Escherichia coli

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 concentrations from 5.4 x 10(-6) M to 2.0 x 10(-5) M, a new peak appears in the position expected for a complex with two tRNATrp molecules bound per enzyme dimer . Sedimentation through gradients lacking tRNATrp favors dissociation of the 1:2 complex but not the 1:1 complex . The data indicate 2 binding sites for tRNATrp on tryptophanyl-tRNA synthetase.

J Biol Chem, 1976 Sep 10, 251(17), 5166 - 77
Progesterone-binding components of chick oviduct . In vitro effects of purified hormone-receptor complexes on the initiation of RNA synthesis in chromatin; Schwartz RJ et al.; We have investigated the manner by which progesterone receptors act to induce initiation of RNA synthesis in a cell-free system derived from chick oviduct . A method utilizing rifampicin enabled us to measure the formation of binary initiation complexes between RNA polymerase and chick oviduct chromatin (Tsai, M.-J., Schwartz, R.J., Tsai S.Y., and O'Malley, B.W . (1975) J.Biol . Chem . 250, 5165-5174) and allowed for the quantitative assessment of RNA chain initiation sites, RNA chain propagation rates, and RNA chain size under conditions which prevent secondary chain reinitiations . We have measured the available initiation sites for transcription in oviduct chromatin prepared from chicks withdrawn from all hormone and then restimulated with a secondary injection of progesterone . Within 1/2 hour after administration of progesterone, the number of initiation sites increased from 8,700 sites/pg of chromatin DNA for the control to 15,500 sites . After 1 hour, the concentration of RNA polymerase needed to saturate chromatin binding sites was increased 60% in comparison to control values, while the number of initiation sites increased 160% . This rapid increment in transcriptional activity preceded temporally the induction of synthesis of ovalbumin mRNA . To test directly the effect of progesterone receptor on transcription, in vitro, a reconstituted cell-free system was employed which contained purified cytoplasmic progesterone-receptor complexes, Escherichia coli RNA polymerase, and chromatin prepared from hormonally withdrawn chick oviducts . Purified progesterone-receptor complex stimulated transcription of oviduct chromatin in vitro by promoting an increase of 3,000 to 5,000 additional sites for RNA chain initiation . These data showed that progesterone receptor can directly increase the number of RNA polymerase binding and initiation sites in the chromatin template in the absence of a detectable change in either the rate of RNA chain propagation or the size of the RNA product . The kinetics of progesterone-receptor stimulation of RNA synthesis in chromatin revealed a t1/2 of 15 min for this effect to occur . This value was identical with the optimal time required for binding of receptor to chromatin . The concentration of receptor required for half-maximal stimulation of RNA chain initiation was approximately 5 x 10(-9) M . This value agreed closely with our previously reported estimates of the affinity (Kd approximately 5 x 10(-9)M) of the progesterone-receptor complex for oviduct chromatin . The stimulatory effect of purified progesterone receptor appeared to be relatively specific for oviduct chromatin in comparison to nontarget tissue chromatins or chick DNA . The data presented here show that steroid hormone-receptor complex can directly regulate gene transcription in vitro in a manner which mimics the events observed in vivo in target cells.

J Biol Chem, 1976 Sep 10, 251(17), 5315 - 20
Oxidative phosphorylation in right-side-out membrane vesicles from Escherichia coli; Tsuchiya T; Oxidative phosphorylation in Escherichia coli membrane vesicles with a right-side-out orientation and loaded with ADP was investigated . Substrates of the electron transport chain could energize the phosphorylation of ADP, with the order of effectiveness being D-lactate greater than reduced phenazinemethosulfate greater than succinate greater than reduced nicotinamide adenine dinucleotide . Inhibitors of D-lactate oxidation, proton conductors, and inhibitor of the Mg2+ATPase (EC 3.6.1.3) all inhibited oxidative phosphorylation when coupled to D-lactate oxidation . ATP synthesis was absent in membrane vesicles prepared from a mutant strain lacking the Mg2+ATPase . Valinomycin or nigericin partially inhibited oxidative phosphorylation in the presence of potassium . Valinomycin plus nigericin completely inhibited ATP synthesis . The effect of various agents on the respiration-dependent establishment of a transmembrane pH gradient was also examined . NaCN and carbonyl cyanide p-trifluoromethoxyphenylhydrazone inhibited the establishment of a pH gradient while dicyclohexylcarbodiimide had no effect . These results are in good agreement with a chemiosmotic model for oxidative phosphorylation.

Biochemistry, 1976 Sep 7, 15(18), 3912 - 7
Allosteric interpretation of Mg2+ binding to the denaturable Escherichia coli tRNAGlu2+; Bina-Stein M et al.; The Mg2+ binding properties of the denaturable tRNAGlu2 from E . coli in 0.1 M Na+, pH7, are characterized by equilibrium dialysis . At 34 degrees C, where the native and denatured conformers are in equilibrium, Mg2+ binding is cooperative . By trapping the tRNA completely in the native conformation at 4 degrees C it is shown that native tRNAGlu2 possesses one strong binding site, K1 = 7.5 x 10(4) M-1 and approximately 36 weak sites with K2 = 8.3 x 10(2) M-1 . A significantly lower affinity for the denatured conformer is indicated . We show that Mg2+ effects an allosteric transition from the low affinity denatured conformational state to the high affinity native state and develop the appropriate equations to fit the Mg2+ binding data with physically meaningful parameters . Our results also suggest the previously reported cooperative cation binding to tRNA arises from a cation induced conformational change to the native tRNA conformation and does not reflect the inherent Mg2+ binding properties of the native conformer.

Biochemistry, 1976 Sep 7, 15(18), 3903 - 12
Magnesium dependence of the association kinetics of Escherichia coli ribosomal subunits; Favaudon V et al.; The magnesium dependence of the Escherichia coli ribosomal subunits association has been investigated by the stopped-flow technique using isolated 30S and 50S particles depleted of polyamines and any initiation factor . Binding of the fluorescent probe bis(8-anilino-1-naphthalenesulfonate) to the ribosomal proteins occurs through biphasic kinetics . A dark reaction corresponding to a very rapid, reversible complexation of the dye molecule is followed by a slow photochemical reaction that gives rise to irreversible addition of the probe . Only the 30S subparticle exhibits a magnesium-dependent conformational change from the kinetic analysis of the dark reaction . The 70S formation kinetics are limited by a conformational change of the 30S subunit if this particle is depleted of Mg2+ (1 mM Mg2+/50 mM K+), while its activated structure is restored by incubation with 8 mM Mg2+/50 mM K+ . No rate-limiting conformation rearrangement of the 50S subunit could ever be evidenced . The Mg2+ dependence of the association kinetics of preactivated ribosomal particles is satisfactorily explained by electrostatic effects and/or formation of salt bridges, in agreement with the results of Wishnia and co-workers (Wishnia, A . Boussert, A., Graffe, M., Dessen, P., and Grunberg-Manago, M . (1975), J . Mol . Biol . 93, 499) . Equilibrium studies indicate that the ribosomal preparations we used are of B type, according to Debey et al . (Debey, P., Hui Bon Hoa, G., Douzou, P., Godefroy-Colburn, T., Graffe, M., AND Grunberg-Manago, M . (1975) Biochemistry 14, 1553) . The addition of spermidine results in a drastic fall of the need of Mg2+ for association, but it does not allow conversion of B-type particles into A-type ones at 25 degrees C . In addition to that, some 30S-bound spermidine appears to be involved directly in the coupling reaction.

Biochemistry, 1976 Sep 7, 15(18), 4053 - 8
Lysine-sensitive aspartokinase of Escherichia coli K12 . Synergy and autosynergy in an allosteric V system; Mazat JP et al.; The interactions of the lysine-sensitive aspartokinase of E . coli K12 with lysine and leucine, as evidenced in the inhibition and binding curves, are well explained by the equations of an allosteric V model . Mathematical treatments of such a model lead to new linearized plots . These representations are applied to our experimental results and allow the direct determination of some parameters of the model (equilibrium constant L' and leucine dissociation constants) . The other parameters are obtained by an optimization method . The theoretical curves drawn according to this model account well for the synergistic inhibition between lysine and leucine and for the role of the two nonequivalent lysine binding sites ("autosynergy").

Biochemistry, 1976 Sep 7, 15(18), 3917 - 24
Changes in tertiary structure accompanying a single base change in transfer RNA . Proton magnetic resonance and aminoacylation studies of Escherichia coli tRNAMet f1 and tRNAMet f3 and their spin-labeled (s4U8) derivatives; Daniel WE Jr et al.; The properties of Escherichia coli tRNAMet f1 and tRNAMet f3 that differ by only one base change, m7G to A at position 47, have been compared structurally by proton magnetic resonance and functionally by the aminoacylation reaction . The NMR spectra of the two tRNA species in the region between 0 and 4 ppm below 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) (methyl and methylene region) were the same except for the absence of the lowest field peak at 3.8 ppm in tRNAMet f3, thus unequivocally identifying this resonance at the methyl group of m7G47 of tRNAMet f1 . The same resonance disappears in tRNAMet f1 spin-labeled at s4U8 and reappears in the diamagnetic reduced spin-labeled tRNAMet f1 from which the average distance between the spin-label and the methyl protons of m7G is estimated to be less than 15 A . The proximity of m7G47 but not T55 to s4U8 in the structure of E . coli tRNAMet f1 in solution is consistant with the crystallographic model for yeast tRNAPhe . A spectral comparison of the hydrogen-bond regions (11-14 ppm below DSS) of tRNAMet f1 and tRNAMet f3 reveals major shifts of four resonances previously assigned to tertiary hydrogen bonds . Of the four, the one at lowest field (14.8 ppm) had been assigned by chemical modification to the tertiary (s4U8-A14) hydrogen bond and the one at 13.3 ppm had been tentatively assigned to the tertiary hydrogen bond G23-m7G47 of the 13-23-47 triple . A more positive assignment of the G23-m7G47 at 13.3 ppm could be made from the additional evidence that this resonance, which was first observed in the difference spectrum between spin-labeled tRNAMet f1 and its reduced form, is the only one missing in the analogous difference spectrum of tRNAMet f3 . At low ionic strength and in the absence of magnesium ions, the differences in the hydrogen-bonded region of the NMR spectra of tRNAMet f1 and tRNAMet f3 are much greater than in the presence of magnesium ions . The optimal magnesium concentration required for maximal initial velocities is also higher for tRNAMet f3 than for tRNAMet f1 . The perturbation caused by the spin-label in destabilizing hydrogen bonds in the region between 13 and 14 ppm is greater for tRNAMet f3 than tRNAMet f1 but the distance relations for the hydrogen bonds in the region between 12 and 13 ppm (the major paramagnetic perturbations) are conserved in the two species . The disruption of one hydrogen bond relative to native tRNAMet f1 either by spin-labeling (s4U8-A14) or by substitution of m7G by A in tRNAMet f3 has little effect on the aminoacyl acceptor activity or the velocity of the aminoacylation reaction at optimal magnesium concentration, but the absence of both tertiary hydrogen bonds in the augmented D-helix region in the spin-labeled tRNAMet f3 results in approximately 60% reduction both in acceptance activity and in initial velocity of the aminoacylation reaction.

Biochim Biophys Acta, 1976 Sep 6, 442(3), 331 - 42
The isolation and properties of a DNA-directed RNA polymerase from yeast mitochondria; Scragg AH; A method is described for the rapid isolation of yeast mitochondrial DNA-directed RNA polymerase . The enzyme obtained had a specific activity of 1.56 nmol UMP incorporated per mg protein in 20 min at 37 degrees C, and is some 95% pure . This purified enzyme upon polyacrylamide gel elctrophoresis consists of a single polypeptide of 68 000 mol . wt . However, the enzyme forms aggregates easily which are affected by ionic strength, an increase decreasing the apparent molecular weight of the aggregates . This property also explains the presence of two peaks of activity upon DEAE-cellulose chromatography . The purified enzyme is still sensitive to rifamycin and to a number of rifamycin derivatives . The enzyme's sensitivity to rifamycin and rifamycin derivatives was compared with Escherichia coli and yeast nuclear RNA polymerases.

Arthritis Rheum, 1976 Sep-Oct, 19(5), 867 - 73
Antibodies to dAT detected by membrane filtration; Lentz K et al.; Sera from patients with active systemic lupus erythematosus caused the retention of the radioactively labeled synthetic polynucleotide 14C-dAT on cellulose nitrate filters . The amount of 14C-dAT bound correlated with the presence of hypocomplementemia and active renal disease . Sera containing only anti-single-stranded DNA antibodies did not bind 14C-dAT but did bind 125I DNA from E coli that had been prefiltered to remove ssDNA contamination . Inhibition experiments also indicated the presence of antigenic differences between 14C-dAT and prefiltered 125I-E coli DNA . The data suggest that dAT carries one of the major antigenic specificities for antinative DNA antibodies in SLE, and that cellulose nitrate filtration fails to remove a significant proportion of contaminating single-stranded DNA determinants in non-synthetic DNA.

P N G Med J, 1976 Sep, 18(3), 157 - 61
Pharmacological investigations of virosecurinine; Hill L et al.; This study reports some biological studies on the alkaloid virosecurinine isolated from the plant Securinega Virosa . Virosecurinine is an isomer of the securinine group of alkaloids present in plants used in traditional medicines in many areas, including the Central District of Papua New Guinea . Virosecurinine is mildly toxic to mice with an LD50 of 73 milligrams per kilogram body weight . Death results from violent tonic convulsions and paralysis similar to those observed with strychnine poisoning . Virosecurinine had no effect on the growth of the following bacteria, E . coli, S . aureus, and B . subtilis, or the fungi, F . monoliforme, P . viridicatum, A . niger, R . solani, R . stolonifer and C . lunata.

J Virol, 1976 Sep, 19(3), 1080 - 9
Isolation of a transcriptive complex from Newcastle disease virions; Colonno RJ et al.; An active transcriptive complex was isolated from purified virions of Newcastle disease virus . After disruption with Triton X-100 and high salt, soluble and particulate fractions were separated by density gradient centrifugation . The transcriptive complex, recovered at a density of 1.275 g/cm3, appeared as a nucleocapsid structure by electron microscopy . When analyzed by polyacryl-amide gel electrophoresis, the nucleocapsids consisted of the nucleocapsid protein, a minor protein of 53,000 molecular weight, and the large L protein . Nucleocapsids possessed less than 1% of the hemagglutinating and neuraminidase activities originally associated with virions . The active complex synthesized predominantly 11 to 20S RNA in vitro and approximately one-fourth of the RNA molecules contained polyadenylic acid segments . In the presence of S-adenosyl-L-methionine, the RNA molecules were capped and methylated at the 5' termini . The transcriptive complex was also capable of methylating exogenous Escherichia coli RNA in the absence of viral RNA synthesis.

Vet Med (Praha), 1976 Sep, 21(9), 527 - 35
{The incidence of porcine Escherichia coli strains suspected to be pathogenic in pigs}; Kruspan J et al.; In the period from 1973 to the first half of 1975 the Central State Veterinary Institute, Bratislava, performed a serological typification of E . coli strains isolated from 1567 dead sucklings and weanlings . The pigs suffering from diarrhoea as the main clinical manifestation of the disease came from 10 districts of the West-Slovakian region . The serological typification of the strains was based on slow agglutination in a test tube and the tests concerned the following 0-antigens: 8, 117, 138, 139, 141, 147, 149, 101 . The E . coli strains suspected to be pathogenic were found in 29.5% of sucklings and in 50.3% of weanlings, on an average for the whole period of study . The individual serological groups of E . coli were represented in the following proportions in sucklings: 0149 (29.8%) of all serological groups studied, 08 (23.9%), 0117 (16.1%), 0147 (12.5%), 0101 (10.1%), 0141 (6.5%), 0138 (1.1%); serological group 0139 did not occur . The following representation was observed in weanlings: 0139 (42.0%), 08 (19.1%), 0117 (13.3%), 0147 (11.9%), 0141 (8.6%), 0101 (3.8%), 0138 (0.9%), 0149 (0.4%) . From the viewpoint of prevention and measures against porcine coli-infections, the most frequent and most important E . coli, strains are mentioned.

J Bacteriol, 1976 Sep, 127(3), 1455 - 64
Organization of the nucleoplasm in Escherichia coli visualized by phase-contrast light microscopy, freeze fracturing, and thin sectioning; Woldringh CL et al.; The organization of the nucleoplasm in Escherichia coli was studied by comparing the results obtained by freeze fracturing and thin sectioning . In addition to exponentially growing cells, we used chloramphenicol-treated cells which show a well-defined nucleoplasm, in the phase-contrast light microscope and can therefore function as a control for treatments necessary for electron microscopy . Two factors were found to determine the visibility of the nucleoplasm in freeze fractures: first, the state of lateral aggregation of deoxyribonucleic and fibrils, which is enhanced by postfixation with OsO4 according to the Ryter-Kellenberger technique; second, the presence of ice crystals . When their formation is prevented by the use of high concentration of freeze-protecting agents, the nucleoplasm appears as a smooth region in cells that have been prefixed . In unfixed cells, however, the freeze-protecting agent causes disappearance of the nucleoplasm by rearrangement of structures within the cell . This observation makes it hard to determine whether the deoxyribonucleic acid in vivo dispersed, as found after glutaraldehyde prefixation, or compact, as after OsO4 prefixation.

Mol Biol (Mosk), 1976 Sep-Oct, 10(5), 1153 - 8
{Peculiarities of DNA winding with bifunctional cations}; Tselikova SV et al.; Addition of the bifunctional cations, bis(2-aminoethyl) disulphide-cystamine, and bis(2-guanidoethyl) disulphide-GED, into water solution of DNA results in a decrease in magnitude of the positive circular dichroism (CD) band . However, unlike the similar effect due to alcaline ions or ions of ammonium and guanidinium the above effect occurs at much smaller, stoichiometric with phosphates, concentrations of the dications . Another peculiarity of GED is the attaining of a plato region for the curve of the CD change with the rise of GED concentration . Since the decrease in positive band CD is connected with increase in the rotation angle between the base pairs, the observed behavior of the bifunctional cations are supposedly due to peculiarities of their winding action upon DNA helix . Reducing the disulphide bond in the cations gives rise to increase in the DNA positive CD band up to the values inherent to those in the presence of the corresponding monocations . The higher efficiency of the bifunctional cations is thus due to the cationic groups belong to one and the same molecule . Such compounds could thus be considered as a simple model of DNA-protein interaction.

Immunology, 1976 Sep, 31(3), 389 - 96
The dissociation of adjuvant properties of mycobacterial components from mitogenicity, and from the ability to induce the release of mediators from macrophages; Rook GA et al.; Twelve preparations from mycobacterial cell walls and culture supernatant fluids were tested for their ability to activate lymphocytes from Balb/c or Nu/Nu mice, and to increase the release of mediators from macrophages in vitro . The peptidoglycolipids (wax D) were B-cell mitogens and induced plaque-forming cells . These properties were lost if the glycopeptide component was removed, leaving the pure lipid, mycolic acid . Neither the glycopeptide fractions, with well-documented adjuvant properties, nor mycobacterial polysacchardie II-activated B cells . Only intact peptidoglycolipid showed synergy with the mitogenic effect of phytohaemagglutinin (PHA) on thymocytes from Balb/c mice . The effect was much smaller than with E . coli lipopolysacharide (LPS) or dextran sulphate . The peptidoglycolipid also enhanced the release of factors from macrophages able to modify the response of Balb/c thymus cells to PHA . In this respect it resembled E . coli LPS . Adjuvant active glycopeptides did not share this property.

Vopr Virusol, 1976 Sep-Oct, (5), 539 - 44
{Fragmentation of human adenovirus type 6 DNA by means of restrictase of E . co R I and BAM I}; Naroditskii BS et al.; The effect of restricting endonucleases of E . co RI and BAMI on DNA of human adenovirus type 6 was studied . E . co RI and BAMI restrictases cleave virus DNA into 4 fragments each . The molecular weight of E . co RI fragments was: A--17.5X10(6), B--3.63X10(6), C--1.56X10(6), D--1.05X10(6), those of BAMI fragments: A--9.7X10(6), B--7X10(6), C--4.09X10(6), D--3.1X10(6) . By means of terminal nucleotidyltransferase and the procedure of partial DNA hydrolysis, the alteration of fragments obtained by the effect of E . co RI enzyme in adenovirus type 6 genome was determined and found to be A--D--C--B.

Mikrobiologiia, 1976 Sep-Oct, 45(5), 917 - 22
{Effect of pancreatic deoxyribonuclease on growth of Escherichia coli}; Beliaeva MI et al.; Cultivation of Escherichia coli K-12 on a medium containing pancreatic deoxyribonuclease at a concentration of 200 visc . units per 1 ml to 1000 visc . units per 1 ml stimulated growth of the cells but only at the lag phase of synchronous growth of the culture.

J S Afr Vet Assoc, 1976 Sep, 47(3), 193 - 5
Pathogenesis and treatment of Escherichia coli infections in calves; Bywater RJ; Two clearly defined types of E . coli infection are recognised and the factors predisposing and giving rise to pathogenicity are discussed . The mode of action of enterotoxins in the secretary mechanism is thought to be through stimulation of adenyl cyclase activity . Treatment and prevention of the disease is considered in relation to the pathogenesis of the infection.

J Med Chem, 1976 Sep, 19(9), 1133 - 7
Phosphorus-nitrogen compounds . 20 . Thiophosphorus hydrazones; Cates LA et al.; Six pyridine-2-carboxaldehyde, one pyridine N-oxide 2-carboxaldehyde, and five diketone thiophosphoric hydrazones, three thiophosphoric hydrazides, and two cupric chelates were synthesized . The chelates and nine of the hydrazones were tested against Ehrlich ascites carcinoma . Seven of these latter agents were administered concurrently with either cupric and/or ferrous salts to mice bearing this tumor . The greatest activity was found with the chelate, cimethyl pyridine-2-carboxyaldehyde phosphorothioic hydrazone-copper (1:1) . The hydrazone portion of this chelate also formed a ligand-copper (2:1) complex . Although all of the hydrazones but one were inactive when evaluated alone, the concurrent injection of cupric ion increased survival times by an avoli alkaline phosphatase was found to be inhibited by two thiosemicarbazones in a manner similar to that previously reported by these agents against alkaline phosphatase derived from Sarcoma 180-6-thiopurine resistant ascites cells . None of the 14 hydrazides or hydrazones tested against E . coli enzyme displayed significant inhibition.

J Biochem (Tokyo), 1976 Sep, 80(3), 463 - 9
Aminoacyl transfer RNA formation . Binding of cations to transfer RNA and its role in aminoacyl transfer RNA formation; Takeda Y et al.; The role of cations (polyamines and Mg2+) in isoleucyl-tRNA formation catalyzed by purified isolecuyl-tRNA synthetase {EC 6.1.1.5} from Escherichia coli was studied . It was found that spermine, spermidine, and Mg2+ bind to tRNA and that when bound to these cations, tRNA acts as substrate of aminoacylation without requiring further cations . These findings suggest that the primary function of cations in aminoacyl-tRNA formation is to bind to tRNA to stabilize its structure, not to bind to the enzyme to activate it.

Cell, 1976 Sep, 9(1), 163 - 9
The relative positions of sea urchin histone genes on the chimeric plasmids pSp2 and pSp17 as studied by electronmicroscopy; Wu M et al.; The relative positions of the sea urchin histone genes and the spacer regions on the chimeric plasmids pS p2 and pSp17 have been mapped by hybridizing total histonemessenger RNA to single strands of the plasmid DNAs . The lengths and spacing between the several RNA:DNA duplex regions on the single strands of DNA were measured by the gene 32-ethidium bromide electron microscope mapping method . We find that the genes are interdigitated with spacer sequences of different lengths; that there are three coding sequences on pSp2, all on the same strand, with the relative order H1, H4, and B4; and that there are two coding sequences on pSp17, both on the same strand, corresponding to the messages denoted B1 and B2-B3, where B4, B1, and B2-3 are electrophoretically resolved components of histone mRNA, all of size intermediate between the larger H1 and the smaller H4 message.

Transplantation, 1976 Sep, 22(3), 229 - 35
Prolongation of allograft survival by combination therapy with anti-thymocyte serum and anti-immunoglobulin; Constantian MB et al.; Heterologous anti-immunoglobulin (AI) is a potent immunosuppressive agent which compares favorably to anti-thymocyte serum (ATS) in inhibiting E and EAC-rosette formation and diminishing the secondary antibody response to sheep erythrocytes and E . coli lipopolysaccharide . AI prolongs H-2 incompatible skin allograft survival than either antiserum used alone, without evidence of toxicity to recipient mice . AI does not cause significant immune complex glomerular or renovascular deposition and may act by interference with antigen binding.

Proc Natl Acad Sci U S A, 1976 Sep, 73(9), 3238 - 42
Specialized transduction of colicin E1 DNA in Escherichia coli K-12; Fukumaki Y et al.; Genetic studies were made on E . coli K-12 TM96, which carries recombinant molecules constructed by in vitro combination of colicin E1 DNA and a DNA fragment of E . coli for guanine synthesis derived from transducing phage . The recombinant molecules existed as stable plasmids within the cell and contained genes for colicin E1 immunity and the guaA enzyme (xanthosine 5'-monophosphate aminase) together with a part of the lambda genome, R through J: (R-A-F-J)+ . A block of the lambda genome, int through Q, was not detected in the recombinant molecule . Thus, this recombinant molecule was named ColEl-coslambda-guaA, and the specialized tranduction of the ColEl-coslambda-guA DNA into various E . coli K-12 cells by lambda phage was described . Lysates prepared by lytic infection of lambda phage onto TM96 or by induction of TM96(lambda) lysogens contained transducing particles which could transduce gua-deleted E . coli to stable guaA+ cells . These transductants were proved to have similar genetic properties as those of TM96 . The frequency of transduction was not affected by the presence of an attachement site for lambda, prophage lambda, colicin E1 plasmids, or the recA property within gua-deleted recipient cells . Transducing particles were resistant to EDTA treatment and most of them had an average density of about 1.472 . This value corresponds to that of lambda phage particles, which contain about 72% of the lenght of lambda DNA.

Proc Natl Acad Sci U S A, 1976 Sep, 73(9), 3156 - 9
DNA dependent synthesis of protein L12 from escherichia coli ribosomes, in vitro; Chu F et al.; The in vitro synthesis of ribosomal protein L12 has been obtained in a coupled system with DNA extracted from the transducing phage lambdarifd18 as template . In addition, a second protein (molecular weight of 16,000) with immunological, chemical, and ribosome-binding characteristics similar to L12 is formed in this in vitro system . The synthesis of both proteins is depressed by one-half when guanosine-5'-diphosphate-3'-diphosphate is added to the reaction mixture.

Proc Natl Acad Sci U S A, 1976 Sep, 73(9), 3103 - 6
Isolation of a set of hybrid lac repressors made in vitro between normal lac repressor and its homogeneous tryptic core; Geisler N et al.; Lactose repressor can be renatured from 8 M guanidine-HCl solution . The renatured repressor is tetrameric and shows DNA binding activity . Thus it becomes possible to obtain hybrid tetramers in vitro between normal repressor and repressor defective in DNA binding by simultaneous denaturation and renaturation . In order to facilitate the separation of the different hybrids, we have used a lac repressor derivative that does not bind DNA, which is missing the amino-terminal 59 residues of the polypeptide chain (homogeneous tryptic core) . The hybrids resulting from the mixed renaturation of homogeneous tryptic core and normal repressor can be separated by electrophoresis on Cellogel . The hybrids have been recovered, and a preliminary characterization of their DNA-binding properties is reported.

Proc Natl Acad Sci U S A, 1976 Sep, 73(9), 3064 - 7
Specific binding of formylated initiator-tRNA to Escherichia coli RNA polymerase; Pongs O et al.; E . coli fMet-tRNAfMEet and E . coli RNA plymerase (RNA nucleotidyltransferase; EC 2.7.7.6; nucleoside-triphosphate:RNA nucleotidyltransferase) form a 1:1 complex with an apparent association constant of 9.0 X 10(6)M-1 at 37 degrees . The affinity of polymerase to tRNA depends on the tRNA as well as the formyl methionine moiety . Core polymerase has a greatly reduced affinity for initiator tRNA . Optimal binding conditions are similar to those that are also optimal for binding initiator tRNA to ribosomes . Binding of initiator tRNA to polymerase stimulates the transcription of lambda plac DNA, as determined in a crude cell-free system for beta-galactosidase (EC 3.2.1.23; beta-D-galactoside galactohydrolase) synthesis as well as in a highly purified transcription system.

Proc Natl Acad Sci U S A, 1976 Sep, 73(9), 3000 - 4
On the mechanism of genetic recombination: electron microscopic observation of recombination intermediates; Potter H et al.; This paper deals with the nature of recombination intermediates . Using the electron microscope to study the DNA of the plasmid colicin E1, we have observed more than 800 molecules that appear to represent intermediates in the process of recombination . Specifically, after isolating colicin DNA and linearizing it with the restriction enzyme EcoRI, we find crossed molecules with twice the normal colicin DNA content . These forms consist of two genome-length elements held together at a region of DNA homology . The molecules can be recovered from wild type and Rec B-C host cells but are not present among the colicin DNA forms isolated from recombination-deficient Rec A cells . We have termed the experimentally observed molecules "chi forms" and believe that they represent the recombination intermediate of the Holliday model.

Proc Natl Acad Sci U S A, 1976 Sep, 73(9), 2987 - 90
Stereochemistry of polymerization by DNA-dependent RNA-polymerase from Escherichia coli: an investigation with a diastereomeric ATP-analogue; Eckstein F et al.; The phosphodiester bond formation by DNA-dependent RNA-polymerase (RNA nucleotidyltransferase, nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) can in principle result in retention, inversion, or racemization of configuration at the alpha-phosphorus of the nucleoside 5'-triphosphate being polymerized . As a first step in elucidating the stereochemistry of this reaction, one diastereomer (A) of adenosine 5'-O-(1-thiotriphosphate) (ATPalphaS) was polymerized with UTP in the presence of poly(dA-dT)-poly(dA-dT) . The resulting polymer was enzymatically cleaved to uridine 2',3'-cyclic phosphorothioate which was determined to be the endo-isomer by comparison with an authentic sample . This shows that no reacemization had occurred and that isomer A of ATPalphaS gives a phosphorothioate diester bond with the R-configuration . Whether this represents inversion of retention of configuration awaits elucidation of the absolute configuration of isomer A for ATPalphaS.

Nucleic Acids Res, 1976 Sep, 3(9), 2387 - 98
A simple method for DNA restriction site mapping; Smith HO et al.; When a DNA molecule, enzymatically labelled with 32p at one end, is partially digested with a restriction enzyme labelled tdna fragments are obtained which form an overlapping series of molecules, all with a common labelled terminus . ta restriction map can then be constructed from an analysis of the size distribution of these molecules . This technique has been used for the restriction site mapping of cloned histone DNA (h22) where as many as 35 cleavage sites may be accurately determined in a single experiment.

Nucleic Acids Res, 1976 Sep, 3(9), 2353 - 66
A comparative X-ray diffraction and circular dichroism study of DNA compact particles formed in water-salt solutions, containing poly(ethylene glycol); Evdokimov YM et al.; Comparative CD and X-ray diffraction studies of DNA compact particules which were obtained in PEG-containing water-salt solutions, have been carried out . Compact particles, formed from native DNA, produce a psi CD spectrum (characterized by a negative band at lambda-270 nm) and a small-angle X-ray diffraction pattern, which shows two reflections: I at 34-40 A and II at 80-90 A (together with its second-order reflection) . Compact particules, formed from DNA molecules with partially disordered secondary structure, do not produce the psi CD spectrum and the reflection I, while the reflection II remains unchanged . It is suggested that the spacing of 34-40 A is associated with a side-by-side packing of DNA fragments in "microcrystallization' regions in compact particules and that such "microcrystallization' accounts for the generation of the psi CD spectrum.

Nucleic Acids Res, 1976 Sep, 3(9), 2223 - 31
RNA chain elongation on a chromatin template; Solage A et al.; The rate of RNA chain elongation has been measured with DNA and chromatin as template . RNA propagation on chromatin is about 50% of the rate found with DNA . Kinetic experiments demonstrate that the inhibition is not due to interference with the addition of the nucleoside triphosphates . Analysis of the dependence of propagation on the Tm of DNA shows that the chromatin proteins interfere with the translocation of the RNA polymerase along the DNA template.

Eur J Pharmacol, 1976 Sep, 39(1), 79 - 90
Release of renal prostaglandins during endotoxin-induced hypotension; Herman AG et al.; The appearance of prostaglandins in dog's blood during endotoxin-induced hypotension was studied by use of the dialysis modification of the blood bathed organ technique . An increase in prostaglandins, mainly E2 and F2alpha was found in renal venous blood, whereas no such increase was seen in blood from the abdominal aorta, the inferior vena cava or the femoral vein . Three possible trigger mechanisms for this increase i.e . hypotension, reduced flow and reflexogenic sympathetic stimulation, have been investigated . It is suggested that, in addition to these three factors, circulating hormones such as noradrenaline, angiotensin or bradykinin, play a role in this release mechanism . Administration of indomethacin produced a restoration of the systemic blood pressure to its pre-endotoxin value; concomitantly a disappearance of the prostaglandins from the circulation was observed . It is concluded that prostaglandins contribute to the hypotension induced by endotoxin . Whether they are beneficial or detrimental remains to be resolved.

Tijdschr Diergeneeskd, 1976 Sep 1, 101(17), 972 - 3
{Scours due to eschierichia coli in piglets (author's transl)}; Bijleveld F et al.; Mortality from scours caused by E . coli was observed among piglets on a pig breeding and multiplying farm in 't Zand over a considerable period . As the usual method of treatment failed to produce the desired results, E . coli vaccine was administered to a number of farrowing sows . Alle piglets born to vaccinated sows were reared without any problems, whereas a number of piglets of non-vaccinated sows died from scours caused by E . coli . Moreover, the growth of piglets of vaccinated sows was superior to that of piglets of non-vaccinated sows . An optimum vaccination programme is described.

J Immunol, 1976 Sep, 117(3), 904 - 10
An in vitro immuno-enzymatic assay of tumor antigens in the mouse with beta-galactosidase; Celada F et al.; A method for detection of the primary binding of soluble tumor-associated antigens by antibodies has been developed by using an enzyme immunoassay (EIA) . A heteroantiserum was produced by injecting tumor cells from a chemically induced murine sarcoma into rabbits, and antibodies reacting with most normal tissue components were removed by exhaustive in vivo absorption . A soluble preparation of tumor cells, obtained by 3 M KCl extraction, was conjugated to beta-galactosidase from Escherichia coli . The antibody binding was measured by determining the enzyme activity that could be separated by anti-antibody coprecepitation . The reaction follows saturation kinetics, and nonlabeled antigen can be readily quantitated by inhibition . The present method detects determinants common to several MC-induced tumors on the same mouse strain but absent in normal cells and nonrelated tumors in addition to individual tumor-specific transplantation antigens . The sensitivity and simplicity of the new method compare favorably with a binding assay that utilizes radioactive iodine as a label . Thus, EIA becomes a flexible tool for the further characterization and purification of these antigens.

J Bacteriol, 1976 Sep, 127(3), 1587 - 9
Method for the isolation of Escherichia coli relaxed mutants, utilizing near-ultraviolet irradiation; Ramabhadran TV; Near-ultraviolet radiation (300 to 380 nm) induces a transient growth inhibition in "stringent" (rel+) strains of Escherichia coli, whereas "relaxed" (rel-) strains are only mildly affected . This difference permits rapid isolation of large numbers of relaxed mutants of E . coli.

J Bacteriol, 1976 Sep, 127(3), 1561 - 3
Isolation of temperature-sensitive mutants of R plasmid by in vitro mutagenesis with hydroxylamine; Hashimoto T et al.; Temperature-sensitive mutants were isolated from a tetracycline resistance plasmid, pSC101, after mutagenesis with hydroxylamine . They were divided into three classes according to their growth characteristics . They include mutants defective in the replication of plasmid deoxyribonucleic acid and strains having a mutation in the drug resistance gene.

J Bacteriol, 1976 Sep, 127(3), 1538 - 42
Missense mutations in the lacZ gene that result in degradation of beta-galactosidase structural protein; Zipser D et al.; Thirty-two missenese mutations were found among more than 200 independently induced mutations in the lacZ gene of Escherichia coli . Twenty of these missense mutations were induced by nitrosguandine, and 12 were induced by aminopurine . The lacZ structural protein was endogenously degradable in seven of the mutant strains; the mutations in these strains were found to lie at only three sites in the lacZ gene . Five of the seven independent mutations were at a single site, and some heterogeneity in the degradation of the lacZ protein was observed within these mutant strains.

J Bacteriol, 1976 Sep, 127(3), 1494 - 1501
Novel mutation that causes a structural change in a lipoprotein in the outer membrane of Escherichia coli; Suzuki H et al.; A novel mutation which caused a structural change in a lipoprotein in the outer-membrane has been found in Escherichia coli K-12 . The lipoprotein of the wild-type strain is known to have a peculiar amino terminal structure: glycerylcysteine with two fatty acids attached by ester linkages and one fatty acid by an amide linkage . In contrast to the wild-type lipoprotein, the mutant lipoproteins is isolated from the E . coli envelope as a dimer of molecular weight of about 15,000 . The dimer can be reduced by mercaptoethanol to the lipoprotein monomer of molecular weight of about 7,500 . The monomer has a free thiol group which is susceptible to monoiodacetie mutant lipoprotein is extremely low in comparison with that into the wild-type lipoprotein . These results suggest that the mutant is defective in transferring a glycerol group to the thiol group of the amino terminal cysteine residue of the lipoprotein . The gene responsible for this modification reaction has been located at 36.5 min on the E . coli chromosome.

J Bacteriol, 1976 Sep, 127(3), 1389 - 99
Septation deficiency and phosphilipid perturbation in Escherichia coli genetically constitutive for the beta oxidation pathway; Vanderwinkel E et al.; Mutants of Escherichia coli defective in the regulation of the fatty acids beta oxidation pathway show an ultrastructural deficiency in septum formation at high growth rate . Several independent pairs of parent and mutant strains have been analyzed biochemically . Each parent strain displays a well-defined pattern of cellular phospholipids, which varies with the growth conditions . High ratios of phosphatidylglycerol to cardiolipin characterize fast-growth conditions . None of the mutant strains, although they grow in mass nearly as rapidly as their respective parents, can reach these high ratios . The beta oxidation pathway regulatory mutation leads to an increased turnover of the glycerol moieties of these phospholipids in the inner as well as in the outer cell membrane.

J Bacteriol, 1976 Sep, 127(3), 1376 - 81
Localization of ampicillin-sensitive sites in Escherichia coli by electron microscopy; Staugaard P et al.; Growth of Escherichia coli B/r ATCC 12407 (doubling time, 65 to 70 min) in the presence of 500 mug of ampicillin per ml for 15 to 20 min induces a sphere alongside the cell . The position was determined with respect to the length axis of the cell by electron microscopy . Although spheres may be found anywhere, some prominent sites do occur . In the shortest cells, which have a length of about 1.5 mum, they are found at the presumed new cell pole . In slightly older cells (length, about 1.8 mum), the position of the sphere is not well defined . Later on spheres occur predominantly at the cell center . In dividing cells (average length, 2.5 mum) a sphere may also occur at about one-quarter of the cell length . The position of the spheres bears resemblance to sites where a pulse of 3H-labeled diaminopimelic acid is incorporated into the peptidoglycan, as has been found by others.

J Bacteriol, 1976 Sep, 127(3), 1298 - 306
Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system: purification and characterization of the phosphocarrier protein; Ullah AH et al.; The Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system consists of three components: a membrane-bound enzyme II, a soluble enzyme I, and a soluble phosphocarrier protein, HPr . The HPr has been purified to homogeneity by a combination of ammonium sulfate precipitations, gel filtration and diethylaminoethyl, carboxymethyl Bio-Gel A, and hydroxylapatite column chromatography . The purified protein is relatively heat stable (ca . 50% activity survives 30 min of boiling) and has a molecular weight of ca . 10,000 (determined by sodium dodecyl sulfate-gel electrophoresis and amino acid analysis) . It contains a single histidine residue per molecule and can be totally inactivated by photooxidation with Rose Bengal dye . Although the mycoplasma HPr is very similar to that of Escherichia coli, it shows no significant association with antiserum produced against E . coli HPr.

J Bacteriol, 1976 Sep, 127(3), 1225 - 38
Regulation of branched-chain amino acid transport in Escherichia coli; Quay SC et al.; The repression and derepression of leucine, isoleucine, and valine transport in Escherichia coli K-12 was examined by using strains auxotrophic for leucine, isoleucine, valine, and methionine . In experiments designed to limit each of these amino acids separately, we demonstrate that leucine limitation alone derepressed the leucine-binding protein, the high-affinity branched-chain amino acid transport system (LIV-I), and the membrane-bound, low-affinity system (LIV-II) . This regulation did not seem to involve inactivation of transport components, but represented an increase in the differential rate of synthesis of transport components relative to total cellular proteins . The apparent regulation of transport by isoleucine, valine, and methionine reported elsewhere was shown to require an intact leucine, biosynthetic operon and to result from changes in the level of leucine biosynthetic enzymes . A functional leucyl-transfer ribonucleic acid synthetase was also required for repression of transport . Transport regulation was shown to be essentially independent of ilvA or its gene product, threonine deaminase . The central role of leucine or its derivatives in cellular metabolism in general is discussed.

J Bacteriol, 1976 Sep, 127(3), 1173 - 6
Mapping of a new genetic locus responsible for ampicillin resistance in Escherichia coli; Buchanan CE et al.; The ampicillin resistance locus of three different ampicillin-resistant, temperature-sensitive Escherichia coli mutants was mapped between proC and purE and does not correspond to any of the known genes in this region . The mutant gene responsible for the temperature sensitivity and consequent morphological changes in each mutant strain was not located in the same 5-min region, even though the two mutants characteristics co-reverted at a very high frequency.

J Bacteriol, 1976 Sep, 127(3), 1157 - 66
Restoration by ribosomal protein S1 of the defective translation in a temperature-sensitive mutant of Escherichia coli K-12: characterization and genetic studies; Ohsawa H et al.; A temperature-sensitive mutant of Escherichia coli was isolated that had a temperature-sensitive defect in ribosomal-wash protein(s) required for translation in vitro of E . coli endogenous messenger ribonucleic acid . It was found that 30S ribosomal protein S1 rescued the defect in the ribosomal-wash protein(s) of the mutant and that the complete restoration to the wild-type level was attained when 1 mol of protein S1 was added to 1 mol of 70S ribosome . The mutation, tss, causing such a defect was mapped at 21 min and was closely linked to the pyrD locus, the region of which was entirely different from that of the other genes coding for the many ribosomal proteins of E . coli . These results indicate that the gene specified by this mutation is involved in the function of the 30S ribosomal protein S1.

J Bacteriol, 1976 Sep, 127(3), 1150 - 6
Deoxyribonucleic acid degradation in vivo and in permeabilized Escherichia coli repair-deficient (recA zab lexA) derivatives; Castellazzi M; There are three mutations (recA, zab, and lexA) each of which suppresses the expression of the Escherichia coli tif mutation and causes high deoxyribonucleic acid (DNA) repair deficiency (Castellazi et al., 1972) . The effect of the zab mutation on DNA stability was investigated . In vivo, a strain carrying the zab-53 mutation shows (i) no spontaneous DNA degradation and (ii) rapid DNA degradation after ultraviolet irradiation, which depends upon the exonuclease V activity coded by recB+/C+genes and which is independent from the correndonuclease II activity coded by uvrA+/B+ . Thus, in regard to DNA stability, the zab mutant behaves like lexA and recA (Howard-Flanders and Boyce, 1966), the latter mutant showing in addition spontaneous DNA breakdown . The degradation patterns of these tif-suppressed strains are shown to be remarkably reproducible in bacteria made permeable to metabolites, by toluene or toluene plus Triton X-100 . The degradation properties reflect the activity of the same biochemical system that works in vivo, in that degradation depends upon the presence of recA, zab, or lexA, ultraviolet irradiation, and exonuclease V activity . In addition, adenosine 5'-triphosphate (1 mM) is required . This assay with permeabilized cells offers a useful tool for studying degradation under controlled conditions, especially by permitting the dissociation of energy-dependent from energy-independent steps.

J Bacteriol, 1976 Sep, 127(3), 1119 - 26
Stringent control of peptidoglycan biosynthesis in Escherichia coli K-12; Ishiguro EE et al.; {3H}Diaminopimelic acid (Dap) was incorporated exclusively into peptidoglycan by Escherichia coli strains auxotrophic for both lysine and Dap . The rate of {3H}Dap incorporation by stringent (rel+) strains was significantly decreased when cells were deprived of required amino acids . The addition of chloramphenicol to amino acid-starved rel+ cultured stimulated both peptidoglycan and ribonucleic acid synthesis . In contrast, a relaxed (relA) derivative incorporated {3H}Dap at comparable rates in the presence or absence of required amino acids . Physiologically significant concentrations of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) inhibited the in vitro synthesis of both carrier lipid-linked intermediate and peptidoglycan catalyzed by a particulate enzyme system . The degree of inhibition was dependent on the concentration of ppGpp in the reaction mixture . Thus, the results of in vivo and in vitro studies indicate that peptidoglycan synthesis is stringently controlled in E . coli.

J Bacteriol, 1976 Sep, 127(3), 1039 - 46
Effect of temperature on motility and chemotaxis of Escherichia coli; Maeda K et al.; The swimming velocity of Escherichia coli at various constant temperatures was found to increase with increasing temperature . The frequency of tumbling had a peak at 34 degrees C and was very low both at 20 and at 39 degrees C . The swimming tracks near the surface of a slide glass showed curves, and the curvature increased the temperature . When the temperature of a bacterial suspension was suddenly changed, a transient change of the tumbling frequency was observed . A temperature drop induced a temporary increase in the tumbling frequency, and a quick rise of temperature, on the other hand, resulted in a temporary suppression of the tumbling . These dynamic responses to sudden changes of temperature was not observed in the smoothly swimming nonchemotactic strains bearing the mutations cheA and cheC and also in a mutant with the metF mutation under a smooth swimming condition.

Monatsschr Kinderheilkd, 1976 Sep, 129(9), 654 - 7
{Screening of newborns for inborn errors of galactose metabolism . Methods and results}; Gitzelmann R; Three inborn errors of galactose metabolism as known today . Only two of them cause illness: the deficiencies of galactokinase and of galactose-l-phosphate uridyltransferase . Both can be detected through mass screening of newborns and are amenable to a simple and successful dietary treatment . Mass screening of newborns for disorders of galactose metabolism can be performed efficiently and inexpensively if it is incorporated in the general newborn screening program.

J Biochem (Tokyo), 1976 Sep, 80(3), 471 - 5
Aminoacyl transfer RNA formation . VII . Lack of correlation between aminoacylation and PPi-ATP exchange catalyzed by isoleucyl-tRNA synthetase of Escherichia coli in the presence of various divalent cations; Takeda Y et al.; Isoleucyl-tRNA formation and isoleucine-dependent PPi-ATP exchange catalyzed by purified isoleucyl-tRNA synthetase {EC 6.1.1.5} of Escherichia coli were studied in the presence of various amounts of either Mg2+, Ca2+, Fe2+, Ni2+, or Cu2+ . In the presence of Mg2+, isoleucine-dependent PPi-ATP exchange was observed in parallel with isoleucyl-tRNA formation, while in the presence of Ca2+, isoleucyl-tRNA formation was observed without isoleucine-dependent PPi-ATP exchange . Moreover, isoleucine-dependent PPi-ATP exchange was much more in the presence of Fe2+ than in the presence of Mg2+, while little isoleucyl-tRNA was formed in the presence of Fe2+ . In the presence of Ni2+ or Cu2+, neither reaction was observed . These data, indicating that formation of an isoleucyl-AMP-enzyme complex is not a necessary step in isoleucyl-tRNA formation, support the existence of a concerted mechanism of isoleucyl-tRNA formation in E . coli.

Biokhimiia, 1976 Sep, 41(9), 1614 - 8
{Participation of 5'-deoxyadenosyl-B12 in regulating methylation of tRNA}; Tarasiavichene LE et al.; The effects of different forms of cobalamines on the activities of tRNA-methylases of Zajdela ascite hepatoma were studied . Of six cobamides studied 5'-deoxyadenosyl-B12 and factor B containing as a ligand HSO3 in the concentrations of 2.4-10(-5) and 4.8-10(-5) M inhibited the tRNA-methylase activity by 21% and 15% correspondingly . The inhibitory effect of 5'-deoxyadenosyl-B12 is probably dependent on the adenosyl part of the molecule . 5'deoxyadenosyl-B12 exerted a selective effect of Zajdela ascite hepatoma tRNA-methylases, inhibiting largely the activity of 5-methyl cytosine methylase during the methylation of the E . coli K12W6 tRNA and yeast tRNA1 Val.

Proc Natl Acad Sci U S A, 1976 Sep, 73(9), 3020 - 4
Identification of enzyme-bound activated CO2 as carbonic-phosphoric anhydride: isolation of the corresponding trimethyl derivative from the active site of glutamine-dependent carbamyl phosphate synthetase; Powers SG et al.; The activated CO2 intermediate formed in the reaction catalyzed by glutamine-dependent carbamyl phosphate synthetase was identified as carbonic-phosphoric anhydride through the use of two independent procedures . The carboxy phosphate intermediate was reduced to formate by treatment with potassium borohydride . Although both free CO2 and the enzyme-bound activated CO2 are reduced to formic acid by borohydride, it was possible to selectively introduce a 14C label into the enzyme-bound activated CO2 and thus into the formic acid derived from it . Such {14C}formate formation required the presence of ATP, KCl, and the enzyme, and evidence was obtained that the {14C}formate found is not derived from carbamyl phosphate or from bicarbonate bound nonspecifically to the enzyme . When the enzyme was treated with L-2-amino-4-oxo-5-chloropentanoate (or cyanate), the formation of {14C}formate was increased about 2-fold, a finding consistent with the previous observation that such treatment effects a similar increase in the bicarbonate-dependent cleavage of ATP catalyzed by the enzyme . When reaction mixtures containing the enzyme, {gamma-32P}ATP, and {14C}bicarbonate were methylated by treatment with diazomethane, a labeled compound was formed which cochromatographed with authentic trimethyl carboxy phosphate . Equimolar quantities of 14C and 32P wer incorporated into the intermediate, thus confirming its identification as carboxy phosphate . Nonenzymatic transphosphorylation from ATP to bicarbonate to form carboxy phosphate was also detected by diazomethane trapping.

J Gen Virol, 1976 Sep, 32(3), 337 - 47
Annealing and hybridization properties of herpes simplex virus type 1 DNA; Cedar H; The hybridization properties of the herpes simplex virus type 1 (HSV) genome have been analysed . The DNA has a kinetic complexity of 1 X 10(-8) . E . cole RNA polymerase was found to initiate synthesis at about 70 sites on the HSV DNA . The in vitro RNA product from this reaction was complementary to about 80% of the HSV genome . The RNA-DNA hybridization rate constant (Kh) was determined using conditions of both RNA excess and DNA excess . Using this rate constant one can analyse the content of HSV sequences in any RNA population.

Mutat Res, 1976 Sep, 36(3), 273 - 82
Mutagenic and inactivating effects of methyl alkylaminosulfonates on Escherichia coli; Sussmuth R et al.; Methyl alkylamino methanesulfonates are mutagenic agents as shown by treating several strains of E . coli at pH 7 . Methyl methylaminosulfonate (CH3-NH-SO3-CH3) was more efficient than methyl ethylaminosulfonate (C2H5-NH-SO3-CH3) which itself was more efficient than methyl isopropylaminosulfonate (C3H7-NH-SO3-CH3) . Methyl methylaminosulfonate seemed to be at least as effective as methyl methanesulfonate (CH3-SO3-CH3) . Methyl methylaminosulfonate produced a yield of up to 1% of auxotrophic mutants . All three new mutagens appeared to react according to the same mechanism by ester fission and methylation of nucleophilic groups as is known for methyl methanesulfonate . The reaction mechanism seems to be of the SN2 type.

Am J Surg, 1976 Sep, 132(3), 316 - 9
Delayed and recurring infection in postoperative abdominal wounds; Sampsel JW; Delayed and recurring wound infection in the abdominal wall of twenty-five patients, producing a variety of signs and symptoms months or years after original operations, were most frequently associated with silk sutures and endogenous infection due to Escherichia coli . The restorative procedures employed at a small community hospital varied from incision and drainage to en bloc wound excision . Timing of operations, culture data, pre- and postoperative antibiotics, and changes in the type of suture material were important adjuncts to therapy.

Proc Natl Acad Sci U S A, 1976 Sep, 73(9), 3146 - 50
A general method for cloning eukaryotic structural gene sequences; Higuchi R et al.; Complementary DNA, transcribed in vitro from purified rabbit globin messenger RNA and made double-stranded, has been inserted into Escherichia coli plasmids pSC101 and pMB9 by the poly(dT)/poly(dA) "tailing" and annealing technique . E . coli transformants given by this DNA preparation have been shown to contain globin sequences by the hybridization of globin RNA to DNA from clones grown and lysed in situ on nitrocellulose filters . An estimate of the amount of inserted globin sequences has been provided by fingerprint analysis of globin mRNA sequences hybridized to the purified plasmid chimeras . Inserted sequences so far subjected to detailed analysis have been ascribed to the rabbit beta globin chain . The susceptibility of inserted beta globin, sequences to the restriction endonuclease EcoRI confirms the existence of a site already found through previous nucleotide sequence analysis.

Cell, 1976 Sep, 9(1), 101 - 16
Identification of a ribonuclease P-like activity from human KB cells; Koski RA et al.; An endoribonuclease which cleaves tRNA precursor molecules has been partially purified from human KB tissue culture cells . This activity is found in cytoplasmic fractions but is not detectable in the nucleoplasm . tRNA precursor molecules from both E . coli and KB cells are cleaved by this novel activity to produce 5' phosphate-terminated oligonucleotides . E coli RNAase P and the KB cell nuclease both make a single endonucleolytic scission in E . coli tRNATyr precursor, thereby separating the 41 extra nucleotides on the 5' end of the precursor molecule from the 5' terminal sequence of the mature tRNATyr molecule . The cleavage products generated from other E . coli tRNA precursors by the KB cell activity are identical in size to those produced by RNAase P . The KB cell endoribonuclease requires Mg2+ and a monovalent cation (Na+, K+, or NH4+) for function . The enzymatic activity has a broad pH optimum, centered near pH 8.0, and the activity is inhibited by tRNA . Several KB cell RNAs with long half-lives in vivo, including 5S and bulk 4S RNA, are not cleaved by this nuclease . The KB cell endoribonuclease resembles E . coli RNAase P in its substrate specificity, pH optimum, ion requirements, and sensitivity to tRNA . These properties and the cytoplasmic localization of the novel endoribonuclease indicate its involvement in the biosynthesis of KB cell tRNA.

Nucleic Acids Res, 1976 Sep, 3(9), 2341 - 52
Novikoff hepatoma deoxyribonucleic acid polymerase . Sensitivity of the beta-polymerase to sulfhydryl blocking agents; Mosbaugh DW et al.; Unlike other beta-class eukaryotic DNA polymerases, the enzyme purified from the Novikoff hepatoma is inhibited by both sulfhydryl blocking agents N-ethylmaleimide (NEM) and p-hydroxymercuribenzoate (pHMB) . The degree of sensitivity varies depending on the enzyme purity, pH of the reaction, and the presence of sulfhydryl reducing agents . Novikoff beta-polymerase activity is unaffected by the presence of 2-mercaptoethanol (2-Me) or dithiothreitol (DTT); however, the combination of 2-mercaptoethanol and NEM or pHMB acts to reverse the inhibition of the sulfhydryl blocking agent . The reversal of inhibition involves more than just a titration of NEM with 2-mercaptoethanol since a) the combination of these two reagents actually stimulates the DNA polymerase, and b) dithiothreitol did not reverse the inhibition . Binding of the polymerase to DNA did not affect the enzyme sensitivity to NEM.

J Virol, 1976 Sep, 19(3), 940 - 9
Enzymatic action of coliphage omega8 and its possible role in infection; Prehm P et al.; The receptor of coliphage omega8 is the O-specific mannan of Escherichia coli O8 in which the trisaccharide alpha-mannosyl-1,2-alpha-mannosyl-1,2-mannose is joined through alpha-mannosyl-1,3-linkages . Coliphage omega8 produces an endo-alpha-1,3-mannosidase which destroys the receptor, liberating a series of oligosaccharides (repeating trisaccharide and multiples) . The enzyme is an integral part of the phage particles and also occurs in a free form in the lysates . Phage particles hydrolyze alpha-1,3-mannosyl linkages in the lipopolysaccharide, the polysaccharide (mannan) moiety, and higher oligosaccharides with an efficiency decreasing in this order . No transmannosylation could be detected . Phage particles also degrade the receptor mannan on whole bacteria, as determined with 14C-labeled E . coli O8 . The values of Km and Vmax were determined with omega8 particles and free enzymes using native lipopolysaccharide and its triethylammonium salt . The latter, which was obtained after electrodialysis, has a micellar weight of 2.5 X 10(5), whereas the native lipopolysaccharide forms supermicelles with micellar weights of several millions . With coliphage omega8 as enzyme and supermicellar lipopolysaccharide as substrate Km=5 X 10(-8) M was obtained . This, together with the fact that omega8 attaches irreversibly to E . coli O8, was used in proposing a hypothesis for the possible role of the enzyme in the first steps of infection with coliphage omega8.

Eur J Biochem, 1976 Sep, 68(1), 81 - 93
Mechanism of discrimination between cognate and non-cognate tRNAs by phenylalanyl-tRNA synthetase from yeast; Krauss G et al.; The interaction between phenylalanyl-tRNA synthetase from yeast and Escherichia coli and tRNAPhe (yeast), tRNASer (yeast), tRNA1Val (E . coli) has been investigated by ultracentrifugation analysis, fluorescence titrations and fast kinetic techniques . The fluorescence of the Y-base of tRNAPhe and the intrinsic fluorescence of the synthetases have been used as optical indicators . 1 . Specific complexes between phenylalanyl-tRNA synthetase and tRNAPhe from yeast are formed in a two-step mechanism: a nearly diffusion-controlled recombination is followed by a fast conformational transition . Binding constants, rate constants and changes in the quantum yield of the Y-base fluorescence upon binding are given under a variety of conditions with respect to pH, added salt, concentration of Mg2+ ions and temperature . 2 . Heterologous complexes between phenylalanyl-tRNA synthetase (E . coli) and tRNAPhe (yeast) are formed in a similar two-step mechanism as the specific complexes; the conformational transition, however, is slower by a factor 4-5 . 3 . Formation of non-specific complexes between phenylalanyl-tRNA synthetase (yeast) and tRNATyr (E . coli) proceeds in a one-step mechanism . Phenylalanyl-tRNA synthetase (yeast) binds either two molecules of tRNAPhe (yeast) or only one molecule of tRNATyr (E . coli); tRNA1Val (E . coli) or tRNASer (yeast) are also bound in a 1:1 stoichiometry . Binding constants for complexes of phenylalanyl-tRNA synthetase (yeast) and tRNATyr (E . coli) are determined under a variety of conditions . In contrast to specific complex formation, non-specific binding is disfavoured by the presence of Mg2+ ions, and is not affected by pH and the presence of pyrophosphate . The difference in the stabilities of specific and non-specific complexes can be varied by a factor of 2--100 depending on the ionic conditions . Discrimination of cognate and non-cognate tRNA by phenylalanyl-tRNA synthetase (yeast) is discussed in terms of the binding mechanism, the topology of the binding sites, the nature of interacting forces and the relation between specificity and ionic conditions.

Eur J Biochem, 1976 Sep, 68(1), 71 - 80
Distinct steps in the specific binding of tRNA to aminoacyl-tRNA synthetase . Temperature-jump studies on the serine-specific system from yeast and the tyrosine-specific system from Escherichia coli; Riesner D et al.; The kinetics of the interaction of tRNASer and seryl-tRNA synthetase from yeast as well as of tRNATyr and tyrosyl-tRNA synthetase from Escherichia coli have been investigated by temperature-jump experiments . It could be shown that complex formation proceeds in two distinct steps . This was demonstrated for both the first and the second binding site . The two-step mechanism was deduced from the characteristic concentration dependence of the relaxation times . Seryl-tRNA synthetase recombines with the first tRNA to form an intermediate complex (kI12, kI21), which is transformed in a fast reaction to the final 1:1 complex (kI23, kI32) . At pH 7.2 with 0.1 M KCl the rate constants are: kI12 = 2.7 X 10(8) M-1 S-1; kI23, kI32) . At pH 7.2 with 0.1 M KCl the rate constants are: kI12 = 2.7 x 10(8) M-1 S-1; kI21 = 220 S-1; kI23 = 760 S-1; kI32 = 330 S-1 . The 1:1 complex can bind a second tRNA . At pH 7.2 without added salt the rate constants are: KII2 = 0.9 X 10(8) M-1 S-1; kII21 = 270 S-1; kII23 = 120 S-1; kII32 = 1250 S-1 . The tyrosine-specific system behaves very similarly to the serine-specific system . Data are given for pH 7.2 (pH 6.0) for the binding of the second tRNA: kII12 = 1 X 10(8) (2.5 X 10(8)) M-1 S-1; kII21 = 470 (170) S-1; kII23 = 150 (530) S-1; kII32 = 1540 (720) S-1 . The kinetic results are discussed in terms of their relevance to the recognition process and their relation to the anticooperative binding behaviour of tRNA to synthetase.

J Bacteriol, 1976 Sep, 127(3), 1315 - 23
Ornithine delta-transaminase activity in Escherichia coli: its identity with acetylornithine delta-transaminase; Billhemier JT et al.; Procedures that have been developed for the purification of acetylornithine delta-transaminase from Escherichia coli W also lead to the simultaneous purification of ornithine delta-transaminase . These two enzymatic activities have the same electrophoretic mobility and are identical immunochemically . Studies of inhibition kinetics demonstrate that the two substrates, acetylornithine and ornithine, compete for the same active site of acetylornithine delta-transaminase; thus, the ornithine delta-transaminase activity in E coli is due to acetylornithine delta-transaminase and not to a separate specific ornithine delta-transaminase.

J Bacteriol, 1976 Sep, 127(3), 1085 - 97
Constitutive and repressivle enzymes of the common pathway of aromatic biosynthesis in Escherichia coli K-12: regulation of enzyme synthesis at different growth rates; Tribe DE et al.; Synthesis of five of the enzymes of the common pathway of aromatic biosynthesis has been shown to be unaffected by either the aromatic amino acids--the product of the first reaction (3-deoxy-D-arabinoheptulosonic acid-7-phosphate) or the product of the last reaction (chorismate)--or by the state of regulator gene loci tyrR . On the other hand, the rate of synthesis of these enzymes, and of several other enzymes for which repression control was inactive because of mutations in relevant regulator genes, was found to change with growth rate . These changes were found to correlate at faster growth rates than those observed in glucose minimal medium with the alterations in the relative frequencies of the corresponding structural genes which occur at these growth rates . It was found that when wild-type cells were grown at these faster growth rates in medium lacking the aromatic amino acids, complete derepression of the tyrosine-inhibitable 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthetase occurred, in strong contrast to the situation when wild-type cells are grown in glucose minimal medium.

J Biol Chem, 1976 Aug 25, 251(16), 5087 - 94
Characteristics of guanosine triphosphate cyclohydrolase I purified from Escherichia coli; Yim JJ et al.; GTP cyclohydrolase I, an enzyme that catalyzes the first reaction in the pathway for the biosynthesis of the pteridine portion of folic acid, was purified from Escherichia coli by 3,900-fold to apparent homogeneity . Its molecular weight is estimated at 210,000 . At relatively high concentrations of salt (e.g . 0.3 M KCl) the enzyme can be dissociated into seemingly identical subunits of 51,000 molecular weight . Removal of the salt allows reassociation . GTP, ATP, and inorganic orthophosphate at concentration of 5 muM, 100muM, and 0.2 mM, respectively, promote the reassociation of the subunits even in the presence of 0.3 M salt . The subunits have little or no catalytic activity . When the enzyme was subjected to electrophoresis on polyacrylamide gel under denaturing conditions (in the presence of sodium dodecyl sulfate) only one protein band was evident; its molecular weight was estimated at 25,500 . Proline was determined as the only NH2-terminal amino acid residue of the enzyme . These observations suggest that the enzyme consists of four identical subunits and that each subunit contains two identical polypeptide chains . Enough GTP was bound to the enzyme to suggest that each polypeptide contains one GTP binding site . The Km value for GTP IS 0.02 MuM . ATP, dGTP, and guanosine 5'-tetraphosphate are competitive inhibitors with Ki values of 0.25 muM, 0.24 muM, and 0.13 muM, respectively . Orthophosphate is an uncompetitive inhibitor . The enzyme is relatively heat-stable; its half-life at 82 degrees is 7 min . Salt (NaCl, KCl, NH4Cl) at a concentration of 0.1 M activates the enzyme by 4- to 5-fold . The only products of the action of the enzyme are formate and the triphosphoester of 2-amino-4-hydroxy-6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropteridine (H2-neopterin-PPP) . The evidence strongly suggests that this single enzyme catalyzes 4 independent chemical reactions in the conversion of GTP to H2-neopterin-PPP.

J Biol Chem, 1976 Aug 25, 251(16), 4839 - 42
5-Iodo-5'-amino-2',5'-dideoxyuridine-5'-N'-triphosphate . Synthesis, chemical properties, and effect on Escherichia coli thymidine kinase activity; Chen MS et al.; 5-Iodo-5'-amino-2',5'-dideoxyuridine-5'-N'-triphosphate (AIdUTP), a phosphoramidate analog of 5-iodo-2',5'-dideoxyuridine 5'-triphosphate (IdUTP), was synthesized and some of its chemical and biological properties were investigated . Although AIdUTP is stable in alkaline solutions, below pH 8 it undergoes degradation by a novel phosphorylysis reaction which exhibits first order kinetics . Inclusion of magnesium ion in the reaction mixture decreased the rate of degradation . Protonation of a group on AIdUTP which has a pKa of 6.10, presumably the secondary ionized oxygen on the gamma phosphate, precedes phosphorylysis . The only detectable reaction products are the nucleoside, 5-iodo-5'-amino-2',5'-dideoxyuridine (AIdUrd), and trimetaphosphate . A mechanism for the acid catalyzed phosphorylysis of AIdUTP is proposed . AIdUTP, like TTP, converts Escherichia coli thymidine kinase into an inactive dimer with a sedimentation coefficient of 5.78 S . AIdUTP is, however, 60-fold more potent as an allosteric inhibitor than is TTP at pH 7.8 . Although the inhibitory effect of TTP is markedly reduced at high pH, the activity of AIdUTP is lowered only slightly . The allosteric effects of AIdUTP also differ from those of IdUTP, which is an inhibitor at low pH but a strong activator above pH 7.4 . 5-Iodo-2'-deoxycytidine 5'-triphosphate, a potent enzyme activator, cannot completely reverse the AIdUTP inhibition, even when present at a 150-fold molar excess.

Biochemistry, 1976 Aug 24, 15(17), 3856 - 63
Proteolytic and chemical modification of colicin E3 activity; Lau C et al.; Proteolyses of colicin E3 by both trypsin and subtilisin yield fragments of various molecular weights . On the basis of sodium dodecyl sulfate gel electrophoresis, tryptic cleavage yields peptides of molecular weight about 42 000 and 18 000, while the comparable pieces in a subtilisin digest have apparent weights of about 36 000 and 24 000 . The digests lose almost all of their in vivo cell killing activity but the in vitro activity leading to ribosomal inactivation is augmented . Trypsin-treated colicin E3 shows a 20-30-fold increase in its ability to release the 52 nucleotide fragment from the 16S ribosomal ribonucleic acid (rRNA) and this activity is associated with the smaller fragment . Subtilisin-treated colicin E3 is only about two to three fold more active than the native protein in vitro, and the peptides obtained upon cleavage cannot be separated by gel filtration or polyacrylamide gel electrophoresis without sodium dodecyl sulfate . However, in the presence of 0.1% sodium dodecyl sulfate, subtilisin-treated E3 shows a 20-30-fold augmentation in in vitro activity which is again associated with the smaller fragment extracted from the sodium dodecyl sulfate gel . Amino terminal end-group studies showed that the two larger fragments and intact E3 have the same N-terminal residue, valine . These fragments presumably originate from the amino end of the native protein . The smaller tryptic fragment has an N-terminal alanine, while the smaller subtilisin piece has an N-terminal leucine . In addition, modification of a single carbosyl group in intact colicin E3 abolishes more than 90% of the in vivo activity with a simultaneous increase in in vitro activity . This carboxyl group is located in the larger fragments obtained in both trypsin and subtilisin cleavage . Binding of E3 to sensitive cells is drastically reduced or eliniated by this chemical modification and by both of the limited proteolytic cleavages.

Biochemistry, 1976 Aug 24, 15(17), 3710 - 6
The effect of Mg(II) on the spectral properties of Co(II) alkaline phosphatase; Anderson RA et al.; Alkaline phosphatase of Escherichia coli, isolated by procedures which do not alter its intrinsic metal content, contains 1.3 +/- 0.3 g-atom(s) of magnesium and 4.0 +/- 0.2 g-atoms of zinc per mol of molecular weight 89 000 (Bosron et al., 1975) . Substitution of Co(II) for Zn(II) and/or Mg(II) results in spectral properties which can be correlated with enzymatic activity . Magnesium does not activate the apoenzyme but augments the activity of 2-Co(II) enzyme almost 3-fold and that of the 4-Co(II) enzyme 1.3-fold . The magnesium-induced increase in activity of the 2-Co(II) enzyme is accompanied by spectral changes which are consistent with an alteration from largely octahedral-like to pentacoordinate-like coordination geometry . Magnesium increases the intensity of the absorption and magnetic circular dichroism (MCD) signals of the 4-Co(II) enzyme but without evidence of changes in coordination geometry . Cobalt when bound to the magnesium sites results in octahedral-like EPR spectra, unperturbed by phosphate which significantly affects cobalt at the pentacoordinate-like sites . In the absence of magnesium, 6 g-atoms of cobalt are required to maximize the spectral properties, but activity does not increase further after the addition of only 4 g-atoms of cobalt, while activity is optimal with only 2 g-atoms of cobalt . Hydrogen-tritium exchange measurements indicate that magnesium also stabilizes the dynamic structural properties of the apo- and 2-Co(II) enzymes but has little effect on the structure of 4-Co(II) phosphatase . The response to magnesium of both the spectral properties and enzymatic activities of cobalt alkaline phosphatase demonstrates that magnesium regulates cobalt (and zinc) binding and modulates the activity of the resultant products.

Biochemistry, 1976 Aug 24, 15(17), 3704 - 10
Cobalt(III) affinity-labeled aspartokinase . Formation of substrate and inhibitor adducts; Wright JK et al.; The kinase active site of the aspartokinase-homoserine dehydrogenase enzyme complex of Excherichia coli has been affinity labeled both with substrates aspartate and adenosine triphosphate and feedback inhibitor threonine . Co(III) exchange-inert adducts of aspartokinase and inhibitor or substrates were produced in situ by oxidation of Co(II) with H2O2 . Emzyme-Co(III)-adenosine 5'-triphosphate (ATP), enzyme-Co(III)-aspartate, and enzyme-Co(III)-threonine ternary adducts were produced in this manner . The formation of the enzyme-Co(III)-threonine adduct leads us to conclude that threonine inhibits the kinase activity of this enzyme complex by binding in the first coordination sphere of the catalytic metal ion cofactor, a conclusion which is consistent with evidence derived from previous nuclear magnetic resonance data obtained in this laboratory . The quaternary adducts formed by H2O2 oxidation in the presence of aspartokinase, Co(II), ATP, aspartate, and threonine comprised a mixture of both ezyme-Co(III)-ATP-aspartate and enzyme-Co(III)-ATP-threonine adducts . The formation of the quaternary aspartate-containing adduct was unexpected, since the presence of threonine was expected to prevent access of the aspartate to the active site; most significantly however, the the sum of the numbers of aspartate plus threonine molecules incorporated per active site is one . We believe that this shows direct steric overlap between the metal-adjacent binding sites for aspartate and threonine . Aspartate or threonine can not occupy the kinase active site simultaneously; this conclusion is consistent with the direct competitive inhibition of aspartate by threonine observed in steady-state kinetic studies.

Biochemistry, 1976 Aug 24, 15(17), 3678 - 85
Methionyl-tRNA synthetase from Escherichia coli: active stoichiometry and stopped-flow analysis of methionyl adenylate formaiton; Hyafil F et al.; Native dimeric methionyl-tRNA synthetase and its monomeric proteolytic fragment are shown to form and to bind 1 mol of methyionyl adenylate per polypeptide chain . Moreover, at 25 degrees C, each monomer of the dimeric native enzyme behaves independently, exhibiting the same parameters for the methionine activation reaction as does the monomeric modified enzyme . These results were obtained using several independent methods including equilibrium and nonequilibrium dialysis, active site and tryptophan fluorescence titrations, and stopped-flow by fluorescence . Stopped-flow resolution of the reversible methionine activation reaction also demonstrates that methionine and ATP-Mg2+ react without coupling to form a ternary enzyme-methionine-ATP-Mg2+ complex . This complex readily converts to enzyme-methionyl approximately adenylate-PP-Mg2+ with a standard free energy close to zero . It is concluded that the uncoupled enzyme-methionine-ATP-Mg2+ complex may resemble the transition state of the reaction at the expense of the additional state of the reaction at the expense of the additional synergistic binding energy provided by reciprocal coupling, within the site, of the methionine molecule with the adenosine and PP-Mg2+ parts of the ATP-Mg2+ molecule (Blanguet, S., Fayat, G., and Waller, J . P . (1975), J . Mol . Biol . 94, 1.).

Biochim Biophys Acta, 1976 Aug 23, 441(2), 201 - 12
Partial purification and properties of diglyceride kinase from Escherichia coli; Schneider EG et al.; Diglyceride kinase (diacylglycerol kinase, E.C . 2.7.1.-), an enzyme localized in the inner membrane of Escherichia coli, has been purified about 600-fold . The purified enzyme exhibits an absolute requirement for magnesium ion; its activity toward both lipid and nucleotide substrates is stimulated by diphosphatidylglycerol or other phospholipids . Adenine nucleotides are much better substrates for the enzyme than are other purine or pyrimidine nucleotides . The purified enzyme preparation catalyzes the phosphorylation of a number of lipids, including ceramide and several ceramide and diacylglycerol-like analogs . The broad lipid substrate specificity of diglyceride kinase suggests that this enzyme may function in vivo for the phosphorylation of an acceptor other than diacylglycerol.

Mol Gen Genet, 1976 Aug 19, 147(2), 225 - 32
Modulation of RNA polymerase specificity by ppGpp; Travers A; ppGpp alters the initiation specificity of RNA polymerase holoenzyme in vitro in a direction which mimics the stringent response in vivo . The transition temperature for opening rRNA promoters is increased by the nucleotide, that for opening phi80 promoters is unaffected . This implies that RNA polymerase can discriminate between different types of promoter . ppGpp may act by effecting a structural change in the enzyme.

Mol Gen Genet, 1976 Aug 19, 147(2), 139 - 43
Altered synthesis and stability of RNA polymerase holoenzyme subunits in mutants of Escherichia coli with mutations in the beta or beta' subunit genes; Takto M et al.; Bacteria with specific temperature sensitive lethal mutations in the gene for the beta' subunit of RNA polymerase synthesize both the beta and beta' subunits at a several fold higher rate at 42 degrees C than wild-type cells relative to total protein . Synthesis of the alpha and sigma subunits proceeds at essentially the wild-type rates under these conditions . In contrast, a mutant with a temperature sensitive lethal mutation in the beta subunit gene synthesizes beta and beta' at 42 degrees C at slightly lower rates than wild-type, while alpha and sigma synthesis is not significantly altered . In all of the mutants at 42 degrees C, newly synthesized alpha subunits are stable, while the beta, beta' and sigma subunits are rapidly degraded . The apparent uncoupling of betabeta' and alpha subunit synthesis seen in the beta' mutants at 42 degrees C might suggest that the synthesis of these subunits is at least in part controlled by different mechanisms.

Mol Gen Genet, 1976 Aug 19, 147(2), 129 - 38
An investigation of the binding sites of proteins S8, L23 and L24 on the ribosomal RNAs of Escherichia coli by electron microscopy; Sloof P et al.; An electron microscopic method was used to investigate the binding regions of proteins S8 on 16S RNA, and proteins L23 and L24 on 23S RNA . Regions of the RNA that were not stabilised by the protein were completely denatured in 80% dimethyl-sulfoxide . The lengths of these denatured RNA regions were compared with that of the whole denatured RNA . Conclusions are drawn concerning the approximate location of the three proteins and these results are correlated with both RNA structural data and RNA sequence data on the RNA binding regions of the proteins.

Biochim Biophys Acta, 1976 Aug 18, 442(2), 216 - 26
Consequences of modification on the association of DNA polymerase with the cell membrane; Campana T et al.; 1 . This is a report of a possible causal relation between the structure of the membrane-bound DNA polymerase and the mutator characteristic of Exherichia coli, mut T1 . The characteristics of the membrane-bound polymerase are compatible with its identification as DNA polymerase II, and enzyme which has been determined to be genetically closely linked to the mut T1 gene . Several genes concerned with membrane structure are also known to be linked to the mutator locus . 2 . Differences exist between membrane-bound polymerase activity of a wild-type E . coli K-12 and of an isogenic strain harboring the mutator gene . (a) The cell membranes of the mutator strain retain 5--6 times as much activity as do membrane complexes from wild-type cells . (b) The DNA polymerase activity of the membranes from the mut T1 strain is less sensitive to inhibition by the sulfhydryl-binding reagent, N-ethylmaleimide . (c) The membrane-DNA polymerase complex of mut T1 cells uses endogenous, membrane-bound DNA for replication-repair in preference to exogenous DNA . 3 . The differences described are specific to structural differences in the membrane complex . When DNA polymerase activity is solubilized from the complexes, the enzymes of the two strains exhibit similar characteristics . These results are consistent with the thesis that an alteration in membrane structure is the basis of mut T1 activity . The results do not support any hypothesis that mut T1 phenotype is a reflection of mutations in the structural gene for DNA replicase (polymerase) or its components.

Biochim Biophys Acta, 1976 Aug 18, 442(2), 197 - 207
Further purification and characterization of T4 endonuclease V; Yasuda S et al.; T4 endonuclease V, which is involved in repair of ultraviolet-damaged DNA, has been purified 3600 fold from T4D-infected Escherichia coli . The enzyme shows optimal activity at pH 7.2 and does not require added divalent ions . Endonuclease V attacks both native and heat-denatured DNA provided that the DNA has been irradiated, and the enzyme activity is dependent on the dose of ultraviolet irradiation . The rate and the extent of the reaction are greater with irradiated native DNA although the Km values for the two types of DNA are the same (2.25 - 10(-5) M) . The enzyme is readily inactivated by heat and is sensitive to p-chloromercuribenzoate . Endonuclease V-treated irradiated DNA is degraded by spleen phosphodiesterase only when the DNA has been treated with alkaline phosphatase, suggesting that the enzyme produces 5'-phosphoryl termini.

Biochim Biophys Acta, 1976 Aug 18, 442(2), 162 - 73
The ATP dependence of the incision and resynthesis steps of excision repair; Masker WE; Escherichia coli made permeable by treatment with toluene can perform a mode of DNA synthesis that is stimulated by ultraviolet radiation and closely resembles the resynthesis step of excision repair . If ultraviolet-irradiated toulene-treated cells are incubated in an assay mixture with ATP but without the four deoxyribonucleoside triphosphates (dNTPs) or NAD, accumulations of single-strand breaks in the DNA are detected by alkaline sucrose gradient analysis . A second incubation with the dNTP'S and NAD but without ATP produces nonconservative DNA synthesis in strains with normal levels of DNA polymerase I . However, in PolA strains, ATP must be present during the second incubation in order to produce measurable amounts of ultraviolet-stimulated DNA synthesis . These results suggest that in strains deficient in DNA polymerase I there may be two ATP-dependent steps in this repair pathway, one required for incision and one associated with resynthesis.

Biochim Biophys Acta, 1976 Aug 18, 442(2), 154 - 61
Repair of x-ray-induced single strand breaks in toluenized Escherichia coli cells; Waldstein EA et al.; We have used sedimentation in alkali to estimate the repair of X-ray-induced single strand breaks in the DNA of irradiated toluenized Escherichia coli cells . Extensive repair requires no exogenous cofactors except ATP although other individual NTPs (except U) or dNTPs can substitute for ATP . There is no repair in polA or resA cells and since nicotinamide mononucleotide (NMN) inhibits repair in wild type cells we interpret the results as indicating that both ligase and polymerase I are needed for repair but that the amount of any gap filling is small and extensive repair replication is not necessary.

Eur J Biochem, 1976 Aug 16, 67(2), 591 - 601
Isolation and characterization of a growth-cycle-reflecting, high-molecular-weight protein associated with Escherichia coli ribosomes; Subramanian AR et al.; A high-molecular-weight, acidic protein, acidic protein whose amount per ribosomal particle depends on the growth cycle is shown to be associated with Escherichia coli ribosomes . The protein is associated with ribosomes from cells harvested at different phases of the growth cycle, but a large increase in its amount is seen with ribosomes from post-exponential phase cells . The protein is only slightly washed off the ribosomes by 1 M NH4CL . When ribosomes are dissociated it remains entirely with the 30-S subunits . We have purified the protein to homogeneity . It has a molecular weight of 70 000 and its amino acid composition showed some resemblance to that of ribosomal protein S1 . However, the two proteins ar-shown variation in the acetylation of L12 during the growth cycle, indicated that certain proteins of (or associated with) E . coli ribosmes may carry specific biochemical roles connected with cellular adaption toward the stationary phase.

Eur J Biochem, 1976 Aug 16, 67(2), 373 - 8
Binding of aminoacyl-tRNA to reconstituted subparticles of Escherichia coli large ribosomal subunits; Kazemie M; The activity of 50-S subunits to stimulate the binding of aminoacyl-tRNA to the 30-S subunits was lost when the particles were washed with LiCl concentrations higher than 1.0 M {Kazemie, M . (1975) Eur . J . Biochem . 58,501-510} . The particles could regain this activity when they were incubated with the corresponding LiCl washes . This effect of LiCl washes was used as an assay for identification of 50-S subunit proteins which are essential for binding of aminoacyl-tRNA . A protein mixture containing mainly Ll, Lll and L16 can reactivate 50-S "core" particles, consisting of 10 proteins and 23-S RNA, to bind aminoacyl-tRNAs in the presence of 30-S subunits . Besides 5-S RNA, protein L24 has a stimulatory effect on the binding of aminoacyl-tRNA . Proteins L2, L20 and L23 are possibly required for maintaining the 50-S subunits in the correct conformation for binding of aminoacyl-tRNAs.

Eur J Biochem, 1976 Aug 16, 67(2), 603 - 13
Specificity and properties of the destabilization, induced by initiation factor IF-3, of ternary complexes of the 30-S ribosomal subunit, aminoacyl-tRNA and polynucleotides; Risuleo G et al.; Initiation factor IF-3 causes the destabilization of preformed ternary complexes of 30-S ribosomal subunit, codons and aminoacyl-tRNAs or peptidyl-tRNA . This destabilization is dilution-dependent and affects all ternary complexes with the exception of those containing the initiator fMet-tRNA, which remain more resistant to IF-3-induced destabilization under the various conditions studied . Several possible reasons for this specificity have been examined . It was found that the basis for the specificity is not: (a) an intrinsic greater stability of the ternary complexes containing fMet-tRNA, (b) the amoung of aminoacyl-tRNA bound to the ribosome, (c) the conditions under which the ternary complex is made or (d) the formylation of the amino group . On the other hand, the nature of the polynucleotide in response to which the ternary complex is formed was found to influence the amount of aminoacyl-tRan bound to the ribosome, and to some extent the amount of aminoacyl-tRNA which can be relased . The ternary complex containing the mischarged initiator tRNA fVal-tRNAfMet displays greater resistance to the IF-3-induced destabilization than the complex containing fVal-tRNAVal . These results indicate that the specificity of the IF-3 activity is due to the special structural feature of the initiator tRNA molecule and to some extent to the nature of the polynucleotide . The IF-3-induced destabilization of ternary complexes was found to be little affected by variations in reaction conditons, so that this IF-3 activity can be used to measure the stoichiometric binding of IF-3 to the ribosome over a broad range of pH and K+ and Mg2+ concentrations . Several antibiotics have been tested for their capacity to interfere with this reaction; only high concentrations of tetracycline blocked this IF-3 activity.

Biochem J, 1976 Aug 15, 158(2), 451 - 6
The composition of an unusual precursor of 50 S ribosomes in a mutant of Escherichia coli; Markey F et al.; Escherichia coli strain 15--28 is a mutant which during exponential growth contains large amounts of a '47S' ribonucleoprotein precursor to 50S ribosomes . The '47S particles' are more sensitive to ribonuclease than are 50S ribosomes . The 23 S RNA of 47S particles may be slightly undermethylated, but cannot be distinguished from the 23S RNA of 50S ribosomes by sedimentation or electrophoresis . Isolated particles have 10--15% less protein than do 50S ribosomes; proteins L16, L28 and L33 are absent . Comparison with precursor particles studied by other workers in wild-type strains of E . coli suggests that the assembly of 50S ribosomes in strain 15--28 is atypical.

Biochim Biophys Acta, 1976 Aug 13, 440(2), 403 - 11
Effects of colicins K and E1 on the glucose phosphotransferase system; Jetten AM; 1 . Glycerol-grown cells of Escherichia coli and its mutant uncA, treated with colicin E1 or K, exhibited a several-fold higher level of alpha-methylglucoside uptake than untreated cells . This stimulation was independent of the carbon source present during the uptake test . In a mutant strain that has elevated levels of alpha-methylglucoside accumulation the addition of colicin E1 or carbonylcyanide m-chlorophenylhydrazone (CCCP) did not further enhance the uptake . 2 . Colicins K and E1 decreased the apparent Km for alpha-methylglucoside uptake significantly and increased the V about twofold . The exit of the glucoside was severely inhibited by the colicins . 3 . In the presence of colicins, alpha-methylglucoside is still accumulated via the phosphoenolpyruvate-phosphotransferase system since no accumulation or phosphorylation occurs in an enzyme I mutant . The colicins increased the relative intracellular concentration of phosphorylated alpha-methylglucoside, possibly by inhibiting the dephosphorylation reaction, and caused an excretion of this compound . 4 . The results are interpreted as indicating that energization of the membrane has an inhibitory effect on the phosphotransferase system . Possible modes of action are discussed.

J Biol Chem, 1976 Aug 10, 251(15), 4713 - 21
Effects of estrogen on gene expression in chick oviduct . The role of chromatin proteins in regulating transcription of the ovalbumin gene; Tsai SY et al.; Histones, extractable non-histone proteins, and a tightly bound non-histone protein DNA complex were fractionated from chromatins which were isolated from estrogen-stimulated and hormone-withdrawn chick oviducts . Reconstitution of homologous constituents was performed and RNA was transcribed from these reconstituted chromatins using Escherichia coli RNA polymerase . DNA complementary to ovalbumin mRNA was then used as a hybridization probe to estimate the concentration of ovalbumin messenger RNA sequences in these in vitro transcripts . Our results demonstrated that 0.011% of the in vitro RNA transcripts produced from reconstituted estrogen-stimulated chromatin was ovalbumin mRNA sequences as compared to 0.0015% obtained for reconstituted hormone-withdrawn chromatin . Thus, the ratio of ovalbumin mRNA sequences in the in vitro transcripts of reconstituted stimulated and withdrawn chromatins was 8 to 1, identical with the value obtained from native estrogen-stimulated and hormone-withdrawn chromatins . Furthermore, the number of initiation sites for RNA synthesis on reconstituted and native chromatins are indistinguishable . Thus, the specificity of transcription of isolated chromatins appears to be conserved upon dissociation and fractionation of the chromatin proteins followed by reconstitution of these constituents to DNA . The effect of chromatin proteins on gene expression was further examined by reconstituting components from different development stages . Reconstitution of extractable non-histone proteins from estrogen-stimulated chromatins to tightly bound non-histone protein . DNA complexes from hormone-withdrawn chromatins resulted in the synthesis of a substantial amount of ovalbumin mRNA sequences . Conversely, when extractable non-histone proteins from withdrawn chromatins were reconstituted to tightly bound non-histone protein DNA complexes from estrogen-stimulated chromatins, very low levels of mRNAov sequences were detected . In contrast, interchange of the histones and tightly bound non-histone protein DNA complexes from hormone-withdrawn and estrogen-stimulated chromatins during reconstitution did not affect the level of mRNAOV sequences produced . Therefore, the extractable non-histone proteins of chromatin appear to be extremely important in regulating specific gene expression.

Mol Gen Genet, 1976 Aug 10, 147(1), 99 - 102
The relation of the genes envA and ftsA in Escherichia coli; Wijsman HJ et al.; The sequence of three genes involved in cell division in E . coli has been determined to be ftsA-envA-azi by three-point transduction experiments . An ftsA envA double mutant strain forms filaments at the restrictive temperature of 42 degrees C, and not chains, but, like the chain forming envA parent strain, is hypersensitive to rifampicin.

Mol Gen Genet, 1976 Aug 10, 147(1), 91 - 7
Regulatory mutants of dihydrofolate reductase in Escherichia coli K12; Sheldon R et al.; Trimethoprim inhibits dihydrofolate reductase . Mutations conferring trimethorpim-resistance on E coli K12 result in either an altered reductase with decreased affinity for the drug, or in 2-30 fold higher levels of the enzyme . Studies of the latter class of mutants indicate that dihydrofolate reductase is regulatdd by a diffusible molecule, and is probably under negative control . The regulatrory mutants, some of which are temperature-sensitive, act cis.

Mol Gen Genet, 1976 Aug 10, 147(1), 79 - 82
Effect of chloramphenicol and the recB gene product on DNA metabolism in Escherichia coli K12 strains defective in DNA ligase; Morse LS et al.; We have examined DNA strand breakage, DNA degradation, and the rate of DNA synthesis in lig and lig-recB strains of Escherichia coli K12 incubated in the presence and absence of 3 mug/ml chloramphenicol . Substantial DNA strand breakage and DNA degradation is observed in the lig strain upon growth at 40 degrees C; however, such strand breakage and DNA degradation is not observed in th lig-recB strainl Incubation of the lig strain at 40 degrees C in the presence of 3 mug/ml chloramphenicol reduces the amount of DNA strand breakage and DNA degradation to the level observed in the lig-recB strain . Together, these results demonstrate that exonuclease V (the recBC gene product) is responsible for the increased DNA degradation associated with DNA ligase deficiency.

Mol Gen Genet, 1976 Aug 10, 147(1), 53 - 8
Effect of glucose starvation on the expression of transferred tsx genes in Escherichia coli K12 zygotes; Leal J; Escherichia coli K12 Hfr H Tsxs Strs and F- Pro- Tsxr His- Arg- Strr bacteria were conjugated in the absence of arginine with or without glucose . The efficiency of conjugation, measured by the frequency of Pro+ and His+ recombinants was not affected . Arginine starvation alone did not affect the tsxs gene expression which occurred in all the zygotes which had received the gene . In contrast, argine and glucose starvation allows tsxs expression only in those zygotes in which the donor gene had been integrated in the genome . As the glucose starvation brings on a destabilization of the messenger RNA synthesized by the F- cells in absence of arginine, the results can be interpreted as follows: the transferred tsxs genes are transitorily expressed in all the zygotes at the unintegrated state . After this transient period, only thsoe genes integrated in the chromosomes of the zygotes continue to be expressed.

Mol Gen Genet, 1976 Aug 10, 147(1), 29 - 37
A lethal mutation which affects the maturation of ribosomes; Johnson SC et al.; A temperature sensitive mutant of Escherichia coli which fails to recover from prolonged carbon starvation, was found to be irreversibly killed by exposure to a nonpermissive temperature (43 degrees C), with a half-life of about half an hour . This bacteriocidal effect of the temperature could be reversed by a number of antibiotics which block protein synthesis but not by blocking DNA synthesis . At the nonpermissive temperature, RNA and the protein synthetic capacities decrease before the DNA synthetic capacity is decreased . Analysis of ribosomal proteins and methylation of them did not reveal any consistent differences between the parental and mutant strains . Analysis of the ribosomal RNA revealed that it is being synthesized in similar amounts as in the parental strain at the nonpermissive temperature, however, after chase its level is decreased . Moreover, the 17S precursor RNA is slow to mature to 16S rRNA in the mutant strain at the nonpermissive temperature . Thus, these studies suggest that the mutation studied here affects a late maturation step in the synthesis of the rRNA . Therefore the gene is designated rimH (for ribosomal modification) . All the properties bestowee on the mutant strain are caused by a single pleiotropic mutation which maps at min 14 of the E . coli map . Three point transduction crosses suggest the order rimH, leuS, RNA, LIP . This gene maps outside the two known clusters for ribosomal structural genes.

Mol Gen Genet, 1976 Aug 10, 147(1), 115 - 7
N-terminal sequence of phage lambda repressor; Beyreuther K et al.; Lambda repressor was purified from an E . coli strain which produces 150 times more lambda repressor than a single lysogen . The sequence of the fifty N-terminal residues was determined by automated Edman degradation . It contains 43% of all arginine and lysine residues of the chain and constitutes according to the genetic data of Oppenheim et al . (1975) a substantial part of the operator-DNA-binding site of the repressor.

Mol Gen Genet, 1976 Aug 10, 147(1), 103 - 9
Insertion sequence IS2 near the gene for prophage lambda excision; Mosharrafa E et al.; In this study we characterize a variant of the lambdacI857S7 prophage, designated lambdabi2cI857S7, which carries a DNA insertion . The insertion sequence is IS2, and it resides in the antipolar orientation II just upstream from the gene for prophage excision (xis) at 61.6%lambda . This bi2 insertion mutant could prove valuable for studies on possible recombination functions of IS2 DNA and of its effect on the lambda integration and excision functions.

Biochemistry, 1976 Aug 10, 15(16), 3639 - 46
Properties of tRNA species modified in the 3'-terminal ribose moiety in an eukaryotic ribosomal system; Baksht E et al.; Phe-tRNAPhe species modified on the 3'-terminal ribose residue were investigated for their ability to participate in individual steps of the elongation cycle using eukaryotic ribosomes from reticulocytes . None of the Phe-tRNAs used, namely Phe-tRNAPhe-C-C-3'dA, Phe-tRNAPhe-C-C-3'-NH2A, and Phe-tRNAPhe-C-C-Aoxi-red, can function in the overall process . All modified Phe-tRNAPhe species can be bound nonenzymatically to ribosomes . Phe-tRNAPhe-C-C-3'NH2A exhibits exceptionally high binding at low Mg2+ concentration compared with Phe-tRNAPhe-C-C-A binding . Ac-Phe-tRNAPhe species prepared from the three modified tRNAs, when bound to the donor site, were devoid of donor activity . The enzymatic binding of both Phe-tRNAPhe-C-C-3'dA and Phe-tRNAPhe-C-C-3'NH2A is less efficient than that of Phe-tRNAPhe-C-C-A but these Phe-tRNAPhe species have acceptor activity . Phe-tRNAPhe-C-C--Aoxi-red is not a substrate for the EF-I promoted binding reaction and has no acceptor activity . The nonaminoacylated species, tRNAPhe-C-C-A, tRNAPhe-C-C-3'dA, and tRNAPhe-C-C-3'NH2A, bind to the ribosome to a larger extent than the corresponding aminoacylated tRNAs, both in the presence and in the absence of poly(U) . Peptidyl-tRNAPhe-C-C-3'dA bound to the donor site cannot activate the acceptor site for EF-I promoted binding of Phe-tRNAPhe as does peptidyl-tRNAPhe-C-C-A . Further, it was observed that a correct codon-anticodon interaction influences the recognition of the 3' terminus of tRNA . Specificity of eukaryotic ribosomes for the 2'- and/or 3'-aminoacylated tRNA species is discussed and compared with the properties of Escherichia coli system.

Biochemistry, 1976 Aug 10, 15(16), 3579 - 90
Photoinduced cross-linkage, in situ, of Escherichia coli 30S ribosomal proteins to 16S rRNA: identification of cross-linked proteins and relationships between reactivity and ribosome structure; Gorelic L; The kinetics of photoinduced cross-linkage of Escherichia coli 30S ribosomal proteins to the 16S-rRNA molecule in the intact Escherichia coli 30S ribosomal subunit was studied in this report . All of the 30S ribosomal proteins become cross-linked to the 16S rRNA before changes in the sedimentation characteristics of the 30S ribosomal subunit can be detected . The proteins exhibit different reactivities in the cross-linkage reaction . One group of proteins-S3, S7-S9, S11, S12, and S15-S19-is cross-linked to the 16S rRNA by single-hit kinetics, or by photoprocesses of nonunity but low multiplicities . A second group of proteins--S1, S2, S4-S6, S10, S13, S14, and S21--is cross-linked to the 16S rRNA by photoprocesses of a complex nature . A comparison of these data with other properties of the individual 30S ribosomal proteins related to ribosome structure indicated that most of the 30S ribosomal proteins cross-linked to the 16S rRNA by photoprocesses of low multiplicities had been classified rRNA-binding proteins by nonphotochemical methods, and most of the proteins cross-linked to the 16S rRNA by photoprocesses of large multiplicities had been classified as nonbinding proteins . There were certain exceptions to these correlations . Proteins S4 and S20, both RNA-binding proteins, become cross-linked to the 16S rRNA by photoprocessses of large multiplicities, and proteins S3, S11, S12, and S18, none of which have been classified RNA-binding proteins, exhibited low multiplicities in the cross-linkage reaction . All of these exceptions could be explained in terms of limitations inherent in the photochemical methods used in this study and in other types of methods that have been used to study RNA-protein interactions in the 30S ribosomal subunit . The data presented here also suggest that labile RNA-protein cross-links are present in the uv-irradiated 30S ribosomal subunits, and that neither peptide-bond cleavage nor photoinduced modification of the charged side-chain groups in the ribosomal proteins accompanied the cross-linkage reaction . However, some photoinduced RNA-chain breakage might have occurred.

Biochemistry, 1976 Aug 10, 15(16), 3536 - 40
Mechanims of stimulation of in vitro protein synthesis by some copolymers of styrene, vinyluracil, and vinyladenine with maleic acid and acrylic acid; Boguslawski S et al.; Copolymers of vinyl bases with acrylic acid and styrene or 1-vinyluracil with maleic acid were found to stimulate in vitro polyphenylalanine synthesis using a system extracted from Escherichia coli MRE600 . Poly(styrene-maleic acid) was found to inhibit a ribosomal bound ribonuclease . Poly(1-vinyluracil, maleic acid), poly(1-vinyluracil, acrylic acid), and poly(9-vinyladenine, acrylic acid) were not inhibitors of the ribosome bound ribonuclease . The potent (up to fivefold) stimulation by these three polymers is due to the action of the polymers to interfere with ribosomal bound inhibitory protein . A protein, removed by washing ribosomes with 1 M ammonium chloride, characterized by M.J . Miller, A . Niveleau, and A.J . Wahba ((1974) J . Biol . Chem . 249, 3803) has been described as a potent inhibitor of in vitro poly(U)-coded protein synthesis using extracts of Escherichia coli MRE 600.

J Biol Chem, 1976 Aug 10, 251(15), 4481 - 9
The nucleotide sequence in the promoter region of the gene for an Escherichia coli tyrosine transfer ribonucleic acid; Sekiya T et al.; The sequence of the first 29 nucleotides in the promoter region of a tyrosine tRNA gene has previously been determined (Sekiya, T., van Ormondt, H., and Khorana, H.G . (1975) J . Biol . Chem . 250, 1087-1098) . This work has now been extended to give the sequence of a total of 59 nucleotides; the sequence is as follows: (see article) . The general approach used in the determination of the sequence involved the DNA polymerase I-catalyzed elongation of synthetic deoxyribopolynucleotide primers hydridized to the l-strand of phi80psu+III DNA at the appropriate site . Sequencing of the newly added nucleotides was facilitated by the use of a number of techniques including (a) elongation of the primer with the use of all of the four nucleoside 5'-triphosphates but limiting the concentration of one of the triphosphates, (b) insertion of ribonucleotide units at appropriate sites so as to permit subsequent specific cleavages by pancreatic RNase, and (c) two-dimensional fingerprinting of the oligonucleotides in conjunction with partial exonucleolytic degradation, comprehensive nearest neighbor analyses, and the determination of pyrimidine tracts.

J Mol Evol, 1976 Aug 3, 8(2), 117 - 35
Doublet frequencies and codon weighting in the DNA of Escherichia coli and its phages; Elton RA et al.; A compilation of nucleic acid sequences from E . coli and its phages has been analysed for the frequency of occurrence of nearest neighbour base doublets and codons . Several statistically significant deviations from random are found in both doublet and codon frequencies . The deviations in E . coli also appear to occur in lambda and in the coat protein gene of MS2, whereas T4 and other parts of the MS2 genome show different sequence properties . These and other findings are discussed in relation to the hypothesis that rapidity of translation of mRNAs in the E . coli system is dependent on doublet frequency and codon usage patterns.

Biochim Biophys Acta, 1976 Aug 2, 442(1), 24 - 31
Aminoacylation of Phaseolus vulgaris cytoplasmic, chloroplastic and mitochondrial tRNAsPro and tRNAsLys by homologous and heterologous enzymes; Jeannin G et al.; The cytoplasmic prolyl-tRNA synthetase can be separated by hydroxyapatite chromatography, from the enzyme present in the chloroplasts and in the mitochondria (organellar enzyme) . The cytoplasmic lysyl-tRNA synthetase can also be separated from the organellar enzyme . There are two tRNAsPro in the cytoplasm; they can be charged by the cytoplasmic enzyme, but not by the organellar enzyme or the Escherichia coli enzyme . Chloroplasts contain, in addition to the two cytoplasmic tRNAsPro, one chloroplast-specific tRNAPro, which is not recognized by the cytoplasmic enzyme, but can be charged by the organellar or the E . coli enzyme . Mitochondria contain, in addition to the two cytoplasmic tRNAsPro, two mitochondria-specific tRNAsPro, which are not recognized by the cytoplasmic enzyme, but can be charged by the organellar or the E . coli enzyme . There are two tRNAsLys in the cytoplasm . Both can be charged by the cytoplasmic enzyme, but one can be charged by the organellar or E . coli enzyme . Chloroplasts contain in addition to one cytoplasmic tRNALys, one chloroplast-specific tRNALys which can only be charged by the organellar or E . coli enzyme . Mitochondria contain, in addition to one cytoplasmic tRNALys, one mitochondria-specific tRNALys which can only be charged by the organellar or E . coli enzyme.

Mol Gen Genet, 1976 Aug 2, 146(3), 303 - 7
A ribosomal RNA gene of Escherichia coli (rrnD) on lamnda daro E specialized transducing phages; Jorgensen P; Lambda-transducing phages carrying segments of the Escherichia coli chromosome in the aroE-trkA region have been isolated and shown by hybridization to carry an rRNA gene (rrnD) . The most likely gene order is trkA aroE rrnD . The EcoRI and SmaI endonuclease cutting pattern of the rrnD gene is identical with the one of rrnB, differented from rrnC.

Mol Gen Genet, 1976 Aug 2, 146(3), 239 - 45
Isolation and characterization of Co1E2 plasmid mutants unable to kill colicin-sensitive cells; Hallewell RA et al.; After transfer from a mutagenized host, twenty one Co1E2 plasmid mutants were isolated after screening 10,000 clones for abnormal colicin production . Analysis by SDS polyacrylamide slab gel electrophoresis of proteins synthesized after mitomycin C-induction of mutant cultures, indicates that all but two of the mutations are in the structural gene from colicin E2 . Of these, nine produce fragments of colicin in both whole cells and minicells and some are suppressed by nonsense suppressors . Studies with a nonsense mutant producing only a small colicin E2 fragment (Co1E2-421) suggest that colicin E2 in not involved in plasmid DNA replication, in the control of its own synthesis, or required for cell death when cells become committed to colicin production . The two plasmid mutants outside the colicin gene segregate plasmid-free cells at 33 degrees, 37 degrees and 43 degrees . One segregates fairly rapidly (about 4% per generation) though the colicin-producing cells make normal amounts of colicin, whilst the other segregates more slowly and the colicin-producing cells make much reduced amounts of colicin.

Mol Gen Genet, 1976 Aug 2, 146(3), 215 - 31
Fusion of two F-prime factors in Escherichia coli studied by electron microscope heteroduplex analysis; Palchaudhuri S et al.; A fused F prime factor was obtained from a mating of a recA donor carrying an F'- factor containing the genes metBJF, ppc and argECBH (KLF5) with a recA recipient carrying an F' factor containing att80, trp and lac (f155) . lysogenization of this fused F-prime factor with gammacI857hphi80 phage followed by thermoinduction produced the transducing phages phi80 dmetBJF and phi80 dppcargECBH . This kind of fusion provides a general procedure for the construction of transducing phages carrying genes from different regions of the E . coli genome . To understand the mechanism of this fusion, the parental F prime factors (F155 and KLF5) were analyzed by the electron microscope heteroduplex technique . F155 has a length of 176 +/- 3 kilobases including two substitutions . The F sequence 0 F-2.8 F has been substituted by 53 kb of chromosomal DNA including the lac operon and the F sequences 8.5 F-16.3 F has been substituted by 27 kb of a chromosome sequence including att80 and the trp operon KLF5 contains 221 +/- 4 kilobases of DNA (molecular weight, 148 megadaltons) . It contains complete F and the segment of the E . coli chromosome from polA to rif . The F sequence 2.8 F-8.5 F known to be involved in F specific recombination in recA+ and recA backgrounds occurs twice on KLF5, once at each of the junctions of F DNA with chromosomal DNA . The population of closed circular plasmid molecules extracted from KLF5-containing strains is heterogeneous . It is proposed that this heterogeneity is due to intramolecular recombination events occurring in KLF5 between the duplicated 2.8 F-8.5 F sequences . Such recombination can account for the genetic instability of KLF5 observed in both recA+ and recA hosts . The F sequence 2.8 F-8.5 (also called gammadelta) is one of the characterized integration sequences on F . A model for the fusion of the parental F prime factors is proposed in which recombination between gammadelta sequences brings att80 close to the metBJF genes . This is followed by a deletion of an F' lac factor . The resulting fused F' factor still carries two gammadelta sequences and is therefore expected to be unstable . The closed circular molecules isolated from the fused F' containing strains show two different sizes of molecules . Genetic and physical analyses of these molecules are in agreement with the predicted instability of the fused F' factor and the existance of the gammadelta sequence in the phi80 dmet phages isolated from fused F' and previously analyzed by the electron microscope heteroduplex technique.

Biochim Biophys Acta, 1976 Aug 2, 442(1), 71 - 5
2'(3')-O-L-(O-methyl-3-phenyllactyl) adenosine . A potential analog of 2'(3')-O-aminoacyl ribonucleosides; Zemlicka J et al.; The title compound and the corresponding diacyl derivative has been prepared by the reaction of 5'-O-(4-methoxytrityl)adenosine with L-(O-methyl-3-phenyl)lactic acid in the presence of dicyclohexylcarbodiimide in pyridine and subsequent detritylation in 80% acetic acid . Neither compound accepted the N-acetyl-L-phenylalanyl residue from N-acetyl-L-phenylalanyl-tRNA in an Escherichia coli ribosomal system nor did either inhibit the puromycin reaction in the same system.

Biochim Biophys Acta, 1976 Aug 2, 442(1), 50 - 7
A study of DNA spontaneous degradation; Crine P et al.; The intact apurinic site half-life in DNA molecules, at 37 degrees C and pH 7.21, in a medium approximating the intracellular ionic composition, was found to be between 288 and 335 h . The spontaneous degradation of 3H-labelled T7 phage DNA, at 37 degrees C and pH 7.21, in the same medium, was followed by sedimentation analysis on sucrose gradients after denaturation with NaOH; it was found to be equivalent to 0.009 break per h per DNA strand . The number of intact apurinic sites found in this incubated T7 phage DNA was much lower than the theoretical value calculated on the assumption that depurination was the only cause of DNA degradation . We concluded that depurination was a minor cause of DNA degradation in our experimental conditions.

Biochim Biophys Acta, 1976 Aug 2, 442(1), 37 - 49
Synthesis of DNAs complementary to human ribosomal RNAs polyadenylated in vitro; Hell A et al.; Conditions are described under which poly(A) polymerase from Escherichia coli ribosomes will catalyse the addition of AMP residues onto the 3'-ends of human 18 S and 28 S ribosomal RNAs at an average rate of 40 AMP residues per 1000 nucleotides in 20 min . Single-stranded complementary DNAs (cDNAs) can then be transcribed from the polyadenylated RNAs with RNA-directed DNA polymerase from avian myeloblastosis virus . All of the sequences in the RNAs are represented in the cDNAs; measurements of the rates at which the cDNAs hybridize with their template RNAs showed that, when appropriate adjustments for differences in lengths and G + C contents of the reacting sequences are taken into account, the Rot 1/2 values of homologous RNA-cDNA hybridization reactions are directly proportional to the base-sequence complexity of the RNAs.

Biochim Biophys Acta, 1976 Aug 2, 442(1), 123 - 7
The sequence of ppGpp and pppGpp in the reaction scheme for magic spot synthesis; Weyer WJ et al.; The kinetics of ppGpp (guanosine 5'-diphosphate, 3'-diphosphate) ahd pppGpp (guanosine 5'-triphosphate, 3'-diphosphate) synthesis, at the onset of amino acid starvation, and of their decay after inhibiting synthesis, were analysed in Escherichia coli . The pentaphosphate, but not ppGpp, is the first product of the stringent response to amino acid starvation . The pentaphosphate is rapidly converted with first order kinetics (t 1/2 = 6 s) to ppGpp which is broken down less rapidly (t 1/2 = 20 s).

Nucleic Acids Res, 1976 Aug, 3(8), 2079 - 87
On the mechanism of tRNA methylase-tRNA recognition; Gambaryan AS et al.; In order to further elucidate the mechanism of tRNA methylase-tRNA intreaction the methylation of some individual tRNAs separately and by pairs was performed . In conditions of tRNA excess the methylation rates of positionally analogous nucleotides in tRNA molecules are not summed up when two substrates are simultaneously present in the reaction mixture . The inhibitory action of yeast tRNASer, possessing m5c in position 29, on the methylation of C29 in other individual tRNAs was shown . Yeast tRNAVal which possesses an A residue in position 27 was shown to inhibit the methylation of G27 in E . coli tRNAMet . The data obtained confirm the suggestion that tRNA methylases recognizes the tertiary structure of tRNAs . They show also that the recognition and the proper catalytic action are two autonomous processes and that the former at least in its first stage is rather unspecific.

Nucleic Acids Res, 1976 Aug, 3(8), 2055 - 65
Partial purification of a double-stranded RNA specific ribonuclease (RNAse D) from Krebs II ascites cells; Rech J et al.; In a search for eucaryotic enzymes which might process the heterogenous nuclear RNA (HnRNA) from animal cells into messenger RNA, a ribonuclease called RNAse D analogous to E . coli RNAse III in its ability to cleave specifically synthetic or viral double-stranded polyribonucleotides has been detected and extensively purified from the cytosol of Krebs II mouse ascites cells . The purification procedure involved cellular fractionation followed by DEAE-and CM-cellulose chromatography and resulted in an RNAas D preparation contaminated with trace amounts of single-strand specific RNAse (equivalent to less than 0.3 ng per ml) as assayed against poly (rC) . Significant levels of RNAse H activity against poly (rA)-poly (dT) were still present in these preparations.

Proc Natl Acad Sci U S A, 1976 Aug, 73(8), 2776 - 80
Determination of molecular weights by fluctuation spectroscopy: application to DNA; Weissman M et al.; A method for determining molecular weights of macromolecules by measuring spontaneous concentration fluctuations is described . The method is absolute, rapid, and requires no shearing forces on the molecules . We have applied this technique to the determination of molecular weight of DNA molecules . The molecular weight values obtained for T2 phage DNA (1.14 X 10(8)) and replicating Escherichia coli DNA (3.9 X 10(9)) agree with previous results . By monitoring individual molecules, an estimate of the molecular weight of nuclei and individual chromosomal DNA molecules of Drosophila melanogaster was obtained.

J Biochem (Tokyo), 1976 Aug, 80(2), 277 - 82
Conformational changes and reassociation properties of small subunits of Escherichia coli ribosomes; Imamura T et al.; Studies on the reassociation of ribosomal subunits from Escherichia coli were carried out using an ultracentrifuge equipped with a UV absorption scanner, and the conformational changes of ribosomal subunits were followed by circular dichroism measurements . Reassociation between the fractionated 30S and 50 S subunits was promoted by the addition of uncharged tRNA at 4 degrees . Furthermore, a sharp increase in the amount of reassociated products was observed on treatment of the sample solution at temperatures above 25 degrees . Treatment at higher temperature only affected 30 S ribosomal slbunits . From CD measurements of Mg2+ concentration dependence for both ribosomal subunits, a significant decrease in molar ellipticity {theta} at the 264 nm positive peak and a slight change of {theta} at the 222 nm band were observed for both subunits in dissociation buffer . Samples of 30 S subunits pretreated at 37 degrees for 40 min showed a small CD change at the 222 nm band . The same samples showed a slight increase in S value and enhanced ability to form 70 S particles with 50 S subunits . On the other hand, we detected no significant changes in CD spectra, S value or the ability to form 70 S particles with 50 S ribosomal samples subjected to same heat treatments.

J Biochem (Tokyo), 1976 Aug, 80(2), 253 - 8
Propidium diiodide-cesium chloride density gradient centrifugation for buoyant separations of duplex DNA molecules containing single-stranded regions; Fukuda A; At increasing dye concentrations in propidium diiodide-CsCl density gradients, the relative buoyant density shift was largest for open-circular duplex phiX174 DNA (RFII), next largest for single-stranded viral DNA, and least for closed-circular duplex DNA (RFI) . These differential relative buoyant density shifts permitted discrete separations of these phiX DNA forms . Further, in propidium diiodide-CsCl density gradients, the distinctive density of single-stranded DNA permitted separations of rolling-circle intermediates with a single-stranded tail that occur during single-stranded phiX DNA synthesis . It is suggested that single-strandedness in a duplex DNA structure influences its buoyand density shift due to differential dye binding to the single-stranded region and is an additional physical basis for relative buoyant separations of DNA molecules in propidium diiodide-CsCl density gradients.

Zh Mikrobiol Epidemiol Immunobiol, 1976 Aug, (8), 45 - 9
{Phagocytosis of enteropathogenic escherichiae of serogroup 0124:K72(B17)}; Borisov LB et al.; Experiments with phagocytosis of E . coli O124 in the macrophage tissue culture demonstrated a different phagocytability of the S- and R-forms of these bacteria determined by their concentration in the medium . The ingestive and digestive capacity depended on the bacterial virulence . Since R-forms of E . coli O124 lost their virulent properties and macrophage resistance it can be considered that the structure of their lipopolysaccharides served as the chemical substrate responsible for their different phagocytability.

Nucleic Acids Res, 1976 Aug, 3(8), 2129 - 42
Increased isoleucine acceptance by sulfur-deficient transfer RNA from Escherichia coli; Harris CL et al.; Sulfur-deficient tRNA, isolated from Escherichia coli HfrC, rel-, met-, cys-, lambda, after cysteine starvation, was found to have an increased acceptance of isoleucine in proportion to the deficiency of 4-thiouridine . Isoleucine acceptance was not altered in the presence of other amino acids of CTP, and the higher acceptance was observed over a wide range of magnesium, isoleucine, tRNA and enzyme concentrations . The Vmax value for sulfur-deficient tRNA was more than three times greater than observed for normal tRNA . Methylated albumin kieselguhr (MAK) chromatography revealed three isoacceptor peak for normal tRNA, while sulfur-deficient tRNA was missing tRNAile, and exhibited a larger, shifted peaks for tRNA normal tRNA, while sulfur-deficient tRNA was missing tRNAille 2, and exhibited a large shifted peak for tRNAile 3 . Treatment with crude RNA sulfurtransferase both lowered the isoleucine acceptance for sulfur-deficient tRNA to that seen for normal tRNA, and restored the missing isoacceptor on MAK . The possibility that thionucleotides may play a role in the aminoacylation of tRNAile in E . coli is discussed.

Nucleic Acids Res, 1976 Aug, 3(8), 2067 - 77
ATP-analogues as substrates for the leucyl-tRNA synthetase from Escherichia coli MRE 600; Marutzky R et al.; No analogous nucleoside triphosphate was found which acts as well as ATP in binding to and supporting catalysis of leucyl-tRNA synthetase from Escherichia coli MRE 600 . However, there are numerous nucleotides which are able to replace ATP, but with lower efficiency . The 6-amino group of the adenine ring and the 2'-hydroxyl group of the ribose ring are essential for binding and catalytic activity . Alterations in the triphosphate moiety of the molecule can cause drastic changes in Km and/or Vmax, whereas alterations of the imidazole ring and substitutions at the 8-position of the adenine ring cause only minor losses of catalytic activity.

Eur J Biochem, 1976 Aug 1, 67(1), 95 - 104
Raffinose metabolism in Escherichia coli K12 . Purification and properties of a new alpha-galactosidase specified by a transmissible plasmid; Schmid K et al.; The utilization by Escherichia coli K12 of raffinose as sole carbon source depends on a new raffinose transport system, an invertase and an alpha-galactosidase specified by the Raf-plasmid D1021 . The alpha-galactosidase was purified to homogeneity from a mutant strain with constitutive synthesis of the enzyme . alpha-Galactosidase hydrolyzes p-nitrophenyl-alpha-D-galactoside (Km 0.14 mM), methyl-alpha-D-galactoside (Km 30mM), melibiose (Km 3.2 mM) and raffinose (Km 60 mM) . The enzymatic activity is strongly inhibited by Ag+, p-chloromercuriphenyl sulfonic acid and, to a lesser extent, by iodoacetamide . Isoelectric focusing indicates the existence of one form of alpha-galactosidase with an isoelectric point of 5.1 . The purified enzyme has an sw,20 value of 11.7 +/- 0.3S and a molecular weight of 329000 +/- 4000; this value is not reduced at high dilutions . When examined by dodecylsulphate gel electrophoresis, purified alpha-galactosidase yields a single subunit band of molecular weight 82000 suggesting that the intact enzyme consists of four subunits . Amino acid analysis indicates the presence of approximately 712 amino acid residues per quarter molecule including 8 half-cystine residues . No carbohydrate moiety has been detected . High resolution electron micrographs and Markham rotation of alpha-galactosidase show enzyme molecules of approximately 11 x 11 nm containing four globular subunits in a tetragonal arrangement . The plasmid-coded alpha-galactosidase differs from the homologous E . coli enzyme by substrate affinities, cofactor requirements, stability and toluene resistance . It can, therefore, be used as a marker enzyme suitable for the detection in vivo of Raf-plasmids.

Eur J Biochem, 1976 Aug 1, 67(1), 53 - 6
The O9 antigen of Escherichia coli . Structure of the polysaccharide chain; Prehm P et al.; The lipopolysaccharide from Escherichia coli O9:K30- was isolated in about 2% yield with aqueous 45% phenol at 65 degrees C, followed by ultracentrifugation . The polysaccharide moiety was obtained by graded hydrolysis and gel permeation chromatography . It consisted of a mannan which carried on its reducing end the core oligosaccharide of the R1 type . The mannan contained 1 leads to 2 and 1 leads to 3 linkages in a ratio of 3:2, as determined by methylation analysis and mass spectrometry . On periodate oxidation, 58% of the mannose residues were destroyed . Degradation of oligosaccharide mixtures with alpha-mannosidase from jack bean meal, as well as a specific rotation of {alpha}25D = +89 degrees indicated that all mannosyl linkages have the alpha-configuration . Smith degradation resulted in the liberation of mannosyl (1 leads to 3)-mannose (bound to glyceraldehyde), as established by methylation analysis . From these results we conclude that the O9 polysaccharide of E . coli has a pentasaccharide repeating unit of alpha-mannosyl(1 leads to 3)-alpha-mannosyl-(1 leads to 2)-alpha-mannosyl-(1 leads to 2)-alpha-mannosyl-(1 leads to 2)-mannose, which are joined in the polysaccharide through alpha-(1 leads to 3)-mannosyl linkages.

Eur J Biochem, 1976 Aug 1, 67(1), 47 - 51
The role of Escherichia coli dnaG function in coliphage M13 DNA synthesis; Dasgupta S et al.; Examination of the role of Escherichia coli dnaG function in different stages of M13 phage DNA synthesis by ultracentrifugal analysis of intracellular phage DNA in a thermosensitive dnaG mutant shows that: (a) the formation of parental double-strand replicative-form DNA (rfDNA) from the infecting virus is independent of dnaG function; (b) the synthesis of progeny rfDNA requires dnaG product; (c) after a pool of rfDNA is made up, dnaG function is not required for the progeny single-strand DNA (ssDNA) synthesis . The ssDNAs produced under nonpermissive condition are mostly circular and biologically functional.

Eur J Biochem, 1976 Aug 1, 67(1), 37 - 45
The role of Escherichia coli dna A gene and its integrative suppression in M13 coliphage DNA synthesis; Mitra S et al.; An F+ derivative of Escherichia coli E508 thermosensitive in dna A function (involved in DNA synthesis initiation), its revertant and an Hfr derivative of E508 (ts) in which the temperature-sensitive phenotype is suppressed by integrative suppression have been compared for their ability to support M13 phage DNA synthesis at the nonpermissive temperature . Upon infection at the nonpermissive temperature, both the revertant and the Hfr strain support normal phage replication while the temperature-sensitive mutant does not . However, when infection is carried out at a permissive temperature and the temperature is shifted up after infection, phage synthesis occurs in the temperature-sensitive mutant also, but in lesser quantity than in the revertant strain . Analysis of intracellular labeled phage DNA indicates: (a) parental replicative form DNA synthesis is not dependent on dna A function; (b) progeny replicative form DNA synthesis is strongly inhibited in the temperature-sensitive dna A mutant at the nonpermissive temperature; (c) progeny single-strand DNA synthesis does not absolutely require dna A function; (d) progeny single-strand DNA is present in the circular form . The implication of the host DNA replication in M13 DNA synthesis is discussed.

Eur J Biochem, 1976 Aug 1, 67(1), 257 - 65
Polypeptide-chain elongation promoted by guanyl-5'-yl imidodiphosphate; Girbes T et al.; In a purified system from Escherichia coli containing ribosomes complexed with poly(uridylic acid) and N-acetyl-phenylalanyl-tRNA, the nonhydrolyzable analog of GTP, guanyl-5'-yl imidodiphosphate (Guo-5'-P2-NH-P), promotes polypeptide synthesis at a rate several times slower than GTP . The activity is completely dependent on elongation factors EF-T (i.e, EF-Ts + EF-Tu) and EF-G . Examination of individual steps of the elongation cycle in partial reactions shows that Guo-5'-P2-NH-P is as efficient as GTP in promoting the EF-T-dependent binding of phenylalanyl-tRNA to the ribosomal A site . In contrast, Guo-5'-P2-NH-P promotes the translocation-dependent binding of phenylalanyl-tRNA to a ribosome complexed with A-site-bound N-acetyl-phenylalanyl-tRNA much more slowly than GTP . This slow rate of binding is due to the presence of EF-G on the ribosome, and not to sluggish translocation, since (a) the rate remains slow even after translocation of N-acetylphenylalanyl-tRNA is completed, (b) it is greatly speeded up by removal of EF-G from the reaction mixture (after translocation has occurred), and (c) it is slowed down again by readdition of the factor . Moreover, with post-translocated ribosomes and in the absence of EF-G, formation of dipeptide subsequent to the EF-T-dependent binding of phenylalanyl-tRNA is much slower when binding of this substrate has been promoted by Guo-5'-P2-NH-P than it is when promoted by GTP . The results suggest that, during polymerization with Guo-5'-P2-NH-P, EF-G and EF-Tu are slowly released from the ribosome and, consequently, the steps of the elongation cycle subsequent to translocation and aminoacyl-tRNA binding (aminoacyl-tRNA binding and peptide bond formation, respectively) are delayed . Thus, durong elongation cycle, GTP hydrolysis is probably essential for fast release of the factors from the ribosome.

Eur J Biochem, 1976 Aug 1, 67(1), 23 - 9
Geometry of the protein S4 from Escherichia coli ribosomes; Paradies HH et al.; The shape of protein S4 from Escherichia coli ribosomes in solution was determined by hydrodynamic methods and low-angle X-ray scattering . The molecular weight of 24000 determined by low-angle X-ray scattering is within 3% of that found by sedimentation equilibrium analysis and 8% of that determined by amino acid sequence work . The radius of gyration of 3.36 nm, the radius of gyration of the cross section of 0.41 nm and the hydrodynamic studies revealed that protein S4 is not spherical, but rather has a markedly extended shape . Calculations of different conformations, e.g . random coil, based on the parameters evaluated from hydrodynamic methods, revealed a rod-like structure of S4 with a length of 14 nm and a diameter of 1 nm . This is supported by a model of an equivalent scattering particle of uniform density based on all parameters obtained in this study.

Eur J Biochem, 1976 Aug 1, 67(1), 171 - 6
L-Phenylalanine: tRNA ligase of Escherichia coli K10 . The effect of O replaced by S substitution on substrate and ligand binding properties of ATP; Pimmer J et al.; The Km and V values of the tRNA-charging reaction have been measured for L-phenylalanine:tRNA ligase and the geometric isomers of adenosine 5'-O-(1-thio)triphosphate, adenosine 5'-O-(2-thio)triphosphate and for 5'-O-(3-thio)triphosphate . All ATP analogs were found to be substrates with values of Km similar or (up to 10-fold) higher, and with values of V in the range of 10--30% compared with the natural substrate . The dissociation constants of the binary enzyme-nucleotide and ternary enzyme-nucleotide-L-phenylalaninol complexes were analysed as a function of the position of the sulfur atom indicating those phosphate groups which are involved in an enzyme-triphosphate interaction . The results are consistent with a participation of the beta and gamma-phosphate in the binary complex formation and an additional interaction at the positions of the alpha and beta-phosphate groups in the ternary complexes.

Eur J Biochem, 1976 Aug 1, 67(1), 145 - 53
Effects of antibodies to various molecular forms of a mutationally altered Escherichia coli alkaline phosphatase on its activation by zinc; Pages JM et al.; Immunogenic and antigenic properties of a Zn2+ -deficient alkaline phosphatase produced in a mutant (U-47) of Escherichia coli have been studied . The native U-47 enzyme, that exists in a monomerdimer equilibrium, was used as immunogen . From the antisera obtained, four antibody populations directed to the various molecular forms of U-47 enzyme have been purified by affinity chromatography using specific antigens coupled to glutaraldehyde-activated beads of indubiose . 70% of the total antibody obtained was directed both to the monomeric and the dimeric forms, 9% was directed to the dimer but showed a low affinity for the monomer; 10% and 11% were specifically directed respectively to the monomer and the dimer . Each antibody population purified had a specific effect on the catalytic activity of the Zn2+ -activated U-47 enzyme . The anti-monomer-dimer and the anti-dimer-monomer inhibit to the same extent whereas the specific anti-monomer does not alter the activity significantly and the specific anti-dimer causes a 30% activation . The catalytic activity of the alkaline phosphatase produced in wild-type strains was also reduced by these anti-U-47 enzyme antibodies . However, whereas the anti-monomer had again very little effect, the anti-dimer-monomer and the anti-monomer-dimer inhibited this enzyme to different extents . The specific antidimer also inhibited this wild-type alkaline phosphate . Antibodies of high affinity to the dimeric form of U-47 enzyme, i.e . specific anti-dimer or anti-dimer-monomer, caused a 30% activation when they were added prior to the reactivation process by Zn2+ . Specific anti-monomer strongly inhibited this reactivation process . The Fab fragment of the anti-wild-type phosphatase antibody, under the same conditions, caused a 300% activation . The extents of interactions of the various molecular forms of U-47 enzyme and of the wild-type enzyme with the anti-monomer-dimer and with the anti-dimer have been determined . U-47 enzyme monomeric form has three determinants exposed and the dimeric form has five determinants exposed for interacting with the anti-monomer-dimer antibody, the free wild-type enzyme has only two determinants exposed to this antibody . These determinants might be close to the active site or in another critical location since this antibody can reduce the catalytic activity of the wild-type enzyme to half the original value . The anti-dimer antibodies can interact with three determinants exposed at the surface of the free Zn2+ -reactivated U-47 enzyme and the non-covalent binding of one mole of inorganic phosphate results in the exposure of one more antigenic determinant.

Eur J Biochem, 1976 Aug 1, 67(1), 115 - 22
The energetics of Escherichia coli during aerobic growth in continuous culture; Farmer IS et al.; 1 . The energetics of Escherichia coli W growing aerobically in continuous culture have been investigated . Conditions were chosen such that growth was limited by the availability of carbon or oxygen (energy-limited cultures), or of ammonium of sulphate ions (excess energy cultures) . 2 . Under glycerol-limited conditions YmaxO2 (true molar growth yield with respect to oxygen) and YmaxATP (true molar growth yield with respect to ATP equivalents) were 50.9 g cells-mol O-02(-1) and 12.7 g cells-mol ATP equivalents-1 respectively; these values were not substantially altered during growth limited by oxygen, ammonium or sulphate . In contrast, M (the energy requirement for maintenance purposes) increased from approximately 2 mmol ATP equivalents-h-1-g cells-1 during energy-limited growth to 16.8 and 30.8 mmole ATP equivalents-h-1-g cells-1 when growth was limited by ammonium and sulphate ions respectively . 3 . Replacement of glycerol by other limiting carbon sources caused YmaxATP to alter within the range 13.9 (glucose) to 7.1 (acetate) g cells-mol ATP equivalents-1 in the order glucose greater than galactose greather than arabinose greater than fructose greater than glycerol greater than fumarate greater than lactate greater than pyruvate greater than acetate . In each case the experimental value of YmaxATP was less than or equal to 55% of the theoretical value calculated from the known energy requirements for the biosynthesis of cell materials . 4 . It is concluded from these results that neither M nor Ymax ATP are constant values for E . coli . M varies with the energy supply, being highest under excess energy growth conditions where it may reflect energy wastage by the cell . On the other hand, YmaxATP varies with the nature of the growth substrate and thus reflects the different energy requirements for the synthesis of cell material from different carbon sources.

Arch Microbiol, 1976 Aug, 109(1-2), 143 - 6
DNA synthesis by Escherichia coli B/r/l synchronized and grown under conditions of slow growth; Buckley DE et al.; Escherichia coli B/r/l was synchronized by a novel method and its growth was followed in a minimal salts medium containing glucose, acetate, aspartate or succinate as the sole carbon source . Thymine incorporation experiments showed agreement with the Cooper-Helmstetter model for DNA synthesis, during the division cycle, both in glucose grown culture with a doubling time 57.5 min and in acetate, aspartate and succinate where the doubling time was extended up to 90 min . The ratio C/C + D was identical or close to that predicted by the model . Prolonged growth of the synchronized cultures prior to each experiment was practised in order to ensure their physiological state without causing any considerable deterioration of synchrony.

Med Biol, 1976 Aug, 54(4), 243 - 53
Effect of endotoxin and trypsin on the blood pressor response to catecholamines in normo- and hypothermic rabbits; Csako G et al.; The action of endotoxin, trypsin or hypothermia on the vascular reactivity to catecholamines was investigated in rabbits, and with trypsin in normothermic dogs as well . In rabbits, LD10 of Escherichia coli 0111 endotoxin increased the blood pressor effect of i.v . adrenaline or noradrenaline with maxima at 60-90 min . LD50 endotoxin elicited a vascular hyporeactivity to catecholamines within an hour . 22 hours later, however, a hyperreactivity to catecholamines developed . At this time, repeated administration of LD50 endotoxin did not reduce the increased catecholamine responsiveness . Trypsin (i.v . 1.5 mg/kg) also potentiated the pressor effect of catecholamines in rabbits and dogs with maxima at 5-10 min . Higher trypsin doses induced a hyporeactivity . LD50 endotoxin in a single or a repeated dose after 22 hours did not decrease the blood pressure of cooled rabbits and failed to alter the vascular reactivity to adrenaline or noradrenaline . The blood pressure effect of trypsin differed in character in normo- and hypothermic rabbits, depending on the depth of cooling . Low body temperature eliminated the potentiating effect of trypsin to the blood pressor action of catecholamines . In normothermic rabbits pretreated with amino-pyrine and phenylbutazone, the blood pressure and the catecholamine potentiation effects of endotoxin or trypsin were inhibited or considerably reduced . The results support the significance of altered vascular reactivity to catecholamines under different pathologic conditions where endotoxin and/or proteases may occur.

Lab Invest, 1976 Aug, 35(2), 171 - 8
Platelet inhibition of renal cortical fibrinolytic activity in the rabbit; Bergstein JM; A fibrin slide test was utilized to study the effect of platelets and endotoxin on renal cortical fibrinolysis in rabbits . Cortical lysis was absent in kidney from animals made markedly thrombocytopenic with goat antirabbit platelet serum or endotoxin; light microscopy was normal and immunofluorescent microscopy for fibrin was negative . No loss of lysis was detected in kidney of animals given goat anti-rabbit albumin or isotonic saline . Plasma from animals with endotoxin or antiplatelet antibody-induced thrombocytopenia demonstrated an increased capacity to inhibit cortical fibrinolytic activity of normal rabbit kidney . Antiplatelet antibody could be substituted for the preparatory injection of endotoxin in the generalized Shwartzman reaction; a parallel rise in lysis inhibitory titer was detected in plasma from animals prepared with antibody or endotoxin . Although lysis was absent following incubation of normal renal cortex with washed platelets, no inhibition of lysis was found after incubation of normal cortex with endotoxin, histamine, serotonin, platelet membranes, or granules . Using a modified fibrin slide technique, a diffusable platelet inhibitor of lysis could be demonstrated . These studies indicate that platelets contain an inhibitor to renal cortical fibrinolysis which is released into the circulation upon platelet destruction . The findings also suggest that platelet destruction is the mechanism through which endotoxin inhibits cortical lysis.

J Virol, 1976 Aug, 19(2), 756 - 9
Genetic analysis of heterogeneous DNA circles formed after prophage Mu induction; Parker V et al.; Induction of a Mu prophage in Escherichia coli Hfr strains lyosgenic for Mu cts62 leads to the generation of F' episomes . Each episome thus formed carries at least one copy of the Mu genome . These results suggest that integration of Mu is mandatory for the formation of the heterogeneous circles during the lytic cycle . The circles may be precursors for phage maturation.

J Bacteriol, 1976 Aug, 127(2), 998 - 1014
Specialized transduction of D-serine deaminase genes: formation of lysogens that yield high lambda-d dsd/lambda ratios and formation of a dimeric lambda-d dsd; Palchaudhuri S et al.; We have obtained two classes of double lysogens that on induction yield higher titers of lambda-d dsd transducing phage than of helper phage . One class was obtained by lysogenization of strain EM6116 (dsddelta attlambdadelta HfrC) with lambda-dsd type 2 (dsdC+ dsdO+ dsdA+, head-tail substitution) . In the absence of either a normal attlambda or the homology of a chromosomal dsd region, the transducing phage integrated at other sites, at least one of which, in strain EM6177, is near the origin of HfrC . On induction, strain EM6177 yields a phage burst of 20 to 50 with a lambdadsd:lambda ratio of 10(4):1 . The asnychronously high yield of lambda dsd is attributed to an efficiency of excision greater than that of lambda . The other class was obtained by lysogenization of strain EM1407 (dsdA attlambda+) with lambda-dsd type 2 (dsdO6 dsdA, partial deletion of dsdC) . The DNA of mature lambda-dsd type 2 is a complete dimer . It lacks nearly all the phage late genes and b2 and carries about five bacterial genes . It could not be packaged as a monomer but is just within the packaging size limit as a dimer . Models for the derivation of these lambda dsd phages and the high-yielding lysogens are presented.

J Bacteriol, 1976 Aug, 127(2), 988 - 97
Electron microscope study of a plasmid chimera containing the replication region of the Escherichia coli F plasmid; Guyer MS et al.; pML31, a plasmid chimera constructed to contain the replication genes of an Flac plasmid, has been studied by electron microscope methods . Heteroduplex analysis shows that the only F sequence present in pML31 is that with corrdinates 40.3-49.3F . This region has previously been identified as essential for plasmid maintenance . The sequence of pML31, which was derived originally from R6-5, carries the km gene(s) and an inverted duplication of a 1.0-kilobase sequence . On the basis of length measurements, the repeated sequence is different from IS1, IS2, IS3, and an inverted repeat associated with the km gene(s) of plasmid JR67.

J Bacteriol, 1976 Aug, 127(2), 923 - 33
Genes for the alpha and beta subunits of the phenylalanyl-transfer ribonucleic acid synthetase of Escherichia coli; Comer MM et al.; The phenylalanyl-transfer ribonucleic acid synthetase of Escherichia coli is a tetramer that contains two different kinds of polypeptide chains . To locate the genes for the two polypeptides, we analyzed temperature-sensitive mutants with defective phenylalanyl-transfer ribonucleic acid synthetases to see which subunit was altered . The method was in vitro complementation; mutant cell extracts were mixed with purified separated alpha or beta subunits of the wild-type enzyme to generate an active hybrid enzyme . With three mutants, enzyme activity appeared when alpha was added, but not when beta was added: these are, therefore, assumed to carry lesions in the gene for the alpha subunit . Two other mutants gave the opposite response and are presumably beta mutants . Enzyme activity is also generated when alpha and beta mutant extracts are mixed, but not when two alpha or two beta mutant extracts are mixed . The inactive mutant enzymes appear to be dissociated, as judged by their sedimentation in sucrose density gradients, but the dissociation may be only partial . The active enzyme generated by complementation occurred in two forms, one that resembled the native wild-type enzyme and one that sedimented more slowly . Both alpha and beta mutants are capable of generating the native form, although alpha mutants require prior urea denaturation of the defective enzyme . With the mutants thus characterized, the genes for the alpha and beta subunits (designated pheS and heT, respectively) were mapped . The gene order, as determined by transduction is aroD-pps-pheT-pheS . The pheS and pheT genes are close together and may be immediately adjacent.

J Bacteriol, 1976 Aug, 127(2), 904 - 16
Envelope-associated folded chromosomes for Escherichia coli: variations under different physiological conditions; Korch C et al.; The folded chromosome of Escherichia coli has been investigated under various lysis and physiological conditions . A new gradient system was devised that allows excellent separation between unlysed cells and envelope-associated and envelope-free chromosomes . Isotope incorporation experiments showed that the fraction often called "membrane-bound nucleoids" contains cell wall in addition to nucleic acids, membranes, and proteins . The amount of lysozyme added and the lysozyme digestion time were found to be important when comparing the rate of sedimentation of envelope-associated chromosomes obtained under various physiological conditions . Amino acid-starved cells were found to be much harder to lyse with lysozyme than exponentially grown cells, The difference in sedimentation coefficient of envelope-associated chromosomes described earlier (Ryder and Smith, 1974) was not detected when the latter two types of cells had been given equivalent, but not identical, lysozyme treatment such that detergent-mediated lysis proceeded at the same rate . Analysis of pulse- and uniformly labeled chromosomes from amino acid-starved cultures revealed no preferential labeling of either envelope-associated or -released nucleoids . Nor was there a difference in sedimentation coefficient between uniform and pulse-labeled envelope-associated nucleoids . These results are in disagreement with the models for chromosome replication of Worcel and Burgi (1974) and Ryder and Smith (1974), respectively . Growing cells on carbon sources poorer than glucose demonstrated that the replicating chromosomes sediment faster than the bulk of envelope-associated nucleoids . The slower the growth rate, the greater this difference became . An alternative hypothesis regarding chromosome replication and its association with the cell envelope is presented.

J Bacteriol, 1976 Aug, 127(2), 881 - 9
Nonintegrated plasmid-chromosome complexes in Escherichia coli; Kline BC et al.; A number of plasmid systems have been examined for the ability of their covalently closed circular deoxyribonucleic acid (CCC DNA) forms to cosediment in neutral sucrose gradients with the folded chromosomes of their respective hosts . Given that cosedimentation of CCC plasmid and chromosomal DNA represents a bound or complexed state between these replicons, our results can be expressed as follows . (i) All plasmid systems complex, on the average, at least one plasmid per chromosomal equivalent . (ii) Stringently controlled plasmids exist predominantly in the bound state, whereas the opposite is true for plasmids that exist in multiple copies or are under relaxed control of replication . (iii) The degree to which a plasmid population binds to host chromosomes appears to be a function of plasmid genotype and not of plasmid size . (iv) For the colicin E1 plasmid the absolute number of plasmids bound per folded chromosome equivalent does increase as the intracellular plasmid/chromosome ratio increases in cells starved for required amino acids or in cells treated with chloramphenicol; however, the ratio of bound to free plasmids remains constant during plasmid copy number amplification.

J Bacteriol, 1976 Aug, 127(2), 706 - 18
Physiological and genetic regulation of the aldohexuronate transport system in Escherichia coli; Nemoz G et al.; In Escherichia coli K-12, the specificity of the aldohexuronate transport system (THU) is restricted to glucuronate and galacturonate . There is a relatively high basal-level activity in uninduced wild-type or isomeraseless strains . Supplementary activity is obtained with the inducers mannonic amide (five-fold), galacturonate (fourfold), fructuronate (fivefold), and tagaturonate (sevenfold) . Specific THU- mutants were selected as strains unable to grow on either aldohexuronate but able to grow on fructuronate or tagaturonate . The remaining transport activity in uninduced and induced THU- starins represents less than 20% of that found in the wild type . Conjugation and transduction experiments indicate that all of the THU- mutations are located in a unique locus, exuT, half-way between the tolC (59 min) and argG (61 min) markers . exuT is closely linked to the uxaC-uxaA operon (60 min) and to the regulatory gene exuR (60 min), which controls the above-mentioned operon and the uxaB operon (45 min) . Growth on either aldohexuronate and transport activity are fully recovered when exuT mutants are allowed to revert to exuT+ on galacturonate or glucuronate . Reversion on glucuronate alone may lead to the mutational derepression of the 2-keto-3-deoxygluconate transport system, which is uninducible in the wild type, which also takes up glucuronate, and whose structural gene belongs to the kdg regulon . Such strains, which remain unable to grow on galacturonate, are exuT and kdgR (constitutive allele of the regulatory gene kdgR of the kdg regulon) . THU activity is superrepressed in an exuR mutant in which the uxaC-uxaA operon and the uxaB operon are superrepressed; exuR+/exuR merodiploids are also superrepressed . In a thermosensitive exuR mutant in which the above-mentioned operons are constitutive at 42 degrees C, the THU activity is fully derepressed at this temperature . On the basis of these and other results, it is concluded that THU is coded for by the structural gene exuT, which is negatively controlled by the exuR gene product and which probably belongs to an operon distinct from the uxaA-uxaC operon.

J Bacteriol, 1976 Aug, 127(2), 698 - 705
Identification of a genetic locus affecting chromosome stabiltiy and cellular survival in a dnaB mutant; Schendel PF; A mutation has been identified in an Escherichia coli K-12 strain carrying dnaB42 . This mutation potentiates both deoxyribonucleic acid degradation and cell death at nonpermissive temperatures . It is located 2 min away from dnaB between malB and metA.

J Bacteriol, 1976 Aug, 127(2), 691 - 7
Expression and regulation of lactose genes carried by plasmids; Guiso N et al.; A number of plasmids carrying the lactose character have been studied . All of the plasmids examined so far code for proteins essential for lactose utilization, i.e., beta-galactosidase and galactoside permease . None of them carries enzymatically or immunologically detectable thiogalactoside transacetylase . The expression of the two enzymes is both negatively and positively controlled: they are inducible by different galactosides and are sensitive to catabolite repression . Since the plasmid-coded lactose systems have many features in common with the Escherichia coli lactose operon, it is suggested that the plasmids could have acquired the lactose genes from an E . coli chromosome.

J Bacteriol, 1976 Aug, 127(2), 1022 - 3
Role of Rec pathways on sensitivity of Escherichia coli to near-ultraviolet and visible light; Gopalakrishna K et al.; In Escherichia coli lack of the RecRC or RecF pathway is found to cause sensitivity to near-ultraviolet and visible light . Resistance to this light is restored in the RecBC-defective strain carrying either the sbcB (Rec+) or xonA (Rec-) mutation . The sensitivity, therefore, is not found to correlate with the degree of recombination proficiency as measured by genetic crosses.

Immunology, 1976 Aug, 31(2), 225 - 32
Biological effects of Escherichia coli lipopolysaccharide (LPS) in vivo . II . Selection in the mouse thymus of PHA- and con A-responsive cells; Adorini L et al.; The effect of LPS injection on mouse thymocytes was examined by in vitro stimulation with PHA and Con A . Thymocytes from LPS treated mice were found more responsive than normal thymocytes to PHA and Con A . The increase of responsiveness was different for PHA and Con A, and dependent on the dose of LPS injected . This dose dependency suggests that LPS in vivo modifies the thymocyte population by progressively eliminating T1 cells and selecting T2 cells . These effects seem to be mediated by corticosteroids because they were prevented by adrenalectomy, a finding in agreement with the notion that the injection of cortisone induces similar cell changes in the thymus.

Cell, 1976 Aug, 8(4), 605 - 9
Multiple modes of ribosomal RNA transcription in vitro; Travers A; The rate of rRNA synthesis from E . coli DNA in vitro can exhibit an unusual temperature dependence . Instead of the typical sigmoid curve, at least five to six pronounced peaks of rRNA synthesis are detectable over a 30degrees range in temperature . rRNA synthesis from performed initiation complexes exhibits a similar pattern and shows that the number of polymerases able to initiate rRNA synthesis increases with each successive peak . These results are interpreted in terms of a model which proposes that the rRNA cistrons are served by multiple promoter sites (subpromoters) whose activation energies form a graded series . Thus the number of polymerases initiating rRNA synthesis could be controlled by regulating the number of active subpromoters.

Am J Vet Res, 1976 Aug, 37(8), 905 - 6
Serum antibody to Escherichia coli heat-labile enterotoxin in cattle and swine; Whipp SC et al.; Antibody titers to Escherichia coli heat-labile enterotoxin (LT) were measured in serum samples collected from mature cows, butcher pigs, mature sows and adult sheep, horses, dogs, cats, turkeys, and chickens . The frequency of LT antitoxin titers was greatest in sows (94%) and less in cows (38%) . Titers were higher in swine than in cattle . There were no LT antitoxin titers in serums from sheep, horses, dogs, cats, turkeys, and chickens . It was concluded that LT-producing Escherichia coli are prevalent in the swine population, but much less so in cattle and the other species examined.

Am J Dis Child, 1976 Aug, 130(8), 823 - 7
Factors relating to intelligence in treated cases of spina bifida cystica; Hunt GM et al.; Analysis of results on 83 survivors of spina bifida cystica showed the following: (1) in the seven children who had had central nervous system (CNS) infection, intelligence was impaired, six being severely retarded . (2) In the nine children who did not suffer CNS infection or require a shunt, intelligence was normal . The need for a shunt was related to radiological appearance (craniolacunae) and to the sensory level at birth . (3) In the 67 children who did not suffer CNS infection but did require a shunt, intelligence was related to sensory level found at birth and to thickness of the pallium measured within four weeks of birth . Their intelligence did not relate to the occipitofrontal circumference at birth, or to its increase before the insertion of the shunt . Intelligence did not relate to the function of the shunt at the time of assessment or to the number of times it had been revised.

Mutat Res, 1976 Aug, 36(2), 147 - 64
Intragenic mutational spectra and hot spots; Clarke CH et al.; In this review we outline the various factors which may contribute to the non-randomness of intragenic mutational spectra and the occurrence of hot spots . These factors include sample size limitation, particularly for sites of low mutability, and possible regions of low recombination potential . In addition, the nature of the gene product places great restraint on the detectability of either frameshift and premature chain-terminating mutations on one hand, or of the majority of missense mutations on the other . The nature of the Genetic Code itself also limits the mutational spectrum in so far as specific base pair substitutions lead only to a limited number of detectable amino acid replacements . Mutational hot spots may be a special example of the influence of neighbouring base pairs in the mutability of any given base pair . This is apparently true for frameshift mutations which tend to occur in runs of repeated base pairs or base pair doublets . Neighbouring base effects could operate not only at the level of initial reactivity with a mutagen, but also subsequently at the levels of DNA repair, recombination or replication . In some cases rare or modified bases may be responsible for neighbour effects . We suggest specific experimental approaches which seem likely to aid in the elucidation of these problems.

Mutat Res, 1976 Aug, 36(2), 135 - 46
On the lack of host-cell reactivation of UV-irradiated phage T5 II . Further characterization of the repair inhibition exerted by T5 infection; Chiang T et al.; Experiments reported in the preceding paper {4} had shown that host-cell reactivation (HCR) of UV-irradiated phage T1 in excision-repair proficient Escherichia coli cells is inhibited by superinfection with phage T5 . Theoretical considerations have led to predictions concerning the dependence of repair inhibition on the multiplicity of superinfecting T5 phage and on the UV fluence to which they were exposed . These predicitions have been supported by experimental results described in this paper . The fluence dependence permitted calculation of the relative UV sensitivity of the gene function responsible for repair inhibition; it was found to be about 2.3% that of the plaque-forming ability of phage T5 . The T5-inhibitable step in excision repair occurs early in the infective cycle of T1 . Furthermore, experiments involving the presence of 400 mug/ml chloramphenicol showed that HCR inhibition of T1 is caused by a protein produced after the FST segment of T5 (i.e . the first 8% of the T5 genome) has entered the host cell . A previously described minor T1 recovery process, occurring in both excision-repair-proficient and -deficient host cells, is inhibited by T5 infection due to a different substance, which is most likely associated with the "second-step-transfer" region of T5 DNA (involving the remainder of the genome) . Superinfection with T4v1 phage resulted in HCR inhibition of T1, resembling that observed after T5 superinfection . The discussion of these results suggests that inhibition of the bacterial excision repair system by T5 or T4 infection occurs at the level of UV-endonucleolytic incision, and that lack of HCR both in T-even phages and in T5 can be explained in the same manner.

Mutat Res, 1976 Aug, 36(2), 121 - 34
On the lack of host-cell reactivation of UV-irradiated phage T5 . I . Interference of T5 infection with the host-cell reactivation of phage T1; Chiang T et al.; UV-irradiated phage T5, in contrast to T1, T3 and T7, fail to display host-cell reactivation (HCR) when infecting excision-repair proficient Escherichia coli cells . Possible causes of this lack of HCR (which T5 shares with the T-even phages) have been investigated by studying HCR of T1 under conditions of superinfection by T5 . Repair-proficient B/r cells were infected at low multiplicity with UV-irradiated phage T1 in the presence of 1.8 mg/ml caffeine and were superinfected after 15 min with heavily UV-irradiated T5 amber mutants at highly multiplicity . The caffeine, which is later diluted out, prevents any T1 repair prior to T5 superinfection, and UV (254 nm) irradiation of T5 with 144 J/m2 reduces the ability of this phage to exclude T1, thus permitting a reasonable fraction of the mixedly infected complexes to produce T1 progeny . Under these conditions, T5 superinfection causes loss of HCR in about 90% of the T1-producing complexes . Superinfection with unirradiated T5 likewise inhibits HCR of T1, but superinfection with irradiated T3 (a host-cell reactivable phage) does not . This indicates that the observed HCR inhibition of T1 results from T5 infection rather than from competition of irradiated foreign DNA for the excision-repair enzymes of the bacterial host . Employment of appropriate T5 amber mutants has shown that "first-step transfer" (FST) of T5 DNA (involving only 8% of the T5 genome) is sufficient for HCR inhibition, but that transfer of the remainder DNA in addition inhibits a previously described minor T1 recovery process . HCR inhibition of T1, and thus presumably lack of HCR in T5 itself, is ascribed to a substance which is produced either post infection by a gene located in the FST segment of the T5 genome, or which is transferred from extracellular T5 together with the FST DNA.

J Immunol, 1976 Aug, 117(2), 674 - 8
Biologic properties of nontoxic derivatives of a lipopolysaccharide from Escherichia coli K235; McIntire FC et al.; Lipopolysaccharide (LPS)2 from Escherichia coli K235 was treated with o-phthalic anhydride to obtain a high degree of esterification of available hydroxyl groups, leaving a free carboxyl for each hydroxyl esterified (SPLPS) . Although there was no demonstrable loss of fatty acids, this conversion of LPS to a polyanionic molecule altered dramatically the spectrum of biologic properties, most of which are normally attributed to the lipid A (LA) moiety . Mitogenicity for mouse B cells was decreased several hundred-fold; reaction with antibodies to LPS was abolished; pyrogenicity and toxicity were decreased by factors of 10(5) and 10(4); the ability to induce the Shwartzman reaction in rabbits was decreased 500-fold, and the ability to stimulate production of interferon in mice was decreased by more than 2 x 10(3) . However, despite the loss of these properties, SPLPS retained the ability to act as an immunologic adjuvant . The nature of the anionic group is important, e.g., sodium succinyl-LPS (SuLPS) is 10-fold more pyrogenic and toxic than sodium phthalyl-LPS (SPLPS) . Data on another LPS derivative, from which ester-linked fatty acid residues were removed before phthalylation, suggest that the ester-linked fatty acid groups in the lipid A moeity of SPLPS may not be necessary for its immunologic adjuvant effect.

J Immunol, 1976 Aug, 117(2), 433 - 9
The inhibitory effect of chemotactic factors on erythrophagocytosis by human neutrophils; Musson RA et al.; The effect of various chemotactic factors on phagocytosis of sheep erythrocytes sensitized with IgG and complement (EAC1423) by human neutrophils (PMN) was studied . The bacterial chemotactic factor, a butanol extract of an Escherichia coli culture filtrate, the pronase sensitive and insensitive chemotactic substances isolated from this extract, C5a, a chemotactic fragment of the fifth component of complement, and a chemotactic synthetic peptide, formlymethionyl-leucine all significantly reduced the initial rate of ingestion at concentrations that are chemotactic for PMN . The bacterial chemotactic factor produced a 45 to 75% decrease in the initial rate of ingestion, but did not affect adherence of the EAC1423 and PMN . This same substance had no consistent effect on the total number of EAC1423 ingested at 90 min or longer or on the time interval from the addition of the EAC1423 to the start of ingestion . The inhibitory effect of the bacterial factor increased as the amount of bacterial factor was increased and the inhibitory effect was reversible . Inhibition by bacterial factor could be decreased by increasing the concentration of EAC1423; and if the concentration was increased 3-fold, the inhibition was abolished . These results suggest that inhibition by bacterial factor is competitive in nature.

Ann Microbiol (Paris), 1976 Aug-Sep, 127B(2), 133 - 49
{Carboyydrates localization on ultrathin sections of "Brucella" and "Escherichia" cells in smooth (S) and rough (R) phase (author's transl)}; Dubray G; Carbohydrates localization on ultrathin sections of Brucella abortus, melitensis and Excherichia coli cells has been studied by the periodic acid thiocarbohydrazide-silver proteinate (PATAg) and phosphotungstic acid (PTA) methods . Carbohydrates are mainly localized on the cell envelope of Brucella and Escherichia but there are several differences between these two bacteria . The differences are discussed and a Brucella polysaccharide envelope model is proposed . The PATAg method gives the same silver grain deposits on Brucella S and R and Escherichia S and R . The phosphotungstic acid method differenciates Brucella S and R cells by lack of contrast on the outer leaflet of the outer membrane of the latter, but does not differenciate E . coli S or R cells . The Brucella outer membrane contains less polysaccharides than that of E . coli . There is a seemingly symetric distribution of polysaccharide in the Brucella outer membrane as compared to an asymetric distribution in E . coli . The peptidoglycan of Brucella reacts strongly as compared with that of E . coli . The polysaccharides present a dispersed pattern in Brucella cytoplasm, whereas in E . coli they appear as dense, strongly reactive clusters very close to the cytoplasmic membrane.

J Med Chem, 1976 Aug, 19(8), 994 - 8
Synthesis and biological properties of some spin-labeled 9-aminoacridines; Sinha BK et al.; Five spin-labeled 9-aminoacridines, each bearing either a 4-(2,2,6,6-tetramethyl-1-piperidinyloxy) or a 3-(2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety in the 9 position, have been synthesized and assayed for biological activity in three different test systems . Sedimentation velocity measurements indicated that the labels caused unwinding of calf thymus DNA . Those acridines which contained both 6-chloro and 2-methoxy substituents were less toxic to leukemia L1210 in static culture than the corresponding unsubstituted analogues . While the unsubstituted aminoacridines were quite good inhibitors of Escherichia coli DNA-primed RNA polymerase, the 6-chloro-2-methoxy-substituted compounds stimulated this enzyme system . In the presence of E.coliDNA, the ESR spectrum of 4-{(6-chloro-2-methoxy-9-acridinyl)amino}-2,2,6,6-tetramethyl-1-piperidinyloxyl (12) became broad and highly asymmteric with a maximal hyperfine splitting of 57.5 G . This observation suggests that when 12 intercalates into DNA the piperidinyl moiety that bears the nitroxide group becomes highly immobilized . These results suggest that the spin-labeled 9-aminoacridines will be useful probes for nucleic acids.

Proc Natl Acad Sci U S A, 1976 Aug, 73(8), 2793 - 7
Transfer of tRNAs to somatic cells mediated by Sendai-virus-induced fusion; Kaltoft K et al.; tRNAs from yeast (tRNAPhe and 4S RNA) and Escherichia coli (suIII+ tRNAITyr) have been transferred to mouse cells by means of a two-step transfer procedure {Loyter, Zakai, and Kulka (1975) J . Cell Biol . 66, 292-304; Schlegel and Rechsteiner (1975) Cell 5, 371-379} . In the first stage of the transfer tRNAs were incorporated into rabbit red blood cells (RBCs) . Thereafter, the loaded erythrocytes were fused with recipient mouse cells by means of Sendai virus . At least 0.3-0.4% of the total input of tRNA used to load the RBCs could be transferred to mouse cells . Of the tRNA incorporated in the mouse cells, at least 50% could be recovered in the form of intact tRNA molecules when yeast 4S RNA was used . With E . coli suIII+ tRNAITyr a rather smaller proportion of the tRNA remained intact (33%) . Although the loading of tRNA into RBCs is not essential for its uptake, we find that the transfer is four times more efficient with RBCs as a vehicle for the injection . Significantly, a fraction (2%) of the recipient cells possessed much more incorporated tRNA than the average cell when Sendai virus and loaded RBCs were used . Such cells were not found in control experiments in which tRNA uptake was induced by Sendai virus alone.

J Gen Microbiol, 1976 Aug, 96(2), 191 - 201
Control of the sequential utilization of glucose and fructose by Escherichia coli; Clark B et al.; In Escherichia coli (ATCCI5224; ML308), glucose and fructose phosphotransferase systems (PT-systems) are constitutive but activities are increased five and 10-fold respectively by aerobic growth on their respective substrates in defined media . In mixtures, glucose is used preferentially and the fructose PT-system activity is kept at its minimum; but, on glucose exhaustion, it overshoots its steady-state level and growth continues on fructose without lag . Cyclic AMP prevents overshoot . Continuous cultures operating as turbidostats on mixtures of glucose and fructose do not use fructose if sufficient glucose is present to support growth . If less glucose is available, it is all used and sufficient fructose is metabolized concurrently to maintain the growth rate characteristic of glucose . Both PT-systems are inhibited by hexose phosphates . Presence of homologous substrate specifically sensitizes each PT-system to inactivation by N-ethylmaleimide (NEM) . Glucose diminishes the ability of fructose to sensitize its PT-system to NEM . This effect parallels the inhibition of fructose utilization by glucose and suggests that glucose denies fructose access to the fructose-specific part of the PT-system.

Chem Biol Interact, 1976 Aug, 14(3-4), 347 - 56
Copper toxicity: evidence for the conversion of cupric to cuprous copper in vivo under anaerobic conditions; Beswick PH et al.; We have determined the toxicity to cells of Escherichia coli B of cupric copper applied under aerobic and anaerobic conditions in two ways . The growth of cells in liquid medium incorporating cupric copper shows differential inhibition, comparing aerobic and anaerobic conditions--toxicity being greater under anoxia . The growth of colonies upon agar plates incorporating cupric copper does not show such a differential effect . We conclude that colonies on plates are largely anoxic even when incubated aerobically . EPR spectra of cells obtained at various times after application of cupric copper under anoxic conditions indicate the conversion of a considerable proportion of the Cu(II) to a non-paramagnetic species, probably Cu(I) . We demonstrate that Cu(I) is more toxic than Cu(II) to cells when applied under anoxic conditions and conclude that the difference in toxicity of Cu(II) applied to cells under aerobic and anaerobic conditions results from the greater extent of reduction of Cu(II) to Cu(I) under anaerobic conditions.

Chem Biol Interact, 1976 Aug, 14(3-4), 207 - 16
Mechanism of the inhibition of transcription by PR toxin, a mycotoxin from Penicillium roqueforti; Moule Y et al.; PR toxin, a mycotoxin synthesized by Penicillium roqueforti, impairs the transcriptional process in liver cells; the two main RNA polymerase systems (enzymes A and B) are affected by PR toxin . The toxin does not require an enzymic conversion before interfering with in vitro RNA synthesis . Addition of ammonium sulphate completely prevents the inhibition of transcription by PR toxin . In vitro results, using RNA polymerase purified from E . coli, suggest that PR toxin impairs the activity of the RNA polymerase itself . Regarding the step of the transcription process affected, it is shown that PR toxin inhibits both initiation and elongation of the polynucleotide chain.

Acta Med Okayama, 1976 Aug, 30(4), 245 - 55
Measurement of endotoxin . I . Fundamental studies of radioimmunoassay of endotoxin; Kimura H; A method for estimating endotoxin by radioimmunoassay was recently introduced . The present paper describes improvements in the speed and sensitivity on this endotoxin measurement . Antigen was purified from E . coli O111: B4 (B) lipopolysaccharide by centrifugation and dialysis . Purified anti-endotoxin antibody was prepared from immunized rabbit serum . A radioimmunoassay system was established with the antigen and antibody . Dextran-coated charcoal was used to separate the antibody-bound antigen from free antigen . Experimental studies were also performed on possible factors related to the antigen-antibody reaction . Accurate measurements on quantities as low as 100 pg/ml (10ng/ml in the plasma) were performed by the dextran-coated charcoal method, and the reaction time was reduced to 2 hr at 4 degrees C . This new method does not require strict sterilization or aseptic handling, and therefore is quite practical for quantitative measurements of endotoxin.

J Exp Med, 1976 Aug 1, 144(2), 358 - 70
In vitro studies on the T-lymphocyte population of human milk; Parmely MJ et al.; Human milk lymphocytes (ML) can be partially purified and propagated in vitro as a means of assessing their immunological function . When exposed to a variety of stimuli known to activate T lymphocytes, ML respond in a unique manner that indicates a selected population of immunocompetent cells . ML are hyporesponsive to to nonspecific mitogens and respond in a reduced manner to histocompatibility antigens on allogeneic cells . In most cases, they are completely unresponsive to C . albicans although blood lymphocytes from the same patients respond to the antigen . The Kl capsular antigen of E . coli induces significant proliferation in lymphocytes obtained from milk, but fails to stimulate blood lymphocytes . This dichotomy of reactivity does not appear to result from suppressive factors or cells in milk or insufficient adherent cell function . Rather it appears to reflect the accumulation of particular lymphocyte clones in the breast and the local nature of mammary tissue immunity at the T-lymphocyte level.

Zentralbl Bakteriol {Orig A}, 1976 Aug, 235(4), 399 - 403
Antigenic degradation in Escherichia coli; O'Farrell SM et al.; Strains of Escherichia coli isolated in a maternity unit were examined serologically . On primary isolation the strains were shown to be O-antigenically distinguishable, although having the same H antigen, biotype and antibiogram . Subsequent detailed serological studies revealed that the colonial variants derived from them showed similar antigenic diversity, irrespective of the original antigenic structure.

Eur J Biochem, 1976 Aug 1, 67(1), 177 - 86
Effect of inhibitors on the substrate-dependent quenching of 9-aminoacridine fluorescence in inside-out membrane vesicles of Escherichia coli; Singh AP et al.; The effect of various inhibitors on the substrate-dependent quenching of the fluorescence of 9-aminoacridine was measured in inside-out membrane vesicles of Escherichia coli . The rate of fluorescence quenching in the presence of inhibitors was dependent on the rate of electron transfer through the respiratory chain with NADH, succinate, D-lactate or DL-glycerol 3-phosphate as substrates . Several patterns of response were given by the inhibitors . Inhibitors competitive with substrate, or those acting only on the dehydrogenases, gave a direct relationship between the extent of inhibition of oxidase activity and the rate of quenching . A biphasic relationship was given by 2-heptyl-4-hydroxyquinoline N-oxide and piericidin A which was due to these compounds acting both as inhibitors of the respiratory chain and, at higher concentrations, as uncoupling agents . Uncouplers inhibited fluorescence quenching with minimal inhibition of oxidase activity . The transmembrane pH difference was calculated from the extent of fluorescence quenching and the intravesicular volume . The maximum pH difference of 3.3--3.7 units was generated by each of the substrates tested.

Immunology, 1976 Aug, 31(2), 217 - 24
Biological effects of Escherichia coli lipopolysaccharide (LPS) in vivo . I . Selection in the mouse thymus of killer and helper cells; Baroni CD et al.; In the present study we have investigated the biological effects on thymus lymphocytes resulting from Escherichia coli lipopolysaccharide (LPS) treatment in young adult mice . It has been established that LPS induces the following effects: (a) a dose-dependent reduction of thymus weight contemporaneous with a rise in the anti-LPS antibody response; (b) an increase of killer activity of thymus cells; (c) an enhancement of thymocytes helper activity; (d) a reduction of theta-positive cells in the thymus; (e) a cellular depletion in the thymus cortex . These data, indicating that LPS selects in the thymus a population of cells more efficient in expressing both killer and helper functions, are interpreted as caused by an increased rate of cortisol secretion induced by the LPS treatment.

Schweiz Med Wochenschr, 1976 Jul 31, 106(31), 1056 - 9
{Gynecologic diseases in the diabetic woman}; Rey-Stocker I et al.; Gynecologic affections such as diabetic vulvitis, vulvo-vaginal mycosis, some dysfunctions of the ovary, recurrent abortion, secondary frigidity and sometimes carcinoma of the endometrium, may be the first signs of a disorder of carbohydrate metabolism . Contraception is particularly indicated in diabetic women . The methods are discussed.

Biochemistry, 1976 Jul 27, 15(15), 3281 - 90
Transcription of isolated mouse liver chromatin; Bacheler LT et al.; Analysis of RNA transcription from isolated mouse liver chromatin has been undertaken by means of RNA-excess hybridizations with small amounts of radioactive DNA . This analysis indicates that mouse liver chromatin is a restricted template for the in vitro synthesis of RNA complements to repetitive DNA, but more RNA species are synthesized than are found in the RNA isolated from mouse liver nuclei . Extraction with 0.5 M NaC1 destroys the template restriction of isolated chromatin . RNA synthesized in vitro from DNA or chromatin templates by Escherichia coli RNA polymerase, as well as in vivo mouse liver nuclear RNA, were each hybridized to 125I-labeled DNA of high, intermediate, or low reiteration frequency . Chromatin-primed and nuclear RNA saturate a smaller portion of each DNA fraction than does DNA-primed RNA . However, chromatin-primed RNA saturates more high and low reiteration frequency DNA than does nuclear RNA . Simultaneous hybridization of nuclear-and chromatin-primed RNA with 125I-labeled DNA indicates that chromatin-primed RNA contains all of the sequences present in nuclear RNA . Extraction of chromatin with 0.5 MNaC1 leads to removal of histone F1, as well as a wide variety of non-histone proteins . When used as a template for in vitro RNA synthesis, such salt-extracted chromatin produced RNAs that hybridize as large a portion of each DNA fraction as does DNA-primed RNA.

Biochemistry, 1976 Jul 27, 15(15), 3362 - 6
Messenger ribonucleic acid metabolism in mammalian mitochondria . Isolation and characterization of polyribosomes from Ehrlich ascites mitochondria; Lewis FS et al.; The Mg2+ precipitation procedure of R . D . Palmiter ((1974) Biochemistry 13, 3606) has been used for preparative scale isolation of polysomes from Ehrlich ascites mitochondria . Digitonin-washed metochondria used for isolating the polysomes contain no detectable reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase and over 200-fold reduced hexokinase activity . The mitochondrial polysomes exhibit a heterogeneous sedimentation and appear to contain highly aggregated particlses ranging over hexamers . These polysomes are sensitive to RNase, (ethylenedinitrilo)tetraacetic acid and puromycin . Mitochondrial polysomes are active in portein synthesis when supplied with supernatant enzymes from the homologous mitochondrial source or from Escherichia coli . Cytoplasmic enzymes, however, appear to be completely inactive . Protein synthesis by mitochrondrial polysomes is sensitive to chloramphenicol and resistant to cycloheximide and emetine . The procedure yields particles containing intact rRNAs . The extent of cytoplasmic RNA contaminating the total mitochondrial RNA or mitochondrial polysomal RNA has been estimated to be negligible.

Biochemistry, 1976 Jul 27, 15(15), 3320 - 30
Nuclear magnetic resonance investigation of the base-pairing structure of Escherichia coli tRNATyr monomer and dimer conformations; Rordorf BF et al.; The structures of the Escherichia coli tyrosine tRNA monomer and dimer have been investigated by high-resolution nuclear magnetic resonance (NMR) . At 23 degrees C the monomer contains 26 +/- 2 base pairs and the low-field NMR spectrum (11.7-15 ppm) can be accounted for in terms of the cloverleaf structure (23 base pairs) and three additional resonances that are assigned to tertiary structure base pairs . Assignments suggested for the various resonances are consistent with thermal denaturation studies in low-salt solutions . Under these conditions the temperature dependence of the spectrum can be interpreted in terms of sequential unfolding of the cloverleaf structure with the minor and dihydrouridine stems melting first, followed by the T psi C stem, the anticodon stem, and finally the amino acceptor stem . Certain features of the tertiary structure of tRNATyr are similar to other tRNA, but some details of the folding must be different, since no resonance from the S4U8-A14 tertiary base pair is observed . The tRNATyr dimer contains only 20 +/- 2 base pairs per tRNA (40/dimer) at 23 degrees C and a good account of the low-field NMR spectrum can be given in terms of a secondary structure in which bases of the T psi C stem and loop are involved in inter-molecular base pairing . Formation of the dimer requires opening of the hU and T psi C stems, but not the anticodon or amino acid acceptor stems, and this fits well with relative stabilities observed for these stems in the monomer . The model also provides an explanation for the formation of 2n-mers, that were stable enough to be separated by gel electrophoresis at room temperature (10 mM Mg2+) . Experimental conditions required for interconversion of monomer and dimer are also described.

Biochemistry, 1976 Jul 27, 15(15), 3254 - 8
Conformational transition of Escherichia coli RNA polymerase induced by the interaction of sigma subunit with core enzyme; Wu FY et al.; The isolated sigma subunit of Escherichia coli RNA polymerase has been labeled covalently with a fluorescent probe, N-(1-pyrene)maleimide . The labeled sigma subunit (PM-sigma) still retained its biological activity in stimulating transcription of T7 DNA by core enzyme . When a stoichiometric amount of core enzyme was added to a solution of PM-sigma, there was a decrease in fluorescence intensity without shifts in emission maxima of PM-sigma . The kinetics of the interaction between the sigma subunit and core enzyme was investigated with the stopped-flow technique by monitoring the fluorescence quenching . A biphasic change of fluorescence intensity with respect to time was observed when PM-sigma was rapidly mixed with an excess of core enzyme . The kinetic data can be analyzed in terms of a mechanism in which a fast bimolecular binding of sigma to core enzyme is followed by a relatively slow isomerization of the holoenzyme formed . From the best-fit kinetic parameters, an overall binding constant of less than or equal to 3X10(-10)M was estimated for the PM-sigma core complex, in agreement with that obtained by the fluorimetric titration . In addition, we have studied the effect of temperature on the rate constant associated with the conformational change of the holoenzyme, which shows a temperature transition around 20 degrees C . The nonlinear Arrhenius plot obtained implies that the conformational transition is complex and may be composed of several processes . The activation energy for the "overall" conformational change was estimated to be 6.7 kcal/mol . The kinetic evidence for the conformational transition of holoenzyme induced by the interactions of sigma subunit with core enzyme presented here further supports the proposition that the sigma subunit acts on core enzyme to trap a unique conformation of RNA polymerase which recognizes the proper promoters and initiates the synthesis of specific RNA chains.

Biochemistry, 1976 Jul 27, 15(15), 3220 - 8
Transition from noncooperative to cooperative and selective binding of histone H1 to DNA; Renz M et al.; A transition from noncooperative to cooperative binding of DNA and histone h1 occurs between 20 and 40mMNaCI in 5 mM Tris-HCI, pH 7.5 . Below 20mM NaCI in mixtures in H1 and excess DNA, H1 binds to all of the DNA molecules, causing them to sediment faster, and does not distinguish between DNA molecules that differ in size or base composition . However, at NaCI concentrations above the narrow transition range, H1 binds to only some of the DNA molecules and leaves the rest free . If the DNA molecules in a mixture are the same size, H1 selectively binds those that have the highest content of adenosine (A)+thymidine (T) . By means of competion experiments at salt concentrations spanning the transition range, it is demonstrated that H1 selectivity requires cooperativity . A high degree of selectivity based on A + T content can be produced by cooperative binding: for average DNA sizes of 2 X10(6) daltons, more than ten molecules of calf lymphocyte DNA (57% a+ t) are chosen per molecule of Escherichia coli DNA (50% A+T).

J Biol Chem, 1976 Jul 25, 251(14), 4372 - 8
Cytidine 5'-triphosphate synthetase of calf liver . Size, polymerization, and reaction stoichiometry; McPartland RP et al.; Calf liver CTP synthetase was purified 2000-fold from cytosol . The formation of 1 mol of CTP from UTP was accompanied by the cleavage of 1 mol of ATP to ADP and the formation of 1 mol of L-glutamate from L-glutamine . The stoichiometry of the liver enzyme reaction was identical with that found for the Escherichia coli B enzyme (Levitzki, A., and Koshland, D.E., Jr . (1971) Biochemistry 10, 3365-3371) . The liver enzyme was also similar to the bacterial enzyme in that it polymerized in the presence of kinetically saturating levels of ATP and UTP . In the absence of nucleotides the enzyme had a sedimentation coefficient of 6.8 and an average molecular weight of 133,000 as determined by sedimentation and molecular sieving . In the presence of ATP and UTP, the enzyme had a sedimentation coefficient of 10.1 and a molecular weight, as determined by molecular sieving, of 263,000 . The molecular weights of the two forms of the liver enzyme are about 25% higher than those of the corresponding forms of the bacterial enzyme (Long . C.W., Levitzki, A., and Koshland, D.E., Jr . (1970) J . Biol . Chem . 245, 80-87).

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