Biochim Biophys Acta, 1977 Feb 16, 474(4), 629 - 38 Interaction of DNA with DNA binding proteins . II . Displacement of Escherichia coli DNA unwinding protein and the condensed structure of DNA complexed with protein HD; Zentgraf H et al.; Three DNA binding proteins from Escherichia coli cells have been complexed with single-stranded phage fd DNA . Electron microscopy reveals granular substructures in the complexes formed with protein HD . In complexes of DNA unwinding protein with fd DNA both protein HD and phage-coded gene 5 protein partially displace the unwinding protein which results in the formation of structures characteristic for the DNA complexes formed with either protein HD or gene 5 protein alone . Combination of protein HD with double-stranded phage T7 DNA leads to a progressive folding and condensing of the genome . The structures observed are discussed in relation to current concepts of the packing of DNA in protein complexes. Biochim Biophys Acta, 1977 Feb 16, 474(4), 609 - 18 A specific polyadenylase from Escherichia coli K12; Antoniades D et al.; A polyadenylase, degrading specifically poly(A) sequences was isolated from Escherichia coli K12 . The enzyme was purified about 850 times to practically electrophoretic homogeneity . It was free of poly(A) polymerase activity, as well as of the well known E . coli RNAases I and II . It is stimulated by bivalent cations like Mg2+ and Mn2+ and splits poly(A) to 3'-AMP and therefore it can be considered as an exonuclease . The enzyme does not degrade any other ribohomopolymer or RNA. Mol Gen Genet, 1977 Feb 15, 150(3), 325 - 8 Relative order of glg mutations affecting glycogen biosynthesis in Escherichia coli K12; Latil-Damotte M et al.; Mutations affecting the glycogen biosynthesis in E . coli can be mapped at three different loci, glg A, glg B and glg C lying between asd and mal A . Transduction tests suggest the following order for the genes in this region: mal A--glg A--glg C--glg B--asd. Mol Gen Genet, 1977 Feb 15, 150(3), 317 - 24 Escape synthesis of RNA polymerase subunits and termination factor rho following induction of prophage lambda in Escherichia coli; Nakamura Y et al.; Synthesis of RNA polymerase subunits and of transcription termination factor p was studied after thermoinduction of prophage lambdac1857 located at several unusual sites on the chromosome of Escherichia coli . When a lysogen carrying the prophage at the bfe gene was induced at 42 degrees C, the rate of synthesis of core polymerase subunits (alpha, beta and beta') rapidly decreased, followed by a marked increase after about 10 min . The latter increase was observed specifically in the "bfe lysogen" and not in any of the other lysogens tested . Similarly, the rate of synthesis of p factor increased appreciably in the induced ilv lysogen carrying the prophage at the ilv gene, and possibly in the bfe lysogen as well, but not in other lysogens examined . Taken together with other evidence, these results suggest that the enhanced syntheses of beta and beta' subunits of RNA polymerase and of p factor observerd represent "escape synthesis", resulting from the close linkage of the prophage genome to the respective structural genes . In contrast, omega factor synthesis was stimulated upon induction of any of the lysogens used without respect to the site of prophage location, suggesting the involvement of an entirely different mechanism. Mol Gen Genet, 1977 Feb 15, 150(3), 285 - 92 Re-examination of F plasmid replication in a dnaC mutant of Escherichia coli; Van Brunt J et al.; The replication of an F' plasmid in a dnaC mutant, thermolabile for initiation of chromosomal replication, has been re-examined using a novel DNA-DNA annealing assay . Plasmid replication ceases rapidly at non-permissive conditions, consistent with a direct role for the dnaC product in the replication of F. Mol Gen Genet, 1977 Feb 15, 150(3), 249 - 55 ppGpp cycle in Escherichia coli; Kari C et al.; Kinetics of accumulation and degradation of ppGpp and pppGpp were analysed in spoT+ and spoT strains of Escherichia coli . The experimental data in this paper indicate that on degradation ppGpp is not converted to pppGpp but instead is converted to GDP which is in turn phosphorylated to GTP . In addition the data are consistent with the idea the pppGpp is a direct precursor of ppGpp . We propose that ppGpp is metabolised according to the following pathway: GTP-pppGpp--ppGpp--GDP--GTP, which we call the ppGpp cycle . Coupled with the observations in spot strains we assume that ppGpp blocks its own synthesis by inhibiting the synthesis of pppGpp but not the interconversion of the two nucleotides. Mol Gen Genet, 1977 Feb 15, 150(3), 237 - 48 Induction of protein X in Escherichia coli; Little JW et al.; Certain treatments that damage DNA and/or inhibit replication in E . coli have been reported to induce synthesis of a new protein, termed protein X, in recA+ lexA+ strains . We have examined some of the treatments that might induce protein X and we have, in particular, tested the hypothesis of Gudas and Pardee (1975) that DNA degradation products play an essential role in the induction process . We confirmed that UV irradiation, nalidixic acid treatment, or thymine starvation result in protein X synthesis in wild type strains . However, we found that UV irradiation, unlike nalidixic acid, also induced protein X in recB strains, in which little DNA degradation occurs . Furthermore, we found that the presence of DNA fragments resulting from host-controlled restriction of phage lambda DNA did not affect protein X synthesis . We conclude that no causal relationship exists between the production of DNA fragments and induction of protein X . The presence of the plasmid R46, which confers enhanced mutagenesis and UV resistance on its host, did not affect protein X synthesis . Growth in the presence of 5-bromouracil, which does not result in production of degradation fragments, resulted eventually in a low rate of protein X synthesis . In dnaA mutants, deficient in the initiation of new rounds of replication, UV irradiation induced protein X, again unlike nalidixic acid . Thus, the inhibition of active replication forks is not an essential requirement for protein X induction. Eur J Biochem, 1977 Feb 15, 73(1), 33 - 8 Simple isolation method and assay for T4 DNA ligase and characterization of the purified enzyme; Knopf KW; A method for the isolation of T4-amber-N82-induced DNA ligase is described which results in a nearly homogeneous enzyme preparation after two column chromatographic steps . The enzyme is detected during the purification by its ability to form a stable acid-precipitable enzyme-adenylate complex . Some properties of the assay, such as the effect of salt, temperature and incubation time, are presented . The isolated enzyme and its adenylate complex are characterized by acrylamide gel electrophoresis under native and denaturing conditions, as well as by isoelectric focusing . The purified enzyme exhibits a molecular weight of approximatel 60000 . Isoelectric focusing yields at least 5 protein components, which are able to form an enzyme-adenylate complex . The main activity possesses a p1 of 6 . The enzyme preparation is capable of repairing T5+ DNA known to contain about 4 or 5 single-strand breaks, to circularize lambda DNA and to join Hind111 and EcoR1 fragments. Eur J Biochem, 1977 Feb 15, 73(1), 297 - 306 Processing by ribonuclease II of the tRNATyr precursor of Escherichia coli synthesized in vitro; Kitamura N et al.; The tRNATyr precursor molecule, synthesized from phi 80 psu3+ DNA (containing a single tRNA gene) by DNA-dependent RNA polymerase and q factor, was about 205 nucleotides long . The main product of its digestion with a ribonuclease tii preparation from Escherichia coli showed the same electrophoretic mobility as tRNAtyr precursor isolated in vivo and was found to be identical to it when analysed using fingerprint techniques . This intermediate precursor synthesized in vitro was converted further by processing with ribonuclease P into an RNA identical size to mature tRNATyr . It was concluded that the initiation of transcription of the tRNATyr gene in vitro occurs at the same site as that of transcription in vivo and a termination occurs at about 80 nucleotides beyond the CCA end of tRNATyr. Eur J Biochem, 1977 Feb 15, 73(1), 179 - 84 Transcription of double-stranded RNA by Escherichia coli DNA-dependent RNA polymerase; Sugiura M et al.; Double-stranded RNA of some virus genomes can be used as template for the DNA-dependent RNA polymerase purified from Escherichia coli . The RNA synthesis requires all four nucleoside triphosphates and manganese ions and is dependent on the presence of sigma subunit . The reaction is inhibited by rifampicin, streptolydigin and ethidium bromide, but not by DNase and actinomycin D which does not bind to double-stranded RNA . The template activity of double-stranded RNA from various viruses is different in each case . The order of template efficiency is Penicillum chrysogenum virus greater than cytoplasmic polyhedrosis virus greater than rice dwarf virus greater than reovirus . The product obtained using cytoplasmic polyhedrosis virus double-stranded RNA as template is single-stranded and hybridizes specifically to the denatured template RNA . One of the major 5'-starting nucleotide sequences of the product RNA is pppA-A-Y-- . These results indicate that transcription in vitro of double-stranded RNA by E . Coli RNA polymerase is initiated at specific sites on the template. Int J Cancer, 1977 Feb 15, 19(2), 236 - 9 Polyoma genome transcription in transformed mouse cells growing in culture and as tumors in syngeneic mice; Grady LJ et al.; The strands of the polyoma genome coding for early and late viral RNA were separated by means of asymmetric cRNA synthesized under high salt conditions by Escherichia coli RNA polymerase . Each strand was then employed in RNA-DNA hybridization experiments to determine the degree to which it is transcribed in transformed cells under several conditions . Except for a concanavalin-A-selected revertant, approximately one-quarter of the early strand was expressed in all of the situations investigated . In contrast, while no significant late strand transcription was detected in transformed cells in culture, the tumors induced by these cells contained transcripts complementary to about 12% of this strand . The results are discussed in terms of current knowledge of the amount of the virus genome required to transform cells. Biochem J, 1977 Feb 15, 162(2), 309 - 20 Transport of galactose, glucose and their molecular analogues by Escherichia coli K12; Henderson PJ et al.; 1 . Strains of Escherichia coli K12 were made that are unable to assimilate glucose by the phosphotransferase system, since they lack the glucose-specific components specified by the genes ptsG and ptsM . 2 . Derivative organisms lacking the methyl galactoside or galactose-specific transport system were examined for their ability to transport galactose, d-fucose, methyl beta-D-galactoside, glucose, 2-deoxy-D-glucose and methyl alpha-D-glucoside . 3 . Galactose, glucose and to a lesser extent fucose are substrates for both transport systems . 4 . 2-Deoxyglucose is transported on the galactose-specific but not the methyl galactoside system . 5 . The ability of sugars to elicit anaerobic proton transport is associated with the galactose-specific, but not with the methyl galactoside transport activity . Hence a chemiosmotic mechanism of energization is likely to apply to the former but not to the latter . Alternatively the methyl galactoside system may be switched off under certain conditions, which would indicate a novel regulatory mechanism . 6 . Details of the procedure for the derivation of strains may be obtained from the authors, and have been deposited as Supplementary Publication SUP 50074 (8 pages at the) British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem . J . (1977), 161,1. Eur J Biochem, 1977 Feb 15, 73(1), 115 - 24 Detergent-resistant phospholipase A of Escherichia coli K-12 . Purification and properties; Nishijima M et al.; Detergent-resistant phospholipase A, which is tightly bound to the outer membranes of Escherichia coli K-12 cells, was purified approximately 2000-fold to near homogeneity by solubilization with sodium dodecylsulfate and butan-1-ol, acid precipitation, acetone fractionation and column chromatographies on Sephadex G-100 in the presence of sodium dodecylsulfate and on DEAE-cellulose in the presence of Triton X-100 . The final preparation showed a single band in the sodium dodecylsulfate gel system . The enzyme hydrolyzes both the 1-acyl and 2-acyl chains of phosphatidylethanolamine or phosphatidylcholine . It also attacks 1-acyl and 2-acylglycerylphosphorylethanolamine . Thus, this enzyme shows not only phospholipase A1 and lysophospholipase L1 activities but also phospholipase A2 and lysophospholipase L2 activities . The enzyme lost its activity completely on incubation at 80 degrees C for 5 min at either pH 6.4 or pH 8.0 . It was stable in 0.5% sodium dodecylsulfate at below 40 degrees C . The enzyme was inactivated on incubation for 5 min at 90 degrees C in 1% sodium dodecylsulfate/1% 2-mercaptoethanol/4 M urea . The native and inactivated enzymes showed different protein bands with RF values corresponding to Mr 21 000 and Mr 28 000 respectively, in a sodium dodecylsulfate gel system . Triton X-100 seemed to protect the enzyme from inactivation . The purified enzyme was fully active on phosphatidylethanolamine in the presence of 0.0002% or 0.05% Triton X-100 . The enzyme requires Ca2+ . From its properties this enzyme seems to be identical with the enzyme purified from crude extracts of Escherichia coli B by Scandella and Kornberg . However, it differs from the latter in its positional specificity and susceptibility to sodium dodecylsulfate . Possible explanation of the difference of positional specificity of the two preparations is also described. Biochim Biophys Acta, 1977 Feb 14, 465(1), 118 - 30 Unmasking of an essential thiol during function of the membrane bound enzyme II of the phosphoenolpyruvate glucose phosphotransferase system of Escherichia coli; Haguenauer-Tsapis R et al.; The addition of N-ethylmaleimide (MalNEt), or of fluoro dinitrobenzene to a suspension of Escherichia coli during the phosphorylating uptake of methyl-alpha-D-glucopyranoside (Me-Glc), a glucose analog, stops uptake and phosphorylation and causes the loss of previously accumulated sugar and of its phosphate ester . After removal of the reagents, the phosphotransferase system remains irreversibly inactive . Pretreatment of the bacteria with the same reagents under the same conditions of concentration, pH, temperature and for the same length of time causes very little inactivation . Mercuric chloride, a reversible inactivator, prevents the phosphotransferase system from reacting simultaneously with MaINEt or with fluorodinitrobenzene . This protection strongly suggests that all three reagents react with the same site, presumably an -SH group . The change which makes this site available to the reagents depends on the phosphorylative uptake of Me-Glc . Preload of the cells and efflux of Me-Glc do not achieve the same change . The rate of inactivation is directly proportional to the rate of phosphorylative uptake . When the Km of phosphorylative uptake is modified by an uncoupling agent, the substrate concentration allowing half maximal rate of inactivation by MaINEt changes accordingly . The reactive sites of the phosphotransferase system can also be made accessible to the -SH group reagents by fluoride inhibition of phosphoenolpyruvate synthesis . This suggests that the inactivator resistent form is an "energized form" of the enzyme . The unmasking of the reactive site is not due to a change in transmembrane penetration of the reagents since incubation of toluene treated cells with MaINEt in the presence of phosphoenolpyruvate fails to inactivate the phosphotransferase activity, while incubation with MaINEt plus Me-Glc causes fast inactivation. J Biol Chem, 1977 Feb 10, 252(3), 1002 - 6 Aminoacyl adenylate, a normal intermediate or a dead end in aminoacylation of transfer ribonucleic acid; Lagerkvist U et al.; The shape of the time curve for the aminoacylation of tRNA has been investigated using five different amino acid:tRNA ligases . Four of these enzymes showed a lag in the time curve during the early phase of the first catalytic turnover of the enzyme . In each case, the lag period could be abolished by preincubating the ligase with amino acid, ATP, and Mg2+ under conditions known to give an aminoacyl adenylate-enzyme complex . With all five ligases the steady state rate of transfer from the preformed aminoacyl-adenylate complex to tRNA was approximately the same as that of the overall reaction. J Biol Chem, 1977 Feb 10, 252(3), 814 - 9 Alteration of the kinetic parameters for aminoacylation of Escherichia coli formylmethionine transfer RNA by modification of an anticodon base; Schulman LH et al.; Treatment of Escherichia coli formylmethionine tRNA with 2 M sodium bisulfite, pH 7.0, in 10 mM MgCl2 at 25 degrees results in formation of uridine/bisulfite adducts at U18 in the dihydrouridine loop, U37 in the anticodon, and U48 in the variable loop . Two products, corresponding to the two diastereoisomers of 5,6-dihydrouridine-6-sulfonate, are formed at each reactive site in the tRNA . Although none of the modifications cause complete loss of methionine acceptor activity, the modified tRNA is amino-acylated at a reduced rate and has a decreased affinity for E . coli methionyl-tRNA synthetase . Aminoacylation of {35S}bisulfite-labeled tRNAfMet with a limiting amount of purified enzyme followed by separation of the acylated and unacylated molecules and structural analysis has shown that the presence of a specific diastereoisomer of the uridine/bisulfite adduct in the anticodon base U37 alters the kinetic parameters for aminoacylation of tRNAfMet. Biochim Biophys Acta, 1977 Feb 9, 480(2), 479 - 88 Studies on aspartase . IV . Reversible denaturation of Escherichia coli aspartase; Tokushige M et al.; Aspartase (L-aspartate ammonia lyase, EC 4.3.1.1) of Escherichia coli, denatured in 4 M guanidine-HCl, was renatured in vitro by simple dilution with a concomitant restoration of the activity . While the native enzyme exhibited a marked negative Cotton effect centered at 233 +/- 1 nm in optical rotatory dispersion, the enzyme denatured in 4 M guanidine-HCl retained little optical activity . Upon dilution of the denatured enzyme, however, more than 90% of the ordered structure was recovered in 1 min, while the restoration of the activity proceeded much more slowly . Estimation of molecular weights by gel permeation chromatography indicated that the tetrameric enzyme is subject to reversible dissociation into monomeric subunits under the experimental conditions . Various environmental factors such as temperature, pH and protein concentration exhibited profound influence on the rate and extent of the reactivation . In order to examine the correlation between the restoration of the activity and the quaternary structure, electron microscopic inspection of the kinetic processes of reversible denaturation was attempted . Upon dilution of the denatured enzyme at 4 degrees C, neither the activity nor tetrameric images were detected over several min . Upon the temperature shift up to 25 degrees C, however, the activity regain was rapidly proceeded concomitant with the appearance of tetrameric molecules . These results are compatible with the possibility that the subunit assembly is an essential prerequisite, thought not sufficient, for enzyme activity. Biochim Biophys Acta, 1977 Feb 7, 459(2), 225 - 40 Energy transduction in Escherichia coli . The effect of chaotropic agents on energy coupling in everted membrane vesicles from aerobic and anaerobic cultures; Hasan SM et al.; 1 . The transduction of energy from the oxidation of substrates by the electron transport chain or from the hydrolysis of ATP by the Mg2+-ATPase was measured in everted membrane vesicles of Escherichia coli using the energy-dependent quenching of quinacrine fluorescence and the active transport of calcium . 2 . Treatment of everted membranes derived from a wild-type strain with the chaotropic agents guanidine-HC1 and urea caused a loss of energy-linked functions and an increase in the permeability of the membrane to protons, as measured by the loss of respiratory-linked proton uptake . 3 . The coupling of energy to the quenching of quinacrine fluorescence and calcium transport could be restored by treatment of the membranes with N,N'-dicyclohyexylcarbodiimide . 4 . Chaotrope-treated membranes were found to lack Mg2+-ATPase activity . Binding of crude soluble Mg2+-ATPase to treated membranes restored energy-linked functions . 5 . Membranes prepared from a wild-type strain grown under anaerobic conditions in the presence of nitrate retained respiration-linked quenching of quinacrine fluorescence and active transport of calcium after treatment with chaotropic agents . 6 . Everted membrane vesicles prepared from an Mg2+-ATPase deficient strain lacked respiratory-driven functions when the cells were grown aerobically but were not distinguishable from membranes of the wild-type when both were grown under anaerobic conditions in the presence of nitrate . 7 . It is concluded (a) that chaotropic agents solubilize a portion of the Mg2+-ATPase, causing an increase in the permeability of the membrane to protons and (b) that growth under anaerobic conditions in the presence of nitrate prevents the increase in proton permeability caused by genetic or chemical removal of the catalytic portion of the Mg2+-ATPase. Mol Cell Biochem, 1977 Feb 4, 14(1-3), 59 - 65 Specific features of the structural organization of the mitochondrial genome in rat liver; Shugalii AV et al.; The nature of intramolecular heterogeneity of mtDNA in the liver of white rats has been studied . The peculiarities of the melting curve, and the possibility of DNA fractionation of nucleotide compounds with hydroxylapatite (HA) column chromatography has shown the presence of sequences differing in the mean nucleotide content . A section of about 350 pairs in size repeated four times was found in the reassociation of most thermolabile fraction with a mean composition of 28% GC . These sections are well seen on the denaturation map of the recorded molecules formed in the range of temperature transition 'helix-coil' . The distance between the centers of fusible sections (in percentage of total length) is 32.5, 32, 14.0 and 21.5. Mol Cell Biochem, 1977 Feb 4, 14(1-3), 47 - 54 The genetic system of kinetoplasts in trypanosomatides; Zaitseva GN et al.; In the present report, the genetic system of Crithidia oncopelti kinetoplast is used as a model for investigation of kinetoplast DNA (kDNA) structure, its transcription, protein synthesizing apparatus of the kinetoplast and the protein synthesis controlled genetically by kDNA . It was shown that kDNA of C . oncopelti can be isolated from cells or from kinetoplast fraction in the form of a network complex structure consisting of a lot of circular molecules . These minicircles have a contour length of about 0.83 micronm and molecular weight of 1.6 X 10(6) . The kDNA was demonstrated to be of higher AT content type than nuclear DNA . Besides, kDNA is characterized by a lesser degree of clustering of pyrimidines as compared with the nuclear one . The isolated kinetoplasts of C . oncopelti were shown to exhibit activity of DNA dependent RNA polymerase . The effect of some antibiotics and intercalating substances on RNA synthesis in kinetoplasts and mitochondria appears to be identical . Kinetoplasts of C . oncopelti have their own protein synthesizing system, whose components (ribosomes, rRNA, proteins, factors of incorporation) differ from those of the cytoplasm . Inhibition of translation by some antibiotics and of transcription by acriflavin allowed the suggestion that several proteins of kinetoplast ribosomes may be synthesized within this organoid . It was shown then that kDNA may be involved in the formation of the protein synthesizing apparatus in the kinetoplast. Mol Cell Biochem, 1977 Feb 4, 14(1-3), 37 - 45 The character of protein-nucleic interaction in relation to the mtDNA-membrane complex; Kazakova TB et al.; Specific sites that interact with structural proteins of the mitochondrial inner membrane were found in mitochondrial DNA (mtDNA) of rat liver . Analysis of the isolated DNA fragments revealed their capacity to form a complex with membrane proteins in vitro and allowed the detection of a protein with a molecular weight 40,000 . The size of the fragments was found to be 12-18 nucleotide pairs with an average molecular weight 10,000 MtDNA sites recognized by membrane protein proved to be quite unique in having a secondary structure, a high content of AT sequences (82%) and oligopyrimidine blocks . It was shown that the light mtDNA strand, rich in adenine, is 60% more active in the binding with membrane mitochondria than the heavy one. Mol Cell Biochem, 1977 Feb 4, 14(1-3), 31 - 6 The structure of animal mitochondrial DNA (base composition, pyrimidine clusters, character of methylation); Vanyushin BF et al.; Base composition, content of pyrimidine isopliths and the degree of methylation of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) from various vertebrates and protozoon Crithidia oncopelti have been studied . MtDNAs from mammals (ox, rat) do not differ in fact in the GC content from the respective nDNA . The GC content in mtDNA from fishes (sheat fish) and birds (duck, chicken) is 1.5-2.5 mole % higher than in the respective nDNA . Kinetoplast DNA (kDNA) from Crithidia oncopelti (GC = 42.9 mole %) differs significantly in base composition from nDNA (GC = 51.3 mole %) . All the mtDNA and kDNA studied differ from the respective nDNA by a lower degree of pyrimidine clustering . The amount of mono and dipyrimidine fragments in mtDNA is more than 30 mole %, whereas in nDNA it does not exceed 23 mole % . The quantity of long pyrimidine clusters (hexa and others) is 2-4 times lower in mtDNA than in nDNA . The lower degree of clustering of pyrimidine nucleotides seems to be a specific feature of all the mtDNA studied . This may be indicative of common traits in the organization and origin of mtDNA . All mtDNA of vertebrates contain 5-methylcytosine as a 'minor' base (1.5- 3.15 mole %) and surpass by 1.5-2 times the respective nDNA in the methylation degree . It has been found that in animals mtDNA is species specific as far as the 5-methyl-cytosine content is concerned . In mitochondria and nuclei of rat liver certain DNA methylase activity has been detected, which provides in vitro the methylation of cytosine residues both in homologous DNA and various heterologous DNAs . The specificity of methylation in vitro of cytosine residues in the same heterologous DNA from E . coli B varies with the source of enzymes . The mitochondrial enzyme methylates cytosine as the lone monopyrimidine residue, whereas the nuclear enzyme methylase cytosine in the di- and tripyrimidine fragments. Arch Microbiol, 1977 Feb 4, 112(1), 103 - 7 Alterations of alkaline phosphatase activity during adaptation of Escherichia coli to phosphite and hypophosphite; Lauwers AM et al.; When Escherichia coli cells were grown in media containing either phosphite or hypophosphite as the sole source of phosphorus, the responded to this situation primarily in the same way as phosphate-limited cultures: The activity of alkaline phosphatase increased drastically, which under natural conditions would enable the cells to compensate for the shortage of phosphate . Subsequent transfers, however, resulted in a quite different response: While the phosphatase activity of phosphate-limited cells stays at a high derepressed level, its increase was followed by a gradual decline in organisms grown on phosphite of hypophosphite . After eight to ten transfers on these P-compounds, phosphatase activity was back to its initial, repressed, low level, indicating that the cells were fully adapted to these substrates . Adaptation to either PO3-3 or PO3-2 was completely abolished if the cells were again grown with PO3-3 as P-source, whereafter the entire process of adaptation had to be repeated . The observed adaptation pattern, reflected by the alterations of phosphatase activity, was qualitatively equal with PO3-3 and PO3-2, but quantitatively different, because the response to hypophosphite gave much higher values than the increase obtained with phosphite . Phosphite-adapted cells are not simultaneously adapted to hypophosphite, but their response to the latter was less intense than observed after direct transfers from PO3-4 to PO3-2 . Adaptation to hypophosphite, however, led simultaneously to phosphite adaptation, so that these cells can utilize both P-compounds as a substitute for phosphate. Biochim Biophys Acta, 1977 Feb 4, 464(3), 562 - 70 ATP-dependent proton translocation and quenching of 9-aminoacridine fluorescence in inside-out membrane vesicles of a cytochrome-deficient mutant of Escherichia coli,; Singh AP et al.; 1 . ATP-dependent proton translocation and ATP-dependent quenching of the fluorescence of 9-aminoacridine were measured in inside-out vesicles derived from a cytochrome-deficient mutant of Escherichia coli . 2 . ATP-dependent quenching of fluorescence was inhibited by nigericin gramicidin, NH4Cl, and carbonylcyanide-m-chlorophenylhydrazone . Inhibition was also produced by the ATPase inhibitors N,N'-dicyclohexylcarbodimide (DCCD) and diphenyl phosphorazidate (DPA), and by the respiratory chain inhibitors piericidin A, 2-heptyl-4-hydroxyquinoline N-oxide, and An2+ . The inhibition of ATP-dependent fluorescence quenching by the ionophores, uncouplers, and respiratory chain inhibitors was not due to an effect on ATPase activity which was insensitive to these agents . 3 . By use of the ATPase inhibitors DCCD and DPA, or by replacing ATP with GTP, ITP and CTP, a correlation between the ATPase activity and the rate of ATP-dependent membrane energization, as measured by fluorescence quenching, was obtained. Biochim Biophys Acta, 1977 Feb 3, 474(3), 363 - 77 Triton X-100 activates nucleoside triphosphate-dependent, recBC-dependent DNA synthesis in toluene-treated Escherichia coli; Harper DJ et al.; Escherichia coli cells whose chromosome replication has been terminated in vivo, either by growth into stationary phase or by incubation of a mutant carrying a temperature-sensitive initiation mutation under restrictive conditions, are inactive in in vitro DNA synthesis as measured in toluene-treated cells . Addition of the non-ionic detergent Triton X-100 to such inactive systems results in a marked stimulation of ATP-dependent in vitro DNA synthesis . This Triton-stimulated DNA synthesis appears to proceed by a semi-conservative mechanism, in that DNA synthesized in vitro in the presence of a density labeled precursor bands in CsCl equilibrium centrifugation at a hybrid density . Neutral sucrose gradient centrifugation demonstrates that most of this hybrid material exhibits a molecular weight in excess of 1 X 10(7) . Triton-stimulated synthesis requires the presence of DNA polymerase III, as does normal in vivo replication . We show here, however, several anomalous properties of the DNA synthesis in the Triton/toluene system . In particular, Triton-stimulated synthesis is absent in cells harboring a recB mutation which lack the ATP-dependent exonuclease V, an enzyme implicated in recombinational repair synthesis in vivo . Furthermore, the ATP requirement for Triton-stimulated synthesis is relatively non-sepcific, and a variety of nucleoside triphosphates can effectively substitute for ATP . Finally, despite their high molecular weight in neutral sucrose gradient centrifugation, Triton-stimulated DNA synthesis generates DNA molecules of low molecular weight (less than 500 000) as determined by alkaline sucrose gradient centrifugation . In contrast, DNA synthesis in the normal toluene-treated cell system is not dependent on recB activity, shows a nearly absolute requirement for ATP which cannot be replaced by other nucleoside triphosphates, and produces molecules of far greater molecular weight as measured on alkaline sucrose gradients . Taken altogether the data strongly suggest that Triton activates an unusual form of DNA synthesis in toluene-treated cells which shows both repair and replicative aspects . These results caution against the use of Triton-activated toluene-treated cells system, for studying simple replicative DNA synthesis. J Virol, 1977 Feb, 21(2), 694 - 712 Replication process of the parvovirus H-1 . VI . Characterization of a replication terminus of H-1 replicative-form DNA; Rhode SL III; The linear duplex replicative form (RF) DNA of the parvovirus H-1 has been characterized with respect to cleavage by the bacterial restriction endonuclease of Escherichia coli, EcoRI . RF DNA has a single cleavage site 0.22 genome length from the left end of the molecule . The molecular weight of H-1 RF DNA determined by gel electrophoresis is 3.26 X 10(6) . H-1 RF DNA has been found to dimerize by hydrogen-bounded linkage at the molecular left end, and in some molecules the viral strand is covalently linked to the complementary strand . Some 10% of monomeric RF DNA also has a covalent linkage between the viral and complementary strands at the left end . The EcoRI-B fragment, containing the left end of the RF molecule, appears to be a replication terminus by its labeling characteristics for both RF and progeny DNA synthesis . These findings suggest that the left end of H-1 RF DNA has some type of "turn-around" structure and that this end is not an origin for DNA synthesis. Eur J Biochem, 1977 Feb, 72(3), 465 - 78 Studies on the sequence of the 3'-terminal region of turnip-yellow-mosaic-virus RNA; Silberklang M et al.; A fragment representing the 3'-terminal 'tRNA-like' region of turnip yellow mosaic (TYM) virus RNA has been purified following incubation of intact TYM virus RNA with Escherichia coli 'RNase P' . This fragment, which is 112+3-nucleotides long has been completely digested with T1 RNase and pancreatic RNase and all the oligonucleotides present in such digests have been sequenced using 32P-end labelling techniques in vitro . The TYM virus RNA fragment is free of modified nucleosides and does not contain a G-U-U-C-R sequence . Using nuclease P1 from Penicillium citrinum, the sequence of 26 nucleotides from the 5' end and 16 nucleotides from the 3' end of this fragment has been deduced . The nucleotide sequence at the 5' end of the TYM virus RNA fragment indicates that this fragment includes the end of the TYM virus coat protein gene. Genetics, 1977 Feb, 85(2), 203 - 8 Complementation test between alkaline phosphatase regulatory mutations phoB and phoRc in Escherichia coli; Pratt C et al.; A phoRc and a phoB mutation belong to the same complementation group suggesting that there is a single positive control gene for alkaline phosphatase synthesis. Genetics, 1977 Feb, 85(2), 193 - 201 Regulation of newly evolved enzymes . III Evolution of the ebg repressor during selection for enhanced lactase activity; Hall BG et al.; The evolution of lactose utilization by lacZ deletion strains of E . coli occurs via mutations in the ebg genes . We show that one kind of mutation in the regulatory gene ebgR results in a repressor which retains the ability to repress synthesis of ebg enzymes, but which permits 4.5-fold more ebg enzyme synthesis during lactose induction than does the wild-type repressor . A comparison between the growth rate of various ebg+ strains on lactose and the amount of ebg enzyme synthesized by these strains shows that the rate of enzyme synthesis permitted by the wild-type repressor is insufficient for growth on lactose as a sole carbon source by a cell with the most active ebg lactase yet isolated . We conclude, therefore, that the evolution of lactose utilization requires both a structural and a regulatory mutation. J Gen Microbiol, 1977 Feb, 98(2), 485 - 91 Monitoring enzyme synthesis as a means of studying peptide transport and utilization in Escherichia coli; Bell G et al.; A new method has been developed for measuring peptide transport in aminoacid auxotrophs of Escherichia coli by following induction of beta-galactosidase . Appearance of the enzyme was determined after addition of inducer and peptides to amino-acid starved bacteria . For a given number of lysine equivalents, the rate and the extent of enzyme synthesis were the same for lysine and lysyl peptides; similar results were found for glycine and glycl peptides . Saturation constants for peptide transport were determined from the exogenous peptide concentration that gave half maximal rates of enzyme synthesis . The saturation constants, studies with mutants defective in peptide transport, and detection of competition between peptides for uptake, all endorsed earlier conclusions from growth tests about the structural specificities for peptide transport . The new method is quicker, more sensitive and more informative than growth tests. Appl Environ Microbiol, 1977 Feb, 33(2), 482 - 4 Correction for the inherent error in optical density readings; Lawrence JV et al.; Except at very low levels, uncorrected photometric determination of bacterial cell densities showed a decreasing proportionally to actual cell density or dry weight . A standard curve was prepared to convert photometric readings to truly proportional optical density values . With one dry weight determination, optical density values may be converted to absolute dry weight values. Proc Natl Acad Sci U S A, 1977 Feb, 74(2), 492 - 5 Isolation of a multi-enzyme complex of fatty acid oxidation from Escherichia coli; Binstock JF et al.; A multi-enzyme complex of fatty acid oxidation has been isolated from E . coli B cells and has been purified to near homogeneity by a simple two-step procedure . The complex exhibits thiolase (EC 2.3.1.9), enoyl-CoA hydratase (EC 4.2.1.17), and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activities towards short-, medium-, and long-chain substrates . The complex has been estimated to have a molecular weight of approximately 300,000 and is apparently composed of two types of subunits with molecular weights of 78,000 and 42,000. Proc Natl Acad Sci U S A, 1977 Feb, 74(2), 487 - 91 Functional expression of cloned yeast DNA in Escherichia coli; Ratzkin B et al.; A collection of hybrid circular DNAs was constructed in vitro using the poly(dA-dT) "connector" method: each hybrid circle contained one molecule of poly(dT)-tailed DNA of plasmid ColE1 (made linear by digestion with EcoRI endonuclease) annealed to a poly(dA)-tailed fragment of yeast (Saccharomyces cerevisiae) DNA, produced originally by shearing total yeast DNA to an average size of 8 X 10(6) daltons . This DNA preparation was used to transform E . coli cells, selecting colicin-E1-resistant clones that contain hybrid ColE1-yeast DNA plasmids . Sufficient numbers of transformant clones were obtained to ensure that the hybrid plasmid population was representative of the entire yeast genome . Various hybrid ColE1-yeast DNA plasmids capable of complementing E . coli auxotrophic mutations were selected from this population . Plasmid pYeleu 10 complements several different point or deletion mutations in the E . coli or S . typhimurium leuB gene (beta-isopropylmalate dehydrogenase); plasmids pYeleu11, pYeleu12, and pYeleu17 are specific suppressors of the leuB6 mutation in E . coli C600 . Plasmid pYehis2 complements a deletion in the E . coli hisB gene (imidazole glycerol phosphate dehydratase) . Complementation of bacterial mutations by yeast DNA segments does not appear to be a rare phenomenon. Proc Natl Acad Sci U S A, 1977 Feb, 74(2), 452 - 6 Lipid and protein segregation in Escherichia coli membrane: morphological and structural study of different cytoplasmic membrane fractions; Letellier L et al.; Lipid and protein segregations can be induced in E . coli cytoplasmic membranes by conformational transitions of their lipid hydrocarbon chains from a disordered to an ordered state . For E . coli strain K 1059 (an unsaturated fatty acid auxotroph) supplemented with linolenic acid, the segregation leads to large areas of membrane surfaces having distinctly different morphological characteristics (smooth compared with strongly particulated fracture faces, as visualized by freeze fracture electron microscopy) . The different regions are physically separated by osmotic lysis of spheroplasts at temperatures below those of the order-disorder transition of the lipid hydrocarbon chains . The analysis of the different cytoplasmic membrane fractions provides a direct demonstration and allows a direct analysis of the segregation . As compared to the nonfractionated membranes, the membrane regions corresponding to the smooth fracture surfaces are poor in proteins, rich in lipids, and enriched in saturated fatty acids, while the membrane regions corresponding to the strongly particulated fracture surfaces are rich in proteins, poor in lipids, and enriched in unsaturated fatty acids . Quantitative information about the extent of these segregations is obtained from high-angle x-ray diffraction of the different membrane fractions and of the corresponding total lipid extracts. Proc Natl Acad Sci U S A, 1977 Feb, 74(2), 442 - 6 Preparation and characterization of a modified form of beta2 subunit of Escherichia coli tryptophan synthetase suitable for investigating protein folding; Hogberg-Raibaud A et al.; Globular proteins often appear to consist of distinct compact "domains," and the assumption is frequently implicitly made that these domains correspond to intermediate structures in the folding process . If this assumption is correct, the polypeptide fragment that builds up a domain should be able to spontaneously fold into its native conformation even when isolated . In an attempt to isolate and study such a fragment, the beta2 subunit of tryptophan synthetase {tryptophan synthase, L-serine hydro-lyase (adding indoleglycerol-phosphate), EC 4.2.1.20} has been subjected to controlled proteolysis . The resulting protein is shown to be a dimer, the protomer of which contains two nonoverlapping polypeptide chains of molecular weights 12,000 and 29,000 . Though inactive, the nicked protein is shown to be in a conformation that closely resembles that of the original enzyme, since it still can form an enzyme-bound intermediate of the catalytic reactions . The fluorescence of this intermediate is used to characterize the binding sites for the cofactor (pyridoxal-P) and substrates, which are shown to exist on the nicked protein . The possibility is discussed of using the fragments isolated from the nicked protein to study individual steps of the enzymatic reaction, intracistronic complementation, and the folding process in the normal beta2 subunit. Proc Natl Acad Sci U S A, 1977 Feb, 74(2), 437 - 41 Isomeric aminoacyl-tRNAs are both bound by elongation factor Tu; Hecht SM et al.; Recent suggestions that elongation factor Tu (EF-Tu) is specific for 2'-O-aminoacyl-tRNA, as compared with the 3'-isomer, prompted us to assay {3H}aminoacyl-tRNAs from Escherichia coli terminating in 2'- or 3'-deoxyadenosine for binding to EF-Tu to determine the possible positional specificity of the factor . Binding of modified aminaocyl-tRNAs to EF-Tu-GTP was measured both as a function of the ability of EF-Tu-GTP to diminish the rate of chemical deacylation of {3H}aminoacyl-tRNAs and by gel filtration of the individual ternary complexes . Fifteen different tRNA isoacceptors were tested by the deacylation procedure, including three (tRNAAsp, tRNACys, and tRNATyr) for which isomeric modified aminoacyl-tRNAs were available . All of the modified aminoacyl-tRNAs were protected fromdeacylation, although generally to a lesser extent than the corresponding unmodified species . Six modified tRNA isoacceptors (including tRNATrp and tRNATyr, for which both modified aminoacyl-tRNAs were accessible by enzymatic aminoacylation) were used in gel filtration experiments to permit direct measurement of the individual aminoacyl-tRNA-EF-Tu-GTP complexes . These experiments were also done in the presence of equimolar amounts of the corresponding unmodified {14C}aminoacyl-tRNAs, and the relative affinities for a limiting amount of EF-Tu-GTP were measured . The results were completely consistent with those obtained by the deacylation procedure and indicated that EF-Tu can bind to both positional isomers of aminoacyl-tRNA with no obvious preference for either. J Biochem (Tokyo), 1977 Feb, 81(2), 381 - 8 Effects of polyamines on the activities of Escherichia coli ribonuclease I and II; Kumagai H et al.; The effects of polyamines on the breakdown of synthetic polynucleotides {poly(A), poly(C), and poly(U)} by E . coli ribonuclease I {ribonucleate 3'-oligonucleotidohydrolase, EC 3.1.4.23} and ribonuclease II {EC 3.1.4.1} have been studied . The degradation of poly(C) by RNase II was stimulated by spermine and spermidine, while that of poly(A) by RNase II was not affected by polyamines . Under our standard experimental conditions, the breakdown of poly(U) by RNase II was inhibited slightly by polyamines . The stimulatory effect of spermine and spermidine on the breakdown of poly(C) occurred in the absence of monovalent cations but not in the absence of divalent cations . When polyamines were used as a stimulant of RNase II, the ratio of poly(C) degradation to poly(U) degradation was greater in the presence of inhibitors such as poly(G) than in their absence . Although the breakdown of all synthetic polynucleotides by RNase I was stimulated by polyamines, the degree of stimulation by polyamines was in the order poly(C)greater than poly(A)(see text)poly(U) . However, the difference in degree of stimulation among polynucleotides decreased as monovalent cation concentration was increased. Infect Immun, 1977 Feb, 15(2), 676 - 8 Improved minca medium for the detection of K99 antigen in calf enterotoxigenic strains of Escherichia coli; Guinee PA et al.; The K99 antigen of Escherichia coli can be detected more readily in cultures grown on Minca medium at 37 degrees C for 6 to 8 h or grown on Minca plus 1% Iso VitaleX for 20 to 24 h. Hoppe Seylers Z Physiol Chem, 1977 Feb, 358(2), 197 - 208 Immunochemical studies on phenylalanyl-tRNA synthetase from Escherichia coli; Hennecke H et al.; Antibodies against the alpha and beta subunits of phenylalanyl-tRNA synthetase were fractionated by ion exchange chromatography into different classes and then digested with papain to yield the respective Fab fragments . The preparations obtained were used to investigate (i) whether the alpha and beta polypeptides share any common antigenic determinants and (ii) whether immunological methods are able to resolve the catalytic function of the subunits of this enzyme (or principally of oligomeric enzymes) . As to the first problem, immunodiffusion and complement fixation experiments showed that there is no immunological relatedness between the subunits which argues against the existence of sequence homoligies . As to the second question investigated, it was found that any binding of immunoglobulins of Fab fragments to the alpha or the the beta subunit affects enzyme activity either in the direction of activation or inhibition . These results therefore show that the immunological approach is not appropriate for resolving subunit-specific funcitons, possibly as a consequence of conformational changes induced in the enzyme by the binding of the immunoglobulins of Fab fragments. Am J Physiol, 1977 Feb, 232(2), E180 - 5 Glucose and lactate kinetics after endotoxin administration in dogs; Wolfe RR et al.; We studied the effects of E . coli endotoxin on the glucose and lactate kinetics in dogs by means of the primed constant infusion of {6(-3)H} glucose and Na-L-(+)-{U-14C} lactate . The infusion of endotoxin induced a transient hyperglycemic level, followed by a steady fall in plasma glucose to hypoglycemic levels . The rate of appearance (Ra) and the rate of disappearance (Rd) of glucose were both significantly elevated (P less than .05) for 150 min after endotoxin, after which neither differed from the preinfusion value . The metabolic clearance rate of glucose was significantly elevated at all times 30 min postendotoxin . By 30 min postendotoxin, Ra and Rd of lactate, plasma lactate concentration, and the percent of glucose turnover originating from lactate were significantly elevated and remained so for the duration of the experiment . We concluded that after endotoxin hypoglycemia developed because of an enhanced peripheral uptake of glucose and a failure of the liver to maintain an increased Ra of glucose . We also concluded that lactate became an important precursor for gluconeogenesis and an important metabolic substrate. Nucleic Acids Res, 1977 Feb, 4(2), 491 - 9 Isolation of ribosomal protein-RNA complexes by nitrocellulose membrane filtration: equilibrium binding studies; Spicer E et al.; E . coli ribosomal proteins are retained by nitrocellulose filters . In contrast, 16S RNA passes through nitrocellulose filters . We have found that specific protein-RNA complexes involving single proteins also pass through nitrocellulose filters . Thus, by utilizing radioactively labeled r-proteins, nitrocellulose filtration can be used to study directly and sensitively the stoichiometry of r-protein-RNA association . The filtration process maintains near equilibrium conditions, making it applicable to weak as well as strong protein-RNA associations . We have used nitrocellulose filtration to obtain saturation binding curves for the association of proteins S4, S7, S8 and S20 with 16S RNA . In each case, the stoichiometry of binding was one mole of protein or less per mole of RNA . The stoichiometry of protein S8 binding to 16S RNA measured by filtration is comparable to that observed by sucrose gradient centrifugation . Association constants for the binding of proteins S4, S8 and S20 to 16S RNA have been determined by analysis of the saturation binding curves and were found to range from .3-6 X 10(7)M-1. Nucleic Acids Res, 1977 Feb, 4(2), 445 - 55 On the mechanism of oligonucleotide-primed RNA synthesis . II . Synthesis of specific primer-initiated RNA copies suitable for DNA sequence analysis; Van Kreijl CF et al.; The effect of temperature and primer concentration on oligonucleotide-primed transcription has been studied using the separated strands of a well-defined natural DNA as template . Results were similar to those obtained in the homopolymer-directed model systems . At high temperature and excess primer concentration mainly primer-initiated RNA copies are synthesized . Omission of one ribonucleoside triphosphate also makes the termination specific . The unique RNA fragments thus obtained have been used to determine the perfectly-repeated sequence of 68 base pairs in this DNA. Nucleic Acids Res, 1977 Feb, 4(2), 425 - 44 On the mechanism of oligonucleotide-primed RNA synthesis . I . Model studies with deoxyhomopolymer templates and Escherichia coli RNA polymerase; Van Kreijl CF et al.; We have studied the effect of temperature, primer chain length and primer concentration on the oligonucleotide-primed transcription by Escherichia coli RNA polymerase . Our experiments with the homopolymer model systems poly(dT) .oligo(A)n, poly(dA) .oligo(U)n and poly(dA) .oligo(dT)n lead to three main conclusions . First, de novo chain initiation on single-stranded templates is preferentially suppressed at higher temperatures . Second, stable annealing of template and primer is neither a prerequisite nor does it stimulate the primer-dependent transcription . Third, formation of the ternary enzyme-template-primer complex is a rate-limiting step in the oligonucleotide-primed RNA synthesis . The maximal rate of primer-stimulated RNA synthesis, moreover, is strongly dependent on the nature of the primer and decreases in the order oligo(A)-primed poly(A) synthesis greater than oligo(U)-primed poly(U) synthesis greater than oligo(dT)-primed poly(U) synthesis . We attribute this to differences in the rate at which the first nucleotide is added to the primer . Raising temperature and primer concentration renders transcription in the model systems almost completely primer-dependent . This can be useful in a transcription approach to DNA sequence analysis. Mutat Res, 1977 Feb, 42(2), 191 - 204 Lethla effect of nitrous acid on Escherichia coli; Nagy Z et al.; The effect of nitrous acid (NA) on viability, integrity of cellular DNA and on membrane transport were studied in 5 strains of Escherichia coli . Stationary phase cells, grown on mineral salts medium, were exposed to NA . The viability of strains decreased in thefollowing order: W3110 wild-type greater than WP2 wild-type, WP2 uvrA greater than NG30 recA greater than P3478 polA . Alterations were found in the DNA sedimentation profile in alkaline sucrose gradient . Disturbance of DNA synthesis was measured by 3H-labelled thymidine ({3H}Thd) incorporation . No degradation of DNA was found after NA treatment . Low doses of NA caused significant inhibition of leucine and glucose transport into whole cells . The results are interpreted in terms of the multi-target action of NA causing the death of cells. Leber Magen Darm, 1977 Feb, 7(1), 1 - 2 {Current concepts in the pathophysiology of the diarrhea (author's transl)}; Ewe K et al.; Diarrhea can be defined as increased frequency of bowel movements (greater than 3 per day) plus decreased consistency of stools (volume greater than 200 ml per defecation) . Two pathogenetic mechanisms have been intensively investigated and partially elucidated within the last years: 1 . Secretion of electrolytes and water by way of induction of an augmented synthesis of cAMP in the mucosa cell . Cholera enterotoxin and other bacterial toxins as well as VIP (vasoactive intestinal peptide) cause diarrhea by this mechanism . 2 . Certain substances such as dihydroxylated bile acids, diphenolic laxatives and probably fatty acids cause leakage of the tight junctions between mucosal cells and cause leakage of electrolytes and water back into the intestinal lumen. J Infect Dis, 1977 Feb, 135(2), 313 - 7 Enterotoxigenic Escherichia coli isolated from food; Sack RB et al.; Approximately 8% of 240 isolates of Escherichia coli obtained from food of animal origin in the United States were found to be enterotoxigenic, as determined in adrenal cells, rabbit ileal loops, and assays in infant mice . These organisms were of diverse serotypes that are not included among the so-called enteropathogenic serotypes and would not have been identified by usual laboratory methods . These enterotoxigenic E . coli are of potential importance to public health. J Dairy Sci, 1977 Feb, 60(2), 289 - 93 Immune responses of the bovine fetus; Conner GH et al.; The bovine fetus is capable of mounting an antibody response when a bacterial antigen (killed Escherichia coli) or viral antigen (live reovirus) is deposited into the amniotic fluid . Time required for the fetus to respond to bacterial antigen given orally (amniotic fluid) is approximately 10 to 14 days and 8 to 10 days for viral antigen . Calves vaccinated prenatally with E . coli from 9 to 102 days before birth and deprived of colostrum survived oral challenge doses of viable E . coli which killed calves not vaccinated prenatally . One mechanism of protection was the local production of antibody in the gastrointestinal mucosa where immunofluorescent techniques showed immunoglobulins IgG, IgM, and anti-E . coli antibody in the duodenum, jejunum, and ileum as well as in the jejunal lymph node . Prenatal vaccination has been used in the field for prevention of colibacillosis . However, the occurrence of some stillbirths and premature births indicates the need for further research before there can be widespread field application of the technique. J Bacteriol, 1977 Feb, 129(2), 916 - 25 Spermidine-Deoxyribonucleic acid interaction in vitro and in Escherichia coli; Rubin RL; The binding of spermidine to deoxyribonucleic acid (DNA) was studied by equilibrium dialysis in a wide range of salt concentrations . The association constants ranged from 6 x 10(5) M-1 in 1 mM sodium cacodylate, pH 7.5, to 3 x 10(2) M-1 in 0.3 M NaCl . MgCl2 reduced spermidine-DNA interaction even more than NaCl so that in moderate-ionic-strength solutions (0.3 M NaCl, 0.002 M MgCl2) there was little detectable binding . Low-ionic-strength media were used to isolate DNA from Escherichia coli by a method shown to minimize loss of spermidine from the DNA . Considerable spermidine was associated with E . coli DNA, but control experiments indicated that complex formation had taken place during or after lysis of the cells . Exogenous DNA or ribonucleic acid added to spheroplasts at the time of their lysis caused most of the cellular spermidine to be scavenged by the extra nucleic acid . The data suggest that spermidine is relatively free in the cell and thereby capable of strong (high-affinity) associations with nucleic acids only after the ionic strength of the cell environment is lowered. J Bacteriol, 1977 Feb, 129(2), 908 - 15 Effects of galU mutation on flagellar formation in Escherichia coli; Komeda Y et al.; Two mutants of Escherichia coli strictly deficient in uridine-diphosphoglucose pyrophosphorylase activity (galU) were found to have very small numbers of flagellar filaments and hooks . In these mutants, both the rate of flagellin (flagellar protein) synthesis and the amount of messenger ribonucleic acid specific for flagellin were considerably lower than in the parental strains . Motile revertants from the galU mutants were isolated and were found to carry a suppressor mutation, which was mapped in the flaH cistron . These strains formed swarms under conditions of catabolite repression; their intracellular concentration of cyclic adenosine 5'-monophosphate was the same as that in the parental strains . These results suggest that the outer membrane affects flagellar formation through the flaH gene product. J Bacteriol, 1977 Feb, 129(2), 714 - 7 Delayed ultraviolet light-induced cessation of respiration by inadequate aeration of Escherichia coli; Joshi JG et al.; Inadequately aerated Escherichia coli B/r cultures did not shut their respiration off 60 min after ultraviolet light (52 M/m2 at 254 nm) as they did when well supplied with oxygen . Since cessation of respiaration is associated with cell death, the result suggested that oxygen toxicity by superoxide radicals generated by cell metabolism might be responsible for cell death . The specific activity of superoxide dismutase, which scavenges O2- radicals, increased twofold after 90 min of adequate aeration, but the specific activity of catalase remained constant . Respiration and viability of irradiated cells were affected not at all by the presence of superoxide dismutase and only slightly by the presence of catalase . Metal ions such as Mn2+ and Fe2+ inducers of superoxide dismutase, had no effect on respiration and viability . When irradiated cells were incubated under N2 for 90 min, the respiration, growth, and viability time-course responses were the same as for the cells not exposed to anareobiosis . We conclude that superoxide anions generated at the time of irradiation play no part in cessation delays the ultraviolet light-induced synthesis of proteins responsible for the irreversible cessation of respiration. J Bacteriol, 1977 Feb, 129(2), 702 - 6 Transient rates of synthesis of five amionacyl-transfer ribonucleic acid synthetases during a shift-up of Escherichia coli; Reeh S et al.; The steady-state levels of a number of aminoacyl-transfer ribonucleic acid synthetases are known to be positively correlated with growth rate in Escherichia coli . To describe the regulation of these enzymes during a nutritional shift-up, use was made of the recent identification of polypeptide chains of several synthetases in whole cell lysates resolved by the O'Farrell two-dimensional gel system . A culture growing in acetate minimal medium was shifted to glucose-rich medium and pulse labeled with {3H}leucine and {3H}isoleucine for 30- or 6-s intervals during the 20 min after the shift . After mixing with a uniformly {35S}sulfate-labeled reference culture, the samples were subjected to two-dimensional gel electrophoresis . The 3H/35S ratio in the resolved synthetase polypeptides provided an accurate estimation of their transient rates of synthesis . Five aminoacyl-transfer ribonucleic acid synthetases (those for argnine, glycine, isoleucine, phenylalanine, and valine) exhibited an increase in formation within 30 to 90 s after the shift-up . The magnitude of the increases corresponded to the final steady-state values and were reached within 2 to 3 min . The addition to rifampin revealed that the increase in the differential rate of valyl-transfer ribonucleic acid synthetase formation was the result of increased messenger ribonucleic acid transcription and not of a relaxation of some translation restriction. J Bacteriol, 1977 Feb, 129(2), 698 - 701 Ultraviolet induction of prophage lambda during inhibition of deoxyribonucleic acid synthesis by hydroxyurea; Lydersen BK et al.; Hydroxyurea inhibited synthesis of certain deoxyribonucleic acid (DNA) precursors and causes the cessation of DNA synthesis . It did not cause induction of lambda . Superinfection of an irradiated lysogen with lambdaind- could prevent induction, but the percentage of cells protected decreased as the time between irradiation and superinfection increased . The presence of hydroxyurea did not increase the time during which cells could be rescued by superinfection . The accumulation of DNA precursors after ultraviolet or ionizing radiation was not necessary for the induction of lambda prophage to occur. J Bacteriol, 1977 Feb, 129(2), 658 - 67 Escherichia coli membrane proteins with an affinity for deoxyribonucleic acid; Kohiyama M et al.; From the membrane fraction of Escherichia coli K-12 strain, four protein fractions (peaks I, IIa, IIb, and III) which have affinity for deoxyribonucleic acid (DNA) have been isolated . The molecular weights of these proteins are between 12,000 and 8,000 . Only the peak III fraction contains a protein that binds preferentially to single-stranded DNA, whereas the others contain proteins that bind also to double-stranded DNA . The binding activity of the peak IIb protein is inhibited in the presence of polyuridylic acid . Peak I and peak IIa protein fractions behave like hydrophobic proteins. J Bacteriol, 1977 Feb, 129(2), 651 - 7 Accumulation of nucleotides by starved Escherichia coli cells as a probe for the involvement of ribonucleases in ribonucleic acid degradation; Cohen L et al.; The acid-soluble ribonucleic acid degradation products formed by Escherichia coli cells starved for a carbon source have been identified . They comprise oligonucleotides, nucleoside diphosphates, 5'- and 3'-nucleoside monophosphates, nucleosides, and free bases . The majority of these products are excreted phates, nucleosides, and free bases . The majority of these products are excreted into the medium, and only small and constant amounts are kept in the pool . During carbon starvation at elevated temperatures, mutants deficient in ribonuclease I do not form oligonucleotides and 3'-nucleoside monophosphates, and mutants that contain a modified form of polynucleotide phosphorylase do not accumulate nucleoside diphosphates . 5'-Nucleoside monophosphates do accumulate, however, in a mutant containing thermoabile ribonuclease II, under conditions where more than 95% of all enzyme activity had been destroyed . The data presented confirm the participation of ribonuclease I and polynucleotide phosphorylase in the final steps of ribonucleic acid degradation and indicate that an exonuclease forming 5'-nucleoside monophosphates is also involved. J Bacteriol, 1977 Feb, 129(2), 606 - 15 Escherichia coli K-12 structural kdgT mutants exhibiting thermosensitive 2-keto-3-deoxy-D-gluconate uptake; Lagarde AE et al.; A specific method is described for selecting thermosensitive mutants of Escherichia coli K-12 able to grow on 2-keto-3-deoxy-D-gluconate (KDG) and D-glucuronate at 2, but not at 42 degrees C . The extensive analysis of one such mutant is consistent with the conclusion that the carrier molecule responsible for KDG and glucuronate uptake becomes thermolabile . (i) Growth on a variety of carbon sources is perfectly normal at 28 and 42 degrees C, whereas in the same temperature range it gradually diminishes on KDG and glucuronate . (ii) The apparent Km value for KDG is about twofold in the range 25 to 40 degrees C . In the same temperature range, the Vmax values for KDG influx are higher for the mutant compared with those of the wild-type strain, but the optimum temperature is 34 degrees C instead of 38 degrees C . On the contrary, the Vmax values for glucuronate influx are lower for the mutant than for the parental strain, and the optimum temperature for both strains is shifted beyond 40 degrees C . (iii) The activation energies for KDG and glucuronate uptake are about twofold higher in the mutant than in the wild-type strain . (iv) Kinetics of counterflow under deenergized conditions (overshoot) at different temperatures indicate that the defect is located in the translocation step rather than in the processes involved in energy coupling . (v) The first-order rate constants for thermal denaturation are, respectively, 2.5- and 5-fold higher at 40 and 30 degrees C in the mutant than in the wild-type strain, and the activation energy for thermal denaturation is lower . (vi) The carrier molecule in the mutant is also much more sensitive to denaturation by N-ethylmaleimide . (vii) Four independent thermosensitive mutations and one revertatn were located by transduction in or near the kdgT locus, defined previously as the site of nonconditional KDG transport-negative mutations . These results support the conclusion that kdgT represents the structural gene coding for the KDG transport system. J Bacteriol, 1977 Feb, 129(2), 580 - 8 Influence of the stringent control system on the transcription of ribosomal ribonucleic acid and ribosomal protein genes in Escherichia coli; Dennis PP; The fraction of the total ribonucleic acid (RNA) synthesis rate that is messenger RNA (mRNA) for ribosomal protein (r-protein) and ribosomal RNA (rRNA) has been estimated in valS(Ts) rel+ stringent and valS(Ts) relA1 relaxed strains of Escherichia coli during a partial inhibition of valyl-transfer RNA aminoacylation . The partial inhibition was accomplished by shifting the strains from the permissive growth temperature of 29.5 degrees C to the semipermissive temperature of 35.5 degrees C . The RNA synthesized at the elevated temperature was pulse labeled with {3H}uracil . The fraction of the total incorpoarted 3H radioactivity in r-protein mRNA or in rRNA was estimated by specific hybridization to the transducing phages gammaspc1, which carries about 15 r-protein genes and lambdailv5, which carries an rRNA transcription unit . The results clearly demonstrate that the rel gene influences the fraction of the total RNA synthesis rate that is r protein mRNA and rRNA; in the rel+ strain they are significantly increased relative to control cultures . This indicates that the expression of the genes coding for the RNA and protein component of the ribosome are most likely regulated at the level of transcription . Furthermore, it appears that the distribution of functioning RNA polymerase between rRNA genes, r-protein genes, and other types of genes is influenced by the rel gene control system; presumably this influence is mediated through the unusual nucleotide guanosine tetraphosphate. J Bacteriol, 1977 Feb, 129(2), 569 - 73 Control of cell division in Escherichia coli: effect of amino acid starvation; Ron E et al.; The effect of amino acid starvation on cell division was studied in cells of Escherichia coli B . In this bacterial strain, deprivation of a required amino acid resulted in synchronous cell division upon restoration of the amino acid . This synchronization was apparently due to a shift forward in the cell cycle during the starvation . As a consequence, the cells divided at a size that was smaller than normal. J Bacteriol, 1977 Feb, 129(2), 1129 - 40 Further mapping of IS2 and IS3 in the lac-purE region of the Escherichia coli K-12 genome: structure of the F-prime ORF203; Deonier RC et al.; The sequence organization of the F-prime ORF203 was determined by heteroduplex analysis . This large, type II F-prime (Scaife, 1967) contains lac, proC, and purE genes derived from the W1485 subline of Escherichia coli K-12 . The IS3 and IS2 elements previously found in the lac-proC-purE region derived from the 58-161 subline (Hu et al., 1975) are also present in the same locations in the bacterial deoxyribonucleic acid (DNA) from the W1485 subline . Recombination between the IS2 region of F and an IS2 element located between lac and proC on the bacterial DNA apparently led to the formation of the perental Hfr, OR21 . IS2 is thus directly repeated, with one copy of each element appearing at each of the two junctions between F and the bacterial sequences on ORF203 . The F plasmid is found together with ORF203 in the plasmid DNA, and this probably forms from ORF203 by recombination between the directly repeated IS2 elements . ORF203 appears to have been excised from the Hfr chromosome by recombination between the IS3 sequence alpha3beta3 located counterclockwise of lac and the directly repeated IS3 sequence alpha4beta4 located clockwise of purE. J Bacteriol, 1977 Feb, 129(2), 1072 - 7 EcoRI cleavage sites in the argECBH region of the Escherichia coli chromosome; Devine EA et al.; The EcoRI cleavage of deoxyribonucleic acids (DNAs) from lambdadarg phages, carrying argECBH, has been examined . The phages are derived from the heat-inducible, lysis-defective strain lambda y199, and their bacterial DNA, including argECBH, is derived from Escherichia coli K-12 . Such cleavage of the phage DNAs, in each case, produces the D, E, and F segments of lambda . Additionally, these DNAs yield segments, ordered from left to right, of length (in kilobases {kb}) determined by electron microscopy and 0.7% agarose slab gel electrophoresis as follows: lambdadarg13 (ppc argECBH bfe), 13.9, 2.8, 1.5, and 5.6; lambdadarg14 (ppc argECBH), 3.0, 2.0, 17.3, and 6.2; and lambdadarg23 (argECBH), 18.4 and 6.2 . For lambdadarg13 sup102 DNA, the segment analogous to the 13.9-kb segment measures 12.2 kb . The direction from left to right corresponds to the clockwise orientation of the E . coli genetic map . The EcoRI segments define five cleavage sites near the arg region of the E . coli chromosome . For each of the DNAs, the arg genes occur on the largest segment produced . The 17.3-kb segment, being entirely bacterial, represents the argECBH-bearing EcoRI segment of the E . coli chromosome . The location of the arg genes was demonstrated electron microscopically in heteroduplex experiments. J Bacteriol, 1977 Feb, 129(2), 1034 - 44 Mutants of Escherichia coli "cryptic" for certain periplasmic enzymes: evidence for an alteration of the outer membrane; Beacham IR et al.; Mutants in which the expression of periplasmic enzymes by whole cells is reduced (termed "cryptic") are also found to show greatly reduced uptake of labeled adenosine 5'-monophosphate (5'-AMP), providing a rapid assay for crypticity . The crypticity of 3'- and 5'-nucleotidase has been examined as a function of substrate concentration . The Km for 3'- or 5'-AMP increases in the cryptic mutants when whole cells are used as the enzyme source . The Vmax is not altered . Electrophoretic analysis of protein prepared from cell envelopes showed that three cryptic mutants have a polypeptide absent from the outer membrane and a relatively high proportion of a polypeptide in the inner membrane . Analysis of the molar ratios of constituent sugars of the lipopolysaccharides showed no differences between three cryptic mutants and the parent strain . One cryptic mutant (3--41), however, has altered sensitivity to phage T4 . By selection for phage resistance, derivatives of the cryptic mutants that are deoxycholate sensitive have been obtained . These mutants are no longer cryptic . We suggest that cryptic mutants have an altered outer membrane, with decreased permeability to 3'- and 5'-AMP, as a result of an altered polypeptide. J Bacteriol, 1977 Feb, 129(2), 1020 - 33 Establishment of exponential growth after a nutritional shift-up in Escherichia coli B/r: accumulation of deoxyribonucleic acid, ribonucleic acid, and protein; Brunschede H et al.; The accumulation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein was followed in cultures of Escherichia coli B/r during exponential growth in different media and for 2 h after a nutritional shift-up from succinate minimal medium (growth rate {mu1} = 0.67 doublings per h) to glucose plus amino acids medium (mu2 = 3.14 doublings per h) . During postshift growth of the culture, the amounts of RNA (R), DNA (D), and protein (P) increased such that the ratios of the increments (delta R/delta P; delta D/delta P) were constants (k1, k2) . This implies that the rates of accumulation of nuclei1:k2:1 . These constants change from their preshift value to their final postshift value (i.e., k1 and k2) within a few minutes after the shift . k1 is a function of the activity of ribosomes, whereas k2 is related to the initiation of rounds of DNA replication . These parameters and the observed change in the doubling time of RNA (= mu2/mu1) were used to derive kinetic equations that describe the accumulation of DNA, RNA, protein, and cell mass during the 2- to 3-h transition period after a shift-up . The calculated kinetics agree closely with the observed kinetics. Eur J Biochem, 1977 Feb, 72(3), 559 - 69 The functional and fluorescence properties of Escherichia coli RNA polymerase reacted with fluorescamine; Damjanovich S et al.; 1 . Fluorescamine (4-phenylspiro{furan-2,(3)1'-phthalan}-3,3'-dione) reacts rapidly with Escherichia coli RNA polymerase and produces a fluorescent derivative which is inactivated to an extent dependent upon reagent concentration . Excess fluorescamine is rapidly hydrolysed . Reaction is with xi-amino gruops of lysine residues in all subunits as revealed by gel electrophoresis and fluorescence scanning . 2 . The extent of inactivation and fluorescence yield are diminished in the presence of added template, a finding which provides evidence for the existence of reactive and essential amino groups which can be at least partially shielded by DNA in the binary complexes . The relative decrease of fluorescence is greatest in the betabeta' subunits . Holoenzyme and core enzyme show essentially the same behavior . 3 . The inactivation of activity by fluorescamine is primarily at the level of initiation . Template binding and chain propagation are less affected . 4 . The enzyme derivatized by fluorescamine shows an intense fluorescence with a peak at 490 nm and an excitation maximum at 390 nm . The fluorescence lifetime is in the range of 3-8 ns and the emission is highly polarized . In reactions carried out at high ionic strength the fluorescence yield is approximately double that at low ionic strength and insensitive to the presence of template . 5 . Energy transfer is observed between the derivatized enzyme as donor and ethidium bromide as acceptor in the presence of template to which both the enzyme and intercalating dye are bound . The transfer efficiency is a function of the relative concentrations and of the conditions of reaction with fluorescamine . An average transfer distance of approx . 4-5 nm has been calculated suggesting a close proximity between bound polymerase and helical regions of the template. Eur J Biochem, 1977 Feb, 72(3), 515 - 23 Selective inhibition of tRNATyr transcription by guanosine 3'-diphosphate 5'-diphosphate; Debenham P et al.; Guanosine 3'-diphosphate 5'-diphosphate (ppGpp) selectively reduces the synthesis of su+III tRNA from omega 80 psu+III DNA relative to the synthesis of omega 80 RNA in a system in vitro containing DNA and Escherichia coli RNA polymerase holoenzyme as the sole macromolecular components . The response of su+III tRNA synthesis to increasing salt and to temperature in the presence of ppGpp suggests that the nucleotide may reduce the affinity of the enzyme for su+III promoters . The Ki for the selective inhibition of tRNA synthesis by ppGpp is 4 muM in contrast to the value of 150 muM for the inhibition of rRNA synthesis. JACEP, 1977 Feb, 6(2), 62 - 5 Necrotizing fasciitis: a persistent surgical problem; Defore WW Jr et al.; Necrotizing fasciitis is a rare but specific clinical entity which, if not diagnosed early and treated aggressively, is rapidly fatal . The disease was first described during the Civil War and continues to be associated with a 50% mortality . The infectious process is diagnosed by the presence of a widespread fascial necrosis with extensive undermining of the adjacent tissues . The initial mechanism of injury as well as the location and etiologic agent of the suppurative fasciitis may vary . A review of 41 cases of necrotizing fasciitis occurring over a 22-year period disclosed an overall mortality of 39% . Most often, the mortality was related to the severity of the associated diseases and a failure to recognize the disease process promptly . The rate may be lowered by early recognition and prompt surgical intervention coupled with intensive supportive therapy. J Virol, 1977 Feb, 21(2), 560 - 4 Reproductive fitness of P1, P2, and Mu lysogens of Escherichia coli; Edlin G et al.; P1, P2, and Mu lysogens of Escherichia coli reproduce more rapidly than nonlysogens during aerobic growth in glucose-limited chemostats . Thus, prophage-containing stains of E . coli are reproductively more fit than the corresponding nonlysogens . If mixed populations are grown by serial dilution under conditions in which growth is not limited, both the lysogen and nonlysogen manifest identical growth rates . The increased fitness of the lysogens in glucose-limited chemostats correlates with a higher metabolic activity of the lysogen as compared with the nonlysogen during glucose exhaustion . We propose that P1, P2, Mu, and lambda prophage all confer an evolutionarily significant reproductive growth advantage to E . coli lysogenic strains. J Virol, 1977 Feb, 21(2), 554 - 9 Increased reproductive fitness of Escherichia coli lambda lysogens; Lin L et al.; Lambda lysogens of Escherichia coli reproduce more rapidly than nonlysogens during aerobic growth in glucose-limited chemostats . If the environment is changed to anaerobic growth, the situation is reversed, and the lysogen reproduces more slowly than the nonlysogen . Based on a tetrazolium dye assay, the increased fitness of the lambda lysogen during aerobic growth seems to result from a continued high metabolic rate as glucose becomes limiting, whereas the metabolic rate of the nonlysogen declines . The lambda rex gene is required for the growth advantage of lysogens since lack of rex function causes lambda lysogens to lose their reproductive advantage over nonlysogens. J Med Chem, 1977 Feb, 20(2), 233 - 6 P-Aminobenzoic acid derivatives as inhibitors of the cell-free H2-pteroate synthesizing system of Escherichia coli; Thijssen HH; A heterogeneous series of compounds, derived from p-aminobenzoic acid (PABA), has been investigated for their PABA-antagonistic potency in a cell-free H2-pteroate synthesizing system of E . coli . A prerequisite of compounds, other than sulfones or sulfonamides, to compete with PABA for the enzyme H2-pteroate synthetase appeared to be the presence of a p-aminobenzoyl moiety . Substitution of the carboxyl group of PABA by an ester, an amide, or a ketone function, however, strongly reduces the ability to interact with the PABA binding site on the enzyme . This decrease in affinity probably has to be ascribed to the inability to create a sufficient negative charge in the carbonyl part of these p-aminobenzoyl derivatives . The relatively high affinities of L-PABG (16), PABP (22), and the alpha-phenyl derivative of 22, as compared with the other substituted p-aminobenzamides and p-aminobenzene-1-alkanones, are explained by assuming that these compounds, besides interfering with the PABA receptor site, also interact with an accessory area on the enzyme. J Lab Clin Med, 1977 Feb, 89(2), 308 - 15 Biological activities of tritiated endotoxins: correlation of the Limulus lysate assay with rabbit pyrogen and complement-activation assays for endotoxin; Tomasulo PA et al.; Tritiated endotoxins were prepared by three different methods . The biological activities of the tritiated endotoxins were determined by the Limulus amebocyte lysate assay, a rabbit pyrogen assay, and a complement-activation assay and were compared to native, unlabeled endotoxin . All three tritiated endotoxin preparations manifested adequate biological activity in each of the three assay systems, and all three assays ranked the biological activity of the different endotoxin preparations in the same order . Endotoxin tritiated by the Wilzbach procedure retained most of its biological activity and also had the highest specific radioactivity . The good correlation between the Limulus lysate, rabbit pyrogen, and complement-activation assays suggests that the same active site of the endotoxin molecule is identified by the three different assays. J Hyg (Lond), 1977 Feb, 78(1), 95 - 8 Escherichia coli serotypes in the faeces of healthy adults over a period of several months; Shooter RA et al.; The faeces of nine subjects eating mainly at home were collected at regular intervals over periods ranging from 2--5 months . Although a large number of serotypes of E . coli were isolated, the variety per subject was lower than is usually found . In most subjects only a limited number of serotypes persisted over most of the periods of study while many serotypes were only isolated on single occasions. J Cell Biol, 1977 Feb, 72(2), 292 - 301 Ultrastructure of a periodic protein layer in the outer membrane of Escherichia coli; Steven AC et al.; Matrix protein (36,500 daltons), one of the major polypeptides of the Escherichia coli cell envelope, is arranged in a periodic monolayer which covers the outer surface of the peptidoglycan . Although its association with the peptidoglycan layer is probably tight, the periodic structure of the peptidoglycan . Although its association with the peptidoglycan later is probably tight, the periodic structure is maintained in the absence of peptidoglycan, and is therefore based on strong protein-protein interactions . A detailed analysis of the ultrastructure of the matrix protein array by electron microscopy and image processing of specimens prepared by negative staining or by freeze-drying and shadowing shows that the molecules are arranged according to three fold symmetry on a hexagonal lattice whose repeat is 7.7 nm . The most pronounced feature of the unit cell, which probably contains three molecules of matrix protein, is a triplet of indentations, each approx . 2 nm in diameter, with a center-to-center spacing of 3nm . They are readily penetrated by stain and may represent channels which span the protein monolayer. J Am Vet Med Assoc, 1977 Feb 1, 170(3), 340 - 2 Effect of oral inoculation of Escherichia coli on colostral antibody production in cattle; Ward AC et al.; Oral inoculation of approximately 1.2 x 10(9) viable Escherichia coli to pregnant cows resulted in increased blood serum and colostral whey titers to the "O" antigen . The antibody titers were more pronounced in colostral whey and were correlated with the inoculum strain of Escherichia coli . There was no correlation between antibody titers of the colostrum ingested and the resulting serum antibody titers of the calves . The incidence of diarrhea in calves did not correlate with the antibody titer in the colostrum . The occurrence of diarrhea was significantly greater in calves that did not ingest colostrum until they were 12 hours old, compared with calves that had free access to their dams and suckled within an hour of birth. Fertil Steril, 1977 Feb, 28(2), 182 - 5 Isolation of a spermatozoal immobilization factor from Escherichia coli filtrates; Paulson JD et al.; A low-molecular weight spermatozoal immobilization factor (SIF) has been isolated from Escherichia coli cultures . This factor is stable to heating, freezing, and lyophilization, and immobilizes but does not kill spermatozoa . The concentration of spermatozoa influenced the effectiveness of the SIF in immobilizing spermatozoa, indicating that bacterial infections could have a greater influence on the oligospermic ejaculate than on a normal specimen . SIF is a stable factor and binds to spermatozoa via a reversible mechanism. Acta Pathol Microbiol Scand {C}, 1977 Feb, 85(1), 65 - 72 In vitro stimulation of human lymphocytes by Bordetella Pertussis; Andersen V et al.; Bordetella pertussis (B.p.) induces blast transformation of human lymphocytes; whole killed B.p . are more efficient than extracts obtained by sonication . Similar responses were obtained with each of the four strains used in the Danish pertussis vaccine . B.p . with low amounts of Protective Antigen and Histamine-Sensitizing Factor also induced lymphocyte transformation, but were less toxic to the lymphocytes at high concentrations . The supernatants of B.p . cultures were purified with respect to Lymphocytosis Promoting Factor; evidence is presented that these purified fractions possess T-lymphocyte mitogenic activity . Lymphocytes from all normal humans were stimulated by B.p., including cells from cord blood . Cells from childbearing women, obtained immediately after delivery, showed a general depression of lymphocyte transformation including the response to B.p . Children with whooping cough had a lower lymphocyte response to B.p . than healthy children . A highly significant correlation was observed between the responses to B.p . and to E . coli in the adults and newborn examined . It is concluded that the major part of the lymphocyte transformation induced by B.p . is non-specific. J Infect Dis, 1977 Feb, 135(2), 195 - 200 Variation in enterotoxigenicity of Escherichia coli; Echeverria P et al.; The possibility that the variable severity of diarrheal disease due to enterotoxigenic Escherichia coli might be explained by quantitative differences in the activity of heat-labile enterotoxin was examined . The amount of toxin secreted by 13 enteropathogenic strains of E . coli was quantitated by measurements of the toxin-dependent increase in adenosine 3':5'-cyclic phosphate (cyclic AMP) in Chinese hamster ovary cells . The activity ranged from 150 pmol of cyclic AMP/ml per mg of protein to 4,040 pmol/ml per mg . With three representative strains there was good correlation (r = -0.999; P less than 0.05) between the amounts of cyclic AMP accumulated intracellularly (4,040, 2,071, and 470 pmol of cyclic AMP/ml per mg of protein) and the ability of the filtrate to distend the rabbit ileal loop, as measured by the amounts of toxin required to produce half-maximal distension (50% effective dose, which had values of 11.5, 27.5, and 38.5 mg, respectively) . The observed strain-to-strain variation in toxin activity may explain the variation in severity of diarrheal disease caused by enterotoxigenic E . coli. J Bacteriol, 1977 Feb, 129(2), 959 - 66 Functional mosaicism of membrane proteins in vesicles of Escherichia coli; Adler LW et al.; Membrane vesicles of Escherichia coli prepared by osmotic lysis of lysozyme ethylenediaminetetracetate (EDTA) spheroplasts have approximately 60% of the total membrane-bound reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (ED 1.6.99.3) and Mg2+-adenosine triphosphatase (ATPase) (EC 3.6.1.3) activities exposed on the outer surface of the inner membrane . Absorption of these vesicles with antiserum prepared against the purified soluble Mg2+-ATPase resulted in agglutination of approximately 95% of the inner membrane vesicles, as determined by dehydrogenase activity, and about 50% of the total membrane protein . The unagglutinated vesicles lacked all dehydrogenase activity and may consist of outer membrane . Lysozyme-EDTA vesicles actively transported calcium ion, using either NADH or adenosine 5'-triphosphate (ATP) as energy source . However, neither D-lactate nor reduced phenazine methosulfate energized calcium uptake, suggesting that the observed calcium uptake was not due to a small population of everted vesicles . Transport of calcium driven by either NADH or ATP was inhibited by simultaneous addition of D-lactate or reduced phenazine methosulfate . Proline transport driven by D-lactate oxidation was inhibited by either NADH oxidation or ATP hydrolysis . These results suggest that the portion of the total population of vesicles capable of active transport, i.e., the inner membrane vesicles, are functionally a homogeneous population but cannot be categorized as either right-side-out or everted, since activities normally associated with only one side of the inner membrane can be found on both sides of the membrane of these vesicles . Moreover, the data indicate that oxidation of NADH or hydrolysis of ATP by externally localized NADH dehydrogenase or Mg2+-ATPase establishes a protonmotive force of the opposite polarity from that established through D-lactate oxidation. J Bacteriol, 1977 Feb, 129(2), 948 - 58 Constitutive mutations in the controlling site region of the araBAD operon of Escherichia coli B/r that decrease sensitivity to catabolite repression; Colome J et al.; Strains of Escherichia coli B/r containing a deletion of the regulatory gene araC are Ara- . Slow-growing revertants of these strains were isolated and designated aralc because they contain a second mutation in a controlling site, aral, that allows for a low level of constitutive expression of the araBAD operon (Englesbert et al., 1969) . We mutagenized aralc delta C strains and selected mutants that grow faster in mineral L-arabinose medium . The new mutations, called araXc, map very close to the original aralc mutations and are in the controlling site region between araB and araC . The aralcXc delta C strains have a higher constitutive level of expression of the araBAD operon than the aralc delta C parents . The araXc mutations are cis acting and decrease the araBAD operon's sensitivity to catabolite repression . The araBAD operon is expressed equally well in ara delta C and ara C cya crp backgrounds . The repressor form of ara C protein is able to repress the constitutive synthesis due to the ara Xc allele. Eur J Biochem, 1977 Feb, 72(3), 571 - 81 Purification and properties of a periplasmic protein related to sn-glycerol-3-phosphate transport in Escherichia coli; Boos W et al.; Protein GLPT, a periplasmic protein previously recognized as closely related to the active transport of sn-glycerol-3-phosphate in Escherichia coli was isolated by the cold osmotic shock procedure . It was purified by Sephadex chromatography and isoelectric focussing . The purified protein does not exhibit any detectable binding activity toward sn-glycerol-3-phosphate . It has no activity as a glycerol phosphatase nor as a glycerol kinase . Polyacrylamide gel electrophoresis in the presence of dodecylsulfate of the protein subsequent to treatment in urea, boiling in dodecylsulfate and crosslinking indicates that it occurs as an oligomeric protein composed of four identical subunits of 40 000 molecular weight . Membrane vesicles of wild-type strains that contain protein GLPT in whole cells loose it during vesicle preparation . However, they still exhibit high transport activity toward sn-glycerol-3-phosphate . Membrane vesicles prepared from glp T mutants that may or may not contain protein GLPT do not transport sn-glycerol-3-phospahte . We conclude from these results that protein GLPT does not participate in the energy-dependent active transport through the cytoplasmic membrane but could be involved in facilitating the diffusion of sn-glycerol-3-phosphate through the outer layers of E . coli. Acta Pathol Microbiol Scand {B}, 1977 Feb, 85B(1), 103 - 7 K antigen determination of Escherichia coli by counter-current immunoelectrophoresis (CIE); Semjen G et al.; The application of the counter-current immunoelectrophoresis (CIE) for determination of E . coli acidic polysaccharide K antigen is described . The most appropriate dilutions of antigens and antisera were established after examination of six different K antigens with homologous OK antisera . According to this result all test strains for acidic polysaccharide K antigens, i.e . K1 to K57, K62, K74, K82, K53, K84, K92, K93, K94, K95, K96, K97, K98 and K100, were examined and OK antisera suitable for the CIE test were selected . The following reciprocal cross-reactions were found: K2 and K62; K7 and K56; K12 and K82; K13, K20 and K23; K18 and K22; K16 and K97, and K53 and K93 . The CIE method is now used as a routine test in our laboratory. J Med Microbiol, 1977 Feb, 10(1), 77 - 85 Evaluation of methods for the determination of Q and K antigens of an 02:K1(L) strain of Escherichia coli; Deb JR et al.; Tests made on ten colonies from a strain of Escherichia coli O2:K1 demonstrated that bacterial agglutination tests were reliable for identifying the O antigen of serogroup O2 but were unreliable for identifying the K1 antigen . The granular nature of K agglutination was not a reliable characteristic of the L type of K antigen . In contrast, indirect haemagglutination, immunodiffusion and immunoelectrophoresis tests with bacterial extracts gave consistent results with all colonies . The polysaccharide K1 antigen formed a long anodic precipitation line with two peaks, indicating its heterogenous nature, and partial fusion of this line with the O-antigen precipitation line suggested the presence of common serological determinants . In addition, a heat-labile protein antigen, possibly another K antigen, was identified by indirect haemagglutination tests and may have produced a short anodic precipitation line . The results also showed that the K1 antigen was still produced after storage of a culture for 12 years on Dorset-egg medium. J Natl Cancer Inst, 1977 Feb, 58(2), 239 - 43 Effects of streptovaricins and their degradation products on RNA-directed DNA polymerase of Rauscher leukemia virus; Li LH et al.; The activities of streptovaricin complexes, streptovaricins, streptovals, and streptovarinic degradation products were elevated against RNA-directed DNA polymerases of Rauscher leukemia virus, DNA-dependent DNA polymerase of bacterial and mammalian cells, and DNA-dependent RNA polymerases of mammalian origin . The activities of streptovaricins were also listed for comparison purposes . The effects of streptovaricin complexes on viral DNA polymerases varied significantly from lot to lot, and streptovaricin complex lot 7 was the most active . All the streptovals and streptovaricin degradation products except varicinal A showed a marked improvement (twofold to tenfold) in activity against the viral enzyme over the parent streptovaricins . None of these compounds, however, displayed any significant effect on either the DNA polymerase of L1210 leukemia cells and Escherichia coli or the RNA polymerase of isolated nuclei of mouse liver . As a result of tests in these systems, some specific inhibitors of RNA-directed DNA polymerases of Rauscher leukemia virus were selected. J Gen Microbiol, 1977 Feb, 98(2), 519 - 28 Evaluation of the role of methional, 2-keto-4-methylthiobutyric acid and peroxidase in ethylene formation by Escherichia coli; Primrose SB; During growth of Escherichia coli strain SPA O in the presence of methionine, an intermediate accumulates in the medium . This intermediate reacts with 2,4-dinitrophenylhydrazine, and can be degraded to ethylene either enzymically or photochemically, the latter being stimulated by the addition of a flavin . The pH optimum for the photochemical degradation of this intermediate and 2-keto-4-methylthiobutyric acid (KMBA) is pH 3 whereas the optimum for methional is pH 6 . The enzyme which converts the intermediate to ethylene also converts KMBA to ethylene and has many of the properties of a peroxidase including inhibition by catalase, cyanide, azide and anaerobiosis . The enzyme which synthesizes the intermediate is not known but requires oxygen and pyridoxal phosphate . A pathway for ethylene biosynthesis is proposed in which methionine is converted to KMBA which can be degraded either by peroxidase or in a flavin-mediated photochemical reaction . Its relevance to the properties of other ethylene-producing bacteria and to the proposed pathway of ethylene release by higher plants is discussed. Proc Natl Acad Sci U S A, 1977 Feb, 74(2), 505 - 9 Membrane-associated assembly of M13 phage in extracts of virus-infected Escherichia coli; Wickner W et al.; Assembly of coliphage M13 is known to occur as the viral DNA crosses the cytoplasmic membrane, shedding its virus-coded DNA unwinding protein and acquiring from the membrane approximately 2400 copies of the major coat protein . Conditions are described in which extracts of M13-infected E . coli and membranes prepared from such extracts will support virus assembly at a rate equivalent to that of intact cells . Extracts prepared from cells infected with temperature-sensitive M13 mutants in genes 1, 3, 4, or 5 are temperature-sensitive in this cell-free assembly reaction . Phage assembly in vitro requires magnesium and as yet an unidentified heat-stable cofactor of low molecular weight . The rate of virus assembly is approximately linear with respect to extract concentration over a 10(4)-fold range, consistent with the observation that the entire M13 assembly activity copurifies with the cell membrane fraction. J Biochem (Tokyo), 1977 Feb, 81(2), 371 - 9 Nucleoside triphosphate phosphohydrolase associated with cytoplasmic polyhedrosis virus; Shimotohno K et al.; Nucleoside triphosphate phosphohydrolase {EC 3.6.1.15} activity was found to be included in silkworm cytoplasmic polyhedrosis (CP) virus, which synthesizes mRNA carrying the 5'-terminal modification . This enzyme releases orthophosphate from the gamma-position in a nucleoside triphosphate, leaving nucleoside diphosphate . The rate of hydrolysis of ATP is faster than that of any other ribonucleoside triphosphate . Deoxy ATP is hydrolyzed rather faster than ATP . However, polynucleotides carrying triphosphate at the 5'-terminus, that is, 4S RNA which was synthesized by E . coli RNA polymerase {EC 2.7.7.6} using calf thymus DNA as a template, and the phage Q beta RNA (30S), are not effective substrates for this enzyme . Although the CP virion loses the viral genome and one kind of protein component on proteolytic treatment with pronase, the partially degraded virion still retains phosphohydrolase activity . The phosphohydrolase must therefore be associated firmly with the virion . This enzyme does not require the presence of nucleic acid for its function . Phosphohydrolysis of ATP by this enzyme activity represents a first step in the synthesis of the 5'-terminal modified mRNA of CP virus. Infect Immun, 1977 Feb, 15(2), 396 - 401 Bacteriostatic effect of human milk and bovine colostrum on Escherichia coli: importance of bicarbonate; Griffiths E et al.; At pH 7.4 and in the presence of NaHCO3, human milk and bovine colostrum inhibited the growth of Escherichia coli O111 . Adding sufficient iron to saturate the iron-binding capacity of the lactoferrin present in the milk or colostrum prevented bacteriostasis . At pH 6.8 neither molk nor colostrum inhibited E . coli 0111 . Adjusting the pH to 7.4 with NaHCO3 resulted in the development of bacteriostatic activity . Adjusting the pH to 7.4 with NaOH was ineffective . Dialyzed colostrum and milk inhibited bacterial growth at pH 6.8 in the absence of added NaHCO3; addition of citrate or iron abolished bacteriostasis . The chromatographic elution profile of tyrosyl-transfer ribonucleic acid (tRNA) from iron-replete E . coli differs significantly from that of tyrosyl-tRNA from iron-deficient organisms . Examination of the elution profile tyrosyl-tRNA from E . coli 0111 growing in colostrum without added NaHCO3 showed that such bacteria were fully replete in iron . The nature of the elution profile of tyrosyl-tRNA also showed that iron was freely available to the bacteria when citrate was added to dialyzed colostrum but not available in its absence, even at pH 6.8 . Results support the idea that the bacteriostatic action of milk and colostrum, due to the combined action of antibody and lactoferrin, depends on the addition of bicarbonate to counteract the iron-mobilizing effect of the citrate normally present in these secretions. J Bacteriol, 1977 Feb, 129(2), 967 - 72 Isolation of an Escherichia coli mutant deficient in thioredoxin reductase; Fuchs J; A mutant of Escherichia coli defective in thioredoxin reductase has been isolated and partially characterized . This mutant has no detectable thioredoxin reductase activity in vitro and yet it exhibits no in vivo defect in reduction of ribonucleotides . Evidence is presented that indicates that, in cells permeabilized via ether treatment, ribonucleoside diphosphate reduction can utilize glutathione as an alternate reducing system. J Bacteriol, 1977 Feb, 129(2), 763 - 9 Adenosine 5'-triphosphate synthesis driven by a protonmotive force in membrane vesicles of Escherichia coli; Tsuchiya T; Adenosine 5'-triphosphate (ATP) synthesis energized by an artificially imposed protonmotive force (delta p) in adenosine 5'-diphosphate-loaded membrane vesicles of Escherichia coli was investigated . The protonmotive force is composed of an artificially imposed pH gradient (delta pH) or membrane potential (deltapsi), or both . A delta pH was established by a rapid alteration of the pH of the assay medium . A delta psi was created by the establishment of diffusion potential of K+ in the presence of valinomycin . The maximal amount of ATP synthesized was 0.4 to 0.5 nmol/mg of membrane protein when energized by a delta pH and 0.2 to 0.3 nmol/mg of membrane protein when a delta psi was imposed . Simultaneous imposition of both a delta pH and delta psi resulted in the formation of greater amounts of ATP (0.8 nmol/mg of membrane protein) than with either alone . The amount of ATP synthesized was roughly proportional to the magnitude of the artificially imposed delta p . Although p-chloromercuribenzoate, 2-heptyl-4-hydroxyquinoline-N-oxide, or NaCN each inhibits oxidation of D-lactate, and thus oxidative phosphorylation, none inhibited ATP synthesis driven by an artificially imposed delta p . Membrane vesicles prepared from uncA or uncB strains, which are defective in oxidative phosphorylation, likewise were unable to catalyze ATP synthesis when energy was supplied by an artificially imposed delta p. Res Exp Med (Berl), 1977 Jan 28, 169(3), 213 - 9 {High-voltage electrophoresis of dihydrofolate reductase from Escherichia coli W 3110 (author's transl)}; Schalhorn A et al.; A method for the high-voltage electrophoresis of dihydrofolate reductase from Escherichia coli W 3110 is described . Dihydrofolate reductase catalyses the reduction of dihydrofolic acid to tetrahydrofolic acid . By addition of a tetrazolium salt, tetrahydrofolic acid reacts by formation of a violet water insoluble formazane which is an indicator for the enzyme . Besides several unspecific bands, two isoenzymes of the dihydrofolate reductase from Escherichia coli W 3110 are found which are specificially inhibited by the folate antagonists methotrexate and trimethoprime in a concentration of 0, 1muM, 1 muM respectively. Science, 1977 Jan 28, 195(4276), 393 - 4 Plasmid detection and sizing in single colony lysates; Barnes WM; A simple and contained procedure for the rapid assay of the presence and size of plasmids similar to Col El is described . Bacteria are picked from an agar plate with a toothpick, lysed with dodecyl sulfate and heat, and placed directly on an agarose gel for electrophoresis. Science, 1977 Jan 28, 195(4276), 391 - 3 Novel screening procedure for recombinant plasmids; Telford J et al.; Lysed bacterial colonies containing potential recombinant plasmids were mixed with molten agar and sealed into slots of an agarose gel . After electrophoresis overnight, the gel was stained with ethidium bromide, which clearly reveals recombinant plasmids . Xenopus laevis ribosomal DNA and histone DNA of Psammechinus miliaris were ligated into pCRI plasmids and screened by this method. Science, 1977 Jan 28, 195(4276), 389 - 91 Nucleotide sequences from the rabbit beta globin gene inserted into Escherichia coli plasmids; Browne JK et al.; A 169-nucleotide region from the rabbit beta globin gene has been sequenced by analysis of complementary DNA's cloned in bacterial plasmids . Comparison of these sequences with those established for this gene by other techniques provides evidence of a high degree of fidelity and allows the unambiguous establishment that these plasmids do not contain harmful sequences. J Biol Chem, 1977 Jan 25, 252(2), 483 - 91 Isolation and purification of double-stranded ribonuclease from calf thymus; Ohtsuki K et al.; A RNase from calf thymus, which specifically cleaves native or synthetic double-stranded RNA molecules endonucleolytically, has been isolated and purified from calf thymus . For optimal activity, the enzyme requires a sulfhydryl reagent and divalent cations; over 95 per cent of the activity is inhibited by 0.5 mm ethidium bromide . The degradation of {3H}poly(C)-poly(I) by purified enzyme preparations yields labeled dinucleotides and octanucleotides; the latter oligonucleotide contained 5'-phosphate and 3'-hydroxyl termini . The enzyme cleaves high molecular weight RNAs such as RNA products formed in vitro by T3 phage-induced RNA polymerase from T3 phage DNA, heterogeneous RNA isolated from duck reticulocyte nuclei, and 45 S RNA isolated from rat liver nucleoli . The mode of degradation of RNA in vitro with the double-stranded RNase is similar to that of Escherichia coli RNase III and appears to act endonucleolytically . The degradation of 45 S RNA with the enzyme results in the production of 29 S and 19 S RNA fragments . These findings suggest that the enzyme may be involved in the processing of high molecular weight precursor RNAs to mRNA or rRNAs in a manner analogous to that reported for RNase III of E . coli. J Biol Chem, 1977 Jan 25, 252(2), 499 - 503 On the role of ATP in phosphodiester bond hydrolysis catalyzed by the recBC deoxyribonuclease of Escherichia coli; Eichler DC et al.; The deoxyribonuclease specified by the recB and recC genes of Escherichia coli (recBC DNase; exonuclease V) has been purified to near homogeneity by a new procedure . Although hydrolysis of even a single nucleotide from a duplex DNA molecule by the pure enzyme is absolutely dependent upon ATP, the extent of phosphodiester hydrolysis is strongly inhibited by ATP concentrations of 0.2 mm or greater, and the initial rate is unaffected . Under these conditions, the extent of DNA hydrolysis is proportional to enzyme concentration . In contrast, neither the rate nor the extent of hydrolysis of single-stranded DNA nor ATP is affected by high concentrations of ATP . The amount of large single-stranded polynucleotide generated by the action of the recBC DNase increases as the ATP concentration increases and, at 0.5 mM ATP, becomes equivalent to the amount of acid-soluble nucleotide formed . These findings suggest that high intracellular concentrations of ATP affect the mechanism of the recBC DNase so as to limit the extent of hydrolysis of duplex DNA, while at the same time favoring the formation of single-stranded regions within the duplex . Such regions may be essential intermediates in the recombination process. J Biol Chem, 1977 Jan 25, 252(2), 471 - 8 Codon-acticodon recognition in the valine codon family; Mitra SK et al.; An in vitro protein-synthesizing system completely dependent on added valine tRNA (valyl-tRNAval) and programmed with RNA from the phage MS2 has been used to investigate the incorporation into MS2 coat protein of valine from isoaccepting valyl-tRNAsval with the anticodons U AC (U represents 5-oxyacetic acid uridine monophosphate), GAC, and IAC in response to the four valine codons GUU, GUC, GUA, and GUG . By examining the incorporation of valine into NH2-terminal and internal positions of three tryptic peptides from the MS2 coat protein it has been established that these anticodons each recognize all four valine codons . We therefore conclude that under our conditions of in vitro protein synthesis the genetic code, as far as the valine codons are concerned, is operationally a two letter code, i.e . the third codon nucleotide has no absolute discriminating function. Biochemistry, 1977 Jan 25, 16(2), 298 - 305 Fluorescence and nucleotide binding properties of Escherichia coli uridine diphosphate galactose 4-epimerase: support for a model for nonstereospedific action; Wong SS et al.; The fluorescence emission spectrum for reduced diphosphopyridine nucleotide (DPNH) in Escherichia coli uridine diphosphate galactose 4-epimerase-DPNH complexes has a maximum at 435 nm, which is about twice as intense when the excitation is at 280 nm as at 340 nm . The fluorescence excitation spectrum monitored at 460 nm has two maxima, one at 340-345 nm and another about twice as intense at 280 nm . The polarization of DPNH fluorescence by these complexes is 0.43-0.44 compared with 0.46 for DPNH immobilized in propylene glycol at -20 degrees C . The small degree of fluorescence depolarization is due to rotational relaxation of the protein, relaxation time 205 ns . The excited-state lifetimes in epimerase-DPNH-nucleotide complexes are 3.5-4.2 ns . The fluorescence data show that the dihydropyridine ring in these complexes is highly immobilized and exhibits no detectable independent motion relative to rotational motions of the protein . The inhibition constants for uridine monophosphate (UMP) and 2,2,6,6-tetramethyl-4-piperidinyl-1-oxyl uridyl pyrophosphate acting as competitive reversible inhibitors of epimerase-DPN+ are 1.2 and 0.2 mM, respectively, at 27 degrees C in 0.1 M sodium bicinate buffer at pH 8.5 . A collection of Ki and Km values for uridine nucleotide inhibitors and substrates indicates that the principle substrate binding interactions involve the nucleotide moieties of substrates . Dissociation constants for uridine nucleotides dissociating from epimerase-DPNH-nucleotide complexes, measured by ultraviolet absorption and fluorescence techniques, are 12 muM for UMP, 14 muM for UDP-hexopyranoses, 4 muM for UDP-pentopyranoses, 27 muM for p-bromoacetamidophenyl uridyl pyrophosphate, 0.14 muM for UDP-4-ketohexopyranose intermediate, and 0.36 muM for UDP-4-ketopentopyranose intermediate at 27 degrees C in 0.1 M sodium bicinate buffer at pH 8.5 . Analysis of th