasmic small RNAs were identified by immunoprecipitation of HeLa cell RNA . Approximately 30% of antisera examined from patients with autoimmune disorders contained anti-RNA antibodies . Two previously undescribed specificities--anti U1 RNA and anti-initiator methionine tRNA--were identified . Anti-RNA antibodies were reactive with gel-purified species as well as with RNA synthesized in vitro using the SP-6 transcription system . Antigenic mapping using two sera specific for the human initiator methionine tRNA revealed separate epitopes, one of which is conserved in formyl-methionine initiator tRNA from Escherichia coli . RNA fragmentation studies further suggested that secondary or tertiary tRNA structure is required for antibody recognition . The mammalian U1 RNA specific antibodies did not precipitate small RNAs of yeast but were highly reactive with yeast ribosomal RNA, thus indicating a possible relationship between these RNA species . Results obtained with these antisera are discussed in terms of higher order structure of small RNA molecules as well as the role of nucleic acid antibodies in the autoimmune phenomenon.

Biochemistry, 1986 Apr 22, 25(8), 1969 - 75
Integrated function of a kinetic proofreading mechanism: double-stage proofreading by isoleucyl-tRNA synthetase; Okamoto M et al.; Experimental measurements for isoleucyl-tRNA synthetase proofreading valyl-tRNAIle in Escherichia coli previously have been incorporated into the conventional Michaelis-Menten model for this system . This model was augmented to include two stages of proofreading--the aminoacyl adenylate and aminoacyl-tRNA stages--and used to predict the values of four additional rate constants that have been determined experimentally . The results suggest that two stages of conventional kinetic proofreading with binding sites designed for isoleucine (the "correct" substrate) are inconsistent with the experimental data, that a double-stage mechanism in which one stage (the "double-sieve") involves a binding site designed for valine (the "incorrect" substrate) and the other involves a binding site designed for isoleucine is consistent with all the experimental data, and that the experimental data are not sufficiently accurate to distinguish the stage at which the double-sieve mechanism operates in vivo . Furthermore, analysis of the model suggests that four parameters have the most questionable values and that experimental refinement of their estimates will be needed to determine which of the two stages involves the double-sieve mechanism.

Biochemistry, 1986 Apr 22, 25(8), 1881 - 6
Use of binding energy in catalysis analyzed by mutagenesis of the tyrosyl-tRNA synthetase; Wells TN et al.; The utilization of enzyme-substrate binding energy in catalysis has been investigated by experiments on mutant tyrosyl-tRNA synthetases that have been generated by site-directed mutagenesis . The mutants are poorer enzymes because they lack side chains that form hydrogen bonds with ATP and tyrosine during stages of the reaction . The hydrogen bonds are not directly involved in the chemical processes but are at some distance from the seat of reaction . The free energy profiles for the formation of enzyme-bound tyrosyl adenylate and the equilibria between the substrates and products were determined from a combination of pre-steady-state kinetics and equilibrium binding methods . By comparison of the profile of each mutant with wild-type enzyme, a picture is built up of how the course of reaction is affected by the influence of each side chain on the energies of the complexes of the enzyme with substrates, transition states, and intermediates (tyrosyl adenylate) . As the activation reaction proceeds, the apparent binding energies of certain side chains with the tyrosine and nucleotide moieties increase, being weakest in the enzyme-substrate complex, stronger in the transition state, and strongest in the enzyme-intermediate complex . Most marked is the interaction of Cys-35 with the 3'-hydroxyl of the ribose . Removal of the side chain of Cys-35 leads to no change in the dissociation constant of ATP but causes a 10-fold lowering of the catalytic rate constant . It contributes no net apparent binding energy in the E X Tyr X ATP complex and stabilizes the transition state by 1.2 kcal/mol and the E X Tyr-AMP complex by 1.6 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1986 Apr 22, 25(8), 1870 - 6
L-threonine dehydrogenase from Escherichia coli K-12: thiol-dependent activation by Mn2+; Craig PA et al.; Addition of 1 mM Mn2+ to all solutions in the final chromatographic step used to purify L-threonine dehydrogenase (L-threonine:NAD+ oxidoreductase, EC 1.1.1.103) from extracts of Escherichia coli K-12 routinely provides 30-40 mg of pure enzyme per 100 g wet weight of cells with specific activity = 20-30 units/mg . Enzyme dialyzed exhaustively against buffers containing Chelex-100 resin has a specific activity = 8 units/mg and contains 0.003 or 0.02 mol of Mn2+/mol of enzyme as determined by radiolabeling studies with 54Mn2+ or by atomic absorption spectroscopy, respectively . Dehydrogenase activity is completely abolished by low concentrations of either Hg2+ or Ag+; of a large spectrum of other metal ions tested, only Mn2+ and Cd2+ have an activating effect . Activation of threonine dehydrogenase by Mn2+ is thiol-dependent and is saturable with an activation Kd = 9.0 microM and a Vmax = 105 units/mg . Stoichiometry of Mn2+ binding was found to be 0.86 mol of Mn2+/mol of enzyme subunit with a dissociation constant (Kd) = 8.5 microM . Mn2+ appears to interact directly with threonine dehydrogenase; gel filtration studies with the dehydrogenase plus 54Mn2+ in the presence of either NAD+, NADH, L-threonine, or combinations thereof show that only Mn2+ coelutes with the enzyme whereas all other ligands elute in the salt front and the stoichiometry of the dehydrogenase-Mn2+ interaction is not affected in any instance . A theoretical curve fit to data for the pH-activity profile of Mn2+-saturated enzyme has a pKa = 7.95 for one proton ionization . The data establish L-threonine dehydrogenase of E . coli to be a metal ion activated enzyme.

Biochemistry, 1986 Apr 22, 25(8), 2205 - 11
Restriction endonuclease EcoRI alters the enantiomeric preference of chiral metallointercalators for DNA: an illustration of a protein-induced DNA conformational change; Barton JK et al.; A conformational change in the DNA plasmid ColE1 appears to occur upon specific binding of the restriction endonuclease EcoRI . Enzyme association alters the chiral discrimination found in binding metallointercalators to DNA sites . The complexes tris(1,10-phenanthroline)ruthenium(II), Ru(phen)3(2+), tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II), Ru(DIP)3(2+), and tris(4,7-diphenyl-1,10-phenanthroline)cobalt(III), Co(DIP)3(3+), in general, bind stereoselectively to DNA helices, with enantiomers possessing the delta configuration bound preferentially by right-handed B-DNA . In the presence of EcoRI, however, this enantioselectivity is altered . The chiral intercalators, at micromolar concentrations, inhibit the reaction of EcoRI, but for each enantiomeric pair it is the lambda enantiomer, which binds only poorly to a B-DNA helix, that inhibits EcoRI preferentially . Kinetic studies in the presence of lambda-Ru(DIP)3(2+) indicate that the enzyme inhibition occurs as a result of the lambda enantiomer binding to the enzyme-DNA complex as well as to the free enzyme . Furthermore, photolytic strand cleavage experiments using Co(DIP)3(3+) indicate that the metal complex interacts directly at the protein-bound DNA site . Increasing concentrations of bound EcoRI stimulate photoactivated cleavage of the DNA helix by lambda-Co(DIP)3(3+), until a protein concentration is reached where specific DNA recognition sites are saturated with enzyme . Thus, although lambda-Co(DIP)3(3+) does not bind closely to the DNA in the absence of enzyme, specific binding of EcoRI appears to alter the DNA structure so as to permit the close association of the lambda isomer to the DNA helix . Mapping experiments demonstrate that this association leads to photocleavage of DNA by the cobalt complex at or very close to the EcoRI recognition site.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochim Biophys Acta, 1986 Apr 22, 870(3), 545 - 51
Evidence that centre 2 in Escherichia coli fumarate reductase is a {4Fe-4S}cluster; Cammack R et al.; Redox titrations of the iron-sulphur clusters in fumarate reductase purified from Escherichia coli, monitored by ESR spectroscopy, identified three redox events, similar to those observed in other fumarate reductases and succinate dehydrogenases: Centre 1, a {2Fe-2S} cluster, at g = 2.03, 1.93, appeared on reduction with Em = -20 mV . Centre 3, probably a {3Fe-xS} cluster, at g = 2.02 appeared in the oxidized state with Em = -70 mV . Centre 2 has been observed as an increase in the electron-spin relaxation of Centre 1 . It titrates as an n = 1 species with Em = -320 mV, but in our hands did not appear to contribute significant intensity to the g = 2.03, 1.93 signal . It therefore appears to be an additional centre which undergoes spin-spin interaction with Centre 1 . The reduction of Centre 2 coincided with the appearance of an extremely broad ESR spectrum, observed at temperatures below 20 K, with features at g = 2.17, 1.9, 1.68 . The broad signal was observed in both soluble and membrane-bound preparations . Its midpoint potential was -320 mV . Its integrated intensity was approximately equal to that of Centre 1, if its broad outer wings were taken into account . Consideration of the ESR properties of this signal, together with the amino acid sequence of the frdB subunit of the enzyme, indicates that Centre 2 is a {4Fe-4S} cluster which, in its reduced state, enhances the spin relaxation of the {2Fe-2S} Centre 1.

FEBS Lett, 1986 Apr 21, 199(2), 187 - 92
A hybrid protein between IFN-gamma and IL-2; Seno M et al.; The complementary DNAs encoding human interferon-gamma (IFN-gamma) and human interleukin-2 (IL-2), two different proteins involved in the same immune system, were fused to code a hybrid protein, which was expressed in E . coli to investigate the interactions of these two proteins at the molecular level . Through immunoprecipitation analysis, this protein was revealed to be of about 31 kDa, which was expected from nucleotide sequencing, and to have the antigenicities of both IFN-gamma and IL-2 . The extract from bacteria expressing this hybrid protein showed at least two biological activities: an antiviral activity derived from IFN-gamma and the ability to support the growth of natural killer (NK) cells derived from IL-2 . Comparing the enhancement of NK cell activity of this hybrid protein with IFN-gamma and IL-2, this hybrid protein appears to conserve each activity almost completely without diminishing the other.

J Mol Biol, 1986 Apr 20, 188(4), 555 - 64
Constraints on codon context in Escherichia coli genes . Their possible role in modulating the efficiency of translation; Shpaer EG; The constraints on nucleotide sequences of highly and weakly expressed genes from Escherichia coli have been analysed and compared . Differences in synonymous codon spectra in highly and weakly expressed genes lead to different frequencies of nucleotides (in the first and third codon positions) and dinucleotides in the two groups of genes . It has been found that the choice of synonymous codons in highly expressed genes depends on the nucleotides adjacent to the codon . For example, lysine is preferably encoded by the AAA codon if guanosine is 3' to the lysine codon (AAA-G, P less than 10(-9)) . And, on the contrary, AAG is used more often than AAA (P less than 0.001) if cytidine is 3' adjacent to lysine . Guanosine occurs more frequently than adenosine 5' to all the lysine codons (AAR, P less than 10(-5), i.e . NNG codons are preferred over the synonymous NNA codons 5' to the positions of lysine in the genes . The context effect was observed in nonsense and missense suppression experiments . Therefore, a hypothesis has been suggested that the efficiency of translation of some codons (for which the constraints on the adjacent nucleotides were found) can be modulated by the codon context . The rules for preferable synonymous codon choice in highly expressed genes depending on the nucleotides surrounding the codon are presented . These rules can be used in the chemical synthesis of genes designed for expression in E . coli.

J Mol Biol, 1986 Apr 20, 188(4), 545 - 53
Deletion analysis of RNA polymerase interaction sites in the Escherichia coli lactose operon regulatory region; Yu XM et al.; Two sets of deletions produced in vitro by S1 nuclease were used to study the structure and function of promoters lacPUV5 and lacP115 . The upstream boundary of the RNA polymerase binding site in lacPUV5 was determined by comparing the levels of beta-galactoside expression in vivo programmed from a set of deletions progressively extending into the -35 region of the lacPUV5 promoter . Sequences upstream from base-pair -37 are not necessary for the full functioning of lacPUV5 . A deletion that removes base-pair -37 retains only half of the promoter activity . Deletion of the first T X A base-pair of the consensus -35 region sequence, 5' T-T-T-A-C-A 3', leads to a sixfold reduction of promoter activity . Deletion of the whole -35 region of lacPUV5 leads to at least a 20-fold reduction of its promoter activity . Abortive initiation assays were performed on the fully functional lacPUV5 and two lacPUV5 deletions, which removed part of the -35 consensus sequence, to study their effect on the kinetics of RNA polymerase-promoter open complex formation . These two deletions show a 3.5 to 7-fold reduction in KB . Analysis in vivo of lacP115 showed that sequence information upstream from the -35 region is important for the full functioning of lacP115 . A deletion removing sequences upstream from -41 caused a three- to fourfold reduction in promoter activity, apparently due to reduced transcription initiation . lacP115 has a much lower k2 value than lacPUV5; its KB value is about threefold higher than that of lacPUV5.

JAMA, 1986 Apr 18, 255(15), 2049 - 53
Exercise-induced anaphylactic syndromes . Insights into diagnostic and pathophysiologic features; Casale TB et al.; To differentiate the diagnoses of exercise-induced anaphylaxis and cholinergic urticaria/anaphylaxis, we developed reproducible diagnostic provocative challenges . The data derived from the study of two representative patients, one with cholinergic urticaria and the other with exercise-induced anaphylaxis, suggest approaches to distinguishing these diagnoses . After specific exercise challenges, both patients developed symptoms consistent with anaphylaxis and had associated increases in plasma histamine levels . After passive heat challenges inducing increases in core body temperature more than 0.7 degrees C, only the patient with cholinergic urticaria developed anaphylactic symptoms and had a rise in the plasma histamine level . Neither patient developed symptoms of anaphylaxis when core body temperatures were increased after administration of intravenous endotoxin . Thus, passive heat challenges are extremely valuable in differentiating these two exercise-related syndromes . Although not important in exercise-induced anaphylaxis, specific thermoregulatory mechanisms appear to play an intricate part in the pathophysiology of cholinergic urticaria/anaphylaxis.

J Immunol Methods, 1986 Apr 17, 88(2), 225 - 32
Further studies on the ELISA-spot technique . Its application to particulate antigens and a potential improvement in sensitivity; Franci C et al.; Applications of the ELISA-spot technique to particulate antigens (sheep erythrocytes and E . coli) are reported; sheep erythrocytes were used to ensure a rigorous comparison of the spot assay and the haemolytic plaque assay . The spot technique was also applied to soluble antigens (dextran and trinitrophenyl-lipopolysaccharide) to assess the putative occurrence of non-haemolytic antibodies . In most instances, the spot assay disclosed higher numbers of antibody-secreting cells than the plaque assay . An examination of the kinetics of spot formation demonstrated that spot development was most rapid during the first and second hours of enzyme activity and slowed thereafter, although the numbers of spots at 16 h were higher than those at 2 h . To shorten the assay time a redox reaction (which yields an insoluble formazan) was coupled to the enzymatic reaction . In duplicate assays, this improved technique gave larger numbers of spots in a shorter time, than the conventional assay.

Biochem J, 1986 Apr 15, 235(2), 329 - 36
The effects of endotoxaemia on protein metabolism in skeletal muscle and liver of fed and fasted rats; Jepson MM et al.; The response of muscle and liver protein metabolism to either a single or three successive daily injections of an endotoxin (Escherichia coli lipopolysaccharide, serotype 0127 B8; 1 mg/ml, 0.3 mg/100 g body wt.) was studied in vivo in the fed rat, and at 24 and 30 h after endotoxin treatment during fasting . In the fed rats there was a catabolic response in muscle, owing to a 60-100% increase in muscle protein degradation rate, and a 52% fall in the synthesis rate . Although there was a 20% decrease in food intake, the decrease in protein synthesis was to some extent independent of this, since rats treated with endotoxin and fasted also showed a lower rate of muscle protein synthesis, which was in excess of the decrease caused by fasting alone . The mechanism of this decreased protein synthesis involved decreased translational activity, since in both fed and fasted rats there was a decreased rate of synthesis per unit of RNA . This occurred despite the fact that insulin concentrations were either maintained or increased, in the fasted rats, to those observed in fed rats . In the liver total protein mass was increased in the fed rats by 16% at 24 h, and the fractional synthesis rate at that time was increased by 35% . In rats fasted after endotoxin treatment the liver protein mass was not decreased as it was in the control fasted rats, and the fractional synthesis rate was increased by 22% . In both cases the increased synthesis rate reflected an elevated hepatic RNA concentration . The extent of this increase in hepatic protein synthesis was sufficient at one point to compensate for the fall in estimated muscle protein synthesis, so that the sum total in the two tissues was maintained.

Eur J Biochem, 1986 Apr 15, 156(2), 277 - 84
Isolation and characterisation of a soluble active fragment of hydrogenase isoenzyme 2 from the membranes of anaerobically grown Escherichia coli; Ballantine SP et al.; An active tryptic fragment of membrane-bound hydrogenase isoenzyme 2 from anaerobically grown Escherichia coli has been purified . The soluble enzyme derivative was released from the membrane fraction by trypsin cleavage . The purification procedure involved ion-exchange, hydroxyapatite and gel permeation chromatography . The enzyme derivative was purified 100-fold from the membrane fraction and the specific activity of the final preparation was 320 mumol benzyl viologen reduced min-1 mg protein-1 (H2:benzyl viologen oxidoreductase) . The native enzyme derivative had an Mr of 180,000 and was composed of equimolar amounts of polypeptides of Mr 61,000 and 30,000 . It possessed 12.5 mol Fe, 12.8 mol acid-labile S2- and 3.1 mol Ni/180,000 g enzyme . Antibodies were raised to the purified preparation which cross-reacted with hydrogenase isoenzyme 2 but not with isoenzyme 1 in detergent-dispersed preparations . Western immunoblot analysis revealed that isoenzyme 2 which had not been exposed to trypsin contained cross-reacting polypeptides of Mr 61,000 and 35,000 . Trypsin treatment of the membrane-bound enzyme to form the soluble derivative of isoenzyme 2, therefore, cleaves a polypeptide of Mr 35,000 to produce the 30,000-Mr fragment . Trypsin treatment of the detergent-dispersed isoenzyme 2 produces the same fragmentation of the enzyme . Neither of the subunits of the enzyme revealed any immunological identity with those of hydrogenase isoenzyme 1.

Eur J Biochem, 1986 Apr 15, 156(2), 265 - 75
Purification and properties of membrane-bound hydrogenase isoenzyme 1 from anaerobically grown Escherichia coli K12; Sawers RG et al.; Hydrogenase isoenzyme 1 from the membrane fraction of anaerobically grown Escherichia coli has been purified to near homogeneity . The preparation involved dispersion of the membrane fraction with deoxycholate followed by ammonium sulphate precipitation, ion-exchange, hydroxyapatite and gel filtration chromatography steps . The enzyme was assayed by quantification of the H2:benzyl viologen oxidoreductase activity immunoprecipitated by a non-inhibitory antiserum specific for the enzyme . The enzyme constituted about 8% of the hydrogenase activity found in the detergent-dispersed membranes, the remainder being attributable to hydrogenase isoenzyme 2 . Isoenzyme 1 was purified 130-fold and the specific activity of the final preparation was 10.6 mumol benzyl viologen reduced min-1 (mg protein)-1 (H2:benzyl viologen oxidoreductase) . The final preparation contained polypeptides of apparent Mr 64,000, 31,000 and 29,000 . Antibodies were raised both to the final preparation and to immunoprecipitation arcs containing hydrogenase isoenzyme 1, excised from crossed immunoelectrophoresis plates . The former cross-reacted with all three polypeptides in the enzyme preparation but the latter recognised only the Mr-64,000 polypeptide . Immunological analysis revealed that the polypeptides of apparent Mr 31,000 and 29,000 are fragments of a single polypeptide of Mr 35,000 which is present in the detergent-dispersed membranes . The fragmentation of the Mr-35,000 polypeptide during the preparation correlates with a change in the electrophoretic mobility of the enzyme . A similar electrophoretic mobility change was observed, accompanied by cleavage of the Mr-35,000 polypeptide to one of 32,000 when the enzyme was analysed after exposure of detergent-dispersed membranes to trypsin . The enzyme in the detergent-dispersed membranes consists minimally of two subunits of Mr 64,000 and two subunits of Mr 35,000 . It contained 12.2 mol Fe and 9.1 mol acid-labile S2-/200,000 g enzyme . The enzyme, purified from bacteria grown in the presence of 63Ni, was found to contain 0.64 (+/- 0.20) mol Ni/200,000 g enzyme . A constant ratio of 63Ni immunoprecipitated to hydrogenase isoenzyme 1 activity immunoprecipitated by antiserum specific for the enzyme was observed during the preparation, consistent with Ni being part of the enzyme . The enzyme has a low Km for H2 (2.0 microM) in the H2:benzyl viologen oxidoreductase assay . It catalyses H2 evolution employing reduced methyl viologen as electron donor . It is inhibited reversibly by CO and irreversibly by N-bromosuccinimide.

J Biol Chem, 1986 Apr 15, 261(11), 5175 - 9
Characterization of the F plasmid TraJ protein synthesized in F' and Hfr strains of Escherichia coli K-12; Cuozzo M et al.; Using purified F plasmid TraJ protein (Cuozzo, M., Silverman, P., and Minkley, E . (1984) J . Biol . Chem . 259, 6659-6666), we prepared rabbit anti-TraJ protein antibodies to analyze for the first time the TraJ protein as it is synthesized in normal F' and Hfr conjugal donor strains . Using affinity-purified antibody, we identified the protein on immuno-overlay blots of whole cell proteins separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis . In contrast to the TraJ protein synthesized in large quantity by heat-induced lambda (traJ) lysogens, the TraJ protein synthesized in normal donor cells was soluble, even after sedimentation at 100,000 X g . The soluble protein was found with the cytoplasmic fraction after separation of cytoplasmic and periplasmic proteins . Velocity sedimentation analysis indicated an S20,w of 3.5 for the single molecular species composed of or including all the TraJ polypeptide in crude extracts . Quantitative analyses showed that conjugal donor strains normally contain 2000-4000 TraJ monomers/cell . However, that level depended on other plasmid and chromosomal genes.

J Biol Chem, 1986 Apr 15, 261(11), 4868 - 74
The reaction of ribosomes with elongation factor Tu.GTP complexes . Aminoacyl-tRNA-independent reactions in the elongation cycle determine the accuracy of protein synthesis; Thompson RC et al.; The fidelity of protein synthesis depends on the rate constants for the reaction of ribosomes with ternary complexes of elongation factor Tu (EF-Tu), GTP, and aminoacyl (aa)-tRNA . By measuring the rate constants for the reaction of poly(U)-programmed ribosomes with a binary complex of elongation factor (EF-Tu) and GTP we have shown that two of the key rate constants in the former reaction are determined exclusively by ribosome-EF-Tu interactions and are not affected by the aa-tRNA . These are the rate constant for GTP hydrolysis, which plays an important role in the fidelity of ternary complex selection by the ribosome, and the rate constant for EF-Tu.GDP dissociation from the ribosome, which plays an equally important role in subsequent proofreading of the aa-tRNA . We conclude that the fidelities of ternary complex selection and proofreading are fundamentally dependent on ribosome-EF-Tu interactions . These interactions determine the absolute value of the rate constants for GTP hydrolysis and EF-Tu.GDP dissociation . The ribosome then uses these rate constants as internal standards to measure, respectively, the rate constants for ternary complex and aa-tRNA dissociation from the ribosome . These rates, in turn, are highly dependent on whether the ternary complex and aa-tRNA are cognate or near-cognate to the codon being translated.

J Biol Chem, 1986 Apr 15, 261(11), 4847 - 54
Contrasting effects of Escherichia coli single-stranded DNA binding protein on synthesis by T7 DNA polymerase and Escherichia coli DNA polymerase I (large fragment) . Evidence that binding protein inhibits trans-lesion synthesis by polymerase I; Michaels ML et al.; The effect of Escherichia coli single-stranded DNA binding protein (SSB) on DNA synthesis by T7 DNA polymerase and E . coli DNA polymerase I (large fragment) using native or aminofluorene-modified M13 templates was evaluated by in vitro DNA synthesis assays and polyacrylamide gel electrophoresis analysis . The two polymerase enzymes displayed differential responses to the addition of SSB . T7 DNA polymerase, a enzyme required for the replication of the T7 chromosome, was stimulated by the addition of SSB whether native or modified templates were used . On the other hand, E . coli DNA polymerase I was slightly stimulated by the addition of SSB to the native template but substantially inhibited on modified templates . This result suggests that DNA polymerase I may be able to synthesize past an aminofluorene adduct but that the presence of SSB inhibited this trans-lesion synthesis . Polyacrylamide gels of the products of DNA synthesis by polymerase I supported this inference since SSB caused a substantial increase in the accumulation of shorter DNA chains induced by blockage at the aminofluorene adduct sites.

Virology, 1986 Apr 15, 150(1), 313 - 7
Dependence of MS2 and T4 phage growth upon host amino acid biosynthesis during infections of Escherichia coli; Rojiani M et al.; By manipulating the growth conditions of Escherichia coli both before and after phage MS2 or phage T4 infections, a dependence of phage growth upon postinfection host amino acid biosynthesis can be inferred . Cells grown under repressing conditions for amino acid biosynthesis shifted to amino acid-free medium postinfection (in the absence of further host gene expression) are poor hosts for phage growth, whereas cells grown under derepressing conditions for amino acid biosynthesis are good hosts regardless of postinfection conditions; thus postinfection biosynthesis of amino acids utilizes performed host enzymes which still function to carry out their biosynthetic pathways during phage infections . Phage MS2 also appears to permit the derepression of host amino acid biosynthetic operons during the infection . A functional dependence of MS2 growth upon postinfection host gene function is perhaps the strongest argument that the RNA phage do not shut off host messenger RNA and protein synthesis.

J Immunol, 1986 Apr 15, 136(8), 3025 - 31
Interleukin 1: a common endogenous mediator of inflammation and the local Shwartzman reaction; Beck G et al.; In this study we investigated the role of interleukin 1 (IL 1) in the induction of inflammatory lesions and in the preparation and provocation of the local Shwartzman reaction . Both of these phenomena can be induced with a variety of agents . This suggested to us that a common endogenous mediator may be crucial to the development of these two lesions . When IL 1 was injected intradermally into shaved rabbit backs, 51Cr-labeled neutrophils accumulated at the injection site . Neutrophils began to accumulate less than 1 hr after injection, and the maximum rate of accumulation was observed by 4 hr . This activity was dose dependent . It was calculated that in all animals, 10(-14) mol of IL 1 induced significant neutrophil accumulation, whereas in many animals, as little as 10(-15) mol of IL 1 sufficed . When 4.2 X 10(-9) mol of E . coli 0111:B4 lipopolysaccharide W was injected i.v . 24 hr after an intradermal injection of IL 1 (2.9 X 10(-13) mol), a local Shwartzman reaction was seen 4 hr later at the intradermal injection site . IL 1 injected i.v . 24 hr after an intradermal injection of either IL 1 or lipopolysaccharide also produced a local Shwartzman reaction . These data indicate that IL 1 may be the common endogenous mediator of the inflammatory response, and IL 1 may serve in the same role for the preparation and provocation of the local Shwartzman reaction.

Eur J Biochem, 1986 Apr 15, 156(2), 399 - 405
Galactose- and maltose-stimulated lipoamide dehydrogenase activities related to the binding-protein-dependent transport of galactose and maltose in toluenized cells of Escherichia coli; Richarme G et al.; The binding protein-dependent transport of galactose and maltose occurs at a reduced but significant rate in Escherichia coli cells which have undergone a mild toluenization . Dihydrolipoate and 3-acetyl-NAD produce a severalfold stimulation of these transports in the toluenized cells . In parallel to the stimulation of galactose and maltose transport by dihydrolipoate and 3-acetyl-NAD, there is a stimulation by galactose and maltose of lipoamide dehydrogenase activities which seem to be related to the binding-protein-dependent transport of these sugars . The lipoamide dehydrogenase component of the pyruvate and 2-oxoglutarate dehydrogenase complexes (the lpd gene product) is not involved in this stimulation . These results are discussed in relation to our recent studies showing a possible involvement of lipoic acid and of the 2-oxoacid dehydrogenases in the binding-protein-dependent transports.

J Biol Chem, 1986 Apr 15, 261(11), 4895 - 901
Sequences of Escherichia coli uvrA gene and protein reveal two potential ATP binding sites; Husain I et al.; We have determined the nucleotide sequence of the uvrA gene of Escherichia coli . The coding region of the gene is 2820 base pairs which specifies a protein of 940 amino acids and Mr = 103,874 . The polypeptide sequence predicted from the DNA sequence was confirmed by analyzing the UvrA protein: the sequence of the first 7 NH2-terminal amino acids as well as the amino acid composition of the pure protein agreed with those predicted from the nucleotide sequence . By comparing the sequence of UvrA protein to the amino acid sequences of other ATPases, we found that two regions in the UvrA protein, separated from one another by about 600 amino acids, have the highly conserved G-X4-GKT(S)-X6-I(V) sequence found at the active sites of many, but not all, ATPases . Our findings suggest that UvrA protein may have two ATP binding sites.

J Biol Chem, 1986 Apr 15, 261(11), 4785 - 8
Neurospora crassa mitochondria contain two forms of a 4'-phosphopantetheine-modified protein; Lakin-Thomas PL et al.; When Neurospora crassa was labeled with {14C}pantothenic acid during growth, the mitochondrial fraction contained two bands of radioactivity of Mr 19,000 and 22,000 by sodium dodecyl sulfate gel electrophoresis . The 19-kDa band was converted to the 22-kDa band by four treatments which are characteristic of the cleavage of a thioester bond: dithiothreitol and 2-mercaptoethanol at basic but not neutral pH, alkaline methanolysis, sodium borohydride in tetrahydrofuran, and hydroxylamine at neutral pH . Mitochondrial subfractionation indicated that the 22-kDa form was preferentially associated with the soluble fraction while the 19-kDa form was found in all fractions . Several properties of the mitochondrial protein were similar to the Escherichia coli acyl carrier protein: Mr on sodium dodecyl sulfate gels, decreased electrophoretic mobility under deacylating conditions, isoelectric point, and covalent attachment of 4'-phosphopantetheine . The 19- and 22-kDa bands may therefore represent acylated and deacylated forms of a mitochondrial acyl carrier protein.

J Biol Chem, 1986 Apr 15, 261(11), 4820 - 7
Simultaneous purification of three mitochondrial enzymes . Acetylglutamate kinase, acetylglutamyl-phosphate reductase and carbamoyl-phosphate synthetase from Neurospora crassa; Wandinger-Ness AU et al.; The early enzymes of arginine biosynthesis in Neurospora crassa are localized in the mitochondrion and catalyze the conversion of glutamate to citrulline . The final conversion of citrulline to arginine occurs via two enzymatic steps in the cytoplasm . We have devised a method for the isolation and purification of three of the mitochondrial arginine biosynthetic enzymes from a single extract . Acetylglutamate kinase and acetylglutamyl-phosphate reductase (both products of the complex arg-6 locus) were purified to homogeneity and near homogeneity, respectively . The large catalytic subunit of carbamoyl-phosphate synthetase was also purified to homogeneity . The three enzymes were resolved into separate fractions by chromatography on three dye-ligand affinity resins, which are specific for nucleotide binding enzymes and have a high protein binding capacity . High performance liquid chromatography was employed in the final stages of purification and was extremely effective in fractionating both acetylglutamate kinase and acetylglutamyl-phosphate reductase from proteins with very similar properties, which were not removed by other techniques . The purified proteins were used to raise specific antisera against these proteins . Acetylglutamate kinase and acetylglutamyl-phosphate reductase were shown to be immunologically unrelated . This finding suggests that the arg-6 locus encompasses two nonoverlapping cistrons . The antisera raised against carbamoyl-phosphate synthetase has been shown to cross-react with related enzymes in Saccharomyces cerevisiae, Escherichia coli, and rat liver (Ness, S . A., and Weiss, R . L . (1985) J . Biol . Chem . 260, 14355-14362) . Acetylglutamate kinase is a regulatory enzyme and has been shown to be feedback-inhibited by arginine . We have determined the submitochondrial localization of acetylglutamate kinase and the second arg-6 product, acetylglutamyl-phosphate reductase . Both enzymes were shown to be soluble matrix enzymes . We discuss the relevance of this finding with respect to possible mechanisms for end product inhibition of a mitochondrial enzyme by a cytoplasmic effector.

Biochem Biophys Res Commun, 1986 Apr 14, 136(1), 183 - 92
Biosynthesis of reovirus-specified polypeptides . Molecular cDNA cloning and nucleotide sequence of the reovirus serotype 1 Lang strain s4 mRNA which encodes the major capsid surface polypeptide sigma 3; Atwater JA et al.; Serotype 1 Lang strain s4 mRNA, which encodes the major capsid surface polypeptide sigma 3 of reovirions, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13 . A complete consensus nucleotide sequence for s4 mRNA has been determined from cDNA clones . The Lang strain s4 mRNA is 1196 nucleotides in length and possesses an open reading frame with a coding capacity of 365 amino acids, sufficient to account for a sigma 3 polypeptide of 41,212 daltons . Comparison of the serotype 1 (Lang) s4 sequence with the serotype 3 (Dearing) s4 sequence reveals 94% homology at the nucleotide level; the predicted sigma 3 polypeptides of the Lang and Dearing strains display 96% homology at the amino acid level . Two third base C codons (leu:CUC and ser:AGC) are used about one-tenth as frequently in the reovirus s4 mRNAs as compared to mammalian cellular mRNAs.

J Chromatogr, 1986 Apr 11, 376, 163 - 73
Crossed affinity immunoelectrophoresis of the Escherichia coli pyruvate dehydrogenase complex; Visser J et al.; The crossed immunoelectrophoretic pattern obtained with the intact pyruvate dehydrogenase complex of Escherichia coli can be modified when this technique is combined with affinity gel electrophoresis using reactive dyes coupled to agarose as ligands . The patterns that arise have been interpreted with respect to localization of the three component enzymes . This was realized by using antibodies with different specificity, active enzyme staining and E2-E3 subcomplex behaviour . Dissociation of E1 subunits occurs more easily than that of E3 but remains incomplete in this system . The free reactive Procion Blue-MX dyes tested inactivate the complex even at neutral pH . The dyes react with all three components but E3 (80%) and E2 (15-20%) retain part of their catalytic activity . Modification leads to an enhanced dissociation of E1.

Biochim Biophys Acta, 1986 Apr 11, 881(2), 268 - 75
Purification and some properties of uracil phosphoribosyltransferase from Escherichia coli K12; Rasmussen UB et al.; Uracil phosphoribosyltransferase from Escherichia coli K12 was purified to homogeneity as determined by polyacrylamide gel electrophoresis . For this purpose a pyrimidine-requiring strain harboring the upp gene on a ColE1 plasmid was used, which showed 15-times higher uracil phosphoribosyltransferase activity in a crude extract . When this strain was grown under conditions of uracil starvation, an additional 10-times elevation of the enzyme activity was obtained . The molecular weight of uracil phosphoribosyltransferase was determined to be 75000; the enzyme consists of three subunits with a molecular weight of 23500 . Uracil phosphoribosyltransferase is specific for uracil and some uracil analogues . The apparent Km values for uracil and PRib-PP were 7 microM and 300 microM, respectively . As an effector of enzyme activity, GTP lowered the Km for PRib-PP to 90 microM and increased the Vmax value 2-fold, but had no effect on the Km for uracil . The effect of GTP was found to be pH-dependent . The enzymatic characterization of uracil phosphoribosyltransferase and the observed regulation of its synthesis emphasizes the role of the enzyme in pyrimidine salvage.

Nucleic Acids Res, 1986 Apr 11, 14(7), 2877 - 90
Structure of the uvrB gene of Escherichia coli . Homology with other DNA repair enzymes and characterization of the uvrB5 mutation; Backendorf C et al.; The complete nucleotide sequence of the Escherichia coli uvrB gene has been determined . The coding region of the uvrB gene consists of 2019 nucleotides which direct the synthesis of a 673 amino-acid long polypeptide with a calculated molecular weight of 76.614 daltons . Comparison of the UvrB protein sequence to other known DNA repair enzymes revealed that 2 domains of the UvrB protein (domain I = 6 amino acids, domain II = 14 amino acids) are also present in the protein sequence of the uvrC gene . We show that the structural homologies between UvrB and UvrC are as well reflected by the cross-reactivity of anti-uvrB and anti-uvrC antibodies with UvrC and UvrB protein respectively . In the N-terminal part of UvrB, domain III (17 amino acids) shows a strong homology with one part of the AlkA gene product . Adjacent to domain III, an ATP binding site consensus sequence is found in domain IV . The uvrB5 mutant gene from strain AB1885 has been cloned on plasmid pBL01 . We show that the uvrB5 mutation is due to a point deletion of a CG basepair and results in the synthesis of an 18 kD protein composed of the 113 N-terminal amino acids of the wild type uvrB gene and a 43 amino acid long tail coded in the -1 frame.

Cell, 1986 Apr 11, 45(1), 53 - 64
Extensive unwinding of the plasmid template during staged enzymatic initiation of DNA replication from the origin of the Escherichia coli chromosome; Baker TA et al.; Early in the staged initiation of enzymatic replication of plasmids containing the unique origin of the E . coli chromosome (oriC), the plasmid is converted to a new topological form which is highly underwound, two to 15 times more than native supercoiled DNA . The underwinding reaction precedes priming of DNA synthesis and follows an initial complex formation, requiring ATP and proteins dnaA, dnaB, and dnaC; underwinding depends on the further addition of gyrase and SSB . DnaB protein as a helicase and gyrase as a topoisomerase drive the underwinding with the energy of ATP hydrolysis . The underwound template, extensively single-stranded and complexed with proteins, is an active form for priming by primase and elongation by DNA polymerase III holoenzyme.

Nucleic Acids Res, 1986 Apr 11, 14(7), 3075 - 87
Codon usage and gene expression; Holm L; The hypothesis that codon usage regulates gene expression at the level of translation is tested . Codon usage of Escherichia coli and phage lambda is compared by correspondence analysis, and the basis of this hypothesis is examined by connecting codon and tRNA distributions to polypeptide elongation kinetics . Both approaches indicate that if codon usage was random tRNA limitation would only affect the rarest tRNA species . General discrimination against their cognate codons indicates that polypeptide elongation rates are maintained constant . Thus, differences in expression of E . coli genes are not a consequence of their variable codon usage . The preference of codons recognized by the most abundant tRNAs in E . coli genes encoding abundant proteins is explained by a constraint on the cost of proof-reading.

Nucleic Acids Res, 1986 Apr 11, 14(7), 3089 - 102
An asparagine-rich protein from blood stages of Plasmodium falciparum shares determinants with sporozoites; Stahl HD et al.; We describe a cDNA clone derived from mRNA of asexual blood-stages of the malaria parasite Plasmodium falciparum . This clone, designated Ag319, expresses a P.falciparum antigen fused to beta-galactosidase in Escherichia coli . Human antibodies from Papua New Guinea were affinity-purified by adsorption to extracts of Ag319 immobilized on CNBr-Sepharose . The antibodies reacted predominantly with P . falciparum polypeptides of Mr 220,000 and 160,000, and a number of ill-defined lower molecular weight species . Antibodies reacted in indirect immunofluorescence with all asexual blood-stages although the antigen appeared to be most abundance in the schizont . Surprizingly the antibodies also reacted with sporozoites . The amino acid sequence predicted from the complete nucleotide sequence of this clone is remarkable because 40% of the residues are Asn, and so the antigen has been termed the Asparagine-Rich Protein (ARP) . Like other P . falciparum antigens, ARP contains tandemly repetitive sequences, based on the tetrapeptide Asn-Asn-Asn-Met and we have confirmed that these represent natural epitopes by reaction of the corresponding synthetic peptides with human antibodies . Surprisingly, ARP is also rich in Asn outside the tandem repeats.

Nature, 1986 Apr 10-16, 320(6062), 555 - 6
Crystallographic analysis of mutant human haemoglobins made in Escherichia coli; Luisi BF et al.; The expression of beta-globin in Escherichia coli has enabled us to study the functional role of individual amino-acid residues in haemoglobin (Hb) by site-directed mutagenesis . In contrast to mammalian Hbs, some teleost fish haemoglobins show a drastic lowering of oxygen affinity and cooperativity at low pH, a phenomenon known as the Root effect . We have produced the two mutant haemoglobins Hb Nympheas {Cys(F9)93 beta----Ser} and Hb Daphne {His(H21)143 beta----Arg, Cys(F9)93 beta----Ser} to investigate this allosteric property . Although these substitutions were thought to be responsible for the Root effect, Hb Nympheas and Hb Daphne show an increased oxygen affinity and a reduced effect of pH on oxygen affinity . Our X-ray crystallographic studies show that the hydroxyl group of Ser 93 beta forms a hydrogen bond with Asp 94 beta which is in equilibrium with the salt bridge between Asp 94 beta and His 146 beta . The oxygen-binding properties of Hbs Nympheas and Daphne are accounted for by the partial disruption of the salt bridge.

Nature, 1986 Apr 10-16, 320(6062), 535 - 7
Expression of the HTLV-III envelope gene by a recombinant vaccinia virus; Chakrabarti S et al.; The discovery that the aetiological agent of acquired immune deficiency syndrome (AIDS) is a retrovirus, referred to as human T-lymphotropic virus type III (HTLV-III) or lymphadenopathy-associated virus (LAV) (for review see ref . 1), has raised the possibility of developing a vaccine . In this regard, the envelope (env) proteins of murine retroviruses can induce protective immunity in mice . The HTLV-III env gene specifies a primary polypeptide of approximately 860 amino acids that is glycosylated to form a precursor of relative molecular mass (Mr) 160,000 (gp160), which gives rise to mature membrane-associated proteins of Mr 120,000 (gp120) and 41,000 (gp41) . The HTLV-III env gene has been expressed in Escherichia coli and by simian virus 40 (SV40) vectors but formation of the authentic proteins has not been demonstrated . Here, we describe the expression of the complete env gene by a vaccinia virus vector . Evidence is presented that synthesis, glycosylation, processing and membrane transport of the env polypeptide occurred without other HTLV-III gene functions; the env protein was recognized by sera from unrelated AIDs patients; and a single vaccination with the infectious recombinant vaccinia virus induced antibodies to gp120 in mice.

Biochemistry, 1986 Apr 8, 25(7), 1621 - 6
Kinetic properties of cyanase; Anderson PM et al.; Cyanase is an inducible enzyme in Escherichia coli that catalyzes the hydrolysis of cyanate . Bicarbonate is required for activity, perhaps as a substrate, and the initial product of the reaction is carbamate, which spontaneously breaks down to ammonia and bicarbonate {Anderson, P . M . (1980) Biochemistry 19, 2882} . The purpose of this study was to characterize the kinetic properties of cyanase . Initial velocity studies showed that both cyanate and bicarbonate act as competitive substrate inhibitors . A number of monovalent anions act as inhibitors . Azide and acetate appear to act as competitive inhibitors with respect to cyanate and bicarbonate, respectively . Chloride, bromide, nitrate, nitrite, and formate also inhibit, apparently as the result of binding at either substrate site . Malonate and several other dicarboxylic dianions at very low concentrations display "slow-binding", reversible inhibition which can be prevented by saturating concentrations of either substrate . The results are consistent with a rapid equilibrium random mechanism in which bicarbonate acts as a substrate, bicarbonate and cyanate bind at adjacent anion-binding sites, and both substrates can bind at the other substrate anion binding site to give a dead-end complex.

Biochemistry, 1986 Apr 8, 25(7), 1605 - 11
Ligand interactions at the active site of aspartate transcarbamoylase from Escherichia coli; Dennis PR et al.; The active site of aspartate transcarbamoylase from Escherichia coli was probed by studying the inhibitory effects of substrate analogues on the catalytic subunit of the enzyme . The inhibitors were chosen to satisfy the structural requirements for binding to either the phosphate or the dicarboxylate region . In addition, they also contained a side chain that would extend into the normal position occupied by the carbamoyl group . All the compounds tested showed competitive inhibition against carbamoyl phosphate . The ionic character of the side chain was found to be highly important in determining the affinity of the inhibitor . On the other hand, very little effect on binding was produced by changing the geometry of the functional group from trigonal to tetrahedral . Our findings suggest that the electrostatic stabilization of the negative charge that develops in the transition state may be a major factor in promoting catalysis . From the available X-ray diffraction data, we propose His-134 as the residue most likely to participate in this interaction . These results have significant implications on the design of reversible and irreversible inhibitors to this enzyme.

Biochemistry, 1986 Apr 8, 25(7), 1482 - 94
Continuous association of Escherichia coli single-stranded DNA binding protein with stable complexes of recA protein and single-stranded DNA; Morrical SW et al.; The single-stranded DNA binding protein of Escherichia coli (SSB) stimulates recA protein promoted DNA strand exchange reactions by promoting and stabilizing the interaction between recA protein and single-stranded DNA (ssDNA) . Utilizing the intrinsic tryptophan fluorescence of SSB, an ATP-dependent interaction has been detected between SSB and recA-ssDNA complexes . This interaction is continuous for periods exceeding 1 h under conditions that are optimal for DNA strand exchange . Our data suggest that this interaction does not involve significant displacement of recA protein in the complex by SSB when ATP is present . The properties of this interaction are consistent with the properties of SSB-stabilized recA-ssDNA complexes determined by other methods . The data are incompatible with models in which SSB is displaced after functioning transiently in the formation of recA-ssDNA complexes . A continuous association of SSB with recA-ssDNA complexes may therefore be an important feature of the mechanism by which SSB stimulates recA protein promoted reactions.

Biochemistry, 1986 Apr 8, 25(7), 1509 - 15
Metal ion requirements and other aspects of the reaction catalyzed by M1 RNA, the RNA subunit of ribonuclease P from Escherichia coli; Guerrier-Takada C et al.; M1 RNA, the RNA subunit of ribonuclease P from Escherichia coli, can under certain conditions catalytically cleave precursors to tRNA in the absence of C5, the protein moiety of RNase P . M1 RNA itself is not cleaved during the reaction, nor does it form any covalent bonds with its substrate . Only magnesium and, to a lesser extent, manganese ions can function at the catalytic center of M1 RNA . Several other ions either inhibit the binding of magnesium ion at the active site or function as structural counterions . The reaction rate of cleavage of precursors to tRNAs by M1 RNA is enhanced in the presence of poly-(ethylene glycol) or 2-methyl-2,4-pentanediol . Many aspects of the reaction catalyzed by M1 RNA are compatible with a mechanism in which phosphodiester bond cleavage is mediated by metal ion.

J Mol Biol, 1986 Apr 5, 188(3), 491 - 4
Cell surface exposure of the outer membrane protein OmpA of Escherichia coli K-12; Freudl R et al.; The 325-residue OmpA protein is one of the major outer membrane proteins of Escherichia coli K-12 . A model, in which this protein crosses the membrane eight times in an antiparallel beta-sheet conformation and in which regions around amino acids 25, 70, 110 and 154 are exposed at the cell surface, had been proposed . Linkers were inserted into the ompA gene with the result that OmpA proteins, carrying non-OmpA sequences between residues 153 and 154 or 160 and 162, were synthesized . Intact cells possessing these proteins were treated with proteases . Insertion of 15 residues between residues 153 and 154 made the protein sensitive to proteinase K and the sizes of the two cleavage products were those expected following proteolysis at the area of the insertion . Addition of at least 17 residues between residues 160 and 162 left the protein completely refractory to protease action . Thus, the former area is cell surface exposed while the latter area appears not to be . The insertions did not cause a decrease in the concentration of the hybrid proteins as compared to that of the OmpA protein, and in neither case was synthesis of the protein deleterious to cell growth . It is suggested that this method may serve to carry peptides of practical interest to the cell surface and that it can be used to probe surface-located regions of other membrane proteins.

J Biol Chem, 1986 Apr 5, 261(10), 4723 - 5
Crystallization and preliminary characterization of CheY, a chemotaxis control protein from Escherichia coli; Volz K et al.; Single crystals of the 14.1-kDa cheY gene product from Escherichia coli have been grown from buffered ammonium sulfate solutions using the combined methods of microdialysis and pulsed diffusion . The crystals are of the monoclinic space group P2(l), have cell constants of a = 51.4 A, b = 112 A, c = 51.2 A, and beta = 107.3 degrees, and contain four molecules per asymmetric unit . They are stable to x-ray radiation and diffract beyond 3.0 A resolution.

J Mol Biol, 1986 Apr 5, 188(3), 355 - 67
Transcription of Escherichia coli ara in vitro . The cyclic AMP receptor protein requirement for PBAD induction that depends on the presence and orientation of the araO2 site; Hahn S et al.; The mechanism by which the cyclic AMP receptor protein, CRP, stimulates transcription of the Escherichia coli araBAD promoter was studied in vitro . Under one set of conditions, CRP stimulated by eightfold the rate of RNA polymerase open complex formation on supercoiled DNA template containing the normal wild-type araBAD regulatory region . Since previous studies in vivo had identified an upstream site termed araO2 that is involved in both repression and in the CRP requirement for PBAD induction, we performed similar experiments in vitro . Deletion of araO2 or alterations of its orientation with respect to the araI site by half integral numbers of turns greatly reduced the CRP requirement for induction of PBAD . Linearizing the DNA has the same effect as deleting araO2 from the supercoiled DNA template . The similarity of conditions that relieve the classical repression of PBAD in vivo and the conditions that eliminate the requirement for CRP for maximal activity in vitro suggest a close relationship between repression in the ara system and the role of CRP . At lower concentrations of AraC protein and slightly different conditions than those used in the above-mentioned experiments, CRP does stimulate transcription from linear or supercoiled templates lacking araO2 . On linear DNA under these conditions, one dimer of AraC protein binds to linear araPBAD DNA, but is incapable of stimulating transcription without the additional binding of CRP . The responses of the ara system under the second set of conditions are unlike its behavior in vivo.

J Biol Chem, 1986 Apr 5, 261(10), 4738 - 48
The Escherichia coli dnaB replication protein is a DNA helicase; LeBowitz JH et al.; Genetic and biochemical analyses indicate that the Escherichia coli dnaB replication protein functions in the propagation of replication forks in the bacterial chromosome . We have found that the dnaB protein is a DNA helicase that is capable of unwinding extensive stretches of double-stranded DNA . We constructed a partially duplex DNA substrate, containing two preformed forks of single-stranded DNA, which was used to characterize this helicase activity . The dnaB helicase depends on the presence of a hydrolyzable ribonucleoside triphosphate, is maximally stimulated by a combination of E . coli single-stranded DNA-binding protein and E . coli primase, is inhibited by antibody directed against dnaB protein, and is inhibited by prior coating of the single-stranded regions of the helicase substrate with the E . coli single-stranded DNA-binding protein . It was determined that the dnaB protein moves 5' to 3' along single-stranded DNA, apparently in a processive fashion . To invade the duplex portion of the helicase substrate, the dnaB protein requires a 3'-terminal extension of single-stranded DNA in the strand to which it is not bound . Under optimal conditions at 30 degrees C, greater than 1 kilobase pair of duplex DNA can be unwound within 30 s . Based on these findings and other available data, we propose that the dnaB protein is the primary replicative helicase of E . coli and that it actively and processively migrates along the lagging strand template, serving both to unwind the DNA duplex in advance of the leading strand and to potentiate synthesis by the bacterial primase of RNA primers for the nascent (Okazaki) fragments of the lagging strand.

J Biol Chem, 1986 Apr 5, 261(10), 4698 - 705
The Cpx proteins of Escherichia coli K12 . Immunologic detection of the chromosomal cpxA gene product; Albin R et al.; Previous studies described lacZ'- and cat'-'cpxA fusion genes whose expression restored to normal all the phenotypic defects associated with cpxA mutations (Albin, R., and Silverman, P . M . (1984) Mol . Gen . Genet . 197, 272-279) . Here, we show by DNA nucleotide sequence analysis that the fusion genes encode 241 carboxyl-terminal amino acids of the CpxA polypeptide . Using this information, we constructed a fusion gene containing the same 241 cpxA codons preceded by 1007 codons of beta-galactosidase . The resultant hybrid polypeptide was purified and used to raise an anti-(CpxA polypeptide) antiserum . Using the antiserum, we have identified the chromsomal Escherichia coli K12 cpxA gene product as a 52-kDa polypeptide . The polypeptide showed temperature-sensitive accumulation in a strain carrying both the cpxA2{Ts} and cpxB1 alleles and accumulated to a level higher than normal in cells that carried a high-copy number, cpxA+ plasmid . Immune precipitates of in vitro transcription-translation reactions with cpxA+ plasmids as template also contained a 52-kDa polypeptide, indistinguishable in electrophoretic mobility from the immunoreactive polypeptide synthesized in vivo . Two regions of amino acid sequence at the carboxyl-terminus of the CpxA polypeptide are significantly homologous to corresponding regions of the E . coli K12 EnvZ polypeptide, an inner membrane component that, like the CpxA polypeptide, is required to maintain the protein composition of the cell envelope . The cpxA coding sequence is followed by two repetitive extragenic palindrome sequences in opposite orientation.

J Biol Chem, 1986 Apr 5, 261(10), 4445 - 50
The elongation factor G carries a catalytic site for GTP hydrolysis, which is revealed by using 2-propanol in the absence of ribosomes; De Vendittis E et al.; In the absence of ribosomal particles, elongation factor G (EF-G) promotes very little GTP hydrolysis . After the addition of some aliphatic alcohols to EF-G, the rate of nucleotide cleavage was significantly increased and GTPase activity was easily detectable . The highest stimulation, nearly 16-fold, occurred with 2-propanol at a 20% (v/v) concentration . The reaction showed the characteristics of an enzymatic catalysis, but the rate was three orders of magnitude lower than that of the ribosome-dependent EF-G GTPase activity . Striking similarities between the two activities indicated that the catalysis stimulated by the alcohol was due to EF-G itself . We found that EF-G GTPase activity in the presence of 2-propanol displayed an absolute specificity for GTP as in the presence of ribosomes; the two activities copurified to a constant ratio and exhibited coincident chromatographic and electrophoretic patterns; the temperature for the half-inactivation of EF-G was 59.3 degrees C for both GTPase systems, as well as the kinetic constant for the thermal inactivation process which was found to be 0.05 min-1; and the Km for the GTP in the presence of 2-propanol (59 microM) was similar to that found in the presence of ribosomes . These results indicate that the EF-G molecule carries a catalytic site for GTP hydrolysis, which in the absence of ribosomal particles is activated by an appropriate alcohol/water surrounding medium.

J Biol Chem, 1986 Apr 5, 261(10), 4574 - 8
Sequence of a peptide susceptible to mixed-function oxidation . Probable cation binding site in glutamine synthetase; Farber JM et al.; Mixed-function oxidation of glutamine synthetase from Escherichia coli causes loss of catalytic activity . The inactivation correlates with the loss of 1 of 16 histidine residues/subunit (Levine, R.L . (1983) J . Biol . Chem . 258, 11823-11827) . A cyanogen bromide peptide containing the oxidizable histidine has been isolated . Within the protein, the sequence is Met-His-Cys-His-Met . This hydrophilic sequence likely forms one of the divalent metal-binding sites of glutamine synthetase . Binding of Fe2+ to this site permits generation of an activated oxygen species which reacts with a nearby histidine residue . This site-specific free radical mechanism accounts for the specificity of the mixed-function oxidation.

J Mol Biol, 1986 Apr 5, 188(3), 383 - 92
Changes in the half-life of ribosomal protein messenger RNA caused by translational repression; Cole JR et al.; The half-life of ribosomal protein operon L11 mRNA in vivo was measured during exponential growth by following the kinetics of incorporation of radioactive precursors into L11 mRNA transcribed from multi-copy plasmids . The degree of translational feedback regulation by L1, the L11 operon-specific translational repressor protein, was changed by altering the site on the "L11 mRNA" where L1 interacts . The half-life of the overproduced L11 mRNA increased by about fivefold when translational repression was abolished, while the half-life of mRNA from the spc ribosomal protein operon, which is not translationally regulated by L1, stayed constant . Furthermore, the half-life of L11 operon mRNA carrying an additional mutation in the ribosome binding site that abolishes translation remains short . This indicates that the change in half-life observed during increased gene dosage is due to translational repression by L1 and is probably a consequence of L1 blocking translation of L11 mRNA and not due to some nucleolytic activity mediated by L1.

Science, 1986 Apr 4, 232(4746), 61 - 5
Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells; Souza LM et al.; Experiments were conducted to isolate and characterize the gene and gene product of a human hematopoietic colony-stimulating factor with pluripotent biological activities . This factor has the ability to induce differentiation of a murine myelomonocytic leukemia cell line WEHI-3B(D+) and cells from patients with newly diagnosed acute nonlymphocytic leukemia (ANLL) . A complementary DNA copy of the gene encoding a pluripotent human granulocyte colony-stimulating factor (hG-CSF) was cloned and expressed in Escherichia coli . The recombinant form of hG-CSF is capable of supporting neutrophil proliferation in a CFU-GM assay . In addition, recombinant hG-CSF can support early erythroid colonies and mixed colony formation . Competitive binding studies done with 125I-labeled hG-CSF and cell samples from two patients with newly diagnosed human leukemias as well as WEHI-3B(D+) cells showed that one of the human leukemias (ANLL, classified as M4) and the WEHI-3B(D+) cells have receptors for hG-CSF . Furthermore, the murine WEHI-3B(D+) cells and human leukemic cells classified as M2, M3, and M4 were induced by recombinant hG-CSF to undergo terminal differentiation to macrophages and granulocytes . The secreted form of the protein produced by the bladder carcinoma cell line 5637 was found to be O-glycosylated and to have a molecular weight of 19,600.

Biochim Biophys Acta, 1986 Apr 2, 849(1), 62 - 9
The mechanism of ATP synthase: a reassessment of the functions of the b and a subunits; Cox GB et al.; A model for the mechanism of ATP synthase was proposed previously (Cox, G.B., Jans, D.A., Fimmel, A.L., Gibson, F . and Hatch, L . (1984) Biochim . Biophys . Acta 768, 201-208) in which the b subunit of the Fo of Escherichia coli rotated . The driving force was proposed to be an interaction between two charged residues in the membrane, namely, Lys-23 of the b subunit and Asp-61 of the c subunit . To test this proposal the Lys-23 of the b subunit was replaced by threonine using site-directed mutagenesis . The resulting mutant, although it had an impairment in the assembly of the F1F0-ATPase, was normal with respect to oxidative phosphorylation . The role of the a subunit, which had been previously proposed to be a structural one, was reassessed by examination of the possible secondary and tertiary structure of the analogous proteins from several sources . Not only did these subunits appear to have very similar structures, but in each there was a highly conserved helical arm on one of the transmembrane helices which could form a proton channel if it interacted with the Asp-61 of the c subunit . A revised model is therefore presented in which five transmembrane helices from the a subunit and two from the b subunit are surrounded by a ring of c subunits . The highly conserved nature of the structures of the a, b and c subunits from various organisms suggests that the model may have relevance for ATP synthases from bacterial plasma membranes, mitochondria and chloroplasts.

Surgery, 1986 Apr, 99(4), 421 - 31
Endotoxin-induced prostanoid production by the burn wound can cause distant lung dysfunction; Demling RH et al.; We injected Escherichia coli endotoxin, 2 micrograms/kg, beneath the eschar of sheep with 25% total body surface full-thickness burns to determine whether burn tissue in the presence of endotoxin releases prostanoids, particularly thromboxane A2, (TxA2), and if increased local TxA2 production can lead to distant lung dysfunction . We compared this response to the lung injury produced by the same dose given intravenously . We noted a marked increase in burn tissue TxA2 production after subeschar endotoxin as reflected in significant increases in burn lymph and pulmonary artery TxB2 levels . Pulmonary artery pressure increased from 22 to 38 mm Hg and PaO2 decreased from 89 to 71 torr while lung lymph flow (QL) increased only modestly with no evidence of increased lung permeability . The TxA2 production and the lung response were prevented by the subeschar injection of ibuprofen, 12.5 mg/kg . Circulating endotoxin was noted in only one of five sheep . After intravenous (endotoxin), a significant increase in lung TxA2 production was noted and a characteristic two-phase lung injury was seen with an initial phase basically identical to that seen with the subeschar injection followed by an increase in lung protein permeability . Burn tissue endotoxin can stimulate local TxA2 production leading to distant lung dysfunction without the need for circulating endotoxin . The source of the TxA2 is the burn, while with endotoxemia the source is the lung.

Cancer Res, 1986 Apr, 46(4 Pt 1), 1663 - 8
Inactivation of O6-methylguanine-DNA methyltransferase and sensitization of human tumor cells to killing by chloroethylnitrosourea by O6-methylguanine as a free base; Yarosh DB et al.; Human fibroblasts and tumor cells with constitutive levels of the DNA repair protein O6-methylguanine-DNA methyltransferase were incubated with mM concentrations of the free base O6-methylguanine for up to 24 h . This treatment depleted the cells of their transferase activity, and sensitized the cells to killing by the antineoplastic drug 1-{2-chloroethyl}-1-nitrosourea . Cells constitutively lacking the methyltransferase were not sensitized to cell killing . Cell free extracts incubated with O6-methylguanine also lost methyltransferase activity . Other alkylpurines, such as O6-methylguanosine, S6-methylthioguanine, O6-ethylguanine, and 3-methyladenine, did not have this effect on extracts of human tumor cells, while O6-methylguanosine and O6-methylguanine inactivated purified methyltransferase from Escherichia coli . The data suggest that the free base O6-methylguanine is probably a substrate for the methyltransferase . Calculation of the second order rate constants for free base versus O6-methylguanine in DNA, and experiments in which the free base was mixed with DNA containing O6-methylguanine before reaction with methyltransferase, indicated that the base in DNA is about 4 X 10(7) better as a substrate than is the free base . These results demonstrate that DNA repair capacity of tumor cells can be diminished without DNA damage, and suggest a method for increasing the efficiency of chemotherapy.

J Gen Microbiol, 1986 Apr, 132 ( Pt 4), 989 - 95
An inverted repeat sequence of the IncFI plasmid ColV2-K94 increases multimerization-mediated plasmid instability; Weber PC et al.; A detailed physical map of the region of the IncFI plasmid ColV2-K94 containing the Rep1 replicon, a Tn903 transposon, and an inverted repeat structure (X1) with unknown properties was prepared by cloning restriction fragments into pBR325 . Inserts carrying the 1.2 kb repeated sequence of X1, but not the IS903 sequence of Tn903, had a destabilizing effect on pBR325 and pBR322 plasmid maintenance . One of these derivatives, pWS139, was studied further and was shown to have elevated levels of multimeric DNA forms; this resulted in decreased copy number and plasmid instability, as multimerization reduces the effective number of randomly segregating plasmids per cell . A ColV2-K94 miniplasmid, which has a copy number much lower than that of ColE1-derived vectors, was also less stably inherited if it contained the X1 structure . This destabilizing effect of the X1 repeat sequence was dependent on the RecA function, but not the RecB or the RecC functions of the host . These results suggest that the inverted repeat sequence of the X1 structure serves as a 'hot-spot' for generalized recombination . Thus, when present in cis, this sequence can generate plasmid instability because plasmid molecules are readily converted into multimeric forms through enhanced recombination at this site.

Acta Pathol Microbiol Immunol Scand {C}, 1986 Apr, 94(2), 57 - 62
The indirect leucocyte migration inhibition assay--an endotoxin-sensitive chemokinetic assay . Part II; Remvig L et al.; Depletion of agarose for endotoxins resulted in a low spontaneous migration of polymorphnuclear cells (PMNC) . Re-addition of endotoxin, in casu lipopolysaccharide from E . coli 026:B6 (LPS), enhanced the spontaneous PMNC migration in a two-phased dose-response pattern, reaching maximum migration with LPS 1 x 10(-7) g/ml . Thus, the migration of PMNC under agarose seems to be a chemokinesis . Leucocyte migration inhibition factor (LIF), induced by PPD 50 micrograms/ml at endotoxin-free conditions, significantly reduced the PMNC migration compared to supernatants from control cultures, however not compared to the conventional limit of significance, MI = 0.80 . With increasing PMNC migration there was an insignificant decrease in the MI . Addition of LPS, 1 x 10(-9) g/ml, during LIF induction caused a significant increase in LIF production, an effect which overshadowed the effect of PPD . Thus, the application of the conventional limit of significance, MI = 0.80, may result in false-negative or false-positive conclusions, depending upon the endotoxin contamination . A standardization of the endotoxin content in both steps of the indirect leucocyte migration inhibition assay seems mandatory in order to obtain a reliable and reproducible bioassay.

Z Urol Nephrol, 1986 Apr, 79(5), 277 - 86
{Cellular immunity in dialysis patients using the lymphocyte transformation test (LTT) in comparison to conservatively treated patients with chronic terminal renal failure}; Meissner S et al.; By means of the lymphocyte transformation test (LTT), using the mitogens phytohemagglutinin (PHA), concanavalin A (ConA), lipopolysaccharide (LPS) E . coli and the antigens tuberculin (PPD) and O-streptolysin, a contribution should be made for the judgment of the functional capacity of the immune system--in particular of the cellular immunity--in uraemia patients with and without dialysis therapy . In 33 patients with different duration of the dialysis (0.5 to 130 months) and 15 retention patients who were not yet treated by means of dialysis the LTT was controlled with stimulant agents mentioned above . In these cases was shown that dialysis patients managed metabolically regularly did not show a significant restriction of the cellular immunity in the LTT (PHA-stimulation over 0.55 transformed cells, con-A-stimulation over 0.37 transformed cells) . The not dialysed patients with chronic uraemia showed a distinct diminution of the unspecific T-cell transformation by PHA and ConA, whereas the antigen-induced stimulation (PPD, O-streptolysin) was not disturbed in this case as well . In the two cases the B-cell transformation (on LPS) was not significantly disturbed . There was a good concordance with the clinical findings: scarcely general infects, no shunt infections, relatively many organ losses by rejection after transplantation in the dialysis patients . The not yet dialysed retention patients revealed clinically a higher inclination to an infect . There were no own experiences about the course after transplantation without preceding dialysis . It is discussed in how far also immunological investigations may play a role in the establishment of the optimum management of the dialysis, the moment of the beginning with the dialysis and for the "more individual preparation of the transplantation".

Avian Dis, 1986 Apr-Jun, 30(2), 247 - 54
Morphopathology of the adenohypophysis of chickens in shock induced by Escherichia coli; Fernandez A et al.; We studied modifications in the adenohypophysis of chickens subjected to experimental septic shock by repeated intraperitoneal inoculations with Escherichia coli 026 B6 . We observed vascular modifications characterized by capillary dilation and endothelial defects, together with marked perivascular edema and collagen fibers in the groups receiving the most inoculations . Similarly, there was a proliferation of mononuclear cells, belonging mainly to the mononuclear phagocyte system, and plasma cells and lymphocytes . The lesions found in chickens receiving only one inoculation may be evidence of a morphopathological relationship between shock induced by E . coli and lesions that develop in the swollen-head syndrome.

Antibiot Med Biotekhnol, 1986 Apr, 31(4), 288 - 91
{Genetic and molecular characteristics of the conjugative R-plasmids of S . typhimurium}; Gridnev VA et al.; The molecular genetic properties of R plasmids from 8 strains of S . typhimurium isolated in hospitals of the Far East of the USSR were studied . It was shown that conjugative R plasmids of S . typhimurium were characterized by belonging to the Inc F1 group, rigorous control of replication, the fi+ type and the transfer frequency of 10(-3) to 10(-4) per a donor cell . The plasmid profile of the S . typhimurium strains was represented by 2 plasmids: conjugative with a molecular weight of about 64 MD and nonconjugative with a molecular weight of 2 MD . The conjugative R plasmids of the hospital strain VG 103 of S . typhimurium were characterized by molecular genetic identity . They were derivatives of the pVG 103 plasmid.

Vet Immunol Immunopathol, 1986 Apr, 11(4), 375 - 89
Humoral immunity in the prepatent primary infection of dogs with Echinococcus granulosus; Barriga OO et al.; Twenty-one parasite-naive dogs were infected with 60,000 protoscolices of Echinococcus granulosus . Transformation of peripheral lymphocytes was investigated before and 29 days after the infection, immunoglobulin concentration and anti-hydatid fluid protein (HFP) titers in serum and feces before and at 35 days of infection, skin reactivity to HFP at 36 days, and characteristics of the parasites at 40 days . The infection caused a significant depression of the spontaneous, lipopolysaccharide-stimulated, and purified protein derivative-stimulated blastogenesis . Responses to phytohemagglutinin were unchanged and reactivity to concanavalin A was enhanced with the infection . Only the concentrations of IgG and IgA in the serum and IgA in the feces increased significantly after infection . Fifteen (71%) dogs produced significant serum titers of anti-HFP hemagglutinins but copro-antibodies were detectable in only 3 dogs at minimum titers . Titers were abolished by treatment with 2-mercaptoethanol . The serum of 11 (52%) dogs transferred passive cutaneous anaphylaxis to guinea pigs but none transferred skin reactivity to pups or rabbits . Five and 1 (but not 0.2) micrograms of HFP caused skin reactivity in 4 parasite-naive dogs . Nineteen (90.5%) infected dogs reacted significantly to skin inoculation of 0.2 microgram of HFP at 0.5 hours and 13 (62%) at 6 hours . The 7 dogs with the highest anti-HFP serum titers or the greatest skin reactivity at 6 hours had significantly less mature or fewer tissue parasites, respectively, than the 7 dogs with the smallest responses . Since there was evidence that the specific immunity was still developing at the time of the study, these results indicate that immunological diagnosis of, and artificial immunization against, canine echinococcosis are feasible.

Microbiologica, 1986 Apr, 9(2), 215 - 20
Incidence of enteroinvasive Escherichia coli in Spain . A 14-month prospective study; Pumarola A et al.; Between February 1984 and May 1985, a prospective study was performed in patients with acute enteritis to determine the incidence of enteroinvasive Escherichia coli (ECEI) in our environment . Eight hundred and forty-three strains of E . coli were studied by agglutination with antisera for serogroups O28ac, 029, 0112, 0124, 0136, 0144, 0152 and 0164 . Eleven strains were found to pertain to one or another of these serogroups: 6 to 029, 2 to 0124, 2 to 0164 and 1 to 0143 . Of these, only the two corresponding to 0124 invaded HeLa cells and had a positive Sereny test . These strain were lysine decarboxylase and lactose negative and did not produce gas . In 125 strains that did not belong to any of the enteroinvasise serogroups indicated, but had been isolated from patients with febrile enteritis in which no other enteropathogen could be isolated (32 strains), or had metabolic characteristics of enteroinvasive Escherichia coli (93) strains, the Sereny test and invasion of HeLa cells were studied . In every case, both tests were negative.

J Biochem (Tokyo), 1986 Apr, 99(4), 1137 - 46
Biosynthesis of isoprenoids in intact cells of Escherichia coli; Fujisaki S et al.; Upon rehydration of lyophilized Escherichia coli cells with phosphate buffer containing {14C}isopentenyl pyrophosphate (IPP), 14C was incorporated into the cells . Radioactivity was found in ubiquinone-8, an unidentified precursor of ubiquinone-8, demethylmenaquinone-8 and phosphate esters of all-trans-octaprenol and cis, trans-polyprenols . On rehydration of the cells with the buffer containing geranyl pyrophosphate or farnesyl pyrophosphate in combination with {14C}IPP, higher radioactivity was incorporated into the above products and some radioactivity was found in free prenols . Fractionation of the 14C-labeled cells by sucrose-density gradient centrifugation before and after recultivation indicated that the size of 14C-labeled cells had changed during the recultivation . This shows that radioactivity of {14C}IPP was incorporated into live cells but not into dead cells . The metabolism of the radioactive products in the recultivated cells was examined . It was found that the unidentified precursor was converted to ubiquinone-8, but demethylmenaquinone-8 was not converted to menaquinone-8 . "Lipid intermediates" in peptidoglycan synthesis increased in the logarithmic growth phase and decreased in the stationary phase . In the stationary phase, however, an increase in cis,trans-polyprenyl monophosphates was observed . These observations suggest the operation of the lipid cycle of peptidoglycan synthesis.

EMBO J, 1986 Apr, 5(4), 787 - 91
Early replicative intermediates of Escherichia coli chromosome isolated from a membrane complex; Yoshimoto M et al.; The replication origin region of the Escherichia coli chromosome was isolated from an outer membrane fraction . The chromosome was further purified by centrifugation in a cesium chloride gradient . Early replicative intermediates were enriched in the preparation when cytosine-1-beta-arabinofuranoside was added to the culture at the time of initiation of chromosome replication . DNA fragments with an eye structure having two branches of less than 400-500 bp in length were associated with components that were removed by phenol treatment . We conclude that the replication fork usually proceeds counter-clockwise toward the unc operon in the earliest period of replication.

DNA, 1986 Apr, 5(2), 157 - 65
Molecular cloning of the gene encoding rabbit tumor necrosis factor; Ito H et al.; A rabbit genomic DNA library was screened using a cloned cDNA encoding rabbit tumor necrosis factor (TNF) as a probe . The entire gene sequence appears to be contained within a 3.2-kb Eco RI fragment . Comparison of the nucleotide sequence of the gene with that of the cDNA showed that the gene consists of four exons . The nucleotide sequence of the rabbit TNF gene is very homologous to that of the human TNF gene . Southern hybridization of the genomic DNAs showed that there is likely only a single TNF gene in each of the rabbit and human genomes.

DNA, 1986 Apr, 5(2), 149 - 56
Molecular cloning and expression in Escherichia coli of the cDNA coding for rabbit tumor necrosis factor; Ito H et al.; cDNA clones containing the entire sequence of the precursor of rabbit tumor necrosis factor (TNF) were identified from a cDNA library prepared using poly(A)+RNA of lipopolysaccharide-induced rabbit alveolar cells . Synthetic oligodeoxynucleotides based on the amino acid sequence of rabbit TNF were selected by RNA blot hybridization and used as probes to screen this library . The DNA sequence and the amino acid sequence deduced from it showed very high homology to the reported DNA and amino acid sequences of human TNF . A plasmid containing the tac promoter and cDNA sequence coding for the 154-amino-acid sequence of mature rabbit TNF protein was constructed and expressed in Escherichia coli . The polypeptide produced had the characteristics of purified rabbit serum TNF and caused a necrotic response in a transplanted syngeneic tumor in mice.

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