Mol Gen Genet, 1976 Oct 18, 148(1), 49 - 55 Multiple regulation of nucleoside catabolizing enzymes in Escherichia coli: effects of 3:5' cyclic AMP and CRP protein; Hammer-Jespersen K et al.; The regulation of the synthesis of nucleoside metabolizing enzymes has been studied in cya and crp mutant strains of Escherichia coli . The synthesis of the cyt-enzymes, cytidine deaminase and uridine phosphorylase regulated by the cytR gene product, is activated by the cAMP-CRP complex . On the other hand the synthesis of the deoenzymes: deoxyriboaldolase, thymidine phosphorylase, phosphodeoxyribomutase and purine nucleoside phosphorylase, appears to be increased if an active cAMP-CRP complex cannot be formed . It also seems that nucleosides serve as poor carbon sources for cya and crp mutants; this could not solely be explained by low levels of nucleoside metabolizing enzymes nor by a deficiency in nucleoside uptake . Addition of casamino acids stimulated the growth of cya and crp mutants, with nucleosides as carbon sources . When grown on glucose and casamino acids growth could be stimulated by adenine and hypoxanthine nucleosides; these results suggest an impaired nitrogen metabolism in cya and crp mutants. N Engl J Med, 1976 Oct 14, 295(16), 849 - 53 Infantile diarrhea produced by heat-stable enterotoxigenic Escherichia coli; Ryder RW et al.; Between December, 1974, and August 1975, intestinal illness occurred in 55 of 205 infants admitted to the special-care nurseries of a large children's hospital . Escherichia coli serotype 078:K80:H12, which produced a heat-stable enterotoxin, was isolated from 18 of 25 symptomatic infants as compared with 14 of 55 asymptomatic infants (P less than 0.001) . Colistin administered prophylactically to 24 culture-negative asymptomatic infants did not prevent colonization in 10, whereas colonization did occur in 22 of 56 not receiving colistin (P = 1.0) . This outbreak provides laboratory and epidemiologic evidence that heat-stable enterotoxigenic Esch . coli is pathogenic in human beings and produces infantile diarrhea. Arch Microbiol, 1976 Oct 11, 110(1), 135 - 43 The site for catabolite deactivation in the L-arabinose BAD operon in Escherichia coli B/r; Bass R et al.; A series of deletions beginning in the leu operon and continuing into the araC gene and also into the ara controlling site region were analyzed in reciprocal merodiploids, e.g., F' A2Cc67/B24delta719, F' B24delta719/A2Cc67, for their effects on catabolite deactivation (CD) . The results of these experiments are consistent with placing the catabolite gene activator-cyclic AMP sensitive site in the controlling site region between araB and araO . With a deletion mutant, delta1109, that places araBAD under leu control when transcription begins at leuP, the araBAD operon is immune to CD even though araCGA, araP and araI are intact and functional . To focus attention on the fine structure and related functions of this region we propose that the three proteins that function therein have separate sites of action: araI (initiator-site for activator), araP (promoter-site for RNA polymerase) and ara(CGA) (catabolite gene activator-site for CGA-cAMP) . None of the eighteen initiator constitutive mutants (Ic) tested have any significant effect on catabolite derepression or on the maximal level of expression of the operon supporting the view that the araI site may be distinct from araP and ARA(CGA) . A series of constitutive mutants in the araC gene (Cc) also have no pronounced effect on catabolite deactivation. J Biol Chem, 1976 Oct 10, 251(19), 5986 - 91 Determination of ligand binding: partial and full saturation of aspartate transcarbamylase . Applicability of a filter assay to weakly binding ligands; Suter P et al.; Carbamyl phosphate and succinate each bind to six sites in the hexameric aspartate transcarbamylase from Escherichia coli when both ligands are present in saturating concentrations . Their respective dissociation constants are 2.4 and 1400 muM . Positive homotropic interaction, shown earlier for the association of succinate with the enzyme in the presence of carbamyl phosphate (Changeux, J.-P., Gerhart, J.C., and Schachamn, H.K . (1968) Biochemistry 7, 513-538), is also found for carbamyl phosphate binding in the presence of succinate . Apparent half-of-the-sites saturation, previously described for carbamyl phosphate binding in the absence of succinate (Rosenbusch, J.P., and Griffin, J.H . (1973) J . Biol, Chem . 248, 5063-5066), also occurs when succinate binds to the enzyme in the absence of carbamyl phosphate . A second class of three low affinity sites for carbamyl phosphate could be detected in the enzyme when succinate was absent . These results indicate that aspartate transcarbamylase exists in an asymmetric state under several defined conditions . The results reported were obtained with a highly sensitive filter bonding assay, modified to allow the study of the protein-ligand interactions with dissociation constants in the millimolar range . The assay is described in detail . Its validity is demonstrated by the good correlation of the results obtained with those observed with independent methods. J Biol Chem, 1976 Oct 10, 251(19), 5911 - 20 The NADH dehydrogenase of the respiratory chain of Escherichia coli . I . Properties of the membrane-bound enzyme, its solubilization, and purification to near homogeneity; Dancey GF et al.; The NADH dehydrogenase of the Escherichia coli respiratory chain has been identified by the following properties: (a) its location in membrane vesicles; (b) its inhibition by AMP in a fashion similar to that of the NADH oxidase; (c) its specificity for NADH, but not NADPH, with the same Km for NADH as that of the NADH oxidase; (d) its sensitivity when membrane-bound to inhibition by dicoumarol, rotenone, and 2-heptyl-4-hydroxyquinoline-N-oxide, which are also inhibitors for the NADH oxidase . The NADH-dehydrogenase of the cytosol fraction (assayed as NADH-dichlorphenolindophenol reductase activity) differs substantially from the membrane-bound activity both in substrate specificity and in the inhibitors of the reaction . The respiratory chain NADH dehydrogenase was extracted from isolated membrane vesicle preparations by solubilization in Triton X-100, and was purified in buffers containing that detergent . The purification employed chromatography on DEAE-cellulose, precipitation by 30% ethanol, and chromatography on hydroxyalapatite and DEAE-agarose . The most highly purified preparations of the enzyme were homogeneous in migration on polyacrylamide gels containing Triton X-100, at pH 9.5, where one band accounted for all of the protein and activity . Electrophoresis on polyacrylamide gels containing sodium dodecul sulfate showed 1 band of molecular weight 38,000, which accounted for over 75% of the protein on the gel . Because of requirements for either Triton X-100 or phospholipid for activity of the purified enzyme, it is difficult to estimate the level of purification achieved over isolated membrane vesicles . However, we estimate that the enzyme was purified some 30-fold over membrane vesicles, or some 300-fold over whole cells. J Biol Chem, 1976 Oct 10, 251(19), 5866 - 74 EcoRI endonuclease . Physical and catalytic properties of the homogenous enzyme; Modrich P et al.; A procedure for large scale isolation of Escherichia coli RI endonuclease in high yield has been developed . The purified enzyme is homogeneous as judged by polyacrylamide gel electrophoresis and analytical sedimentation . The denatured and reduced form of the enzyme has a molecular weight of 28,500 +/- 500 . In solution the enzyme exists as a mixture of dimers and tetramers of molecular weights 57,000 and 114,000, respectively . We estimate the dissociation constant for tetramer to dimer transition to be less than or approximately equal to 1 x 10-7 M . Steady state kinetic analysis of the endonuclease with ColE1 DNA as substrate showed that the enzyme obeys Michaelis-Menten kinetics . At 37 degrees the turnover number is four double strand scissons per min, and the Km for ColE1 molecules is 8 x 10(-9) M . At 0 degrees the major product of endonuclease action contains only one single strand break in the RI site, and such molecules can dissociate from the enzyme . In contrast, at 30 degrees to 37 degrees, two single strand breaks are introduced into the RI sequence prior to dissociation of the enzyme . A transient enzyme-bound intermediate containing only one break in the RI site was observed in studies of a single turnover at 30 degrees . Kinetic analysis of this reaction indicates that the first break is introduced into the RI site with the first order rate constant of at least 40 min-1, while the second cleavage occurs with a rate constant of 14 min-1 . Since the turnover number of the enzyme at 30 degress is only 0.72 min-1, these results indicate that the rate-limiting step is release of endonuclear from its DNA product. J Biol Chem, 1976 Oct 10, 251(19), 5976 - 85 Evidence from 13C NMR for protonation of carbamyl-P and N-(phosphonacetyl)-L-aspartate in the active site of aspartate transcarbamylase; Roberts MF et al.; Nuclear magnetic resonance has been used to study the binding of {13C}carbamyl-P (90% enriched) to the catalytic subunit of Escherichia coli aspartate transcarbamylase . Upon forming a binary complex, there is a small change in the chemical shift of the carbonyl carbon resonance, 2 Hz upfield at pH 7.0, indicating that the environments of the carbonyl group in the active site and in water are similar . When succinate, an analog of L-aspartate, is added to form a ternary complex, there is a large downfield change in the chemical shift for carbamyl-P, consistent with interaction between the carbonyl group and a proton donor of the enzyme . The change might also be caused by a ring current froma nearby aromatic amino acid residue . From the pH dependence of this downfield change and from the effects of L-aspartate analogs other than succinate, the form of the enzyme involved is proposed to be an isomerized ternary complex, previously observed in temperature jump and proton NMR studies . The downfield change to chemical shift for carbamyl-P bound to the isomerized complex is 17.7 +/- 1.0 Hz . Using this value, the relative ability of other four-carbon dicarboxylic acids to form isomerized ternary complexes with the enzyme and carbamyl-P has been evaluated quantitatively . The 13C peak for the transition state analog N-(phosphonacetyl)-L-aspartate (PALA), 90% enriched specifically at the amide carbonyl group, is shifted 20 Hz downfield of the peak for free PALA upon binding to the catalytic subunit at pH 7.0 . In contrast, the peak for {1-13C} phosphonaceatmide shifts upfield by about 6 Hz upon binding . Since PALA induces isomerization of the enzyme and phosphonacetamide does not, these data provide further evidence consistent with protonation of the carbonyl group only upon isomerization . The degrees of protonation is strong acids of the carbonyl groups of PALA, phosphonacetamide and urethan (a model for the labile carbamyl-P) have been determined, as have the chemical shifts for these compounds upon full protonation . From these data it is calculated that the amide carbonyl groups of carbamyl-P and PALA might be protonated to a maximum of about 20% in the isomerized complexes at pH 7.0 . The change in conformation of the enzyme-carbamyl-P complex upon binding L-aspartate, previously proposed to aid catalysis by compressing the two substrates together in the active site, may be accompanied by polarization of the C=O bond, making this ordinarily unreactive group a much better electrophile . A keto analog of PALA, 4,5-dicarboxy-2-ketopentyl phosphonate, also binds tightly to the catalytic subunit and induces a very similar conformational change, whereas an alcohol analog, 4,5-dicarboxy-2-hydroxypentyl phosphonate, does not bind tightly, indicating the critical importance of an unhindered carbonyl group with trigonal geometry. J Biol Chem, 1976 Oct 10, 251(19), 5966 - 75 Aspartate transcarbamylase of Escherichia coli . Heterogeneity of binding sites for carbamyl phosphate and fluorinated analogs of carbamyl phosphate; Ridge JA et al.; Some preparations of both native aspartate transcarbamylase from Escherichia coli and catalytic subunit have fewer tight binding sites per oligomer for carbamyl-P than the number of catalytic peptide chains . In contrast, the number of sites for the tight-binding inhibitor N-(phosphonacetyl)-L-aspartate does equal the number of catalytic chains in each case . Binding of the labile carbamyl-P was determined using rapid gel filtration, with conversion to stable carbamyl-L-aspartate during collection . Native enzyme (six catalytic chains) obtained from cells grown under the conditions of J.C . Gerhart and H . Holoubek (J . Biol . Chem . (1967) 242, 2886-2892) has 5.4 tight sites for carbamyl-P at pH 8.0 (KD = 9.9 muM), whereas native enzyme from cells grown with higher concentrations of glucose, uracil, and histidine (to yield more enzyme per unit volume of culture) has only 1.9 tight sites at pH 8.0 (KD = 4.6 muM) and only 2.3 tight sites at pH 7.0 (KD = 2.6 muM) . At pH 8.0, catalytic subunit (three catalytic chains) obtained from the former native enzyme has 2.2 tight sites for carbamyl-P (KD = 2.4 muM) and the number of sites is 2.3 in the presence of 35 mM succinate, whereas catalytic subunit obtained from the latter native enzyme has 1.8 tight sites (KD = 3.6 muM) in the absence of succinate and 2.3 tight sites in its presence . The number of tight binding sites is also less than the number of subunit peptide chains in 19F nuclear magnetic resonance experiments performed with catalytic subunit and two fluorinated analogs of carbamyl-P at comparable concentrations of analogs and active sites . A model is proposed in which incomplete removal of formylmethionine from the NH2 termini of the enzyme under conditions of extreme depression affects affinity for ligands. J Biol Chem, 1976 Oct 10, 251(19), 5921 - 8 The NADH dehydrogenase of the respiratory chain of Escherichia coli . II . Kinetics of the purified enzyme and the effects of antibodies elicited against it on membrane-bound and free enzyme; Dancey GF et al.; The purified respiratory chain NADH dehydrogenase of Escherichia coli oxidizes NADH with either dichlorophenolindophenol (DCIP) . ferricyanide, or menadione as electron acceptors, with values for NADH are similar with the three electron acceptors (approximately 50 muM) . The purified enzyme contains no flavin and has an absolute requirement for FAD, with Km values around 4 muM . The pH optimum of the enzyme appears to be between 6.5 and 7; the optimum is difficult to establish because of nonenzymatic reduction of DCIP at the lower pH values . Potassium cyanide stimulates the DCIP reductase activity about 2-fold, but has no effect on ferricyanide reductase . The enzyme exhibits hyperbolic kinetics with respect to NADH concentration in both the ferricyanide and DCIP reductase assays, but cooperatively is seen in the menadione reductase reaction . NAD+ is an effective competitive inhibitor of the reaction (Ki congruent to 20 muM); in the presence of NAD+, the NADH saturation curve becomes cooperative, even in the DCIP reductase assay . Many adenine containing nucleotides are competitive inhibitors of the enzyme . The apparent Ki values for these nucleotides as inhibitors of the purified enzyme, the membrane-bound NADH dehydrogenase, and the NADH oxidase are equivalent . An examination of inhibitory effects of a series of adenine nucleotides suggests that the inhibitors act as analogues of NAD+, which is the true physiological inhibitor . The results suggest that the enzyme in situ is always partially inhibited by the levels of NAD- in the E coli cell, and thus behaves in a cooperative fashion to changes in the NAD+/NADH ratio . An antibody has been elicited against the purified NADH dehydrogenase . Immunodiffusion and crossed immunoelectrophoresis show that the antibody is directed principally against the NADH dehydrogenase, with some activity against minor contaminants in the purified preparation . The antibody inhibits NADH dehydrogenase activity 50% at saturating levels . When this antibody preparation is used to examine solubilized membrane preparations, two major immunoprecipitates are found . A parallel inhibition of the membrane-bound NADH dehydrogenase and NADH oxidase activities is seen, supporting the hypothesis that the purified enzyme is indeed a component of the respiratory chain-dependent NADH oxidase pathway. J Biol Chem, 1976 Oct 10, 251(19), 5881 - 7 A stereochemical method for detection of ATP terminal phosphate transfer in enzymatic reactions . Glutamine synthetase; Midelfort CF et al.; An isotope scrambling method is described for the detection of transient {Enz:ADP:P-X} formation from {18O}ATP in ATP-coupled enzyme reactions . The method makes use of torsional symmetry of the newly formed (see article) group in ADP . {18 O}ATP labeled in the betagama bridge oxygen was incubated with enzyme and reversible cleavage of the PbetaO -- Pgamma bond was detected by the appearance of 18O in the beta nonbridge oxygens of the ATP pool . Experiments with sheep brain and Escherichia coli glutamine synthetases show that cleavage of ATP of enzyme-bound ADP and P-X requires glutamate . The exchange catalyzed by the E . coli enzyme with glutamate occurs in the absence of ammonia and is partially inhibited by added NH4Cl, as expected if the exchange is in the mechanistic pathway for glutamine synthesis . The results provide kinetic support for a two-step mechanism where phosphoryl transfer from ATP to glutamate precedes reaction with ammonia. Biochemistry, 1976 Oct 5, 15(20), 4356 - 63 Fidelity of chromatin transcription in vitro; Biessmann H et al.; Chromatin and DNA from Schneider's Drosophila melanogaster cell line 2 were transcribed in vitro with Escherichia coli RNA polymerase . Using mercurated UTP as precursor, the newly synthesized RNA could be separated from DNA and endogenous RNA by affinity chromatography on sulfhydryl-Sepharose 6B . Characterization of the transcription products with complementary DNA (cDNA) made from polyadenylated nuclear RNA and with fractionated cDNA probe demonstrated a fair quantitative fidelity in the in vitro transcript from chromatin which was not evident when DNA was transcribed . However, as shown by hybridization to total nuclear RNA, E . coli RNA polymerase transcribed both DNA strands from chromatin in vitro . We conclude that E . coli polymerase is able to distinguish sections of chromatin at which rapid synthesis of RNA occurs in the cell. Biochemistry, 1976 Oct 5, 15(20), 4429 - 32 Changes in rotational motion of a cell-bound fluorophore caused by colicin E1: a study by fluorescence polarization and differential polarized phase fluorometry; Weber G et al.; The stationary fluorescence polarization and the differential phase delay of the polarized components of the fluorescence of 1-phenylnaphthylamine in Escherichia coli suspensions were measured before and after addition of colicin E1 . Both sets of measurements register an increase in the rotational relaxation time of the fluorescent probe when colicin is present . These increases are absent in an E . coli mutant tolerant to colicin E1 . The physical interpretation of the changes demands separate estimation of the fraction f2 of the emitting fluorophores that change their properties upon colicin addition and of the rotational relaxation time p2 of this fraction, following the colicin-induced changes . By themselves, the steady state polarizaiton observations permit only the conclusion that f2 must be in the range of 1-0.06 and the change in p2/pi between 1.5 and a value larger than 10 . Combination of the data of stationary polarization with those of differential phase fluorometry results in an important reduction in the uncertainty:f2 must be in the range 1-0.33 and the change in p2/pi in the range 1.5-2.5. Biochemistry, 1976 Oct 5, 15(20), 4377 - 85 Physical characterization of a ribosomal nucleoprotein complex; Tritton TR et al.; The complex between ribosomal protein L24 and its RNA binding site (that region of the 23S RNA which the protein protects from ribonuclease digestion) has been studied by various physicochemical methods . The RNA is composed of two fragments of about 160 and 140 nucleotides which interact with each other to form the L24 binding site . Circular dichroism spectroscopy suggests that the two interacting fragments have a unique region of secondary structure which is not present in either of the two components alone; hence there are important structural interactions between regions of the RNA which are separated in the primary sequence . Addition of the L24 protein to the RNA site promotes a structural change associated with base unstacking, but with little or no change in the hydrogen-bonded base pairing . Heat activation is not required for complex formation . Thermal denaturation studies reveal a broad featureless transition and the amount of hypochromic change indicates that the RNA site contains less secondary structure than other RNAs such as tRNA and total rRNA . Temperature-jump relaxation measurements on the mechanism of unfolding of the RNA show a concerted melting of the entire secondary and tertiary structure, which is altered upon addition of the protein . A structrual basis for this RNA-protein complex is discussed. Biochemistry, 1976 Oct 5, 15(20), 4370 - 7 Quantitative determination of the number of secondary and tertiary structure base pairs in transfer RNA in solution; Bolton PH et al.; Resonances in the low-field (11-15 ppm) nuclear magnetic resonance spectrum (NMR) of tRNA molecules arise from secondary and tertiary structure base pairs (1 resonance for each base pair) as well as tertiary structure hydrogen bonds . An accurate method for integrating the low-field spectra has been developed and applied to seven different tRNA . In the presence of high levels of magnesium (10 mM free magnesium) the number of resonances (base pairs) per molecule is typically 3-4 more than the number predicted by the cloverleaf model . These results confirm our recent proposal that, under proper conditions, most tRNA exhibit 3-4 tertiary structure interactions in solution, which are also observed in x-ray diffraction studies of yeast tRNAPhe . In addition to common resonances in the 11-15 ppm region, there are common resonances at 10.5 and 9.5 ppm . A critique of methods used to integrate the low-field spectra is given and possible sources of error are indicated . The discrepancy between our present results and previous studies, which indicated that the number of base pairs per molecule was close to the number predicted by the cloverleaf model, can be attributed partly to differences in magnesium concentration and partly to inaccuracies inherent in the integration methods used. Biochim Biophys Acta, 1976 Oct 4, 447(2), 175 - 87 Escherichia coli DNA polymerases II and III: activation by magnesium or by manganous ions; Helfman WB et al.; Escherichia coli DNA polymerases II and III have been extensively studied in vitro when activated with Mg2+ . The Mn2+-activated polymerization reactions are considered here, and shown to differ from the Mg2+-activated reactions . The Mn2+-activated DNA polymerase II reaction requires K+ or spermidine, and the effects of monovalent cation and polyamine are additive . In contrast, the Mg2+-activated reaction does not require, but is stimulated by, K+ or spermidine, in a non-additive manner . Under optimal conditions, DNA polymerase II is activated better with Mn2+ than it is with Mg2+, suggesting a physiological role for the Mn2+-activated enzyme . The observed preference for Mn2+ over Mg2+ in reaction kinetics and at high DNA template concentrations suggest that Mg2+ may preferentially activate the associated exonuclease activity . At 29 degrees C, the Mn2+-activated DNA polymerase III reaction is stimulated by K+ and inhibited by ethanol or phosphatidylethanolamine . In contrast, the latter compounds and Triton X-100 increase the initial rate of the Mg2+-activated reaction, whereas K+ inhibits this reaction at all concentrations . The K+ inhibition is reduced at low Mg concentrations when Mn2+ is also present . After stimulating the initial reaction rate, ethanol causes a rapid decrease in the rate of the Mg2+-activated reaction during incubation at 20 degrees C . At 27 degrees C, all surface-active compounds inhibit the Mg2+-activated reaction . Preincubation of the enzyme at 30 degrees C or below with DNA template and divalent cation increases the initial reaction rate, suggesting that formation of an enzyme-divalent cation-DNA template complex occurs as the first step in DNA polymerase III catalysis . The apparent Km at 21 degrees C for gapped calf thymus DNA was 25 muM with Mn2+ and 125 muM with Mg2+ for DNA polymerase III, and 18 muM at 30 degrees C for DNA polymerase II with either Mn2+ or Mg2+ . Reactions with poly{d(A-T)} were enhanced by Mn2+ relative to Mg2+, and activity with poly(rA)-poly(dT) was Mn2+ dependent for both enzymes. C R Acad Sci Hebd Seances Acad Sci D, 1976 Oct 4, 283(7), 825 - 8 {Ultrastructure of the particles reconstituted by complementation of extracts from chl-r mutants of Escherichia coli K 12}; Mutaftschiev S et al.; By freeze-fracturing it is shown that the vesicles reconstituted by complementation of the chlA and chlB mutants of E . coli K 12 extracts are characterized by an asymmetric membrane bilayer . In a feature quite similar to the original intact plasma membranes, the membrane splits in two halves and the intramembranous particles are asymmetrically distributed on the two facture faces . It is proposed that the process of membrane reconstitution, which is also associated with the restoration of nitrate-reductase activity, relies on a sequence of increasing complexity of the molecular organisation. Nucleic Acids Res, 1976 Oct, 3(10), 2617 - 32 Integration of eukaryotic genes for 5S RNA and histone proteins into a phage lambda receptor; Clarkson SG et al.; Highly purified HindIII restriction fragments of Xenopus laevis 5S DNA and of Psammechinus miliaris histone DNA have been covalently inserted into a derivative of phage lambda . This phage, genetically constructed by Murray et al . (1), contains only a single target for HindIII in the cI gene . Viable hybrid molecules were detected as clear plaque-forming phage after transfection of E . coli, the vast majority of which were shown by hybridization to be recombinants of the desired type . The lambdaSam7 mutation has been introduced into one hybrid phage containing histone DNA, thereby substantially increasing the yield of recombinant DNA. Biokhimiia, 1976 Oct, 41(10), 1859 - 70 {Control of RNA biosynthesis in rat liver . Some features of RNA biosynthesis during prolonged protein synthesis inhibition}; Todorov IN et al.; A drastic inhibition of protein biosynthesis in rat liver in vivo by cycloheximide (CHI) (0.3 mg/100 g of body weight) first caused an increase of RNA synthesis (after 1 hour), which was then followed by its decrease . Partial gradual restoration of the protein synthesis level was shown to be accompanied by a repeated increase of RNA synthesis (12 hs) and its normalisation after 24 hs . The first maximum of RNA synthesis increase in the isolated nuclei system was AU-type RNA synthesis (sensitive to alpha-amanitine), the second one was due to GC-type RNA synthesis (resistant to this toxin) . Purified chromatine template activity in the system with E . coli RNA polymerase (by 14%) an hour after CHI treatment, but 3 hrs later was decreased and subsequently restored (12 hrs after CHI injection) . The changes of RNA biosynthesis induced by prolonged protein synthesis inhibition suggest the existence of continuous RNA synthesis control in nuclei . This control is realized by translation system using the feed back principle. Am J Vet Res, 1976 Oct, 37(10), 1189 - 93 Blood leukocytes, neutrophil phagocytosis, and plasma corticosteroids in colostrum-fed and colostrum-deprived calves; LaMotte GB et al.; Blood leukocyte patterns, neutrophil phagocytosis of killed Escherichia coli in vitro, and plasma corticosteroids were studied in colostrum-fed (CF) and colostrum-deprived (CD) calves during the 1st 144 hours after birth . There was a marked increase in neutrophil numbers between 6 and 12 hours in CF but not CD calves, apparently as a result of colostrum ingestion . Phagocytosis was inactive at birth but increased quickly thereafter . Phagocytosis was more efficient in CF than in CD calves . Plasma corticosteroid concentrations were high at birth and decreased quickly thereafter in both groups of calves. Vet Med (Praha), 1976 Oct, 21(10), 597 - 607 {The effect of furazolidone or carbadox in the starter mixture for early-weaned piglets}; Dvorak M et al.; For a period of 14 days, piglets from six litters, weaned between the 25th and 28th day of age, were fed the COS 2 starter containing either a premix with furazolidone or carboadox of Czechoslovak origin . Bentonite hydrosilicate was used as a carrier in both cases . Furazolidone administered in the dose of 200 mg per 1 kg of feed prevented diarrhoea, insignificantly increased body weight gain, and decreased the consumption of feed per 1 kg of gain from 5.6 kg in the control to 4.0 kg in the test animals . Carbadox administered in the dose of 50 mg per 1 kg of feed suppressed the signs of enteritis in comparison with the control piglets, significantly increased body weight gains, and reduced feed consumption to 1.9 kg per 1 kg of gain . No differences were recorded in the concentration of blood glucose, total protein, and total cholesterol in plasma . The control piglets showed increased parameters of the adrenocortical function . The proportion (percentage) of haemolytic E . coli in rectum was affected neither by carbadox nor by furazolidone; furazolidone suppressed the occurrence of lactoso-negative strains . An insignificant drop of the number of haemolytic E . coki in the duodenum and jejunum of the furazolidone-and carbadox-treated piglets was observed after 14 days . With their clinical effects, the two substances tested manifest themselves as suitable for the reduction of losses in weaned piglets. Can J Microbiol, 1976 Oct, 22(10), 1522 - 39 Functional and structural differences between photosynthetic and heterotrophic Rhodospirillum rubrum ribosomes and S-100 fractions; Chow CT; Cell-free, protein-synthesizing activity has been tested by using various combinations of the S-100 and ribosome fractions prepared from photosynthetic and heterotrophic Rhodospirillum rubrum . The photosynthetic ribosomes are highly active when combined with either the photosynthetic or the heterotrophic S-100 fractions, whereas the heterotrophic ribosomes are active only when combined with the photosynthetic S-100 fraction . Addition of a photosynthetic pigment-containing fraction to the homologous heterotrophic system is, however, able to stimulate its activity . An inhibitor and an activator involved in cell-free protein synthesis have been isolated from the stationary heterotrophic cells . The inhibitor is a very small, dialyzable compound which inhibits not only the R . rubrum but also the E . coli protein-synthesizing activity in vitro, whereas the activator is a non-dialyzable, small RNA molecule capable of stimulating only the R . rubrum activity . Differences exist between the photosynthetic and the heterotrophic systems in their response to various chemical compounds and to light as well as in their structure. Z Psychosom Med Psychoanal, 1976 Oct-Dec, 22(4), 370 - 7 {Influence of psychological factors on the immune system . Changes in susceptibility to infection after infantile stimulation in relationship to age}; Schlewinski E; NMRI mice of both sexes and in various stages of the suckling period were handled oncea day for three minutes . They were intraperitoneally infected with E . coli O 111 on the eighteenth day of life . Handling carried out from the first to the 18th day led to a significantly higher mortality (P = 1,4%) as compared to the control animals . Differences in mortality were not observed when the stimulation occurred on the 1st to 10th or 10th to 18th day of life . Weight determination carried out just prior to infection showed no remarkable differences between the experimental groups. J Biochem (Tokyo), 1976 Oct, 80(4), 895 - 8 Enzyme-linked sandwich immunoassay of ornithine delta-aminotransferase from rat liver using antibody-coupled glass rods as solid phase; Hamaguchi Y et al.; A macromolecular antigen, ornithine delta-aminotransferase {EC 2.6.1.13} from rat liver (OAT) was assayed by the sandwich procedure using rabbit (anti-OAT) Fab'-beta-D-galactosidase complex and rabbit (anti-OAT) IgG-coupled glass rods as a solid phase . The Fab' fragments of the rabbit (anti-OAT) IgG were conjugated with beta-D-galactosidase {EC 3.2.1.23} from Escherichia coli using N, N'-o-phenylenedimaleimide . The rabbit (anti-OAT) IgG was coupled to the aminoalkylsilyl glass rods using glutaraldehyde . The rabbit (anti-OAT) IgG-coupled glass rods were incubated with OAT and then with the rabbit (anti-OAT) Fab'-beta-D-galactosidase complex . The amount of OAT was determined from the activity of beta-D-galactosidase bound to the glass rods . A minimum of 0.03 fmoles of OAT could be determined by this method and use of the glass rods gave greater reproducibility, and was more sensitive and simpler than use of Sepharose 4B. J Biochem (Tokyo), 1976 Oct, 80(4), 821 - 30 Escherichia coli membrane D-lactate dehydrogenase . Isolation of the enzyme in aggregated from and its activation by Triton X-100 and phospholipids; Tanaka Y et al.; D-Lactate dehydrogenase was obtained in an aggregated form consisting of 2 to 3 molecules of the monomer enzyme after removal of most Triton X-100 from the preparation as described previously (1) . The aggregate dissociated reversibly to the monomeric form after addition of 0.06% or 1.0% Triton X-100 . Formation of these aggregates was confirmed by the finding that the enzyme activity was only partially sensitive to specific antibody . The specific activity of the aggregated enzyme was one-third that of the enzyme with Triton X-100 and it increased approximately 5-fold on addition of phospholipids or cardiolipin of Escherichia coli and lecithin from egg yolk . Both the monomer and micelle forms of Triton X-100 caused activation of the enzyme . The activity of the aggregates after preincubation with Triton X-100 or phospholipids was completely inhibited by specific antibody . The difference in the properties of the aggregated enzyme after preincubation with Triton X-100 and with phospholipids suggested that its interaction with phospholipids was stronger than with Triton X-100 . Kinetic studies also suggested a difference between the interactions of the enzyme with phospholipids and with Triton X-100 . Aggregated enzyme had an apparent Km value for D-lactate similar to that of membrane-bound enzyme after preincubation with phospholipids. Nucleic Acids Res, 1976 Oct, 3(10), 2593 - 603 Specific replacement of Q base in the anticodon of tRNA by guanine catalyzed by a cell-free extract of rabbit reticulocytes; Okada N et al.; Guanylation of tRNA by a lysate of rabbit reticulocytes was reported previously by Farkas and Singh . This reaction was investigated further using 18 purified E . coli tRNAs as acceptors.Results showed that only tRNATyr, tRNAHis, tRNAAsn and tRNAAsp which contain the modified nucleoside Q in the anticodon acted as acceptors . Analysis of the nucleotide sequences in the guanylated tRNA showed that guanine specifically replaced Q base in these tRNAs. J Gen Microbiol, 1976 Oct, 96(2), 383 - 91 Uptake of fructose by the sorbitol phosphotransferase of Escherichia coli K12; Jones-Mortimer MC et al.; Strains of Escherichia coli that are unable to grow on fructose because they lack the phosphoenolpyruvate: fructose phosphotransferases specified by ptsF and ptsX mutate to grow on media containing fructose as sole carbon source, but do not regain the function of either of the missing phosphotransferases . Instead, fructose is taken up and phosphorylated to fructose 6-phosphate by a phosphoenolpyruvate: sorbitol phosphotransferase which, in wild-type cells, is induced by sorbitol but not by fructose, but which is constitutively expressed in these mutants . The regulatory gene srlC controlling enzymes of sorbitol uptake and catabolism has been located on the E . coli genome as part of the linkage group cysI srlC attI86 pheA. J Gen Microbiol, 1976 Oct, 96(2), 269 - 75 Haemagglutinating and adhesive properties associated with the K99 antigen of bovine strains of Escherichia coli; Burrows MR et al.; The K99 antigen common to some bovine strains of Escherichia coli caused mannose-resistant haemagglutination of sheep erythrocytes and was shown to be responsible for the attachment of K99-positive bacteria to calf brush-border preparations because (i) strains grown at 18 degrees C did not produce K99 antigen, cause haemagglutination, or attach to brush borders; (ii) a K12 (K99+) recombinant strain showed both haemagglutinating activity and attachment to brush borders whereas, before it received the K99 plasmid, the recipient strain was negative in both respects; and (iii) cell-free extracts of K99 antigen showed haemagglutinating activity and inhibited the attachment of K99-positive organisms to brush borders . K99 antigen appears to be a virulence determinant in the pathogenesis of neonatal calf diarrhoea . It is readily demonstrated by haemagglutination and brush-border attachment tests. Eur J Biochem, 1976 Oct 1, 69(1), 35 - 44 Active transport by membrane vesicles from anaerobically grown Escherichia coli energized by electron transfer to ferricyanide and chlorate; Boonstra J et al.; Active transport of amino acids by membrane vesicles from Escherichia coli, grown anaerobically on glucose in the presence of nitrate, can be energized under anaerobic conditions by electron transfer in the nitrate respiration system with formate as electron donor and nitrate as acceptor . A high rate of amino acid transport is also obtained under anaerobic conditions by electron transfer from formate to the nitrate analogue chlorate or to the membrane-impermeable electron acceptor ferricyanide . Electron transfer from formate to nitrate results in the generation of an electrical potential as is indicated by the uptake of the lipophilic cation triphenylmethylphosphonium . Ferricyanide accpets electrons from at least two sites of the nitrate respiration system . One of these sites appears to be nitrate reductase, because cytochrome b, reduced by formate, is completely reoxidized by ferricyanide and glutamate transport energized by formate plus ferricyanide and formate plus nitrate are affected by the same electron transfer inhibitors . A second site of electron transfer to ferricyanide appears to be located prior to nitrate reductase in the nitrate respiration system, since formate is oxidized at a higher rate in the presence of ferricyanide than with nitrate while formate/ferricyanide energizes transport of amino acids at a lower rate than formate/nitrate . Moreover, electron transfer inhibitors block electron transfer from formate to nitrate to a significantly higher extent than from formate to ferricyanide . The effects of irradiation of the membrane vesicles with near ultra-violet light suggest that quinones play an essential role in the electron transfer from formate to nitrate or ferricyanide . Irradiation blocks completely formate-dependent nitrate and ferricyanide reduction and active transport driven by formate/nitrate and formate/ferricyanide, but has hardly any effect on the activity of formate dehydrogenase and on ascorbate/phenazine methosulphate/oxygen-driven transport . Similar effects of ferricyanide have been observed in membrane vesicles from E . coli, grown anaerobically in the presence of fumarate . In these membrane vesicles a high rate of lactose and triphenylmethylphosphonium uptake under anaerobic conditions is obtained by electron transfer from glycerol 1-phosphate to fumarate and also to ferricyanide and evidence has been presented for the involvement of cytochromes in these electron transfers. Eur J Biochem, 1976 Oct 1, 69(1), 289 - 97 Accumulation of free ribosomal proteins S1, L7, and L12 in Escherichia coli; Ramagopal S; The total content of free ribosomal proteins in the cells of Escherichia coli was determined to study the nature of intracellular accumulation during growth . Labeled ribosomes and post-ribosomal supernatant were prepared from exponentially growing and stationary-phase cultures . The fraction of free ribosomal protein in the supernatant was estimated by resolving both the acidic and basic proteins separately with two different techniques of two-dimensional gel electrophoresis . Free ribosomal proteins in the cell sap were identified on the basis of coelectrophoresis with authentic ribosomal protein markers, molecular weights and amino acid composition . Among the acidic proteins, S1, L7, and L12 were identified and examined in detail . All three proteins accumulated to significant levels in these cultures . Stationary-phase cells contained 2-4 times more free S1, L7, and L12 than midlogarithmic phase cells . Moreover, free S1, L7, and L12 and ribosome-bound forms were stable during exponential and post-exponential growth of cultures . At this growth transition, non-ribosomal proteins in the supernatant and those associated with the ribosomes showed different characteristics of accumulation . The ratio of L12:L7 in the supernatant did not exhibit a remarkable shift during the growth cycle like the ratio of L12:L7 in ribosomes . In addition, free L12 in the supernatant was not acetylated, although there was a rapid acetylation in the cells. Eur J Biochem, 1976 Oct 1, 69(1), 141 - 51 Protease I from Escherichia coli . Some physicochemical properties and substrate specificity; Pacaud M et al.; Protease I, a periplasmic endopeptidase from Escherichia coli has been further purified by a modified procedure . While the purified protein consists of a single polypeptide chain of about 21000 daltons, its molecular weight in dilute salt solution was estimated to be near 43000, suggesting that the enzyme has a marked tendency to dimerize . It has only one disulphide bond and is very sensitive to urea . In agreement with previous evidence of a chymotrypsin-like specificity, hydrolytic assays of various p-nitrophenyl esters of N-substituted amino acids showed that phenylalanine and tyrosine derivatives are the best substrates for the enzyme . The Km(app) for N-benzoyloxycarbonyl-L-tyrosin-p-nitrophenyl ester at pH 7.5 In 100 mM sodium phosphate buffer at 25 degrees C was found to be 0.2 mM . In contrast to chymotrypsin, protease I is unable to hydrolyse N-acetyl-L-phenylalanine ethyl ester and its tyrosine analogue . Moreover, the enzyme appears devoid of amidase activity and exhibits a low activity upon polypeptides . At 37 degrees C, it cleaves the carboxymethylated B-chain of bovine insulin at four points: Phe25-Tyr26, Phe24-Phe25, Leu15-Tyr16 and Ser9-His10 . From a detailed study of peptides bonds hydrolyzed, it was concluded that protease I has a stringent requirement for both residues forming the scissile bond, and appears to possess an extended hydrophobic binding site. Eur J Biochem, 1976 Oct 1, 69(1), 105 - 15 The action pattern of amylomaltase from Escherichia coli; Palmer TN et al.; Amylomaltase, the inducible 4-alpha-glucanotransferase of Escherichia coli strain ML, has been purified to homogeneity . Its specific activity with a commercial maltose substrate was 500 mkat/kg protein (30 mumol glucose formed min-1 mg protein-1) . The purified enzyme, dependent on buffer concentration, exists in interconvertible low-molecular-weight (apparent molecular weight 71000) and high-molecular-weight (apparent molecular weight 370000) forms . The specificity of amylomaltase has been redefined . Hitherto, the enzyme was thought to be a glucosyltransferase, catalysing the transfer of single glucosyl units, and maltose has been regarded as its most important substrate . Amylomaltase is now shown to exhibit both glucosyl-transfer and 4-alpha-glucanosyl-transfer specificity . 4-alpha-Glucanosyl chains containing up to at least nine glucosyl units can be transferred . However, it is concluded that the transfer reaction by which amylomaltase action was originally expressed, does not take place, i.e., Maltose + maltose in equilibrium Maltotriose + glucose and that maltose has a restricted role as a substrate . This may be due to the inability of maltose to function as a donor substrate, serving only as an acceptor substrate . It is confirmed that when a maltodextrin serves as a donor, that portion of the molecule transfered by the enzyme is that containing the nonreducing-end-group . Enzyme action on chromatographically pure maltose is characterized by a lag phase in the time course of glucose release . The lag pahse is overcome by addition of 'priming' (catalytic) concentrations of maltotriose or higher maltodextrins . An autocatalytic reaction mechanism involving the generation of primer molecules is proposed to explain the action of the enzyme on maltose . The redefined action pattern of amylomaltase is consistent with the redefined role of the enzyme in the utilization of exogenous and endogenous 1,4-alpha-glucans by E . coli. Br J Surg, 1976 Oct, 63(10), 774 - 8 Endotoxin, bile salts and renal function in obstructive jaundice; Bailey ME; It is generally agreed that there is an increased incidence of postoperative renal failure in patients with obstructive jaundice . The proposition that this may be due to endotoxin, and that the endotoxin may be absorbed from the patient's own bowel flora, has been investigated . The study was conducted in patients and in animals . Twenty-four patients with obstructive jaundice were studied . Sixteen had endotoxin in the portal blood at operation and 13 of these had peripheral endotoxaemia at the time of operation or developed it during the 72 hours following operation . Only 3 of the patients had a proved possible site of infective origin for the endotoxaemia, the remainder absorbing the endotoxin from their own gastro-intestinal organisms . Portal endotoxaemia occurred when the serum bilirubin was 8-5 mg/100 ml or greater . There was a highly significant decrease in mean endogenous creatinine clearance postoperatively in patients with endotoxaemia, and expression of this as a percentage postoperative fall compared with preoperative levels enhanced the significance . Jaundiced patients without endotoxaemia had no significant fall in endogenous creatinine clearance . Matched non-jaundiced control patients did not develop portal or peripheral endotoxaemia, and there was no significant fall in postoperative creatinine clearance . Experiments using animals showed that the absence of bile salts in the intestinal tract in obstructive jaundice allows endotoxin to be absorbed, and that this absorption can be prevented by the oral administration of bile salts . The therapeutic implications in patients are discussed. Appl Environ Microbiol, 1976 Oct, 32(4), 465 - 9 Factors affecting catalase level and sensitivity to hydrogen peroxide in Escherichia coli; Yoshpe-Purer Y et al.; Composition of the culture medium, growth phase, and temperature play important roles in the sensitivity of Escherichia coli to H2O2 . The medium and growth phase affected the sensitivity of the cells to H2O2 by modifying the amount of catalase synthesized by them, whereas the effect of temperature was due to the thermolability of the enzyme . Since catalase is unstable in the presence of its substrate, the correlation between the catalase level in the cells and their sensitivity to H2O2 could be observed only when the H2O2 concentration was not excessive in proportion to the amount of catalase. Proc Natl Acad Sci U S A, 1976 Oct, 73(10), 3700 - 4 Prophage lambda induction of Escherichia coli K12 envA uvrB: a highly sensitive test for potential carcinogens; Moreau P et al.; A simple, inexpensive, and sensitive test for potential carcinogens based upon the property of carcinogens to induce prophage lambda is described . By using chemicals activated with microsomal enzymes and E . coli K12 permeable (envA) tester bacteria also deficient in DNA repair (uvrB), the range of carcinogens detected in a lysogenic induction test (inductest) has been extended . We have provided the evidence that, after activation, carcinogenic polycyclic hydrocarbons such as benzo{a5pyrene and 7,12-dimethylbenz{a}anthracene induce prophage lambda . Three variants of the test have been developed (inductests I, II, and III), which are as sensitive as the mutagenicity test of Ames et al . {Ames, B . N., McCann, J . and Yamasaki, E . (1975) Mutat . Res . 31, 347-364} . Inductests II and III provide a quantitative estimation of the inducing activity of a carcinogen . With the latter test, one can determine: (i) the cellular toxic effect of a carcinogen and (ii) the kinetics of appearance and disappearance of active metabolites . For two series of chemicals, aflatoxins and benz{a}anthracenes, there is a good correlation between their carcinogenic activity in rodents and their prophage inducing activity in bacteria . The fact that the majority of the cell population is induced makes it possible to test the inducing activity of carcinogens at the biochemical level, e.g., by measuring lambda repressor inactivation. Proc Natl Acad Sci U S A, 1976 Oct, 73(10), 3676 - 9 Polyclonal activation of bone-marrow-derived lymphocytes from human peripheral blood measured by a direct plaque-forming cell assay; Fauci AS et al.; A culture and assay system for the stimulation of human peripheral blood lymphocytes with polyclonal activators of bone-marrow-derived lymphocytes (B cells), such as pokeweed mitogen and Escherichia coli lipopolysaccharide, and subsequent measurement of single cell antibody production by a hemolysis-in-bel direct plaque-forming cell assay against sheep erythrocytes has been established . The critical culture requirements have been delineated and a new highly sensitive ultrathin gel assay method has been described . Under these conditions a substantial and highly reproducible plaque-forming cell response was detected in normal human peripheral blood . This system can be readily used to explore the complex events associated with activation of human B cells. Proc Natl Acad Sci U S A, 1976 Oct, 73(10), 3529 - 33 The functional repressor parts of a tetrameric lac repressor-beta-galactosidase chimaera are organized as dimers; Kania J et al.; The chimaeric protein repressor-galactosidase, in which fully active lac repressor is covalently linked to the active enzyme beta-galactosidase, was used as a system for probing the quaternary structure of lac repressor . Electron micrographs revealed repressor-galactosidase to be a tetrameric aggregate . When lac repressor, alone, was crosslinked with dimethyl suberimidate, dimers, trimers, tetramers, and oligomers of the protein subunit were produced, whereas crosslinking of the tetrameric repressor-galactosidase resulted in the production of only dimers of the chimaera . Treatment of lac repressor with iodine resulted in the formation of protein dimers; the same result was obtained with repressor-galactosidase . After limited proteolysis of lac repressor, no crosslinking was obtained after treatment with dimethyl suberimidate, whereas iodine still produced a covalent linkage . These results are interpreted as evidence that the lac repressor parts of the tetrameric repressor-galactosidase-chimaera are organized as dimers on the tetrameric-beta-galactosidase core . Because this chimaera has been previously shown to have normal repressor activity {B . Muller-Hill and J . Kania (1974) Nature, 249,561-563}, we conclude that lac repressor still is biologically active as a dimeric aggregate. Proc Natl Acad Sci U S A, 1976 Oct, 73(10), 3511 - 5 Mechanism of DNA elongation catalyzed by Escherichia coli DNA polymerase III, dnaZ protein, and DNA elongation factors I and III; Wickner S; Elongation of a primed single-stranded DNA template catalyzed by E . coli DNA polymerase III (DNA nucleotidyltransferase, deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) requires dnaZ protein and two other protein factors, DNA elongation factors I and III . The reaction occurs by the following mechanism: (i) dnaZ protein and DNA elongation factor III together catalyze the transfer of DNA elongation factor I to a primed DNA template . This transfer reaction requires ATP or dATP in addition to dnaZ protein, DNA elongation factors I and III, and primed template; it does not require DNA polymerase III . (ii) DNA polymerase III binds to the complex of DNA elongation factor I with primed template; it does not bind to primed template which is not complexed with DNA elongation factor I . This binding reaction proceeds in the absence of ATP or dATP as cofactor, dnaZ protein, and DNA elongation factor III and without additional DNA elongation factor I . (iii) The complex of DNA polymerase III, DNA elongation factor I, and primed template catalyzes DNA synthesis upon the addition of dNTPs. Proc Natl Acad Sci U S A, 1976 Oct, 73(10), 3502 - 5 Regulation of ornithine decarboxylase activity by guanine nucleotides: in vivo test in potassium-depleted Escherichia coli; Sakai TT et al.; The observation that guanosine 5'-triphosphate (GTP) is an activator and guanosine 5'-diphosphate-3'-diphosphate (ppGpp) is an inhibitor of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) of Escherichia coli {E . Holtta et al . (1974) Biochem . Biophys . Res . Commun . 59, 1104-1111} has been confirmed . The hypothesis that synthesis of both polyamine and RNA in E . coli is regulated in vivo by these nucleotides was tested in E . coli B-207 . On transfer of this K+-requiring, amino-acid-deficient strigent strain from K+-medium to Na+-medium, the organism stops protein synthesis, maintains a high rate of RNA synthesis, and increases putrescine synthesis from ornithine manyfold . Under these conditions, the cells do not markedly change their contents of GTP and ppGpp . The proposed mechanism of regulation of RNA and putrescine synthesis by guanine nucleotides does not appear to explain the metabolic phenomena observed in this organism during K+ deficiency . Nevertheless, amino acid depletion in K+-medium does result in a marked increase in ppGpp. Proc Natl Acad Sci U S A, 1976 Oct, 73(10), 3492 - 6 Amplification in Escherichia coli of enzymes involved in genetic recombination: construction of hybrid ColE1 plasmids carrying the structural gene for exonuclease I; Vapnek D et al.; Endo-R-HindIII restriction endonuclease fragments obtained from F30 and pMB9 plasmid DNAs were ligated in vitro and used to transform a recB21 recC22 sbcB15 strain of E . coli K-12 . The inability of this strain to stably maintain pMB9 alone permitted the isolation of transformants that carried hybrid plasmids containing the sbcB+ allele . These transformants became sensitive to ultraviolet light and recombination defieient and showed a 25-fold increase in the level of exonuclease I activity . The stability of the sbcB hybrid plasmids and their effects on exonuclease I activity have also been determined in wild-type and recA1 genetic backgrounds . The presence of the plasmids results in a 7-fold increase in the level of exonuclease I in a wild-type strain and 15-fold increase in a recA1 strain . The increased activity in the recA1 mutant appears to be a result of increased plasmid stability in this genetic background. Proc Natl Acad Sci U S A, 1976 Oct, 73(10), 3471 - 5 lac repressor: 3-fluorotyrosine substitution for nuclear magnetic resonance studies; Lu P et al.; This paper describes the isolation of 3-fluorotyrosine-substituted lac repressor, and its 19F nuclear magnetic resonance spectrum . From the spectrum, one can conclude that for each of the four identical subunits of the repressor there are four or five surface tyrosines, two buried or internal tyrosines, and one tyrosine with an phenolic group ionized or involved in a hydrogen bond . Conditions are described that can be used for the 3-fluorotyrosine substitution of a variety of Escherichia coli proteins for 19F nuclear magnetic resonance studies. Proc Natl Acad Sci U S A, 1976 Oct, 73(10), 3423 - 7 Conversion of beta-galactosidase to a membrane-bound state by gene fusion; Silhavy TJ et al.; We have isolated a series of strains in which the lacZ gene has been fused to one of the maltose operons, such that the synthesis of beta-galactosidase (beta-D-galactoside galactohydrolase; EC 3.2.1.23) is inducible by maltose . The most frequent event that generates such fusions results in strains in which an intact lacZ gene has become a part of the malE,F operon . By using a special selection procedure, we have detected much rarer fusion events resulting in an altered beta-galactosidase molecule . In these strains, we presume that there is a hybrid protein molecule produced, comprised of an NH2-terminal amino acid sequence from a maltose transport protein (malF) and a COOH-terminal amino acid sequence from beta-galactosidase . The hybrid protein, which still retains some beta-galactosidase activity, is found in the cytoplasmic membrane . These results provide information on the component of the malF gene essential for incorporation of its product into the membrane. J Virol, 1976 Oct, 20(1), 290 - 306 DNA of minute virus of mice: self-priming, nonpermuted, single-stranded genome with a 5'-terminal hairpin duplex; Bourguignon GJ et al.; The genome of the nondefective parvovirus minute virus of mice (MVM) is a linear DNA molecular weight 1.48 x 10(6), which is single stranded for approximately 94% of its length . In contrast to the genomes from defective parvoviruses MVM DNA does not contain a detectable inverted terminal redundancy . A combination of enzymatic and physical techniques has shown that the molecule contains a stable hairpin duplex of approximately 130 base pairs located at the 5' terminus of the genome . MVM DNA is efficiently utilized as a template-primer by a number of DNA polymerases, including reverse transcriptases . Polymerases lacking 5' to 3' exonuclease activity yield a duplex DNA product with a molecular weight 1.96 times that of the viral genome, in which the newly synthesized complementary strand is covalently attached to the template . This duplex contains an internal "nick" that can be sealed by DNA ligase to produce a self-complementary single-strand circle . The MVM DNA duplex is cleaved twice by EcoR-RI restriction endonuclease to yield three distinct fragments in molar amounts . These results suggest that the initiation of DNA synthesis in vitro occurs at a point within 100 bases of the 3' end of the genome, using the 3' terminus of viral DNA as a primer, and that the sequence of nucleotides in the genome is not permuted. J Bacteriol, 1976 Oct, 128(1), 510 - 3 Thiogalactoside transacetylase of the lactose operon as an enzyme for detoxification; Andrews KJ et al.; Thigalactoside transacetylase, the lacA gene product, confers selective advantage to cells of Escherichia coli K-12 growing on beta-galactosides in the presence of non-metabolizable analogues. J Bacteriol, 1976 Oct, 128(1), 490 - 1 Iron-requiring mutant of Escherichia coli carrying a deletion in the aroG-nadA region of the chromosome; Nagy J et al.; A mutant of Escherichia coli K-12 carrying a deletion in the aroG-nadA region of the genome requires a high concentration of iron for growth . The strain is chromium sensitive, and in the presence of citrate lower concentrations of iron support cell growth . The deletion mutant lost a gene between aroG and nadA that is responsible for the uptake of iron. J Bacteriol, 1976 Oct, 128(1), 487 - 9 Genetic location of the gene (ush) specifying periplasmic uridine 5'-diphosphate glucose hydrolase (5'-nucleotidase) in Escherichia coli K-12; Beacham IR et al.; Further genetic mapping to two- and three-factor crosses show that the ush gene is closely linked to two other genes hemG and pisA, and that the probable gene order is proC-hemG-plsA-ush-gal. J Bacteriol, 1976 Oct, 128(1), 485 - 6 Convenient method for detecting 14CO2 in multiple samples: application to rapid screening for mutants; Tabor H et al.; A procedure is presented for the rapid screening of bacterial colonies to detect mutants unable to produce 14CO2 from a labeled precursor . The method is especially useful for mass screening for mutants that cannot be easily detected by their phenotypic characteristics. J Bacteriol, 1976 Oct, 128(1), 477 - 80 Relationship of Bdellovibrio elongation and fission to host cell size; Kessel M et al.; The extent of Bdellovibrio growth, and hence progeny produced in infected cells, appears to depend upon host cell size as determined from the ratio of ultimitate length of Bdellovibrio to host cell area calculated from light microscopy. J Bacteriol, 1976 Oct, 128(1), 463 - 72 Molecular cloning of an Escherichia coli plasmid determinant than encodes for the production of heat-stable enterotoxin; So M et al.; A conjugative plasmid, ESF0041 was isolated from an enterotoxigenic strain of Escherichia coli from calves . ESF0041 was found to be 65 x 10(6) daltons in mass of a member of the F incompatibility complex . Acquisition of ESF0041 by E . coli K-12 was invariably associated with the capacity to produce heat-stable (ST) enterotoxin . ESF0041 and pSC101 deoxyribonucleic acids were cleaved with EcoRI, and the fragments were ligated with polynucleotide ligase . Transformation of E . coli K-12 with the ligation mixture led to the isolation of an ST+ clone . Further analysis of the plasmid deoxyribonucleic acid from this clone showed that a structural gene(s) associated with ST biosynthesis had been isolated as a 5.7 x 10(6)-dalton ESF0041 fragment in pSC101 . In turn, 5.7 x 10(6)-dalton fragment was ligated to a multicopy COLE1 derivative, RSF2124, so that toxin synthesis was amplified about threefold. J Bacteriol, 1976 Oct, 128(1), 390 - 3 Cysteine and methionine content of the Escherichia coli ribonucleic acid polymerase subunits; Burgess RR et al.; We describe a procedure that allows cysteine and methionine content to be determined on microgram amounts of partially purified protein . The only requirements are that the protein can be obtained as a pure band after electrophoresis on a polyacrylamide gel and that some data on amino acid content be available . This method involves double labeling by growing bacterial cells with {3H}leucine and {35S}SO4 and determining the ratio of these radioisotopes incorporated into the ribonucleic acid polymerase subunits . The relative specific activities of {3H}leucine and {35S}cysteine and methionine are determined from the ratio of these isotopes incorporated into beta-galactosidase, the leucine, cysteine, and methionine contents of which are known . We have used this procedure to determine the sulfur content of the subunits of Escherichia coli ribonucleic acid polymerase . These new data are necessary to quantitate the rates of synthesis of these subunits by in vivo labeling with {35S}SO4. J Bacteriol, 1976 Oct, 128(1), 39 - 48 Structural and regulatory mutations allowing utilization of citrulline or carbamoylaspartate as a source of carbamoylphosphate in Escherichia coli K-12; Legrain C et al.; Escherichia coli mutants lacking carbamoylphosphate synthase require arginine and uracil for growth . It is, however, possible to obtain mutants in which carbamoylphosphate is obtained by phosphorolysis of citrulline or carbamyolaspartate . Citrulline utilizers are argG bradytrophs or strains in which the synthesis of ornithine carbamoyltransferase (either of the F or I type) is specifically depressed by unstable chromosomal rearrangements or stable mutations that presumably affect the operators of those genes . Carbamoylaspartate utilization as a source of carbamoylphosphate appears to require more than one mutation; the best-understood strains are pyrD pyrH or pyrC pyrH mutants in which aspartate carbamoyltransferase activity is high and the pool of cytidine triphosphate (feedback inhibitor of aspartate carbamoyl-transferase) is presumably low and in which channeling of carbamoylaspartate towards pyrimidine biosynthesis is considerably reduced . Selection of enzyme overproducers based on a metabolic dependency for a reversed enzymatic reaction can be regarded as a means for isolating regulatory mutants. J Bacteriol, 1976 Oct, 128(1), 302 - 8 Growth response of Escherichia coli to nutritional shift-up: immediate division stimulation in slow-growing cells; Sloan JB et al.; When Escherichia coli 15T- cells growing exponentially at 70- to 80-min doubling times are subjected to a nutritional shift-up via glucose addition, cell division continues at the preshift rate for about 70 min (rate maintenance) . The same cells growing at doubling times of 120 min or longer, however, begin to divide at a new faster rate immediately upon glucose addition . In both the rate maintenance and immediate division situations, cell mass, as measured by optical density (OD), begins to increase immediately upon shift-up . Consequently, the OD/cell pattern differs in the two growth-rate transitions . During rate maintenance, the OD/cell ratio increases dramatically for 60 to 70 min, and then slows appreciably and approaches the OD/cell characteristic of the new medium . During immediate division situations, the OD/cell increases only slightly for the first 180 +/- min; then the rate of increase accelerates but does not stop at the OD/cell characteristic of the new medium . Immediate division upon nutritional shift-up apparently depends upon initial doubling times in excess of 115 to 120 min and provision of a readily metabolized carbon source supporting doubling times of about 40 min . Similar immediate division occurs in E . coli B/r and K-12. J Bacteriol, 1976 Oct, 128(1), 28 - 34 Characterization of the hybridization between purified 16S and 23S ribosomal ribonucleic acid and ribosomal deoxyribonucleic acid from Escherichia coli; Dennis PP et al.; An assay to distinguish specifically between 16S and 23S ribosomal ribonucleic acid (rRNA) has been developed . The assay involves hybridization of radioactive rRNA to deoxyribonucleic acid (DNA) from lambdailv5 transducing phage, which carries an rRNA transcription unit . Radioactive 16S or 23S rRNA can be specifically and completely competed from hybrids by using highly purified nonradioactive 16S or 23S competitor RNA, respectively . The preparation and purification of 16S rRNA and 23S rRNA are described in detail . The hybridization assay is extremely sensitive and efficient; 65 to 70% or more of the input radioactivity hybridizes to specific DNA in the absence of homologous competitor RNA, and at saturation virtually all of the specific DNA sequences are hybridized to rRNA . The results indicate that: (i) the 16S rRNA and 23S rRNA prepared as described are greater than 99% pure, (ii) 16S RRNA and 23S rRNA hybridize with equal efficiency and in equal molar amounts of lambdailv5 DNA; (iii) at saturation, about one molecule of 16S and one molecule of 23S rRNA are hybridized per genome equivalent of lambda ilv 5 DNA; (iv) essentially no cross-hybridization occurs between 16S and 23S rRNA; and (v) the sequence homology between 16S and 23S rRNA is negligible. J Bacteriol, 1976 Oct, 128(1), 264 - 70 Recognition properties of the beta subunit of Escherichia coli ribonucleic acid polymerase; Tessman ES et al.; Changes in the phage protein patterns obtained by gel electrophoresis of extracts from phage S13 and phiX174 infection of rifampin-resistant hosts suggest that the beta subunit of ribonucleic acid polymerase of Escherichia coli has a function in the recognition of promoter or terminator sites or both . The altered protein patterns also provide information on the location of some ribonucleic acid polymerase recognition signals in S13 deoxyribonucleic acid . There is a promoter site before gene A, which lies either in gene H or between H and A . There is evidence for a promotor between genes C and D or in gene C . There is either a terminator or a promoter somewhere between the end of gene D and the beginning of gene F. J Bacteriol, 1976 Oct, 128(1), 257 - 63 Isolation of pseudorevertants of lac oc mutants: selection system for superoperator mutations; Pfahl M et al.; A selection procedure and rapid screening test are described that allow the isolation of rarely occurring pseudorevertants of lac oc mutants . These techniques were developed with the aim of isolating superoperator (os) mutants in which a secondary mutation in the operator would increase the affinity of the repressor of superoperator, thereby overcoming the effect of the oc mutation . The occurrence of superoperator mutants is predicted on the basis of a probable twofold symmetry in the lac repressor-operator recognition . Over 2,300 oc pseudorevertants were isolated and screened . In vitro measurements of the affinity of lac repressor for the operator region indicate that, among these, one oc pseudorevertant probably results from an oc mutation . The selection procedure also yielded other types of regulatory mutants that behave as promoter mutants and as i gene mutants in which the repressor is capable of overcoming an oc mutation. J Bacteriol, 1976 Oct, 128(1), 165 - 9 Prediction of the rate of glucose utilization from cellular levels of glucose 6-phosphate and fructose 1,6-diphosphate in Escherichia coli; Dietzler DN et al.; A comprehensive equation, upsilon = VM/{1 + (A0.5/Fru-P2)n} { 1 + (Glc-6-P/I0.5)}, has been proposed to represent the quantitative interrelationships between the rate of glucose utilization and the levels of glucose-6-phosphate and fructose-1,6-diphosphate in the intact Escherichia coli cell . This comprehensive equation was derived from empirical equations that describe the relationship between the rate of glucose utilization and one of these hexose phosphates in metabolic situations where the other hexoses phosphate was not altered . In the experiments described in this report, treatment of nitrogen (NH4+)-starved cultures of E . coli W4597 (K) with various concentrations of sodium azide altered the levels of both hexose phosphates as well as the rate of glucose utilization . In each case the observed rate and the rate predicted by the comprehensive equation agreed closely, substantiating the validity of this comprehensive relationship as a quantitative indicator of metabolic events in the intact cell . The mechanism of metabolic regulation that is represented by this equation is discussed in light of the cellular levels of adenosine 5'-triphosphate and phosphoenolpyruvate observed in these experiments. J Bacteriol, 1976 Oct, 128(1), 130 - 43 Independence of deoxyribonucleic acid replication and initiation from membrane fluidity and the supply of unsaturated fatty acids in Escherichia coli; Thilo L et al.; Mutant derivatives of the unsaturated fatty acid auxotroph K1062 were employed to investigate whether the supposedly membrane-bound bacterial replication machinery requires for its replicatory functions a fluid membrane environment as is known for several membrane-associated protein functions . Temperatures Tt for fluid reversible nonfluid phase transitions of membrane phospholipids are raised from below 18 to 38 degrees C when mutant cells are supplemented with elaidate instead of with oleate . In this experimental system current or synchroneously initiated new rounds of DNA replication are shown in vivo to continue 8 degrees below Tt, provided appropriate corrections for the concurrent cellular metabolic breakdown are considered . Temperature rate profiles for in vitro deoxyribonucleic acid replication rates measured in lysates of either oleate- or elaidate-supplemented cells yield congruent Arrhenius plots without discontinuities at corresponding Tt positions . We conclude that neither the start nor the propagation of replication forks depends on a fluid membrane . The capacity for the assembly of new replication complexes was studied in replication-aligned cells either shifted from oleate to elaidate (at temperatures below Tt for newly synthesized phospholipids) or starved for oleate . Regardless of whether unsaturated fatty acids are exchanged or completely withheld, new replication complexes can be normally assembled and initiated . These results do not support the conclusions reached by Fralick and Lark (1973) that the availability of unsaturated fatty acids is a prerequisite for the assembly of a functional replication complex. Wien Klin Wochenschr, 1976 Oct 1, 88(78), 585 - 8 {Splenectomy for traumatic rupture of the spleen in childhood and its sequaelae}; Passl R et al.; This study was undertaken in order to assess the clinical and immunological consequences of splenectomy for traumatic reasons in childhood . Immunological testing of 22 persons 1 to 20 years subsequent to removal of the spleen for traumatic rupture between the ages of 3 and 6 revealed diminished or absent agglutinins in 10 cases and diminished or absent opsonins to E . coli in 16 cases . All patients were in good health and no clinical evidence of increased susceptibility to severe infections was found postoperatively . It is, therefore, assumed that the diagnosed defects had been compensated for by other immunological mechanisms . In contrast to the opinions of other authors, it is concluded that splenectomy in childhood between the ages of 3 and 6 does not appear to carry greater risks than in later years. Mutat Res, 1976 Oct, 37(1), 11 - 18 Mechanism of the mutagenic action of hydroxylamine . X . Certain specificities in the mutagenesis of N-hydroxy and N-methoxy analogs of cytosine and adenine derivatives; Budowsky EI et al.; In contrast with N4-methoxycytidine, N6-methoxyadenosine and the corresponding 2'-deoxynucleosides, N4-hydroxycytidine readily penetrates cells of Escherichia coli . Apparently this explains the non-mutagenicity of the N-methoxy compounds when added to an E . coli suspension, and the potent mutagenic effects of the N4-hydroxy analogs under the same conditions . 1-Deazaadenosine and N9- and N1-hydroxyalkyl-substituted adenines and cytosines, inhibitors of adenosine and cytidinedeaminases, are also incapable of crossing the E . coli cell wall. J Clin Invest, 1976 Oct, 58(4), 971 - 9 The role of lysosomal elastase in the digestion of Escherichia coli proteins by human polymorphonuclear leukocytes: experiments with living leukocytes; Blondin J et al.; Human polymorphonuclear leukocyte (PMN) elastase has been implicated in various pathological conditions . However, its physiological role remains undefined . One possible function of this enzyme may be digestion of bacterial proteins after phagocytosis . To test this hypothesis, we prepared Escherichia coli labeled with {3H}arginine and treated these bacteria with a lipid-soluble, active-site-directed chloromethyl ketone inactivator of pancreatic and granulocyte elastases (carbobenzoxy-L-glycyl-L-leucyl-L-alanine chloromethyl ketone, dissolved in methanol) . Control bacteria were treated with methanol alone . When E . coli pretreated with the inactivator were incubated with solutions of porcine pancreatic elastase or with PMN granule extract, release of trichloroacetic acid-soluble radioactivity was significantly lower than in the control E . coli . Similar results were obtained when treated and control E . coli were fed to viable human PMN . In contrast, release of trichloroacetic acid-soluble radioactivity from E . coli containing {3H}thymidine was not affected by pretreatment of bacteria with elastase inactivator before feeding them to PMN, suggesting that phagocytosis of E . coli had not been inhibited by the chloromethyl ketone . When treated and control bacteria were fed to PMN, no significant difference was observed in the activity of lysosomal beta-glucuronidase recovered from post-granule supernatant fractions of homogenized leukocytes, suggesting that lysosomal degranulation had not been suppressed by the inactivator . However, elastase activity of the same fractions was depressed if the leukocytes had phagocytized chloromethyl ketone-treated E . coli, suggesting that inhibition of PMN elastase had occurred . We conclude that PMN elastase participates in digestion of E . coli proteins by human PMN. Biokhimiia, 1976 Oct, 41(10), 1905 - 6 {The formation of ATP from adenosine 5'-phosphoroimidazolide and pyrophosphate catalyzed by valyl-tRNA-synthetase}; Biriukov AI et al.; The formation of 32P-ATP from adenosine 5'-phosphoroimidazolide and 32P-pyrophosphate brought about valine: tRNA-ligase of E . coli is demonstrated in the standard conditions of pyrophosphate exchange for aminoacyl-tRNA synthetases . The role the enzyme as specific matrix is suggested. Aust Vet J, 1976 Oct, 52(10), 438 - 41 Changes in intestinal structure and function of neonatal calves infected with reovirus-like agent and Eschericia coli; Halpin CG et al.; Measurements of villus/crypt length ratio and mucosal beta-galactosidase activity were made on calves less than 3 weeks of age which had diarrhoea associated with reovirus-like agent and E . coli . In calves with diarrhoea, the villus/crypt length ratios at all sites examined along the small intestine were less than in normal calves of similar age . This was attributed to a reduction in length of vili in calves infected with the reovirus-like agent . The activity of mucosal beta-galactosidase in the intestine of calves with diarrhoea was less than in normal calves, at all sites examined . A relationship existed between beta-galactosidase activity in vitro and lactose hydrolysis in vivo . It was concluded that calves with diarrhoea associated with reovirus-like agent, have a reduced ability to utilize dietary lactose. Eur J Biochem, 1976 Oct 1, 69(1), 249 - 55 Nuclear-magnetic-relaxation studies of the interaction of inhibitor with the threonine-sensitive aspartokinase of Escherichia coli; Tilak A et al.; The nature of the feedback inhibition of the bifunctional enzyme, aspartokinase I-homoserine dehydrogenase I of Escherichia coli was studied using 13C nuclear magnetic resonance (NMR) . Since aspartokinase is activated by Mn(II), the interaction of the inhibitor L-threonine (specifically enriched to 90% 13C in the carboxyl carbon) with the metal-enzyme complex was studied . Spin-lattice (T1) and spin-spin (T2) relaxation times were determined by the partially relaxed Fourier transform method and line-width measurements respectively at 20 MHz . The pronounced broadening of the DL-threonine carboxyl peak in the presence of the Mn(II)-enzyme complex indicates that an L-threonine binding site is close to the metal binding site of the kinase active site . The non-identity of (T1)*M and (T2)*M indicates that conditions of fast exchange prevail . The (T1)*M/(T2)*M ratio was used to estimate a correlation time of 2.0 ns for the dipolar interaction at 25 degrees C . An estimate for the distance between Mn(II) and the threonine carboxyl carbon of 4.4 A (0.44 nm) was obtained . This 13C NMR study has thus located one of the two classes of threonine regulatory sites which exist per subunit; the threonine site identified here is at the aspartokinase active site, adjacent to the catalytic metal site. Proc Natl Acad Sci U S A, 1976 Oct, 73(10), 3438 - 42 Novel structure at 5'-ends of nascent DNA chains; Siegmann DW et al.; Because of their association with protein short nascent DNA chains in Escherichia coli can be separated from other cellular DNA by chromatography on hydroxylapatite . Protein-free DNA chains of less than 500 nucleotides in length are resistant to degradation from the 5'-end by alkaline phosphatase {orthophosphoric-monoester phosphohydrolase (alkaline optimum); EC 3.1.3.1} and spleen phosphodiesterase (oligonucleate 3'-nucleotidohydrolase; EC 3.1.4.18) . In contrast, DNA chains containing more than 500 nucleotides are degradable . From these results we conclude that short nascent DNA chains are structurally modified at their 5'-ends . The nature of this structure and its possible functions are discussed. J Bacteriol, 1976 Oct, 128(1), 157 - 64 Stimulatory effect of lithium ion on proline transport by whole cells of Escherichia coli; Kayama Y et al.; The effect of monovalent cations on proline transport in whole cells of Escherichia coli K-12 has been examined . Lithium ion added to the uptake medium stimulated proline transport severalfold and K+ and Na+ were slightly effective, whereas Rb+, Cs+, and NH4+ were completely without effect . The stimulatory effect of Li+ on proline transport was not due to an increase in osmolarity of the uptake medium, and d 5 mM p-chloromercuribenzene sulfonic acid completely blocked this effect of Li+ without having any effect on the basal rate of proline transport . The Arrhenius plots for Li+-stimulated transport showed a clear transition point at 35 degrees C in addition to 20 degrees C which was also detectable in the basal transport . Lithium ion stimulated proline transport synergistically in the presence of glucose and succinate as a carbon source . The addition of 2.5 mM KCN or 0.5 mM arsenate did not inhibit this synergistic effect, although the presence of these inhibitors inhibited completely the stimulation of proline transport induced by the addition of carbon source . Carbonylcyanide m-chlorophenylhydrazone and 2,4-dinitrophenol blocked both the basal and Li+-stimulated proline transport . When membrane potential of E . coli cells was measured by the dibenzyldimethylammonium uptake method, the incubation of Li+ with the cells did not affect the preexisting membrane potential . These results suggest that Li+ stimulates proline transport by intact cells of E . coli in a manner somewhat affecting membrane component(s) different from the transport carrier of proline . It is uncertain whether the effect of Li+ is directly involved in the mechanisms of energy coupling of proline transport. J Immunol, 1976 Oct, 117(4), 1336 - 9 Mitogen-stimulated glutaraldehyde-fixed spleen cells: ability to stimulate in the mixed lymphocyte reaction and generate effector cells in cell-mediated lympholysis; Lightbody JJ et al.; Mouse spleen cells treated with glutaralde lose their stimulating ability in the MLR . If the spleen cells are first converted to a blastogenic state by lipopolysaccharide and subsequently fixed with glutaraldehyde, their stimulating capacity is maintained. J Bacteriol, 1976 Oct, 128(1), 242 - 7 Transport of vitamin B12 in tonB mutants of Escherichia coli; Bassford PJ Jr et al.; It is known that the tonB mutation in Escherichia coli is responsible for a defect in the transport of iron chelates . These are transported by systems that involve outer membrane components . We found that tonB mutants were also deficient in the secondary, energy-dependent phase of vitamin B12 transport, although the mutants have normal levels of B12 receptors on their cell surface . In addition, tonB mutants derived from vitamin B12 auxotrophs required elevated levels of B12 for normal growth . Maltose uptake, mediated by another transport system involving an outer membrane component, was unaffected by the tonB mutation. Rev Rhum Mal Osteoartic, 1976 Oct, 43(10), 555 - 60 {Arthritis related to intestinal anastomoses}; Hubault A et al.; Jejuno-colic and jejuno-ileal anastomoses may provoke arthritis . The recently recognized physiopathology is that of arthritis due to immune complexes related to pullulation of Escherichia coli and of Bacilus fragilis . These cases of arthritis, usually sensitive to therapy, have, in some stutborn cases, required the re-establishment of intestinal continuity, which in each case has made the joint phenomena disappear. Proc Natl Acad Sci U S A, 1976 Oct, 73(10), 3418 - 22 Stepwise biosynthesis in vitro of globin genes from globin mRNA by DNA polymerase of avian myeloblastosis virus; Rougeon F et al.; Two approaches have been explored for the synthesis of double-stranded DNA from single-stranded DNA template complementary to rabbit 9S globin mRNA (cDNA) . (i) cDNA was elongated with dCMP or dTMP homopolymeric tracts using terminal deoxynucleotidyltransferase (EC 2.7.7.31; nucleosidetriphosphate:DNA deoxynucleotidylexotransferase) . cDNA-dC, in the presence of an oligo(dG)10 primer, was an efficient template with either DNA polymerase of Escherichia coli (EC 2.7.7.7; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase) or RNA-directed DNA polymerase of avian myeloblastosis virus . cDNA-dT { with an oligo(dA)10 primer} functioned as template only with E . coli polymerase . (ii) cDNA, without homopolymeric tails, was also efficiently copied in the absence of oligonucleotide primer, by DNA polymerase of avian myeloblastosis virus or of E . coli . The product of the reaction consisted of long hairpin molecules which could be converted into DNA duplex (melting temperature, 93 degrees) by digestion with single-strand nuclease S1 . The data indicate that a loop structure on the 3' end of cDNA allowed DNA synthesis to take place by a "self-priming" mechanism . Some of the double-stranded DNA synthesized corresponded to the entire sequence of the 9S mRNA template . The synthesis of full-length double-stranded DNA from mouse globin mRNA and immunoglobulin light chain mRNA is also discussed. Immunology, 1976 Oct, 31(4), 553 - 61 Long-term antibody synthesis in vitro- IV . Independent segregation of antibodies directed to different determinants of an antigen molecule in its native configuration; Conway de Macario E et al.; Independent segregation of antibody populations directed to different portions of E . coli beta-d-galactosidase occurs during the immune response against the enzyme . Anti-enzyme antibodies able to interact and activate a naturally occurring ligand, the mutant-defective enzyme AMEF (Antibody Mediated Enzyme Factor), do not parallel anti-enzyme antibodies which are measured by a coprecipitation assay involving precipitation of the wild-type molecule . Dissociation of the two antibody populations is best achieved in microcultures sustaining long-lasting responses . Similarly, anti-NIP (4-hydroxy-3-iodo-5-nitrophenylacetic acid) antibodies could be elicited without concomitant synthesis of anti-carrier antibodies by short-term challenge in vitro of ovalbumin-NIP-primed lymph nodes with a heterologous conjugate in which the hapten NIP was coupled to a carrier known to be non-immunogenic under the conditions of challenge . The potential applications of these findings are indicated, namely: large-scale production of monospecific antibodies in vitro; and the possibility of studying the regulatory role of antibodies directed towards on portion of the immunogenic molecule on the response to other regions of the same molecule. Immunology, 1976 Oct, 31(4), 533 - 9 Effect of bursa Fabricius extracts on antibody production in bursectomized or bursal cell autografted chickens; Baba T et al.; Restoration and enhancement of immune response against BSA antigen was achieved by 5-day consecutive doses of BF estract from 4-5-week-old chickens, in birds which had been surgically bursectomized or given BF-cell autografts at 17 days of age . A similar 5-day treatment with other tissue extract, i.e . liver, spleen, pancreas or intestine, or with LPS of E . coli, in contrast, failed to provide such restoration or enhancement. Mol Cell Biochem, 1976 Sep 30, 12(3), 147 - 59 Poly ADP-ribosylation of DNA-dependent RNA polymerase I from quail oviduct . Dependence on progesterone stimulation; Muller WE et al.; Progesterone causes in goblet cells of oviducts of estrogen hormone-stimulated immature quails selectively gene activation without affecting DNA synthesis . This biological model has been used to study the influence of poly ADP-ribosylation during the processes of DNA transcription . Administration of progesterone in vivo causes an increase of the activity of RNA polymerase I and II in isolated nuclei . This increase is accompanied by a marked decrease of the specific activity of poly (ADP-Rib) polymerase . After in vitro ADP-ribosylation of nuclear proteins the template capacity of chromatin for ""exogenous'' RNA synthesis (with E . coli DNA-dependent RNA polymerases) as well as for ""endogenous'' RNA synthesis with DNA dependent RNA polymerases II is not affected, whereas the data presented seem to indicate that the capacity for RNA synthesis mediated by ""endogenous'' DNA-dependent RNA polymerase I might be inhibited after ADP-ribosylation . Evidence is presented to show that a considerable amount of poly (ADP-Rib), synthesized by poly (ADP-Rib) polymerase in isolated nuclei, is linked with RNA polymerase I . The rate of synthesis of poly (ADP-Rib) is dependent on the incubation temperature (optimum at 25 degrees C) and it can be inhibited by the specific inhibitors of poly (ADP-Rib) polymerase nicotineamide, thymidine and formycin B . Poly (ADP-Rib) is probably associated with RNA polymerase I through a covalent linkage . ADP-ribosylated RNA polymerase I has been purified 550 fold with respect to the nuclear extract corresponding to a 4,000 fold purification from the whole cell homogenate . The ratio between poly (ADP-Rib), formed during preincubation of nuclei with NAD, and RNA polymerase I remains almost constant during the purification procedures . The extent of ADP-ribosylation of RNA polymerase I decreases during gene expression . Thus we conclude that poly ADP-ribosylation of this enzyme is one of the regulatory mechanisms by which specificity of DNA transcription is achieved. J Biol Chem, 1976 Sep 25, 251(18), 5565 - 74 Effect of estrogen on gene expression in the chick oviduct . Studies on the initiation of RNA synthesis on chromatin in vitro; Tsai MJ et al.; PIP: The effect of estrogen on gene expression in the chick oviduct was investigated . Studies on the effect of the temperature requirement for ribonuclei acid (RNA) chain initiation by Escherichia coli RNA polymerase on deoxyribonucleic acid (DNA), chromatin, and reconstituted chromatin were carried out to better understand the nature of the initiation process . Varying the temperature or ionic strength during preincubations had little effect on the formation of stable preinitiation complexes between RNA polymerase and chromatin . This was in contrast to similar studies performed on native DNA and indicates that initiation sites for RNA synthesis on chromatin are different from those on double-stranded DNA and resemble more closely initiation of RNA synthesis on single-stranded DNA . These observations suggest that the local unwinding of the initiation region which is required for RNA chain initiation on native DNA may not be a prerequisite for RNA initiation on chromatin . Studies on reconstituted chromatin devoid of different classes of chromatin proteins demonstrate that both histone and nonhistone fractions are essential inmaintaining the charcteristics inherent to initiation of RNA synthesis on chrmatin . Removal of moderately lysine-rich histone or arginine-rich histone fractions led to the complete loss of the characteristic ''chromatin type'' initiation pattern for RNA synthesis whereas removing lysine-rich (F1) histone had no effect . Additional studies suggest that initiation sites on chromatin are not located in freely accessible single- or double-stranded regions of DNA . J Biol Chem, 1976 Sep 25, 251(18), 5516 - 21 Synthesis of ribosomal proteins L7L12 in relaxed and stringent strains of Escherichia coli; Morrissey JJ et al.; The control of the synthesis of ribosomal proteins L7L12 (which lack histidine) was examined during growth and histidine starvation of stringent and relaxed histidine mutants . Since no ribosomes are synthesized during starvation, these proteins, in both the stringent and relaxed organisms, accumulated in the supernatant and were shown to possess both biological and physical characteristics typical of normal L7L12 . However, the rate and extent of synthesis of these proteins during starvation is greater in the relaxed strain than in the stringent . These data suggest that the regulation of the synthesis of these proteins, similar to that of ribosomal RNA is regulated by the stringent control system . It was also shown that during normal growth of both organisms, L7L12 is also found in the supernatant as well as on the ribosomes . The L7L12 found in the supernatant under these conditions, however, appears to be different than ribosomal L7L12. J Biol Chem, 1976 Sep 25, 251(18), 5478 - 82 Stereochemistry of the formation of cysteine by O-acetylserine sulfhydrase; Floss HG et al.; The enzymatic preparation of (2S, 3R)- and (2S, 3S)-{3-3H} serine from the corresponding stereospecifically tritiated 3-P-glycerates is described . Following conversion into T-acetylserine, these substrates were used to establish the steric course of the O-acetylserine sulfhydrase reaction . The two samples of cysteine formed in this reaction were analyzed for their configuration at C-3 . The results indicate that the replacement of the acetoxy by a sulfhydryl group in the O-acetylserine sulfhydrase reaction occurs with retention of configuration at carbon atom 3. J Biol Chem, 1976 Sep 25, 251(18), 5522 - 7 Regulation of carbohydrate permeases and adenylate cyclase in Escherichia coli . Studies with mutant strains in which enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system is thermolabile; Castro L et al.; Carbohydrate uptake and cyclic adenosine 3':5'-monophosphate (cyclic AMP) synthesis were studied employing mutant strains of Escherichia coli in which Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system was heat-labile . Partial loss of Enzyme I activity, which resulted from incubation of cells at the nonpermissive temperature, depressed the rate and extent of methyl alpha-glucoside uptake . Temperature inactivation of Enzyme I also rendered cyclic AMP synthesis and the uptake of several carbohydrates (glycerol, maltose, melibiose, and lactose) hypersensitive to inhibition by methyl alpha-glucoside . Protein synthesis did not appear to be required for these effects . The parental strains and "revertant" strains in which Enzyme I was less sensitive to temperature did not exhibit heat-enhanced regulation . Inhibition was abolished by the crr mutation . The results suggest that Enzyme I functions as a catalytic component of the regulatory system . Simple positive selection procedures are described for the isolation of bacterial mutants which are deficient for either Enzyme I or the heat-stable protein of the phosphotransferase system. J Biol Chem, 1976 Sep 25, 251(18), 5558 - 64 Rapid transfer of oxygens from inorganic phosphate to glutamine catalyzed by Escherichia coli glutamine synthetase; Stokes BO et al.; Measurements are reported on certain isotopic fluxes during the net conversion of glutamine, ADP and Pi to glutamate, NH3, and ATP by Escherichia coli glutamine synthetase (adenylylated form, Mn2+ activated) in presence of a hexokinase/glucose trap to remove the ATP formed during the reaction . The results show that the transfer of oxygens from Pi to glutamine is the most rapid of the measured isotopic interchanges, over five oxygens from Pi being transferred to glutamine for each glutamate formed by net reaction . Under similar conditions, the oxygen transfer from Pi to glutamate, was stimulated somewhat by an increase in the glutamate concentration but inhibited by an increase in the ammonia concentration . The enzyme from brain or peas did not show the rapid transfer of 18O from Pi to glutamine shown by the E . coli enzyme . Deductions are also made from the data about the availability of the oxygens of gamma-carboxyl of bound glutamate for reaction . The most logical explanation of the results with the E . coli enzyme is that the gamma-carboxyl group of bound glutamate has sufficient rotational freedom so that under conditions of rapid substrate interconversion either carboxylate oxygen can participate in the reaction . The results with the pea enzyme are consistent with hindered rotation of the gamma-care additional findings make likely a relative order of certain catalytic steps for the E . coli enzyme as follows: ATP release less than NH3 release less than glutamate release less than substrate interconversion less than glutamine release and Pi release and glutamate release less than ADP release. J Biol Chem, 1976 Sep 25, 251(18), 5440 - 7 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase . Purification, properties, and kinetics of the tyrosine-sensitive isoenzyme from Escherichia coli; Schoner R et al.; The tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (7-phospho-2-keto-3-deoxy-D-arabino-heptonate D-erythrose-4-phosphate lyase (pyruvate-phosphorylating), EC 4.2.1.15) was purified to homogeneity from extracts of Escherichia coli K12 . A spectrophotometric assay of the enzyme activity, based on the absorption difference of substrates and products at 232 nm, was developed . The enzyme has a molecular weight of 66,000 as judged by gel filtration on Sephadex G-200, and a subunit molecular weight of 39,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate . This suggests either a rapid monomer-dimer equilibrium, or a very asymmetric shape for the native enzyme . The enzyme shows a narrow pH optimum around pH 7.0 . The enzyme is stable for several months when stored at -20 degrees in phosphate buffer containing phosphoenol-pyruvate . Intersecting lines in double reciprocal plots of initial velocity data at substrate concentrations in the micromolar range suggest a sequential mechanism with-catalyzed reaction . Product inhibition studies specify an ordered sequential BiBi mechanism with a dead-end E-P complex . The feedback inhibitor tyrosine at concentrations above 10 muM exhibits noncompetitive inhibition with respect to erythrose-4-P, and competitive inhibition with respect to the other substrate, P-enolpyruvate . In addition, tyrosine at concentrations of at least 10 muM causes an alteration of one or more than one kinetic parameter of the enzyme. Biochim Biophys Acta, 1976 Sep 24, 444(2), 344 - 8 gamma-Carboxyglutamic acid distribution; Zytkovicz TH et al.; The distribution of the vitamin K-dependent amino acid, gamma-carboxyglutamic acid was examined in proteins from a variety of sources . Proteins examined include purified rat and bovine coagulation proteins, barium citrate-adsorbing proteins from trout plasma, lamprey plasma, earthworm hemolymph, army worm hemolymph, lobster hemolymph, E . coli B/5, soybean leaf, the protein lysate from the hemolymph cell of the horseshoe crab and parathyroid extract . Other purified proteins examined included human alpha-1-antitrypsin, pepsinogen, S-100, fetuin, tropomyosin-troponin and complement protein C-3 . Of these, only the blood-cotting proteins and the vertebrate plasma samples were shown to contain gamma-carboxyglutamic acid. Mol Gen Genet, 1976 Sep 23, 147(3), 337 - 41 Characterization of a ts beta' mutant RNA polymerase of Escherichia coli; Gross G et al.; RNA polymerase isolated from ts XH56, a conditional lethal mutant unable to grow and synthesize RNA at 42 degrees, was found to be temperature sensitive in vitro . The mutation affects the beta' subunit as determined by mixed reconstitution of isolated subunits from wild-type and mutant enzyme . The mutant RNA polymerase is unstable; addition of glycerol stabilizes the enzyme and increases its activity on native DNA . In addition, the mutant enzymes is sensitive to high ionic strength . Both high temperature and high ionic strength do not affect chain elongation; thus, the mutation renders the enzyme unable either to bind to or melt out promotor sites . From these data we conclude that the beta' subunit plays an important role in promotor selection. Mol Gen Genet, 1976 Sep 23, 147(3), 331 - 5 ColE plasmid replication in DNA polymerase I-deficient strains of Escherichia coli; Tacon W et al.; Replication of the non-conjugative plasmids ColE1, ColE2 and Col3 has been examined in a number of DNA polymerase I-deficient strains, two of which contain the amber mutation polA1 along with either of two temperature-sensitive supF amber suppressors . These latter two strains produce reduced amounts of DNA polymerase I polymerizing activity of similar, if not identical properties to that produced by polA+ strains . Our results indicate that the ColE plasmids require different amounts of DNA polymerase I for stable plasmid maintenance . Moreover whereas all three plasmids are maintained in a strain defective in the 5' leads to 3' exonuclease activity of DNA polymerase I, ColE2 and ColE3 are not stably maintained between 30 degrees and 43 degrees in a number of DNA POLYMERASE I-deficient strains that are temperature-sensitive for ColE1 replication. Mol Gen Genet, 1976 Sep 23, 147(3), 307 - 14 Mapping of the polA locus of Escherichia coli K12: orientation in the amino- and carboxy-termini of the cistron; Kelley WS et al.; Three mutations of the polA cistron, the structural gene for DNA polymerase I of E . coli, have been ordered by three factor transductional crosses . The three mutant polymerase species have altered properties which may be ascribed to defects located in different portions of the polypeptide chain . Our data indicate that the amino terminal end is encoded by the end of the polA cistron nearer to metE and that transcription and translation proceed clockwise on the E . coli circular map towards the rha locus. Mol Gen Genet, 1976 Sep 23, 147(3), 263 - 9 Heptose-deficient mutants of Escherichia coli K12 deficient in up to three major outer membrane proteins; van Alphen W et al.; Mutants of Escherichia coli K12, deficient in up to three major outer membrane proteins b, c and d have been constructed . Mutants that lack the lipopolysaccharide sugar heptose are deficient in protein b . All heptose-deficient strains are supersensitive to lysozyme, various antibiotics and detergents . They excrete the periplasmic enzyme ribonuclease I . Mutants deficient in proteins c and/or d have the same sensitivity towards these compounds as the parent strain . Cells of single, double and triple mutants are all rod-shaped . Electrophoretic analysis of cell envelope proteins indicates that in some mutants the protein deficiency is partially compensated for by increased amounts of one or two of the other major outer membrane proteins . Heptose-deficient strains have an increased amount of 2-keto-3-deoxyoctonate. Mol Gen Genet, 1976 Sep 23, 147(3), 243 - 50 Evidence that the gene uvrB is indispensable for a polymerase I deficient strain of Escherichia coli K-12; Morimyo M et al.; Conclusive evidence in presented to show that the gene uvrA is dispensable, but the uvrB is indispensable for an Escherichia coli strain carrying gene polA1 . We constructed strains E139 (sup-126 polAl uvrB59) and E159 (sup-126 polAl uvrA43) where mutations polAl, uvrB59 and uvrA43 are amber mutations and mutation sup-126 is an amber suppressor mutation effective at 30 degrees C but not at 42 degrees C . At 42 degrees C, strain E139 is inviable but strain E159 viable whereas both are viable at 30 degrees C . Revertants of E139 viable at 42 degrees C occurred spontaneously at a frequency of about 3 X 10(-4) . One of the revertants was shown to be caused by suppressor mutation, designated spu, rather than back mutation of the gene uvrB59 or polAl or amber suppressor mutation . Viabilities of the revertants varied from 10(-3) to 1.0 at 42 degrees C compared with those of 30 degrees C . At 42 degrees C, all the revertants with normal viabilities at 42 degrees C were non-filamentous in contrast to the filamentous character of E139 . However, strain E159 was viable at 42 degrees C despite its filamentous character . We conclude that the gene uvrB is involved not only in excision repair but also in normal growth in a polA background. Biochim Biophys Acta, 1976 Sep 21, 448(1), 189 - 91 Permeability to cobalt ions and action of colicin K in a mutant that overproduces cardiolipin; Lusk JE; A mutant of Escherichia coli that produces excess cardiolipin becomes less capable of transporting Co2+ . Cardiolipin therefore does not act as an ionophore under these conditions . Colicin K brings about the typical increase in permeability to Co2+ in the mutant. Biochemistry, 1976 Sep 21, 15(19), 4347 - 52 Circular dichroism study of the interaction of glutamyl-tRNA synthetase with tRNAGlu2; Willick GE et al.; The interaction of glutamyl-tRNA synthetase with tRNAGlu2 has been studied . The enzyme was purified to apparent homogeneity, and consists of a single chain with a molecular weight of 59 000 . The sedimentation coefficient (sdegrees20,w) was found to be 3.7 S and suggests this enzyme is quite asymmetric . The enzyme binds 1 mol of tRNAGlu2 and has a binding constant of 5 X 10(6) M-1 at pH 7.0 in 0.1 M sodium chloride . A circular dichroic study of the interaction under the same solvent conditions implied both the synthetase and tRNAGlu2 underwent a change in conformation as the complex was formed . In the case of the enzyme there appears to be some loss of alpha-helical structure . The tRNAGlu2 results can be interpreted to indicate a change in the conformation of one or more of the helical regions of this molecule . A residue in the anticodon loop, 5-methylaminomethyl-2-thiouridine, has a distinct circular dichroic band at 340 nm in the free tRNAGlu2 . As the complex is formed this band is shifted to the blue . This was interpreted to indicate that the enzyme forms a hydrogen bond with this residue in the anticodon loop, with a change in the conformation of the loop possibly also having occured. Biochemistry, 1976 Sep 21, 15(19), 4222 - 7 Purification of thymidylate synthetase from enzyme-poor sources by affinity chromatography; Slavik K et al.; The adsorption of thymidylate synthetase from Escherichia coli B to aminoalkyl-Sepharose with the increasing length of carbon chain (2--6 carbon atoms) was investigated . A correlation was found between the chain length and adsorption effectiveness, increasing from the two- to the six-carbon chain . A hydrophobic chromatography of the enzyme on aminobutyl-Sepharose gave about 20-fold purification . A new affinity chromatography carrier was synthesized containing tetrahydromethotrexate linked to aminoethyl-Sepharose via its carboxylic groups . The carrier adsorbed the enzyme from the crude preparation only in the presence of deoxyuridine 5'-monophosphate (dUMP) in a concentration of 2 X 10(-5) M . The specifically adsorbed thymidylate synthetase was eluted with sacharose-containing buffers in which dUMP was omitted . The purification procedure was applied to a crude thymidylate synthetase preparation from resting E . coli, calf thymus, Sarcoma 180, and Gardner lymphosarcoma . The purified enzyme from all mentioned sources showed one protein band on disc electrophoresis corresponding to enzymatic activity . The formation of a reversible noncovalent complex enzyme-tetrahydromethotrexate-dUMP on the affinity column is supposed. Eur J Biochem, 1976 Sep 15, 68(2), 481 - 7 Molecular model for 5-S RNA . A small-angle x-ray scattering study of native, denatured and aggregated 5-S RNA from Escherichia coli ribosomes; Osterberg R et al.; A tertiary structural model is suggested for Escherichia coli 5-S RNA that consists of one large and two small double helices arranged in the form of the letter Y . This model is consistent with the small-angle X-ray scattering data of native 5-S RNA, measured in the angular range 20 less than or equal to 140 mrad . The radium of gyration is 3.61 +- 0.1 Nm . Denatured 5-S RNA yields a much lower radius of gyration, 2.7 nm, which might indicate that during denaturation one minor double-helical arm of the Y-shaped structure partially collapses into single-stranded areas . At high concentrations (60 mg/ml) of 5-S RNA, the X-ray scattering data indicate that 5-S RNA is aggregated. Biochim Biophys Acta, 1976 Sep 14, 445(2), 437 - 45 The effect of trinitrophenylation on subunit interactions in L-asparaginase; Parrott CL et al.; L-Asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) from Escherichia coli B was modified by treatment with 2,4,6-trinitrobenzene-1-sulfonic acid at pH 7.5 . The introduction of 13 trinitrophenyl groups into one mol of the tetrameric enzyme (TNP 13-asparaginase) results in a loss of 67% of the catalytic activity while the presence of 20 groups (TNP 20-asparaginase) reduces the enzymatic activity by 88% . The modified proteins are homogeneous as judged by disc gel electrophoresis and by the monodisperse boundary in the analytical ultracentrifuge having a sedimentation coefficient of 7.2 S . The rate of dissociation of the TNP 13-asparaginase is twice as fast and TNP 20-asparaginase three times as fast as that of unmodified asparaginase in 4 M urea . Trinitrophenylated subunits in 8 M urea can reassociate into the tetramer after removal of urea by dialysis or by dilution . hybridization of unmodified and TNP subunits indicates that that trinitrophenyl derivatives qualify as suitable variants for studying subunit interactions in oligomeric proteins. Biochim Biophys Acta, 1976 Sep 14, 445(2), 406 - 19 Purification and properties of a new aminopeptidase from Escherichia COLI K12; Yang LM et al.; An aminopeptidase (EC 3.4.11.-) capmable of hydrolyzing L-alanyl-beta-naphthyl-amide and certain other aminoacyl beta-naphthylamides was purified to homogeneity from extracts of Exherichia coli K-12 . The enzyme, designated aminopeptidase II, is a monomeric protein of mol . wt . 100 000 . It exhibits a broad pH optimum in the range pH 7.0--9.0 . Although Zn2+, Fe3+ and Cr3+ are strong inhibitors of enzyme activity, a metal requirement for catalysis could not be firmly established . Neither sulfhydryl reagents nor serine protease inhibitors affected enzyme activity. Biochim Biophys Acta, 1976 Sep 14, 445(2), 280 - 5 NADP+-specific isocitrate dehydrogenase of Excherichia coli . III . Two-step purification employing affinity chromatography; Hy M et al.; The NADP+-specific isocitrate dehydrogenase (threo-DS-isocitrate:NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) of Excherichia coli has been purified to electrophoretic homogeneity by a two-step purification procedure employing affinity chromatography . The overall yield of enzyme was 30% with specific activity 125 mumol/min per ng protein . Electrophoretic homogeneity of the isocitrate dehydrogenase was deterimed in analytical polyacrylamide gels in a Tris/acetate/EDTA buffer system at pH 7.5 and in a citrate/phosphate buffer system at pH 6.0. Biochim Biophys Acta, 1976 Sep 13, 440(3), 723 - 32 ATP concentration in Escherichia coli during oxygen toxicity; Mathis RR et al.; Escherichia coli, strain E-26, grown in defined salts medium with glucose as the sole carbon and energy source, contained 1.50+/-0.16-10(6) molecules of ATP/cell . ATP was extracted with HC104 and assayed with a Dupont Luminescence Biometer using the luciferin-luciferase assay . Exposure during exponential growth at 37degreesC to 4.2 atm of oxygen resulted in complete growth cessation within 5 min, and to cyclic changes in cellular ATP concentration over a 2 h period . However, significant decrease in cellular ATP concentration occurred after growth inhibition in hyperbaric oxygen; hence, lack of ATP was not the cause of growth inhibition from oxygen toxicity. J Biol Chem, 1976 Sep 10, 251(17), 5195 - 9 Two substrate binding sites on tryptophanyl transfer ribonucleic acid synthetase of Escherichia coli; Muench KH; Tryptophanyl-tRNA synthetase of Escherichia coli has 1.8 binding sites for L-tryptophan with Kdiss of 12 x 10(-5) M as shown by equilibrium dialysis . The results are in accord with the known structure of the enzyme, and alpha2 dimer of 74,000 molecular weight, and with 2 binding sites for tryptophanyl-ATP ester . Ordinary sucrose density gradient centrifugation reveals a complex composed of one tRNATrp bound per enzyme dimer . When tRNATrp is mixed throughout the gradient at concen