Mol Gen Genet, 1979 Jun 20, 173(3), 345 - 7 Decreased transfer of pyrimidine dimers from parental to daughter DNA strands in UV-irradiated Escherichia coli deficient in DNA polymerase III; Tomilin NV et al.; The number of pyrimidine dimers (sites sensitive to UV-endonuclease from M . luteus) transferred at 43 degrees to daughter DNA strands during postreplication repair in UV-irradiated E . coli uvr A polCts was found to be decreased as compared to that after repair at 32 degrees . This indicates the involvement of DNA polymerase III in the sister DNA recombination in UV-irradiated E . coli. Mol Gen Genet, 1979 Jun 20, 173(3), 333 - 8 Studies of colicin induction with an imm- col+ mutant of the plasmid colicin E1; Inselburg J; A mutant of a derivative of the colicin E1 plasmid has been isolated that does not confer immunity to colicin E1 on its host (imm-) although it is still capable of producing colicin (col+) . Cells carrying the col+, imm- plasmid are capable of forming colonies and grow best in liquid culture in the presence of trypsin . The induction of colicin synthesis by ultraviolet light has been analysed using this mutant plasmid . The results suggest that a) the expression of the col+ gene may be delayed for many generations after the inducing stimulus, b) although induced cells are usually killed they can reproduce and c) the capacity to produce colicin can be propagated and segregated into the progeny of an induced cell. Mol Gen Genet, 1979 Jun 20, 173(3), 323 - 31 Regulation of the L-arabinose operon in strains of Escherichia coli containing ColE1-ara hybrid plasmids; Wallace LJ et al.; Hybrid plasmids were constructed from fragments of F'ara episomes formed by the restriction endonuclease EcoRI and a linear form of the plasmid ColE1 created by cleavage with EcoRI . Hybrid plasmids were constructed containing the entire ara region or the ara region with various parts deleted . E . coli K12 host strains were constructed which contained different deletions of the ara region . The hybrid plasmids were transferred to those strains whose ara deletion complemented that of the plasmid . The initial differential rates of synthesis of L-arabinose isomerase, the product of the araA gene, were determined for the Ara+, plasmid containing strains . These studies demonstrated that strains containing delta(araOIBA)718 produce elevated levels of araC protein, suggesting the araC promoter has been altered by this deletion . Evidence is also presented which suggests that araC protein activates the ara-BAD operon to higher levels when it is present in cis rather than trans . Amplification of the products of the cloned genes is observed when compared to haploid levels in some cases. Mol Gen Genet, 1979 Jun 20, 173(3), 339 - 43 Expression of the lac operon in RNA polymerase mutants of Escherichia coli K12; Mandecki W et al.; Three temperature-sensitive mutant strains for RNA polymerase beta or beta' subunits (carrying mutations tsx, A2R7 and R120) were used in order to investigate the dependence of the induced lac expression on stimulation by cyclic AMP after the shift to non-permissive temperature . High temperature lowered the rate of beta-galactosidase synthesis . However, the low rate of synthesis could be strongly increased by cyclic AMP (30, 2.4 and 5.7-fold increases for tsX, A2R7 and R120 mutants, respectively) . At the permissive temperature stimulation by cyclic AMP was less than 1.4-fold (minimal medium supplemented with glycerol) . The results suggest that the maximal expression of the lac operon is saturated, that is, a hypothetical increase in RNA polymerase or cAMP-CRP concentration in the cell with not enhance the expression . The concept of saturation explains why it was possible to increase the beta-galactosidase synthesis in conditions of limited promoter binding activity of RNA polymerase through increase in concentration of cyclic AMP-CRP complex in the cell (addition of cyclic AMP) to the values higher than that observed on glycerol. Biochim Biophys Acta, 1979 Jun 19, 578(2), 365 - 71 Shape of proteins S3 and S17 from the small subunit of Escherichia coli ribosome; Franz A et al.; The shapes of proteins S3 and S17 purified from the 30 S subunit of Escherichia coli A19 were studied by hydrodynamic methods . The proteins have s020,w values of 2.1 +/- 0.1 S and 1.2 +/- 0.1 S and D020,w values of 7.4 +/- 0.5 . 10(-7) cm2/s and 11.4 +/- 0.6 . 10(-7) cm2/s . The respective molecular weights determined by sedimentation equilibrium are 25 800 +/- 500 and 9900 +/- 300 . The intrinsic viscosity values for the two proteins are 5.8 +/- 0.3 ml/g and 4.2 +/- 0.2 ml/g . From these hydrodynamic parameters a slightly elongated shape for S3 and a globular shape for S17 have been concluded. Biochim Biophys Acta, 1979 Jun 19, 578(2), 337 - 45 Quaternary structure of DNA-dependent RNA polymerase from Escherichia coli . Measurement of distances by fluorescence energy transfer; Stender W et al.; Distances between the subunits in Escherichia coli RNA polymerase (core and holo enzyme) and the rifamycin binding site have been determined using the nonradiative energy transfer technique . The appropriate donor and acceptor labels have been chosen in order to optimize the spectral overlap and maximize the energy transfer . Spacer linked derivatives of rifamycin SV possessing nitrobenzo-oxadiazole groups (energy acceptor) were synthesized for this purpose . The donor label, acetylaminoethylaminonaphthalene sulfonate, was introduced into the intact enzyme, and the subunits were separated . Enzyme molecules selectively labelled on one kind of subunit were produced by mixed reconstitution techniques employing labelled and non labelled subunits . The labelled beta'-subunit could not be prepared in sufficient amounts . Energy transfer distances between the enzyme-bound rifamycin derivative and the subunits were determined to be approximately 5.9 nm for sigma, 7.2 nm for alpha 2 and 6.1 nm for beta. Biochemistry, 1979 Jun 12, 18(12), 2632 - 6 Biosynthesis of the pyrimidine moiety of thiamin in Escherichia coli: incorporation of stable isotope-labeled glycines; White RH et al.; Methods are described for the cleavage, extraction, and subsequent gas chromatographic-mass spectrometric analysis of the pyrimidine moiety of thiamin as 2-methyl-4-amino-5-{(ethylthio)methyl}pyrimidine . The methods are of a general nature and can be applied to any system . Using these methods to evaluate the incorporation of 13C-, 15N-, and 2H-labeled glycines into the pyrimidine moiety of thiamin by Escherichia coli, we established that the nitrogen and carbon atoms of glycine are incorporated as a unit into the pyrimidine . 13C- and 15N-labeled glycines are incorporated at greater than 60% but deuterium from {2-(2)H2}glycine was incorporated at only 18% . A detailed analysis of the mass fragmentation pattern of the pyrimidine derivative has established that the glycine nitrogen atom supplies the N-1 of the pyrimidine and that the C-1 and C-2 of the glycine supplies the C-4 and C-6 of the pyrimidine, respectively . This evidence is consistent with the substitution of a C2 unit between the C-5 and C-4 of the 4-aminoimidazole ribonucleotide precursor during the biosynthesis of the pyrimidine moiety of thiamin in E . coli. Biochemistry, 1979 Jun 12, 18(12), 2520 - 5 Shapes of proteins L1, L9, L25, and L30 from the 50S subunit of the Escherichia coli ribosome, determined by hydrodynamic studies; Giri L et al.; Proteins L1, L9, L25, and L30, purified by a nondenaturing method from the 50S ribosomal subunit of Escherichia coli A19, have been characterized . The four proteins were studied under conditions which resemble those used for reconstitution experiments . These proteins have S020,W values of 2.0 S, 1.8 S, 1.8 S, and 1.0 S and D20,W values of 8.4 X 10(-7), 9.0 X 10(-7), 14.0 X 10(-7), and 15.0 X 10(-7) cm2/S . Apparent specific volumes at 20 degrees C are 0.738, 0.733, 0.700, and 0.735 mL/g for the four proteins . The respective molecular weights determined by sedimentation equilibrium are 25 000, 17 300, 12 000, and 6500 . The intrinsic viscosity values for the four proteins are 4.0, 5.5, 3.6, and 3.2 mL/g . From these hydrodynamic parameters L1 and L9 appear to have globular or at most only slightly elongated shapes, whereas L25 and L30 appear to be definitely globular. Nucleic Acids Res, 1979 Jun 11, 6(7), 2611 - 26 1H NMR studies on the conformational characteristics of 2-thiopyrimidine nucleotides found in transfer RNAs; Yokoyama S et al.; The molecular conformations of naturally occurring 2-thiopyrimidine nucleosides (5-methylaminomethyl-2-thiouridine, 5-methoxycarbonylmethyl-2-thiouridine and 2-thiocytidine) and 5'-mononucleotides (5-methylaminomethyl-2-thiouridine 5'-monophosphate and 2-thiocytidine 5'-monophosphate) in 2H2O solution were elucidated by analyses of the proton NMR spin-coupling constant, nuclear Overhauser effect, and lanthanide-induced shifts and relaxation enhancements . As monomers, these nucleotides are almost exclusively in the 3E-gg-anti form, even in the absence of ordinary stabilizing factors of this form; i . e., base-stacking and base-pairing interactions with other nucleotide units . This inherent conformational rigidity of the 2-thiopyrimidine units probably contributes to stability of the conformation of tRNA. Nucleic Acids Res, 1979 Jun 11, 6(7), 2583 - 99 Studies on gene control regions X . The effect of specific adenine-thymine transversions on the lac repressor-lac operator interaction; Sista HS et al.; Chemical and enzymatic methods were used to synthesize a transition (AT to GC) and a transversion (AT to TA) at a lac operator site known to interact with lac repressor through the thymine 5 methyl group . These operators also contained a poly(dA) . poly(dT) tail 8 to 12 base pairs in length at one end . Results suggest that the steric constraints of lac repressor relative to the position of the 5 methyl group are quite critical . For example a seven fold reduction in stability was observed for the transversion . Results also suggest that the operator spans at least 21 base pairs. Nucleic Acids Res, 1979 Jun 11, 6(7), 2569 - 82 Rapid chemical synthesis and circular dichroism properties of some 2'-5'-linked oligoriboadenylates; Markham AF et al.; Specific synthesis of some oligoadenylates including A2'p5'A2'p5'Ap(2'), the 2'-phosphorylated oligoribonucleotide core of the recently discovered protein synthesis inhibitor pppA2'p5'A2'p5'A is described using a novel solid-phase method . The CD spectra of A2'p5'Ap(2'), A2'p5'A2'p5'Ap(2') and A2'p5'A2'p5'A (derived by treatment of the phosphorylated synthetic trimer with E . coli alkaline phosphatase) are presented . Comparison of the latter spectrum with that of A2'p5'A2'p5'A obtained similarly from a biologically derived sample of pppA2'p5'A2'p5'A provides further evidence that this molecule is in fact the first naturally-occurring 2'-5'-linked oligoribonucleotide. Nucleic Acids Res, 1979 Jun 11, 6(7), 2483 - 97 The use of R-looping for structural gene identification and mRNA purification; Woolford JL Jr et al.; A method is presented for the purification of mRNAs and the identification of structural gene sequences in recombinant DNA molecules . RNA is hybridized to double-stranded linear DNA such that R-loops are formed between most DNAs and their complementary RNA sequences . These R-loops are purified from unhybridized RNAs by gel filtration chromatography in the presence of a high concentration of salt . The complementary RNAs are released from the R-loops by heating, and are assayed by gel electrophoresis or cell free translation to determine their purity and to identify the proteins for which they code . We have demonstrated that recombinant DNAs containing sequences for abundant or moderately abundant mRNAs of Saccharomyces cerevisiae can be identified by this means. Nucleic Acids Res, 1979 Jun 11, 6(7), 2453 - 70 A ribonuclease-resistant region of 5S RNA and its relation to the RNA binding sites of proteins L18 and L25; Douthwaite S et al.; An RNA fragment, constituting three subfragments of nucleotide sequences 1-11, 69-87 and 89-120, is the most ribonuclease-resistant part of the native 5S RNA of Escherichia coli, at 0 degrees C . A smaller fragment of nucleotide sequence 69-87 and 90-110 is ribonuclease-resistant at 25 degrees . Degradation of the L25-5S RNA complex with ribonuclease A or T2 yielded RNA fragments similar to those of the free 5S RNA at 0 degrees C and 25 degrees C; moreover L25 remained strongly bound to both RNA fragments and also produced some opening of the RNA structure in at least two positions . Protein L18 initially protected most of the 5S RNA against ribonuclease digestion, at 0 degrees C, but was then gradually released prior to the formation of the larger RNA fragment . It cannot be concluded, therefore, as it was earlier (Gray et al., 1973), that this RNA fragment contains the primary binding site of L18. J Biol Chem, 1979 Jun 10, 254(11), 4698 - 706 Purification and characterization of protease III from Escherichia coli; Cheng YS et al.; An endoproteolytic enzyme of Escherichia coli, designated protease III, has been purified about 9,600-fold to homogeneity with a 6% yield . The purified enzyme consists of a single polypeptide chain of Mr 110,000 and is most active at pH 7.4 . Protease III is very sensitive to metal-chelating agents and reducing agents . The EDTA-inactivated enzyme can be reactivated by Zn2+, Co2+ or Mn2+ . Protease III is devoid of activity toward aminopeptidase, carboxypeptidase, or esterase substrates but rapidly degrades small proteins . When fragments of beta-galactosidase are used as substrates for protease III, the enzyme preferentially degrades proteins with molecular weights of less than 7,000 . Protease III cleaves the oxidized insulin B chain at two sites with an initial rapid cleavage at Tyr-Leu (16-17) and a second slower cut at Phe-Tyr (25-26). J Biol Chem, 1979 Jun 10, 254(11), 4309 - 12 Characterization of a mutant form of ribosomal protein S1 from Escherichia coli; Subramanian AR et al.; An altered form of ribosomal protein S1 from a mutant of Escherichia coli has been isolated and characterized . The mutant protein (denoted m1-S1) has a molecular weight of 57,000 as shown by sodium dodecyl sulfate-gel electrophoresis and the same NH2-terminal sequence as wild type S1 . Protein m1-S1 binds poly(U) in the same manner as protein S1 and is active in protein synthesis with either synthetic or natural mRNA . Thus, about 75% of the sequence of protein S1 (which includes the NH2-terminal region) contains essentially all the functional domains of this protein involved in protein biosynthesis. Mol Gen Genet, 1979 Jun 7, 173(2), 217 - 20 Deletions, insertions and rearrangements affecting rpoB gene expression; Collins J; Through cloning and deletion experiments on ColEl hybrids the rpoB gene (Rifr) was located on a physical restriction map; RNA polymerase binding studies showed no binding site within the structural gene . The genetic data and RNA polymerase binding studies lead to the conclusion that rp/L and rpoB are dependent upon a common promoter. Mol Gen Genet, 1979 Jun 7, 173(2), 197 - 201 Restriction enzyme analysis of the plasmid ColIb DNA; Skorupska A et al.; Plasmid ColIb (61.5 Mdal) was digested with restriction enzymes EcoRI and HindIII . The DNA digestion products were separated by electrophoresis on 1.2% agarose gels . There were identified 22 fragments of ColIb DNA generated by the endonuclease EcoRI and 21 fragments produced by HindIII . Molecular weights of the fragments were estimated . The total molecular weight of the fragments generated by EcoRI was 61.42 Mdal and for HindIII fragments 62.79 Mdal. Mol Gen Genet, 1979 Jun 7, 173(2), 183 - 7 Post-translational modification of Escherichia coli ribosomal protein S6; Reeh S et al.; Escherichia coli has multiple forms of ribosomal protein S6, differing in number of glutamyl resideus at the C-terminal end . Three forms are revealed when crude cell extracts are fractionated by a two-dimensional gel electrophoresis technique . Pulse-chase experiments show that the shortest and most alkaline form of S6 is the first to appear . In about one doubling time this form reaches equilibrium with the two other forms of S6, implicating the existence of an enzyme, which adds glutamic acid residues to S6 . We show that the relative levels of these three S6 forms are not affected by the growth rate of the culture. Nature, 1979 Jun 7, 279(5713), 494 - 500 Unusual location and function of the operator in the Escherichia coli galactose operon; DiLauro R et al.; The operator of the gal operon is located about 60 base pairs preceding the startpoints of the transcription of the two gal promoters . This location contrasts with the location of the operator in other phage or bacterial operons where the repressor binds more closely to the respective transcription initiation sites . Models explaining how the repressor-operator interactions may control the two gal promoters are presented. Nature, 1979 Jun 7, 279(5713), 492 - 4 Modulation of the two promoters of the galactose operon of Escherichia coli; Adhya S et al.; The gal operon of Escherichia coli is controlled by two independent promotors--one is activated and the other inhibited by cyclic AMP and cyclic AMP receptor protein . The two promotors are modulated, however, by the same operator locus and receptor protein. Mol Gen Genet, 1979 Jun 7, 173(2), 189 - 96 Origin and binding specificity of protein(s) coded for by Mu prophages; Schumann W et al.; Crude extracts of bacteria lysogenic for temperature phage Mu contain proteins that retain specifically Mu DNA on nitrocellulose filters . The amount of binding protein is directly proportional to the number of Mu prophages per E . coli genome . Specificity of the binding reaction could be demonstrated by using heterologous DNAs as substrate and by a competition experiment . By using hybrid plasmids containing different amounts of the immunity end and extending to various degrees into MuDNA, it was found that the binding activity is coded for by the left 1,000 nucleotide-pair HindIII fragment . When using these hybrid plasmids as binding substrate, two different binding sites for the immunity product were detected . Joining of the MucI gene to the left lambda early promoter resulted in increased production of immunity protein at elevated temperature . A possible explanation for the relatively low amounts of immunity protein in all of the different strains studied is discussed. Biochim Biophys Acta, 1979 Jun 6, 568(2), 467 - 74 Enzyme-enzyme interaction and the biosynthesis of aromatic amino acids in Escherichia coli; Powell JT et al.; The technique of affinity chromatography has been used to demonstrate that enzymes involved in the biosynthesis of tyrosine and phenylalanine in Escherichia coli undergo reversible interactions . Thus it has been shown that the aromatic amino acid aminotransferase (aromatic-amino-acid: 2-oxoglutarate amino-transferase, EC 2.6.1.57) reacts specifically with chorismate mutaseprephenate dehydrogenase (chorismate pyruvate mutase, EC 5.4.99.5 and prephenate: NAD+ oxidoreductase (decarboxylating), EC 1.3.1.12) in the absence of reactants and with chorimate mutase-prephenatedehydratase (prephenate hydro-lyase (decarboxylating), EC 4.2.1.51) in the presence of phyenylpyruvate . Tyrosine causes dissociation of the aminotransferase: mutasedehydrogenase complex while dissociation of the aminotransferase-mutasedehydratase complex occurs on omission of phenylpyruvate . Only the active form of chorismate mutase-prephenate dehydrogenase participates in complex formation. Biochim Biophys Acta, 1979 Jun 6, 568(2), 428 - 36 The purification of glutamine synthetase from Azotobacter and other procaryotes by blue sepharose chromatography; Lepo JE et al.; We report the facile purification of glutamine synthetase (L-glutamate: ammonia ligase (adenosine 5'-diphosphate-forming), EC 6.3.1.2) in both the adenylylated and unadenylylated form, from Azotobacter vinelandii ATCC 12837 . A general affinity column, which used as an affinity ligand Reactive blue 2 dye (Cibacron blue) covalently linked to Agarose, was employed as an efficient first step of purification . Further purification to electrophoretic homogeneity employed DEAE-cellulose chromatography and an additional Affigel chromatographic step . The method was used successfully to prepare glutamine synthetase from Escherichia coli, Rhodopseudomonas sphaeroides and Anabaena sp . strain CA. Vet Rec, 1979 Jun 2, 104(22), 496 - 500 Intestinal defence of the neonatal pig: interrelationship of gut and mammary function providing surface immunity against colibacillosis; Chidlow JW et al.; The neonatal requirements for maternal passive immunity and the lactation immunobiology with regard to sow immunisation for neonatal protection are reviewed . A vaccination protocol which combines oral and parenteral antigen administration to produce antibody activity mediated mainly by IgM is described . Its efficacy in affording protection to neonatal piglets was tested against a lethal oral infection with a virulent strain of Escherichia coli "Abbottstown" . Piglets suckled on vaccinated or non-vaccinated sows were exposed to an infective challenge in the gastrointestinal tract and the relative pathology in test and control groups observed over the neonatal period . Death ensued in 76 per cent of piglets suckled on control sows and 26 per cent of piglets suckled on sows vaccinated by two intramuscular injections . Litters suckled on orally vaccinated sows were able to resist a similar infective challenge, there being only one fatality out of 42 piglets. Cancer Treat Rep, 1979 Jun, 63(6), 1073 - 9 L-cyst(e)ine requirements of malignant cells and progress toward depletion therapy; Uren JR et al.; The L-cyst(e)ine requirements of normal and malignant cells are reviewed and expanded within the context of establishing whether the measurement of gamma-cystathionase levels constitutes a predictive test for tumor sensitivity to L-cyst(e)ine depletion . The ability of both purified L-cysteine desulfhydrase and gamma-cystathionase to inhibit the growth of the L-cystine-dependent L1210 leukemia in culture is presented, as well as approaches to circumvent the limitations of these enzymes for in vivo therapy . The ability of proparagylglycine to inhibit L-cysteine biosynthesis in vivo is reviewed for its possible use in combination therapy . In addition, the ability of poly D,L-alanine modification of Escherichia coli L-asparaginase to increase the plasma half-life in mice tenfold as well as to decrease the immunogenicity of the enzyme is presented. Tsitologiia, 1979 Jun, 21(6), 722 - 9 {Photoreactivation of cells and phages injuried by ultraviolet radiation in the ecological long-wave band}; Samoilova KA et al.; Photoreactivation (PR) was measured after inactivation by far (254 nm), middle (300-315 nm) and near (315-400 nm) UV radiation of Paramecium caudatum and 8 strains of Escherichia coli differing in PR and dark repair capability . PR volume was high and practically the same after irradiation by far and middle UV, but PR was not observed in near UV-inactivated cells of all the strains . It is proposed that pyrimidine dimers are not significant in near UV lethal lesions in cells, as near UV-irradiated phages (T7 and lambdacI 857) are not photoreactivated in undamaged host bacterial cells. Zentralbl Bakteriol {Orig A}, 1979 Jun, 244(1), 90 - 104 Colicin receptors and the mechanisms of colicin uptake; Kadner RJ et al.; This review deals in detail with the nature, synthesis, physiologic functions, and the regulation of colicin receptors, which represent components of transportsystems, as well as with the two mechanisms of the colicin uptake within the groups A and B of colicins. Zentralbl Bakteriol {Orig A}, 1979 Jun, 244(1), 78 - 89 The concept of bacteriocins; Reeves P; After a short description of the discovery of bacteriocins, especially the colicins in this review the following points are discussed: the classification of colicins especially with the aid of resistant mutants of sensitive indicator strains, the bacteriocin-receptors, the bacteriocin-specificity, and the possible ways of transport of colicins across the outer membrane. Zentralbl Bakteriol {Orig A}, 1979 Jun, 244(1), 105 - 20 Mode of action of colicins Ia, E1 and K; Konisky J et al.; Addition of colicins Ia, E1 or K to sensitive Escherichia coli leads to inhibition of macromolecular synthesis and an uncoupling of electron transport from active transport . Recent results indicate that these colicins affect energy metabolism by interacting directly with the cytoplasmic membrane . This results in a transmembrane flow of ions leading to membrane depolarization . It is proposed that the function of the outer membrane colicin receptor is to mediate access of the colicin molecule to the cytoplasmic membrane. Rev Can Biol, 1979 Jun, 38(2), 97 - 9 {Localization of a gene responsible for ozone sensitivity in Escherichia coli}; Cotes G et al.; A gene, ozrC, responsible for sensitivity to ozone in Escherichia coli, was localized on the E . coli chromosome between argEH and metA by means of analysis of cotransduction frequencies of the gene ozrC with certain gene markers in the malB region of the chromosome. Pediatr Res, 1979 Jun, 13(6), 737 - 41 The effect of human colostrum on neutrophil function; Bjorksten B et al.; Strains of Escherichia coli were opsonized in human colostrum via heat stable opsonins and the classic complement pathway, but colostrum lacked capacity to opsonize E . coli via the alternative pathway . There was no bacteriostatic activity against serum sensitive E . coli strains, although specific antibodies against the strains were present . Neutrophils suspended in colostrum had normal chemotaxis and this was not altered by treating the colostrum with HCl. J Gen Microbiol, 1979 Jun, 112(2), 321 - 8 Location of binding sites on common type 1 fimbriae from Escherichia coli; Sweeney G et al.; Common type 1 fimbriae were isolated from Escherichia coli and their length distribution profile was determined before and after treatment with ultrasound . As fimbriae were shortened, so their haemagglutinating capacity decreased, but their ability to bind to erythrocytes did not decrease to the same extent . Isolated fimbriae did not agglutinate inside-out vesicles prepared from horse erythrocytes or liposomes, suggesting that the binding mechanism was not based on non-specific hydrophobic interactions . The results support a lateral rather than a terminal location for the fimbrial binding site responsible for haemagglutination. Eur J Biochem, 1979 Jun, 97(1), 23 - 8 Peptide bond formation stimulated by protein synthesis factor EF-P depends on the aminoacyl moiety of the acceptor; Glick BR et al.; Elongation factor EF-P is a soluble protein that stimulates peptide bond synthesis catalyzed by the 50-S ribosomal subunit . This factor was previously identified and characterized based on its ability to promote the synthesis of formylmethionine-puromycin . In the present work, we tested the ability of EF-P to promote peptide bond synthesis between ribosome-bound fMet-tRNA and several analogues of the 3' terminus of aminoacyl-tRNA, i.e . the cytidylyl(3'-5')-{2'(3')-O-L-aminoacyladenosines} . EF-P promoted synthesis to the greatest extent with certain acceptors which were otherwise inefficient in the peptidyl transferase reaction . This activity of EF-P could not be replaced by the other soluble proteins known to be involved in polypeptide synthesis, such as EF-Tu, EF-Ts and EF-G . One role of EF-P in protein synthesis may be to allow peptide bond synthesis to occur more efficiently with some aminoacyl-tRNAs that are poor acceptors for the ribosomal peptidyl transferase. Can J Genet Cytol, 1979 Jun, 21(2), 213 - 21 Lethal zygosis in recombination-deficient mutants of Escherichia coli; Nestmann ER; Use of nonselective medium for plating cells following mating has revealed that Rec recipient strains of E . coli may be killed as a result of conjugation . Sensitivity of RecA-, RecB-, and RecC- recipients increases with ratio of donor: recipient cells in mating mixtures and with time of mating . A Rec+ recipient shows no lethal zygosis in these experiments performed without aeration . Cell contact does not seem to be responsible for the sensitivity of Rec- strains, since lethality is prevented when cell contact is permitted but DNA transfer is not . Thus, an event(s) occuring subsequent to entry of donor DNA appears to cause lethality in Rec- recipients. Can J Biochem, 1979 Jun, 57(6), 834 - 42 Elucidation of the quaternary structure of reversibly immobilized alkaline phosphatase derivatives; McCracken S et al.; Escherichia coli alkaline phosphatase has been reversibly immobilized on Sepharose CL-4B through two different methods, both based on a disulfide linkage, under conditions selected to favour the coupling of the enzyme to the solid support through one covalent linkage . The quaternary structure of the reversibly immobilized subunit, produced by dissociation of the matrix-bound dimer, was examined by cross-linking with the bifunctional reagent dimethyl suberimidate . Following release from the solid support, the protein was analysed by sodium dodecyl sulfate gel electrophoresis demonstrating the presence of a sufficient amount of dimeric structures in the immobilized subunit preparation to account for all the enzyme activity observed in this sample . These results suggest that the subunit of alkaline phosphatase may be catalytically inactive . This approach to studying the quaternary structure of immobilized subunit derivatives offers the opportunity to directly determine the homogeneity and structure of matrix-bound 'monomer' preparations and is particularly useful in determining if low levels of catalytic activity observed in some immobilized subunit populations are due to the presence of contaminating oligomeric structures. Can J Biochem, 1979 Jun, 57(6), 813 - 21 Purification and characterization of membrane-bound fumarate reductase from anaerobically grown Escherichia coli; Dickie P et al.; Fumarate reductase has been purified 100-fold to 95% homogeneity from the cytoplasmic membrane of Escherichia coli, grown anaerobically on a defined medium containing glycerol plus fumarate . Optimal solubilization of total membrane protein and fumarate reductase activity occurred with nonionic detergents having a hydrophobic-lipophilic balance (HLB) number near 13 and we routinely solubilized the enzyme with Triton X-100 (HLB number = 13.5) . Membrane enzyme extracts were fractionated by hydrophobic-exchange chromatography on phenyl Sepharose CL-4B to yield purified enzyme . The enzyme whether membrane bound, in Triton extracts, or purified, had an apparent Km near 0.42 mM . Two peptides with molecular weights of 70 000 and 24 000, predent in 1:1 molar ratios, were identified by sodium dodecyl sulfate polyacrylamide slab-gel electrophoresis to coincide with enzyme activity . A minimal native molecular weight of 100 000 was calculated for fumarate reductase by Stephacryl S-200 gel filtration in the presence of sodium cholate . This would indicate that the enzyme is a dimer . The purified enzyme has low, but measurable, succinate dehydrogenase activity. Can J Biochem, 1979 Jun, 57(6), 798 - 805 Aspartate transcarbamoylase: loss of homotropic but not heterotropic interactions upon modification of the catalytic subunit with a bifunctional reagent; Chan WW et al.; The role of conformational changes in the allosteric mechanism of aspartate transcarbamoylase from Escherichia coli was studied by reacting the isolated catalytic subunit with the bifunctional reagent tartryl diazide . Two derivatives differing moderately in substrate affinity were obtained depending on whether the reaction was conducted in the presence or absence of the substrate analogue succinate and carbamoyl phosphate . The modification was not accompanied by aggregation or dissociation . The modified catalytic subunits retained the ability to reassociate with unmodified regulatory subunits and produced hybrids similar in size to the native enzyme . These hybrids were appreciably sensitive to the allosteric effectors ATP and CTP but unlike native enzyme showed no cooperativity in substrate binding . The Michaelis constants of these hybrids for aspartate were intermediate between that of the isolated catalytic subunit and that of the relaxed state . Activation by ATP was caused by a reduction in Km to the value characteristic of the relaxed state whereas CTP inhibited by lowering the Vmax . The properties of the hybrids are strikingly similar to the modified enzyme obtained by Kerbiriou and Herve from cells grown in the presence of 2-thiouracil . However, the crucial modifications are found in the regulatory subunits of the enzyme studied by these authors whereas they are located in the catalytic subunits of the hybrids reported here . Our results suggest that interactions between the catalytic and regulatory subunits have considerable effects on the state of the substrate binding sites in the native enzyme. Can J Biochem, 1979 Jun, 57(6), 749 - 57 Purification and Best Department of Medical Research, University of Toronto, Ont., Canada; Glick BR et al.; Factor EF-P is a nonribosomal (soluble) protein of Escherichia coli that stimulates peptide bond synthesis when certain aminoacyl-tRNA analogues are used . The purification of this protein to apparent homogeneity is described here . EF-P has a molecular weight of about 21 000, a Stokes radius of 27 A (1A = 0.1 nm), and a frictional coefficient of 1.48, suggesting an asymmetric structure . By this and a number of other criteria, EF-P is a new factor that controls peptide bond formation during protein biosynthesis. Can J Biochem, 1979 Jun, 57(6), 653 - 61 Dicarboxylic acid transport in Escherichia coli K12: involvement of a binding protein in the translocation of dicarboxylic acids across the outer membrane of the cell envelope; Bewick MA et al.; We have previously found that the dicarboxylate transport system in Escherichia coli K12 is an active transport system and that at least one binding protein and two cytoplasmic membrane transport components are involved in the uptake of dicarboxylic acids . Recently, through surface labelling studies, some dicarboxylate binding proteins were found to be exposed on the cell surface . In the present paper, we demonstrate that the dicarboxylate transport component located in the outer membrane can be inactivated by two different kinds of nonpenetrating inhibitors, viz . proteases, and diazosulfanilic acid . These inhibitors seem to act on the dicarboxylate binding protein . By adding this protein to inactivated cells or to transport-negative mutants, we have succeeded in reconstituting the dicarboxylate transport system . These findings suggest that the dicarboxylate binding protein found on the cell surface plays an essential role in the translocation of dicarboxylic acids across the outer membrane. Am J Vet Res, 1979 Jun, 40(6), 792 - 4 Endotoxin absorption in hay-fed and lactic acidotic sheep; Huber TL et al.; Absorption of endotoxin from the gastrointestinal tract was evaluated in hay-fed and lactic acidotic sheep duodenally infused with 10 mg of Escherichia coli endotoxin, and in lactic acidotic sheep not infused . The effect of abomasal fluid on biological activity of endotoxin was also evaluated . Leukopenia was the criterion used for detecting endotoxemia . Absorption of endotoxin from the gastrointestinal tract was not detected in either hay-fed or lactic acidotic sheep . Endotoxin appeared to maintain its activity after incubation with abomasal fluid, and the presence of endogenous endotoxin in abomasal contents was indicated . The results indicate that endotoxin of alimentary origin may not be involved in the lactic acidosis syndrome in ruminants. Z Immunitatsforsch Immunobiol, 1979 Jun, 155(5), 387 - 98 Protein I and protein II from the outer membrane of Escherichia coli are mouse B-lymphocyte mitogens; Bessler WG et al.; Protein I from the outer membrane of Escherichia coli is a B-lymphocyte mitogen in mice . Polyclonal activation of mouse splenocytes was demonstrated by 3H-thymidine incorporation into DNA, 3H-uridine incorporation into RNA, and by a hemolytic plaque assay in three inbred mouse strains . B-lymphocytes from LPS responder mice (C57Bl/10, STU/nu/nu) and LPS non-responder mice (C3H/HeJ) both responded well to protein I . The presence of serum was not necessary for mitogenicity; bovine serum albumin exhibited a beneficial effect on serum-depleted cultures . Thymocytes of C3H/HeJ mice were not activated by protein I . Protein II* from E . coli was also tested in our systems and showed a weak B-lymphocyte stimulatory activity . Human peripheral blood lymphocytes were not activated. Z Gesamte Inn Med, 1979 Jun 1, 34(11), 318 - 9 {Mass screening for bacteriuria in students}; Wachtel D; From 1973 to 1978 in 2.6% out of 244 female students and in none out of 160 male students a significant persisting bacteriuria was proved . By the slight rate of falsely positive findings could be demonstrated that the midstream technique gives evident results when persons able to cooperate are sufficiently instructed. Infect Immun, 1979 Jun, 24(3), 965 - 6 Evidence for two heat-stable enterotoxins produced by enterotoxigenic Escherichia coli; Kapitany RA et al.; Heat-stable enterotoxins from a bovine and porcine strain of Escherichia coli were isolated and showed significant differences in amino acid composition and heat stability. Infect Immun, 1979 Jun, 24(3), 895 - 9 B lymphocyte colony formation in renal infection; Miller T et al.; The effect of renal infection on B lymphocyte colony formation has been investigated in the belief that a study of the effect of infection on subpopulations of lymphoid cells might provide direct evidence of the effect of infection on the immmune system . Renal infection was induced in mice, and the specific immune response of the B lymphocyte compartment was quantitated by determining the serum antibody and plaque-forming cell response to infection . Under the conditions of the experiment, the ability of splenic lymphocytes to form B lymphocyte colonies was significantly depressed during the first 7 days of infection, and the results suggest that a study of the responses of lymphoid cells to infection may provide information of diagnostic and prognostic value. Infect Immun, 1979 Jun, 24(3), 793 - 7 Heat-labile enterotoxin production in isolates from a shipboard outbreak of human diarrheal illness; Wachsmuth K et al.; As reported elsewhere, an enterotoxigenic strain of Escherichia coli serotype O25:K98:NM was epidemiologically incriminated as the etiological agent in a shipboard outbreak of diarrheal illness . This enterotoxigenic E . coli strain and possibly other enteric isolates were found to produce heat-labile toxin and not heat-stable toxin . Since previous genetic analyses of enterotoxigenic E . coli strains producing heat-labile and heat-stable toxins have shown a plasmid location for both toxin determinants and since in this outbreak more than one bacterial strain appeared to produce only heat-labile toxin, the possibility of an extrachromosomal heat-labile toxin determinant was investigated . Results of endonuclease cleavage and hybridization experiments, as well as apparent heat-labile toxin phenotypic instability, strongly suggest a plasmid mediation of toxin production . Additionally, the stability of this heat-labile toxin production was evaluated after several traditional methods of bacterial cell preservation. Infect Immun, 1979 Jun, 24(3), 606 - 10 Passive immune hemolysis for detection of heat-labile enterotoxin produced by Escherichia coli isolated from different sources; Serafim MB et al.; Fifty-one strains of Escherichia coli isolated from humans, swine, food, and water and identified as enterotoxinogenic by the Y-1 adrenal cell assay, were examined for heat-labile enterotoxin (LT) production by the passive immune hemolysis test . Cholera antitoxin, anti-choleragenoid and anti-LT were used as antisera . Cholera antitoxin was much more potent than anti-choleragenoid and LT antiserum in the detection of LT-positive strains . All strains isolated from pigs and sausage were negative in tests made with LT antiserum . A few strains isolated from humans, food, and water also gave negative results . These data showed that the passive immune hemolysis test is not as efficient as the Y-1 adrenal cell assay in the detection of enterotoxinogenic E . coli strains. Genetika, 1979 Jun, 15(6), 972 - 88 {Control of plasmid incompatibility: characteristics of the bireplicon hybrid pAS8 and its deletion mutants}; Sakanian VA et al.; The phenomenon of incompatibility has been investigated using deletion mutants of hybrid bireplicon plasmid pAS8 . The hybrid pAS8 displays incompatibility specific for both components of its structure . In contrast to P-specificity of pAS8, functions of ColE1-specificity are not effectively expressed . Expression of ColE1-specificity in pAS8 plasmid and its derivatives is characterized by different directions and this is due to the presence or absence of genes of RP4 replication machinery in the plasmid DNA . Mutant plasmids show different efficiency of P-specificity depending on the extension of deletion in the region of essential genes of the RP4 component . Some of the mutants, in spite of the loss of replication genes, including origin of vegetative replication, are incompatible with the representatives of the Inc P group in both directions of testing . Different character and the level of expression of ColE1- and P-specificity in the pAS8 hybrid and its deletion derivatives are not associated with change in the number of plasmid DNA copies, for all of them are subjects to stringent control of replication . The data suggest the existence of incompatibility functions control mechanism which does not seem to include replication genes . Possible ways of realization of the inc genes functions are discussed. Eur J Cell Biol, 1979 Jun, 19(2), 120 - 30 Structure of the 50S ribosomal subunit from Escherichia coli . Investigation of the intact subunit and core particles by electron microscopy and analogue image processing; Spiess E; Structures of 50S ribosomal subunits, CsCl and ethidium bromide core particles from these subunits have been investigated by electron microscopy and image processing by FAIRS . This method revealed structural details which are obscured in individual images, and enabled to distinguish six crown forms, different in their side protuberances, and two kidney forms . Crown forms were imaged as symmetrical or asymmetrical forms . The latter type was far more frequent in untreated populations than the first . The depletion of proteins by both agents caused stepwise degradation of the side protuberances in the crown forms thereby transforming asymmetrical to symmetrical forms . It is concluded from these findings that asymmetrical and symmetrical forms in untreated populations represent also structurally different particles . From the higher complexity in terms of component composition and structure it is concluded that the asymmetrical crown forms are more likely to represent the native structure of isolated 50S subunits than the symmetrical forms . Existing models for this subunit are discussed in terms of this finding. Eur J Biochem, 1979 Jun 1, 96(3), 535 - 43 The influence of ribonucleoside triphosphates, and other factors, on the formation of very-salt-stable RNA-polymerase . su+III-tRNA(tRNATyr)-promoter complexes; Debenham P; The formation of a stable RNA-polymerase . su+III-tRNA-promoter complex was found to require sigma factor and the incorporation of ribonucleoside triphosphates which match the 5' sequence of the su+III tRNA transcript . This complex, stable to at least 2 M KCl, can be retained on a Millipore filter . Its formation closely parallels the extent of transcription obtained from the su+III tRNA promoter in response both to increasing ionic strength and to temperature during incubation of RNA polymerase with the DNA . The RNA-polymerase . DNA complex retained during this assay therefore appears to relate directly to that formed during promoter-directed transcription . The formation of RNA-polymerase . su+III-tRNA-promoter complexes is sensitive to the presence of ppGpp. Br J Vener Dis, 1979 Jun, 55(3), 214 - 7 Haematuria presenting in outpatients attending a department of genitourinary medicine; Amarasuriya KL; Of all the patients attending a department of genitourinary medicine during a 10-month period, about 2% (1 out of 50) presented with haematuria, or haematuria was discovered on initial examination . In about 25% of cases, the haematuria was due to Escherichia coli infection of the lower genitourinary tract . Gonococcal infection was the next commonest cause; one patient with gonorrhoea presented with frank urethral bleeding . In the remaining patients other causes of haematuria, which included renal cyst . carcinoma of the ureter, bilharziasis, and IgA disease, required more extensive investigations and follow up. Proc Natl Acad Sci U S A, 1979 Jun, 76(6), 2649 - 53 lac repressor changes conformation upon binding to poly{dA-T)}; Kelsey DE et al.; N-(Iodoacetylaminoethyl)-1-naphthylamine-5-sulfonate reacts with Escherichia coli lac repressor to selectively label cysteine-140 with the fluorescent N-(acetylaminoethyl)-1-naphthylamine-5-sulfonate group . The fluorescence intensity of this label decreases by 20% when labeled repressor associates with poly{d(A-T)} . Fifteen base pairs of poly{d(A-T)} per repressor tetramer are required to complete this decrease . Stopped-flow experiments have shown that the repressor undergoes at least two conformational changes as it binds to poly{d(A-T)}, with half-lives of 5.0 +/- 1.2 msec and 3.5 +/- 1.0 sex . Quite likely, these conformational changes serve to strengthen the interaction of repressor with DNA. Proc Natl Acad Sci U S A, 1979 Jun, 76(6), 2615 - 9 Initiation of general recombination catalyzed in vitro by the recA protein of Escherichia coli; McEntee K et al.; Homogeneous recA protein catalyzes the hybridization of single-stranded DNA to homologous regions in duplex DNA . The products are D-loops, which are formed with equal efficiency in linear and supercoiled molecules . This assimilation reaction can be separated into two partial reactions . In the first, recA protein binds to duplex DNA and produces a reA protein-DNA complex . The binding shows a sigmoidal dependence on recA protein concentration, requires ATP, GTP or the gamma-thio analog of ATP, and Mg2+, but does not require hydrolysis of the nucleoside triphosphate . In the second reaction, single-stranded regions of the recA protein-ATP-duplex DNA intermediate hybridize with free complementary single strands to produce D-loop structures . This reaction is coupled to ATP hydrolysis and is analogous to the renaturation of single-stranded DNA catalyzed by the recA protein {Weinstrock, G.M., McEntee, K . & Lehman, I.R . (1979) Proc . Natl . Acad . Sci . USA 76, 126-130} . Hydrolysis of ATP appears to be required in these reactions for dissociation of recA protein from the DNA. J Biochem (Tokyo), 1979 Jun, 85(6), 1527 - 9 Kinetics of selective acylation of sn-glycerol 3-phosphate in the synthesis of phospholipid molecular species; Kito M et al.; The kinetics of the sn-glycerol 3-phosphate acyltransferase {EC 2.3.1.15} reaction support the view that the selective acylation primarily depends on the differences in the affinity of the enzyme for acyl-CoAs. J Bacteriol, 1979 Jun, 138(3), 944 - 8 Biosynthesis of murein lipoprotein in Escherichia coli: effects of 3,4-dihydroxybutyl-1-phosphonate; Chattopadhyay PK et al.; The effects of 3,4-dihydroxybutyl-1-phosphonate, a four-carbon analog of sn-glycerol 3-phosphate, on the biosynthesis of the glyceryl moiety in murein lipoprotein of Escherichia coli were studied . The compound at a concentration of 55 microM strong inhibits in the incorporation of {2-3H}glycerol radioactivity into lipoprotein by virtue of its inhibition of the synthesis of phosphatidylglycerol . On the other hand, the incorporation of prelabeled {2-3H}glycerol radioactivity into lipoprotein was only partially inhbited by 3,4-dihydroxybutyl-1-phosphonate even at a much higher concentration (1 mM) . These data were consistent with the postulated pathway for the biosynthesis of the lipid moiety in lipoportein: cysteine-lipoprotein + phosphatidylglycerol leads to glycerylcystein-lipoprotein + phosphatidic acid. J Bacteriol, 1979 Jun, 138(3), 871 - 7 Characterization of Azotobacter vinelandii deoxyribonucleic acid and folded chromosomes; Sadoff HL et al.; The properties of Azotobacter vinelandii deoxyribonucleic acid (DNA) and folded chromosomes were studied and compared to those of Escherichia coli as a standard . Based on melting temperature and buoyant density measurements, the guanosine + cytosine content of purified A . vinelandii DNA was 65%, whereas that of E . coli DNA was 50% . The results of renaturation studies showed that the unique DNA sequence lengths of the two organisms were similar with Cot1/2 values of 7.3 +/- 0.4 mol.s/liter and 7.5 +/- 0.3 mol.s/liter, respectively, for A . vinelandii and E . coli . Folded chromosomes of A . vinelandii sedimented in a centrifugal field at a rate identical to those derived from E . coli, 1,600 to 1,700S . Based on the DNA content per cell and the mass of a single genome, A . vinelandii contains at least 40 chromosomes per cell. J Bacteriol, 1979 Jun, 138(3), 861 - 70 Chemical heterogeneity of major outer membrane pore proteins of Escherichia coli; Lee DR et al.; Peptide mapping and isoelectric focusing were used to compare the major outer membrane pore proteins from various strains of Escherichia coli K-12, including strains carrying mutations in the nmpA, nmpB, and nmpC genes which result in the production of new membrane proteins . Proteins 1a, 1b, and 2 and the NmpA proteins each gave unique peptide and isoelectric focusing profiles, indicating that these are different polypeptides . The NmpA protein and the NmpB protein appeared to be identical by these criteria . The NmpC protein and protein 2 were nearly identical, although one different peptide was observed in comparing the proteolytic peptide maps of these proteins and there were slight differences in their isoelectric focusing profiles . Antiserum against protein 2 showed partial cross-reactivity with the NmpC protein . These results indicate that the various pore proteins of E . coli K-12 fall into four different classes. J Bacteriol, 1979 Jun, 138(3), 832 - 8 Genes coding for ribosomal proteins S15, L21, and L27 map near argG in Escherichia coli; Kitakawa M et al.; Mutants with alterations in the structural genes for ribosomal proteins S15, L21, and L27 were used in mapping the genes coding for these proteins . Results from P1kc-mediated transductions indicate that the genes for L21 (rplU) and L27 (rpmA) form a gene cluster and are located between argG and gltB at 68.1 min, whereas the gene for S15 (rpsO) is situated close to, but on the opposite side or, argG . The gene order in this region is concluded to be gltB-(rplU, rpmA)-argG-rpsO-mtr. J Bacteriol, 1979 Jun, 138(3), 783 - 7 Genetic mapping of a mutation affecting pyridine nucleotide transhydrogenase in Escherichia coli; Hanson RL et al.; A mutation, pnt-1, causing loss of pyridine nucleotide transhydrogenase activity in Escherichia coli, was mapped by assaying for the enzyme in extracts of recombinant strains produced by conjugation, F-duction, and P1 transduction . The site of this mutation was near min 35, counterclockwise from man, and it co-transduced 59% with man . The mutation was associated with loss from the cell membrane fraction of energy-independent and adenosine 5'-triphosphate-dependent transhydrogenase activities, but reduced nicotinamide adenine dinucleotide dehydrogenase activity was not affected . Strains were constructed which lack phosphoglucoisomerase (pgi-2) and which carry either pnt+ or pnt-1 . Although such strains, when grown on glucose, are expected to produce a large excess of reduced nicotinamide adenine dinucleotide phosphate, the growth rate was not affected by the pnt-1 allele. J Bacteriol, 1979 Jun, 138(3), 770 - 8 Mechanism of export of colicin E1 and colicin E3; Jakes KS et al.; The mechanism of export of colicins E1 and E3 was examined . Neither colicin E1, colicin E3, Nor colicin E3 immunity protein appears to be synthesized as a precursor protein with an amino-terminal extension . Instead, the colicins, as well as the colicin E3 immunity protein, appear to leave the cells where they are made, long after their synthesis, by a nonspecific mechanism which results in increased permeability of the producing cells . Induction of ColE3-containing cells with mitomycin C leads to actual lysis of those cells, as some time after synthesis of the colicin E3 and its immunity protein has been completed . Induction of ColE1-containing cells results in increased permeability of the cells, but not in actual lysis, and most of the colicin E1 produced never leaves the producing cells . Intracellular proteins such as elongation factor G can be found outside of colicinogenic cells after mitomycin C induction, along with the colicin . Until substantial increases in permeability occur, most of the colicin remains cell associated, in the soluble cytosol, rather than in a membrane-associated form. J Bacteriol, 1979 Jun, 138(3), 715 - 20 Restriction enzyme cleavage sites surrounding the structural gene for the lipoprotein of the Escherichia coli outer membrane; Nakamura K et al.; The purified messenger ribonucleic acid (mRNA) for the lipoprotein of the Escherichia coli outer membrane was hybridized with fragments obtained by digestion of E . coli chromosomal deoxyribonucleic acid (DNA) with eight different restriction enzymes . For each restriction enzyme digestion, one specific fragment separated by agarose gel electrophoresis was found to hybridize with the lipoprotein mRNA . From the analysis of restriction fragments generated by double digestions with various combinations of restriction enzymes, cleavage sites for the restriction enzymes near the locus of the lipoprotein structural gene (lpp) were mapped . No restriction fragments of DNA from the E . coli lpp-2 mutant hybridized with the lipoprotein mRNA, confirming that the mutant has a deletion mutation in the vicinity of the lpp gene. J Bacteriol, 1979 Jun, 138(3), 705 - 14 Organization of structural and regulatory genes that mediate tetracycline resistance in transposon Tn10; Jorgensen RA et al.; The location of Tn10 genes encoding tetracycline resistance and its regulation was determined by analyzing the properties of recombinant plasmids carrying partial HpaI digestion products of lambda::Tn10 transducing phage deoxyribonucleic acid . Within a 2,700-base pair region are encoded tetracycline resistance, the structural gene (tet) for a tetracycline-inducible polypeptide, and the regulatory elements for the induction of both the resistance phenotype and the polypeptide . Fusion of different sequences to an HpaI site in the tet gene alters the molecular weight and stability of the polypeptide as well as the tetracycline resistance phenotype of strains producing fusion polypeptides . These results indicate the orientation of the tet gene and support the conclusion that the tet polypeptie is required for tetracycline resistance . A HincII cleavage site immediately upstream from the tet gene is protected by ribonucleic acid polymerase, but only the absence of ribonucleotide triphosphates . The possibility that tet transcription is initiated at this site is discussed. J Bacteriol, 1979 Jun, 138(3), 1033 - 5 Nature of transforming deoxyribonucleic acid in calcium-treated Escherichia coli; Strike P et al.; A study of the reactivation of ultraviolet-irradiated plasmid and phage deoxyribonucleic acid molecules after transformation into Escherichia coli strains indicated that, when double-stranded deoxyribonucleic acid was used as the donor species, it was taken up without conversion to the single-standed form. Cell, 1979 Jun, 17(2), 389 - 97 In vitro processing of B . mori transfer RNA precursor molecules; Garber RL et al.; Ribonuclease P and 3'-5' nuclease, two enzymatic activities necessary for tRNA synthesis in E . coli, are also found in the silkgland cells of Bombyx mori . B . mori subcellular extracts containing RNAase P activity can cleave the E . coli tRNA precursor molecule endonucleolytically at the same site as the E . coli enzyme, and will also cleave in vitro all E . coli tRNA precursors (pre-tRNAs) which the bacterial enzyme recognizes . B . mori RNAase P will not cleave two E . coli RNAase P substrates that are structurally unrelated to tRNA . Pre-tRNAs from B . mori contain extra 5' and 3' nucleotides as judged by RNA fingerprinting and 5' terminal phosphate analysis . Crude silkgland extracts containing both RNAase P and 3'-5' nuclease can remove the 5' and 3' extra nucleotides from B . mori pre-tRNAs, whereas purified fractions containing RNAase P remove only 5' extra nucleotides . Only large silkworm pre-tRNAs were found to be susceptible to cleavage by B . mori RNAase P . This observation and sequence analysis of intermediates of in vitro processing reactions indicate a two-step process of pre-tRNA maturation in which extra 5' nucleotides are first removed by RNAase P and extra 3' nucleotides are then trimmed off by a 3'-5' nuclease. J Dent Res, 1979 Jun, 58(6), 1634 - 9 A comparison of the effects of endotoxin upon fibroblast proliferation and macromolecular syntheses; Singer RE et al.; The effects of Escherichia coli endotoxin upon mouse L929 cell proliferation, DNA synthesis, protein synthesis, and proline incorporation were determined . It was found that a level of endotoxin which inhibited cell proliferation prompted a similar inhibition of DNA synthesis and overall cell protein synthesis . In contrast, endotoxin was shown to inhibit incorporation of proline into cell protein to a significantly greater extent. Fed Proc, 1979 Jun, 38(7), 2134 - 8 Protective effects of supplemental vitamin E against infection; Nockels CF; Vitamin E supplementation (dl-alpha-tocopheryl acetate except where noted) in excess of requirement significantly increased humoral immune response or disease resistance . Mice immunized with sheep red blood cells or tetanus toxoid and fed the supplemental vitamin demonstrated increased plaque-forming cells (PFC) and hemagglutinin (HA) titers . A vitamin E deficiency resulted in decreased PFC and little IgG which was partially corrected by N,N-diphenyl-p-phenylenediamine but not as effectively as by vitamin E . Hens immunized with Brucella abortus and fed different levels of the vitamin produced chicks with increased passive immunity; a biphasic antibody response to the level of the vitamin fed was noted . Vitamin E fed to nonimmunized hens was found to significantly increase the primary immune response of their immunized chicks . Feeding dl-alpha-tocopheryl acetate to guinea pigs immunized with Venezuelan equine encephalomyelitis virus resulted in no increased immunity . Injecting this form of the vitamin resulted in severe tissue reaction . However, injecting dl-alpha-tocopheryl significantly improved hemagglutinin inhibition titers . Chicks and turkeys infected with Escherichia coli and fed supplemental vitamin E had reduced mortality and increased HA titers . Sheep fed vitamin E and challenged with Chlamydia had improved weight gains and no detectable Chlamydia. Gastroenterology, 1979 Jun, 76(6), 1368 - 73 Prophylactic doxycycline for travelers' diarrhea: results of a prospective double-blind study of Peace Corps volunteers in Morocco; Sack RB et al.; A second randomized double-blind study to determine the efficacy of doxycycline, 100 mg daily, for the prevention of travelers' diarrhea was carried out among 50 Peace Corps Volunteers during their first 10 wk in Morocco . The volunteers took either doxycycline or placebo for 3 wk, and were observed for an additional 7 wk . Eleven of 24 taking the placebo and 2 of 26 taking doxycycline had travelers' diarrhea during the treatment period (P less than 0.01) . One week after cessation of the doxycycline, however, persons in that group developed an increase in frequency of travelers' diarrhea (P less than 0.05) so that by 3 wk after the drug was stopped, there were no differences between groups . Enterotoxigenic E . coli, most of which were sensitive to doxycycline, were the most frequently isolated pathogens during the entire study . This study corroborates the effectiveness of doxycycline prophylaxis for travelers' diarrhea. Acta Pathol Microbiol Scand {C}, 1979 Jun, 87C(3), 241 - 50 Fractionation of connective-tissue-activating factors from the culture medium of silica-treated macrophages; Aalto M et al.; The medium of cultured, SiO2-treated peritoneal macrophages contained a factor which enhances the incorporation of labelled proline to collagen and other proteins in granulation tissue slices, cells and polysomes . Simultaneously, the activity of alkaline RNase in the whole medium was decreased in comparison with the corresponding control . Polyvinylpyridine-N-oxide, PVNO, protected the macrophages against SiO2 . Latex-particles and E . coli lipopolysaccharide decreased the RNase activity in the macrophage medium, but unlike SiO2 did not cause liberation of the collagen synthesis-stimulating factor . Fractionation of the medium by gel filtration chromatography showed the SiO2-pretreatment to have caused a very significant decrease in the aggregation state of RNase . The fraction from gel filtration chromatography that contained the SiO2-liberated factor stimulating collagen synthesis also contained the disaggregated RNase . There was no RNase-activity in the control sample . A homogenous protein (mol . wt . 14,300) was isolated with repeated gel filtrations from the medium of silica-treated macrophages . It increased the incorporation of 3H proline and 3H thymidine into cultured granuloma cells. J Bacteriol, 1979 Jun, 138(3), 933 - 43 Aromatic amino acid biosynthesis: regulation of shikimate kinase in Escherichia coli K-12; Ely B et al.; Starvation of cells of Escherichia coli K-12 for the aromatic amino acids results in an increased rate of synthesis of shikimate kinase activity . The two controlling amino acids are tyrosine and tryptophan, and starvation for both results in derepression . The product of the regulator gene tyrR also participates in this control, and shikimate kinase synthesis was depressed in tyrR mutants . Chromatography of cell extracts on diethylaminoethyl-Sephadex allowed partial separation of two shikimate kinase enzymes and demonstrated that only one of these subject to specific repression control involving tyrR . By contrast, chromatography of cell extracts with G-75 or G-200 columns revealed a singl-molecular-weight species of shikimate kinase activity with an apparent molecular weight of 20,000 . The levels of shikimate kinase in a series of partial diploid strains indicated that aroL, the structural gene for the tyrR-controlled shikimate kinase enzyme, is located on the E . coli chromosome between the structural genes proC and purE . By means of localized mutagenesis, an aroL mutant of E . coli was isolated . The mutant was an aromatic prototroph and, by the criterion of column chromatography, appeared to have only a single functional species of shikimate kinase enzyme. J Bacteriol, 1979 Jun, 138(3), 671 - 7 Effects of 8-substituted analogs of cyclic adenosine 3',5'-monophosphate on in vivo and in vitro syntheses of beta-galactosidase in Escherichia coli; Ito T et al.; Several 8-substituted alkylthio and alkylamino cyclic adenosine 3',5'-monophosphate (cAMP) derivatives were tested for their ability to stimulate beta-galactosidase synthesis in Estherichia coli in vivo and in vitro and to inhibit the cAMP phosphodiesterase activity of E . coli . Stimulation of beta-galactosidease synthesis in vivo by cAMP derivatives decreased with increasing length of the unbranched carbon chain of the substituent . On the other hand, the stimulation in vitro was increased as the carbon chain elongated . The 8-decylthio- and 8-dodecylthio-cAMP compounds stimulated beta-galactosidase synthesis almost eight-fold compared with cAMP, whereas 8-undecyl-, 8-dodectyl-, and 8-tridecylamino-cAMP stimulated beta-galactosidase synthesis about threefold . However, in in vitro experiments with a phosphodiesterase-deficient strain of E . coli, the Crooks strain, the stimulatory effects of the derivatives disappeared, except for 8-dodecylthio cAMP which stimulated beta-galactosidase about 1.4- to 1.6-fold . All derivatives were quite resistant to hydrolysis by phosphodiesterase . Most derivatives competitively inhibited the hydrolysis of cAMP by phosphodiesterase. Clin Exp Immunol, 1979 Jun, 36(3), 355 - 63 Immunological and purine enzyme studies on hyperuricaemic and normouricaemic patients with Down's syndrome; Watts RW et al.; Hyperuricaemia in Down's syndrome is unreleated to the activity of phosphoribosylamidotransfrease, which catalyses the activity of the first specific step on the purine biosynthetic pathway, and to the activity of hypoxanthine phosphoribosyltransferase and phosphoribosylpyrophosphate synthetase, abnormalities of which are known to be associated with hyperuricaemia . Immunological studies involving serum immunoglobulins, natural E . coli antibodies, test immunization with pneumococcal polysaccharide type III (PnPS), in vitro lymphocyte transformation to mitogens, and pokeweed mitogen (PWM) induced immunoglobulin production showed no difference between hyperuricaemic or normouricaemic Down's patients and institutionalized controls . The Down's patients had higher serum IgA, IgG and IgE, and some also produced more immunoglobulin in PWM-stimulated lymphocyte cultures when compared to normal healthy controls . However, both patients with Down's syndrome and the institutionalized controls had significantly lower responses to PnPs than normal healthy controls . The only deficiency confined to the Down's patients was a signficant depression in delayed hypersensitivity to dinitrochlorobenzene . These findings indicate that the in vivo abnormality of depressed cellular and humoral immunity in Down's patients is not paralleled by in vitro function as measured by PHA lymphocyte transformation and immunoglobulin production by PWM-stimulated lymphocytes . There is also no apparent link between a putative defect in purine metabolism in Down's patients and any immunological abnormalities. Gene, 1979 Jun, 6(2), 173 - 97 Inceptor and origin of DNA replication in lambdoid coliphages . II . The lambda DNA maximal replication system; Lusky M et al.; In pBR313-lambda dv hydrid plasmids a second system for initiation of DNA replication has been detected in lambdoid replicator DNAs (in the absence of the p0 promoter) . The "maximal" (or "maxi") initiation system depends on the origin of replication (ori) sequence, in conjuction with the "inceptor" (ice) element located in the lambdoid cII genes . Only leftward, but not bidirectional, primer RNA synthesis seems to be initiated at ori in its newly defined boundaries, and it appears to be catalysed by dnaG-coded primase . Only if transcriptionally activated, will ori effectively initiate lambda specific, O and P-dependent "maximal" hybrid-plasmid replication . In addition, it will repress a complete lambda "minimal" initiation system in cis, i.e., if present on the same plasmid molecule . This newly discovered repressive activity of the ori system depends on only three factors: an intact left section of ori, the O product, and transcriptional activation of ori (rightward or leftward) . A repressed minimal initiation system will regain its activity as soon as a segment carrying either part of the O gene or a promoter for transcriptional activation is delected from such a plasmid which was combining both the "mini" and "maxi" systems of lambda replication. Gene, 1979 Jun, 6(2), 123 - 36 Construction and analysis of recombinant lambda phages containing mitochondrial DNA fragments; Kobayashi M et al.; Rat mtDNA has a molecular length of about 16 kilobase (kb) pairs and is cleaved into seven fragments by restriction endonuclease EcoRI . These fragments were cloned in Escherichia coli K-12 host using lambda gtWES.lambda B' (lambda gtWES.lambda B, for short, in this paper) as a vector . Recombinant DNAs containing one or a few fragments of the mtDNA were transfected to CaCl2-treated E . coli, and the plaques containing specific recombinant phages were selected . DNA amplified in the recombinanat phage lambda gt.mt was shown to contain the same restriction endonuclease cleavage sites as those found in the mtDNA . Present results permitted the DNA sequencing of any portion of the mitochondrial genome. J Bacteriol, 1979 Jun, 138(3), 923 - 32 Transient growth inhibition of Escherichia coli K-12 by ion chelators: "in vivo" inhibition of ribonucleic acid synthesis; Collins JJ et al.; The ion chelators picolinic acid, quinaldic acid, 1,10-phenanthroline, and 8-hydroxyquinoline, but not ethylenediaminetetraacetate, ethyleneglycol-bis-(beta-aminoethyl ether)-N,N-tetraacetate, or dipicolinic acid, rapidly but transiently arrest growth of Escherichia coli K-12 . Cells adapt and become resistant to growth inhibition by these agents, a process which requires protein synthesis . Mn2+, at low concentrations, decreases the time required for resumption of growth . Proteins synthesized during the lag are quantitatively and qualitatively different from those synthesized during normal growth . Inhibition of growth can explained by an effect on RNA polymerase, a known metalloenzyme. J Bacteriol, 1979 Jun, 138(3), 726 - 30 Use of chlC-lac fusions to determine regulation of gene chlC in Escherichia coli K-12; Fimmel AL et al.; Gene fusions between the lac structural genes and the chlC locus were isolated, and the regulation of lac gene expression was studied . The fused lac genes were induced by nitrate anaerobically and repressed by the presence of oxygen. Surgery, 1979 Jun, 85(6), 638 - 43 The effect of septic shock on skeletal muscle action potentials in the primate; Trunkey DD et al.; Techniques developed for the in vivo study of cellular physiology have been applied to septic shock in primates . Measurements of skeletal muscle transmembrane resting and action potentials were correlated with an analysis of fluid and electrolyte changes in the intracellular and extracellular compartments of skeletal muscle . The data obtained indicated a marked depletion of muscle extracellular water and an increase in intracellular sodium chloride and water content during shock . The significant decrease of resting membrane potential was associated with a decrease in amplitude of the action potential and prolongation of both the repolarization and depolarization time . In addition, there was a decrease of muscle intracellular potassium concentration during shock . This study demonstrates that the alterations in cellular membranes in hemorrhagic shock and septic shock are similar. Infect Immun, 1979 Jun, 24(3), 760 - 9 Isolation and characterization of homogeneous heat-labile enterotoxins with high specific activity from Escherichia coli cultures; Clements JD et al.; The heat-labile enterotoxin (LT) has been isolated in homogeneous form with high specific activity from three sources: cell-free supernatant, NaCl extract, and whole-cell lysates of an enterotoxigenic Escherichia coli strain . In vitro immunological assays were used in lieu of tedious and highly variable bioassays to recognize fractions with activity . This revealed that the major portion of the LT remained adherent to columns containing agarose, from which it could be eluted quantitatively in practically homogeneous form by galactose . Isolated LT has remarkable similarities to the cholera enterotoxin (choleragen) in both subunit structure and amino acid composition, although there are also notable differences in these two enterotoxins, which are related immunologically and by mode of action . Unlike choleragen, in which the A region is totally nicked, E . coli LT, depending on its source, is activated by proteolytic processing . The activity of LT is equivalent to that of choleragen in bioassays on adrenal cells, in rabbit skin, and in rabbit ileal loops, especially when, depending on the source of material, the LT has been activated by treatment with trypsin . The whole-cell lysate is the richest source of LT. Can J Biochem, 1979 Jun, 57(6), 822 - 33 The reactions of Escherichia coli citrate synthase with the sulfhydryl reagents 5,5'-dithiobis-(2-nitrobenzoic acid) and 4,4'-dithiodipyridine; Talgoy MM et al.; Citrate synthase of Escherichia coli reacts rapidly with 1 equivalent of Ellman's reagent, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), per subunit, losing completely its sensitivity to the allosteric inhibitor, NADH . When the enzyme is treated instead with 4,4'-dithiodipyridine (4,4'-PDS), all activity is lost . Certain evidence in this paper is consistent with the belief that the sulfhydryl group modified by DTNB, and that whose modification by 4,4'-PDS inactivates the enzyme, are the same . (i) Both reagents abolish NADH fluorescence enhancement by the enzyme . (ii) Saturating levels of NADH and some other adenylic acid derivatives inhibit the reactions with both reagents . (iii) When the enzyme is modified with one equivalent of DTNB or 4,4'-PDS, subsequent reactivity toward the other reagent is greatly decreased . (iv) Following modifications, the DTNB and 4,4'-PDS derivatives spontaneously lose thionitrobenzoate (TNB) or pyridine-4-thione (PT), respectively, in reactions which are thought to involve displacement of TNB or PT by a second enzyme sulfhydryl group, so that an enzyme disulfide is introduced . The introduction of the disulfide bond, if this is what occurs, does not lead to cross-linking of citrate synthase polypeptide chains, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing conditions . Certain evidence has also been found, however, that the sites of modification by DTNB and 4,4'-PDS are not the same . (i) DTNB modification desensitizes to NADH but does not inactivate, while 4,4'-PDS inactivates at least 99.9% . (ii) The presumed disulfide from elimination of TNB is also active, while that from PT modification is no more active than the original 4,4'-PDS modified product . (iii) Prior modification of the enzyme with DTNB affords no protection against later inactivation by 4,4'-PDS . The studies therefore indicate a close relationship between the DTNB desensitization and 4,4'-PDS inactivation, but they are unable to identify it exactly . Other properties of the DTNB reaction are also described, and a hypothesis is offered to explain quantitatively the finding that desensitization lags behind modification during the modification of citrate synthase by DTNB. J Bacteriol, 1979 Jun, 138(3), 969 - 75 Structure of common pili from Escherichia coli; McMichael JC et al.; Several important properties of the common pili from Escherichia coli are discussed . These pili were resistant to the gentle Folin-Ciocalteau reagent methods for protein detection and were not readily solubilized by sodium dodecyl sulfate . They were found to contain a reducing sugar but not peptidoglycan . The pilin had multiple conformations in sodium dodecyl sulfate solution, and the appearance of multiple bands on sodium dodecyl sulfate gels did not necessarily indicate heterogeneity of the preparation . The ilus subunit was found to be a different protein than outer membrane III, which has the same apparent molecular weight . In addition, we conformed the results of Brinton (Trans . N.Y . Acad . Sci 27:1003-1054, 1965): that there is a dramatic change in the properties of pili after they are heated at pH values below 2. Virchows Arch A Pathol Anat Histol, 1979 May 31, 382(2), 179 - 89 Extensive hepatic cell necrosis produced by the Shwartzman mechanism; Mori W et al.; Acute, severe, and extensive necrosis of the liver was produced in pregnant and non-pregnant female adult rabbits by the Shwartzman mechanism . Shwartzman reagent (E . coli endotoxin) was administered in various combinations by three routes of injection, the portal vein (mesenteric vein), the bile duct, and the ear vein . Morphologic changes of the extrahepatic organs were minimal . The similarity to massive necrosis in human liver and the effect of pregnancy on hepatic necrosis in rabbit and man were discussed . The lesion is presented as a new animal model for acute massive hepatic necrosis and is proposed as a third category of Shwartzman reaction, designated the univisceral type. Ann N Y Acad Sci, 1979 May 31, 320, 518 - 34 Quantification of total IgM and IgG and specific IgM and IgG to a thymus-independent (LPS) and a thymus-dependent (tetanus toxoid) antigen in the rat by enzyme-linked immunosorbent assay (ELISA); Vos JG et al.; Rat IgM and IgG was determined by mechanized "sandwich" enzyme-linked immunosorbent assay (ELISA) using peroxidase labeled anti-rat-IgM and -IgG . Linear ranges in standard curves of a reference rat serum had a slope similar to the slopes found with sera of 25 rats of various age . IgM and IgG measurements by ELISA in these sera correlated well with results obtained by single radial immuno-diffusion (SRID) . In addition, the precision of the enzyme immunoassay was the same as obtained with the SRID . Compared with SRID, ELISA is less time consuming and the amount of antiserum used in the macro-ELISA is one order of magnitude lower; and again 10 times lower in the mechanized micro-ELISA that is currently being developed . In conclusion, the ELISA is a specific, reliable, sensitive, and economic method for routine measurement of rat serum IgG and IgM e.g . in toxicity studies . In the second part of this study, ELISA and the passive hemagglutination test were compared to determine the primary and secondary antibody response to E . coli lipopolysaccharide (LPS) and tetanus toxoid in rats . In the ELISA, the antigens were bound to the wells of polystyrene microplates . Tetanus toxoid was coated directly, LPS after complexing with methylated bovine serum albumin . After incubation with dilutions of the rat sera, the amount of antibody bound to the solid phase was quantified by means of peroxidase-labeled antiimmunoglobulin . The specificity of the enzyme immunoassay was tested by absorption of the sera with the respective antigens . ELISA proved to be more sensitive than the hemagglution reaction, except when titers were determined during the secondary response to tetanus toxoid . Besides its specificity and sensitivity, ELISA is a convenient method for measuring both IgM and IgG antibodies . Finally, evidence is presented that in the rat, the humoral immune response to LPS is a thymus-independent phenomenon . Thus, by using the antibody response to LPS and tetanus toxoid in function studies of the immune system of the rat, insight can be obtained in the thymus-independent and thymus-dependent humoral immune response. Biochemistry, 1979 May 29, 18(11), 2139 - 45 Evidence for a conformational change in the Escherichia coli maltose receptor by excited-state fluorescence lifetime data; Zukin RS; The initial signaling event during maltose chemoreception in Escherichia coli is identified with a delocalized liqand-induced conformational change in the maltose binding protein . Substantiation for the conformational change involves a new application of the "distant reporter group technique" {Zukin, R.S., Hartig, P.R., & Koshland, D.E., Jr . (1977a) Proc . Natl . Acad . Sci . U.S.A . 74, 1932-1936} utilizing excited-state fluorescence lifetime measurements . Binding of maltose to its receptor results in changes in the microenvironment of the two tryptophan residues of the receptor protein and of an experimentally attached reporter group, 5-(iodoacetamido) fluorescein . The minimum distance between the two typtophans from efficiency of fluorescence energy transfer theory is 17 A; the minimum distance from the farther tryptophan to the fluorescein is 50 A . Thus, the maltose receptor is shown to undergo molecular rearrangements at distant sites upon ligand binding . The general feature of conformational change as the initial signaling event during chemoreception in the enteric bacteria is discussed. J Biol Chem, 1979 May 25, 254(10), 4245 - 52 Purification of the o-dianisidine peroxidase from Escherichia coli B . Physicochemical characterization and analysis of its dual catalatic and peroxidatic activities; Claiborne A et al.; Extracts of aerobically grown Escherichia coli B exhibit both catalase and dianisidine peroxidase activities . Polyacrylamide gel electrophoresis demonstrates two distinct catalases which have been designated hydroperoxidases I and II (HP-I and HP-II) in order of increasing anodic mobility . HP-I has been purified to essential homogeneity and found to be composed of four subunits of equal size . Its molecular weight is 337,000, and it contains two molecules of protoheme IX per tetramer . Its amino acid composition is unusual, for so large a protein, in lacking half-cystine . HP-I is a very efficient catalase with an activity optimum at pH 7.5, a Km for H2O2 of 3.9 mM, and a turnover number of 9.8 x 10(5) per min . It is also a broad specificity peroxidase capable of acting upon dianisidine, guaiacol, p-phenylenediamine, and pyrogallol . Dianisidine acted as a powerful reversible inhibitor of the catalatic activity of HP-I and as a suicide substrate when HP-I functioned in its peroxidatic mode. J Biol Chem, 1979 May 25, 254(10), 4009 - 14 Oxygen-sensitive and -insensitive nitroreduction by Escherichia coli and rat hepatic microsomes; Peterson FJ et al.; Nitrofurazone is shown to undergo an initial 1-electron (oxygen-sensitive) or 2- or more electron (oxygen-insensitive) reduction by partially purified nitroreductases from Escherichia coli . Nitrofurazone (50 micronM) is reduced by the oxygen-sensitive reductase to a nitro anion free radical as indicated by ESR and visible spectroscopy . The visible spectrum of the nitro anion free radical is characterized by an increase in absorption at 406 nm . In the presence of the oxygen-sensitive reductase, nitrofurazone stimulates superoxide formation and oxygen consumption . This enzyme gives a steady state radical concentration which is proportional to the square root of the enzyme concentration, suggesting that the nitrofurazone anion radical is an obligate intermediate in the reduction and that the radical decays by a nonenzymatic second order process . The oxygen-insensitive reductase does not form the nitro anion free radical nor in the presence of nitrofurazone does it stimulate oxygen consumption . Visible spectroscopy shows that nitrofurazone is reduced by the oxygen-sensitive reductase to a species with an absorption maximum at 335 nm, which has been previously identified as the amine . The oxygen-insensitive reductase reduces nitrofurazone to a previously identified cyano derivative with an absorption maximum at 280 nm . Rat hepatic microsomes appear to metabolize nitrofurazone in a manner similar to the oxygen-sensitive E . coli reductase. J Biol Chem, 1979 May 25, 254(10), 3730 - 7 Nucleoside transport in cells and membrane vesicles from Escherichia coli K12; Munch-Petersen A et al.; Osmotic shock treatment of cells of Escherichia coli K12 caused a reduction in the transport of nucleosides into the cells . The strains used carried mutations in the nucleoside catabolizing enzymes . This indicated that the decrease in transport capacity was not due to loss of these enzymes during the shock treatment . Membrane vesicles, prepared from the same strains, showed a limited transport of cytidine, deoxycytidine, and uridine . Transport of purine nucleosides and of thymidine was very low in vesicles lacking the appropriate nucleoside phosphorylases and no significant stimulation was observed if the nucleoside phosphorylases were present in the membrane vesicles . These results all indicate that components outside the cytoplasmic membrane are important for nucleoside transport . Selection for resistance to fluorodeoxycytidine yielded mutants which were unable to transport any nucleoside, even when the nucleoside phosphorylases were present in high amounts . This finding is consistent with a requirement for a specific transport process prior to the initial enzymatic attack on the incoming nucleoside. J Biol Chem, 1979 May 25, 254(10), 3873 - 8 The structure of the RNA binding site of ribosomal proteins S8 and S15; Muller R et al.; Proteins S8 and S15 from the 30 S ribosomal subunit of Escherichia coli were bound to 16 S RNA and digested with ribonuclease A . A ribonucleoprotein complex was isolated which contained the two proteins and three noncontiguous RNA subfragments totaling 93 nucleotides, that could be unambiguously located in the 16 S RNA sequence . We present a secondary structural model for the RNA moiety of the binding site complex, in which the two smaller fragments are extensively base-paired, respectively, to the two halves of the large fragment, to form two disconnected duplexes . Each of the two duplexes is interrupted by a small internal loop . This model is supported by (i) minimum energy considerations, (ii) sites of cleavage by ribonuclease A, and (iii) modification by the single strand-specific reagent kethoxal . The effect of protein binding on the topography of the complex is reflected in the kethoxal reactivity of the RNA moiety . In the absence of the proteins, 5 guanines are modified; 4 of these, at positions 663, 732, 733, and 741, are strongly protected from kethoxal when protein S15 is bound. J Biol Chem, 1979 May 25, 254(10), 3761 - 4 Immunological studies on 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase isoenzymes; McCandliss RJ et al.; An apparently homogeneous preparation of the phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase isoenzyme from Escherichia coli was used as the antigen for antibody production in New Zealand white rabbits . The antibodies were monospecific as judged by immunodiffusion and immunoelectrophoresis . Antigen . antibody complexes maintained full enzyme activity and were inhibited by phenylalanine, indicating that neither the active site nor the feedback-inhibitor binding site is mechanistically connected to amino acid sequences which are antigenic determinants . While phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase could be quantitatively removed from solution by immunoprecipitation with soluble or immobilized antibodies, neither the tyrosine-sensitive nor the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, the other two isoenzymes catalyzing the first step in the biosynthesis of aromatic compounds, formed any detectable complexes with the antibodies . This indicated less structural similarity than would be expected for isoenzymes . Also, the antibodies did not cross-react with 5-dehydroquinate synthase, the enzyme catalyzing the second step of the common aromatic biosynthetic pathway. Biochim Biophys Acta, 1979 May 24, 562(3), 534 - 45 Fractionation and structural elucidation of the active components of aurintricarboxylic acid, a potent inhibitor of protein nucleic acid interactions; Gonzalez RG et al.; Commercially available, as well as synthetically prepared, samples of aurintricarboxylic acid (a widely employed potent inhibitor of protein nucleic acid interactions) consist mostly of a heterogeneous collection of polymers, as shown by fractionation schemes utilizing both dialysis and ultrafiltration, and by molecular weight measurements . 13C-NMR studies suggest that the polymeric material is of the phenol-formaldehyde type; inhibitory assays that depend on the formation of a protein-nucleic acid complex revealed that potency varied directly with the molecular weight of the polymer . Fractions of molecular weight 400 were essentially inactive. Biochim Biophys Acta, 1979 May 24, 562(3), 418 - 28 The effect of differential methylation by Escherichia coli of plasmid DNA and phage T7 and lambda DNA on the cleavage by restriction endonuclease MboI from Moraxella bovis; Dreiseikelmann B et al.; The nucleotide sequence recognized and cleaved by the restriction endonuclease MboI is 5' GATC and is identical to the central tetranucleotide of the restriction sites of BamHI and BglII . Experiments on the restriction of DNA from Escherichia coli dam and dam+ confirm the notion that GATC sequences are adenosyl-methylated by the dam function of E . coli and thereby are made refractory to cleavage by MboI . On the basis of this observation the degree of dam methylation of various DNAs was examined by cleavage with MboI and other restriction endonucleases . In plasmid DNA essentially all of the GATC sequences are methylated by the dam function . The DNA of phage lambda is only partially methylated, extended methylation is observed in the DNA of a substitution mutant of lambda, lambda gal8bio256, and in the lambda derived plasmid, lambdadv93, which is completely methylated . In contrast, phage T7 DNA is not methylated by dam . A suppression of dam methylation of T7 DNA appears to act only in cis dam . A suppression of dam methylation of T7 DNA appears to act only in cis since plasmid DNA replicated in a T7-infected cell is completely methylated . The results are discussed with respect to the participation of the dam methylase in different replication systems. Biochim Biophys Acta, 1979 May 24, 562(3), 400 - 17 Replication of the linear mitochondrial DNA of Tetrahymena pyriformis; Goldbach RW et al.; 1 . Electron micrographs of the linear mtDNA from Tetrahymena pyriformis strain GL show linear molecules with a duplex 'eye' of variable size in the middle . This indicates that replication of this DNA starts near the middle of the molecule and proceeds bidirectionally to the ends, as previously shown for the mtDNA of strain ST (Arnberg, A.C., Van Bruggen, E.F.J., Clegg, R.A., Upholt, W.B . and Borst, P . (1974) Biochim . Biophys . Acta 361, 266-276) . The mtDNAs of these two strains have little base sequence homology beyond the ribosomal RNA cistron (Goldbach, R.W., Bollen-De Boer, J.E., Van Bruggen, E.F.J . and Borst, P . (1978) Biochim . Biophys . Acta 521, 187-197) . 2 . Electron micrographs of mtDNA from strain ST, spread under non-denaturing conditions, contain only molecules with fully duplex ends . mtDNA spread under conditions of early denaturation contains duplex loops on one end (40% of all molecules) or both ends (37%) . The loops are stable to partial denaturation and vary in size from 0.15 to approximately 1.0 micron, most loops measuring 0.25--0.40 micron . No loops are formed with single-stranded DNA under analogous conditions and we conclude from this result that loop formation is based on the presence of straight, rather than inverted, duplications near the ends . 3 . When full-length 3H-labelled mtDNA from strain ST, 32P-labelled at the 5'-termini with T4 polynucleotide kinase, was sedimented in alkaline sucrose gradients, greater than 70% of the 3H and less than 30% of the 32P cosedimented with full-length molecules; the remaining 32P sedimented heterogeneously and predominantly with the DNA less than 10% the size of intact single strands . Brief incubations of full-length mtDNA with DNA polymerase I from Escherichia coli and labelled dNTPs at 15 degrees C did not lead to preferential labelling of terminal EcoRI fragments of the DNA . From these results we infer that the DNA contains nicks or gaps near the termini and that these are not bordered by free 3'-OH groups . 4 . A model is presented in which straight sequence repetitions at the termini of Tetrahymena pyriformis mtDNA are involved in the later stages of replication . This model can also account for the pronounced terminal heterogeneity previously observed in this DNA. Biochim Biophys Acta, 1979 May 24, 562(3), 527 - 33 Polynucleotides XLVII . Synthesis and properties of poly(2-methylthio- and 2-ethylthioadenylic acid) . Formation of non-Watson-Crick type complexes; Fukui T et al.; Poly(2-methyl- and 2-ethylthioadenylic acid) were prepared by polymerization of corresponding diphosphates with Escherichia coli polynucleotide phosphorylase . These polynucleotides have relatively large hypochromicity of 30-35% . Acid titration of these polymers showed abrupt transition at pH 5.34-5.4, which may indicate that the introduction of alkylthio group at 2-position of adenine bases reduced their basicity . Thermal melting of these polymers showed no clear transition points at neutral pH, but in acidic media they have Tm values of 57 and 56 degrees C, somewhat lower than that of poly(A) . Upon complex formation with poly(U), these poly(A) analogs showed only one poly(rs2A) . poly(U) type double-strand complexes, similar to that found in the case of poly(m2A) . poly(U). Mol Gen Genet, 1979 May 23, 173(1), 39 - 50 Expression of Escherichia coli ribosomal protein and RNA polymerase genes cloned on plasmids; Fiil NP et al.; Fragments of lambda drifd 18 DNA with different end-points within the set of structural genes of ribosomal proteins L11 (RPLK), Li (rplA), L10 (rplJ) and L12 (rplL) as well as the beta (rpoB) ANd beta' (rpoC) subunits of RNA polymerase have been cloned on plasmids . These plasmids were transformed in host cells which were mutant for each of the genes, enabling expression of both wild-type (plasmid-borne) and mutant (chromosomal) genes to be differentiated . On the basis of these results we propose the following genetic structure for the region: rplK and rplA are in one operon; rplL, rpoB and rpoC are in a second . Our data suggest the possibility that rplJ is by itself in an operon situated between the other two. Mol Gen Genet, 1979 May 23, 173(1), 15 - 21 Development of a system useful for studying the formation of unstable alleles of IS2; Peterson PA et al.; IS2-induced deletions of the gal control region were isolated in a plasmid carrying gal OP-308::IS2-7 . This contains a 54 basepair long, unstable mini insertion within IS2, thus allowing constitutive expression of the gal structural genes . Deletion PPI is 11.9 kilobasepairs (kb) long and is Gal+ because it has retained the mini insertion . In PP4 7.2 kb DNA material including markers gal OP, chlD and pgl are deleted . PP4 has lost the mini insertion and is therefore Gal negative . DNA sequencing of the newly formed junction in PP4 reveals that the deletion terminates precisely at nucleotide 1 of IS2 and that no DNA sequence homology is involved in this IS2-mediated deletion formation . PPI segregates Gal- clones due to the loss of the mini insertion . One such segregant PPIS and PP4 both give only constitutive Gal+ revertants, which consist of the previously known mini insertions and also a new class of "supermini" inserts within IS2 of about 10 to 20 basepairs long . Therefore, PPIS and PP4 can be used to study various parameters involved in the formation of mini insertions. Biochim Biophys Acta, 1979 May 23, 578(1), 188 - 95 Manual solid phase sequence analysis of polypeptides using 4-N-N,-dimethylaminoazobenzene 4'-isothiocyanate; Chang JY; A manual solid-phase method for sequence analysis of polypeptides is described . The immobilized polypeptide was subjected to stepwise degradation by Edman-type reagent, using the 4-N,N-dimethylaminoazobenzene 4'-isothiocyanate phenyllisothiocyanate double coupling method . The N-terminal amino acids were released (after conversion reaction) as 4-N,N-dimethylaminoazobenzene 4'-thiohydantoin (identified by thin layer chromatography) and phenylthiohydantoin derivatives . The method required 2--10 nmol polypeptide. Biochim Biophys Acta, 1979 May 23, 578(1), 31 - 40 NADP-specific isocitrate dehydrogenase of Escherichia coli . IV . Purification by chromatography on Affi-Gel Blue; Vasquez B et al.; Affinity chromatography on Affi-Gel Blue has been used to purify the NADP-specific isocitrate dehydrogenase (EC 1.1.1.42) from Escherichia coli . The protocol permits rapid purification of the enzyme in milligram quantities with a yield of 50% and is carried out almost entirely at room temperature . The preparation was judged to be homogeneous by non-denaturing electrophoresis at pH 7.5 and denaturing electrophoresis in the presence of sodium dodecyl sulfate . The subunit molecular weight of 53 000, determined by sodium dodecyl sulfate gel electrophoresis, is in reasonable agreement with the value of 46 900 estimated from the amino acid composition data. J Chromatogr, 1979 May 21, 173(2), 281 - 98 Major and modified nucleosides in tRNA hydrolysates by high-performance liquid chromatography; Davis GE et al.; We describe a high-performance liquid chromatographic analytical method that can be readily placed in operation, and which is particularly well suited to scientists investigating tRNA structure, biosynthesis, and function, and for the determination of major and modified nucleosides of tRNA . The method is characterized by the following features: (1) Sensitivity at the nanogram level; (2) High chromatographic resolution and selectivity; (3) Direct measurement of nucleosides with accuracy and precision; (4) Analysis is non-destructive and the high capacity of this chromatographic system allows easy isolation of pure nucleosides for further characterization; (5) Rapid separation and measurement in ca . 1 h after hydrolysis to nucleosides; and (6) Quantitation without use of radiolabeled compounds; however, labeled compounds are readily isolated and measured. Biochim Biophys Acta, 1979 May 17, 553(2), 224 - 34 Nature of the regions involved in the insertion of newly synthesized protein into the outer membrane of Escherichia coli; de Leij L et al.; Outer membrane proteins are synthesized by cytoplasmic membrane-bound polysomes, and inserted at insertion sites which cover about 10% of the total outer membrane when cells grow with a generation time of 1 h . A membrane fraction enriched in outer membrane insertion regions was isolated and partly characterized . The rat at which newly inserted proteins are transferred from such insertion regions into the rest of the outer membrane was found to be very fast; the new protein content of insertion regions and that of the remaining outer membrane equilibrate completely within about 20 s at 25 degrees C . Given the rather rigid structure of the outer membrane and the multiple interactions between outer membrane components and the murein layer, lateral diffusion of newly inserted proteins from insertion sites to the remaining outer membrane is not likely to explain this rapid equilibration . Instead, the data support a model in which insertion regions move along the cell surface, leaving behind stationary, newly inserted outer membrane proteins. Eur J Biochem, 1979 May 15, 96(2), 301 - 9 The synthesis of chloroplast high-molecular-weight ribosomal ribonucleic acid in spinach; Hartley MR et al.; Illuminated suspensions of chloroplasts isolated from young spinach leaves show incorporation of {3H}uridine into several species of RNA . One such RNA species of Mr 2.7 x 10(6) shows sequence homology with both the chloroplast 23-S rRNA (Mr = 1.05 x 10(6)) and 16-S rRNA (Mr = 0.56 x 10(6)), as judged by DNA/RNA competition hybridization . Leaves labelled in vivo with {32P}orthophosphate in the presence of chloramphenicol accumulate labelled RNAs of Mr 1.28 x 10(6), 0.71/0.75 x 10(6) and 0.47 x 10(6) . The 1.28 x 10(6)-Mr RNA shows 80.5% sequence homology with the 1.05 x 10(6)-Mr rRNA and the 0.71/0.75 x 10(6)-Mr RNA mixture shows 76% sequence homology with the 0.56 x 10(6)-Mr rRNA . We conclude that the pathway of rRNA maturation in spinach chloroplasts is similar to that of Escherichia coli. Eur J Biochem, 1979 May 15, 96(2), 403 - 16 Affinity chromatography of tryptophan synthase from Escherichia coli . Systematic studies with immobilized tryptophanol phosphate; Gschwind HP et al.; Inhibition studies and affinity chromatography indicate that derivatives of tryptophanol phosphate are suitable ligands for the affinity chromatography of tryptophan synthase . A phenyl group on the spacer arm strengthens the interaction of immobilized tryptophanol phosphate with the enzyme . The alpha 2 beta 2 complex specifically requires the presence of 0.3--0.5 M phosphate ions for binding . The alpha subunit binds in dilute Tris buffer, but its binding is also enhanced by the presence of phosphate ions . The beta 2 subunit binds unspecifically but strongly to the affinity material and to a variety of other immobilized hydrophobic ligands . Binding studies with suspensions of affinity material show that the alpha subunit interacts rapidly and reversibly . Indoleglycerol phosphate and indolepropanol phosphate release bound alpha 2 beta 2 complex and alpha subunit in a competitive manner, indicating that the interaction occurs biospecifically, i.e . via the active site of alpha subunit . L-Serine is a non-competitive inhibitor of binding . These results are discussed with regard to the composite-active-site hypothesis {T . E . Creighton (1970) Eur . J . Biochem, 13, 1--10} . Both the alpha subunit and the alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli can be obtained with high yields and in homogenous form by absorption to the affinity material from partially purified preparations . Elution is achieved with linear gradients either of indolepropanol phosphate or of indoleglycerol phosphate or, in the case of the complex, of L-serine . At the low concentrations of the complex found in crude extracts of wild-type E . coli cells, the unexpectedly high affinity of the beta 2 subunit for hydrophobic ligands leads to partial dissociation of the complex. Biochemistry, 1979 May 15, 18(10), 2103 - 12 Study of the cytoplasmic and outer membranes of Escherichia coli by deuterium magnetic resonance; Davis JH et al.; The cytoplasmic and outer membranes of Escherichia coli were studied between 0 and 40 degrees C by deuterium magnetic resonance quadrupolar echo spectroscopy . The L51 strain of E . coli was used to incorporate perdeuterated palmitic acid into the membrane phospholipids . The cytoplasmic and outer membranes were separated using standard techniques . The spectrum of each membrane preparation was dominated at high temperatures (greater than or equal to 37 degrees C) by the characteristic liquid-crystalline plateau previously observed for perdeuterated palmitate chains in model phospholipid membranes . At low temperatures, the shape and width of the spectrum were characteristic of the gel phase . The relative intensities of the liquid-crystalline and gel features varied systematically with temperature . A quantitative analysis of the acyl chain orientational order was carried out by using the method of moments . The orientational order at each temperature was greater in the outer membrane sample than in that of the cytoplasmic membrane, indicating that the liquid-crystalline-gel transition region in the outer membrane is shifted to higher temperatures than that of the cytoplasmic membrane by about 7 degrees C . It is clear from the results that most of the phospholipid molecules participate in the phase transition. Biochemistry, 1979 May 15, 18(10), 2028 - 38 Phenylalanyl-tRNA synthetase of Escherichia coli K 10 . Multiple enzyme-aminoacyl-tRNA complexes as a consequence of substrate specificity; Guntner C et al.; The interaction between Phe-tRNA(Phe) or other acyl-tRNA derivatives thereof and phenylalanyl-tRNA synthetase of Escherichia coli K 10 has been investigated by nonequilibrium dialysis, by fluorescence titration in the presence of 2-p-toluidinylnaphthalene-6-sulfonate, by the kinetics of the aminoacylation of tRNA(Phe), and by the kinetics of the catalytic hydrolysis of Phe-tRNA(Phe) . Phe-tRNA(Phe), or derivatives thereof, forms two types of complexes with the synthetase . One type involves the attachment of the phenylalanyl moiety to the phenylalanine-specific site of the enzyme, and the other type, to the tRNA(Phe)-specific binding site . They resemble alternative modes of a destabilized enzyme-product complex and are predicted on the basis of thermodynamic considerations . The two modes of binding of acyl-tRNA compete with each other . The attachment of Phe-tRNA(Phe) to the phenylalanine-specific site dominates . At equilibrium, this complex is present at a fourfold higher concentration than the other type of complex . The HNO2 deaminated Phe-tRNA(Phe) binds exclusively to the site specific for L-phenylalanine . On the contrary, Ile-tRNA(Phe) adds at 94.1% to the tRNA(Phe)-specific site . The association of Phe-tRNA(Phe) with this site leads to enzymatic hydrolysis into L-phenylalanine and tRNA(Phe) . The complex involving the phenylalanine-specific site is hydrolytically unproductive . L-Phenylalanine acts as an activator of the hydrolysis by occupying the amino acid specific site and by shifting the equilibrium between the complexes toward the binding ot Phe-tRNA(Phe) at the tRNA(Phe)-specific site . The association of Phe-tRNA(Phe) at the phenylalanine-specific site does not interfere sterically with the binding of free tRNA(Phe) . The sequential addition of free and aminoacylated tRNA(Phe) exhibits negative cooperativity . Such a mechanism could help to expel the product from the enzyme. Biochemistry, 1979 May 15, 18(10), 2019 - 27 Conformation of Escherichia coli ribosomal protein L7/L12 in solution: hydrodynamic, spectroscopic, and conformation prediction studies; Luer CA et al.; The conformation of Escherichia coli ribosomal protein L7/L12 in solution has been studied using spectroscopic and hydrodynamic methods . Circular dichroism studies in the near-ultraviolet region reveal two bands at 262 and 268 nm originating from the tertiary conformational environment of the phenylalanyl residues . Additional characterization of the phenylalanine environment includes an intrinsic fluorescence emission spectrum arising from the phenylalanine fluorophores . Computer analysis of the far-ultraviolet circular dichroism spectrum suggests that L7/L12 contains as much as approximately 76% alpha helix . Hydrodynamic properties of L7/L12, measured with the purpose of providing relevant shape information, include the frictional coefficient ratio (1.84 +/- 0.03) and intrinsic viscosity (28 +/- 0.4 mL/g) . The experimentally determined frictional coefficient (6.15 +/- 0.15 X 10(-8) has been compared with theoretical calculations of the same value employing two independent methods and assuming various dimensions for the L7/L12 dimer . Combining the experimental results from this work with those available from the literature, and using conformation predictive methods of Chou & Fasman {P . Y . Chou & G . D . Fasman (1974) Biochemistry 13, 211-222, 222-245} and of Maxfield & Scheraga (F . R . Maxfield & H . A . Scheraga (1976) Biochemistry 15, 5138-5153), several possible molecular models of the L7/L12 dimer have been constructed and critically examined . A model which is consistent with all of the available data is proposed. Biochemistry, 1979 May 15, 18(10), 1979 - 84 Subunit interaction in tryptophan synthase of Escherichia coli: calorimetric studies on association of alpha and beta 2 subunits; Wiesinger H et al.; Association of the apo-beta 2 and the holo-(beta-PLP)2 subunits of tryptophan synthase from Escherichia coli (L-serine hydro-lyase (adding indole) (EC 4.2.1.20)) with alpha subunits of the same enzyme has been studied by microcalorimetry . The results obtained from thermometric titrations clearly demonstrate that only the native complex alpha2beta 2 is formed, independent of an excess of alpha protein . The reaction of the holo-(beta-PLP)2 with alpha subunits at 25 degrees C is accompanied by a negative enthalpy change, which is almost twice as large as that for complex formation with the apo-beta 2 protein, thus indicating that the interaction enthalpy becomes more favorable in the presence of the coenzyme pyridoxal 5'-phosphate (PLP) . Both reaction enthalpies show very large negative temperature coefficients, -3600 +/- 100 cal K-1 (Mol of beta 2)-1 being the value for the formation of the apoenzyme and -2300 +/- 100 cal K-1 (mol of beta 2)-1 pertaining to formation of the holoenzyme . The studies on the association of alpha and beta2 subunits in the two buffers revealed that at 25 degrees C approximately 0.75 proton are absorbed in the presence and absence of the coenzyme, whereas at 35 degrees C one proton is taken up from the solution when PLP is present, but two if the apo-beta 2 complex reacts . These results are a clear indication of energetic linkage between intersubunit interaction, hydrogen ion equilibria, and the binding of the coenzyme. Biochemistry, 1979 May 15, 18(10), 2038 - 44 RNA replication: required intermediates and the dissociation of template, product, and Q beta replicase; Dobkin C et al.; Replication complexes containing only one molecule of Q beta replicase and one strand of midivariant RNA (MDV-1 RNA) template were prepared by incubating the replicase with an excess of MDV-1 (-) RNA . In the presence of excess minus strands, these monoenzyme replication complexes were shown to synthesize essentially pure MDV-1 (+) RNA in both the first and second cycles of replication . When an equivalent concentration of mutant MDV-1 (-) RNA was added to this reaction before completion of the first cycle of replication, only wild-type MDV-1 (+) RNA was produced in the first cycle, but both mutant and wild-type MDV-1 (+) RNA were produced in the second cycle of replication . These results demonstrate that a monoenzyme complex is competent to synthesize RNA and, therefore, that a multienzyme replication complex is not a necessary intermediate of replication . The data also imply that after the completion of chain elongation, the product strand is released from the replication complex and that the template and the replicase then dissociate. Biochemistry, 1979 May 15, 18(10), 2012 - 9 Properties of Escherichia coli 16S ribosomal ribonucleic acid treated with 4,5',8-trimethylpsoralen and light; Karathanasis SK et al.; 16S rRNA reacted with the furocoumarin 4,5',8-trimethylpsoralen (trioxsalen) and 360-nm light showed a number of chemical and physical differences from untreated RNA . After extensive irradiation, five molecules of trioxsalen were bound per molecule of RNA . The trioxsalen-treated RNA had an altered ultraviolet absorption spectrum and a distinctive fluorescence emission spectrum . The modified RNA was significantly more resistant to T1 ribonuclease digestion than was control RNA . Treated RNA, when mixed with purified ribosomal proteins, was not functional in the in vitro reconstitution of 30S subunits and yielded more slowly sedimenting particles which were inactive in protein synthesis assays . By contrast, 16S rRNA within the 30S subunit structure did not exhibit these changes when reacted with the same dose of trioxsalen and light, suggesting that the ribosomal proteins were effective in protecting the RNA from interaction with the drug. Biochim Biophys Acta, 1979 May 10, 568(1), 234 - 42 Tritium exchange reactions catalyzed by 2-oxo-4-hydroxyglutarate aldolase from Escherichia coli