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