Escherichia coli

J Biol Chem, 1975 May 25, 250(10), 3660 - 5
Purification of thymidine phosphorylase from Escherichia coli and its photoinactivation in the presence of thymine, thymidine, and some halogenated analogs; Voytek P; Isoelectric focusing was used as the final step in the isolation of thymidine phosphorylase which was found to have an isoelectric point of 4.1 . Analytical acrylamide gel electrophoresis showed the purified enzyme preparation contained one major protein band which stained for thymidine phosphorylase activity and usually a minor, faster migrating band devoid of activity . Inactivation of thymidine phosphorylase alone or in the presence of sensitizers by ultraviolet light, primarily at 253.7 nm, followed first order inactivation kinetics . The rate of inactivation of the enzyme was the same at pH 5 and 7.4 and the addition of various pyrimidine bases and nucleosides enhanced the inactivation rate at both pH values, but to a greater extent at pH 5 . Linear plots of inactivation rates versus concentrations of thymidine or thymine were the same . At 7.8 mM thymidine or thymine, 11- and 4.4-fold increases in photoinactivation of thymidine phosphorylase were observed at pH 5 AND 7.4 RESPECTIVELY . Parabolic curves were obtained with increasing concentrations of either 5-iodo-2'-deoxyuridine or 5-iodouracil . 5-Iodouracil at 5.2 mM caused 212- (pH 5) and 100- (pH 7.4) FOLD INCREASES IN THE RATES OF PHOTOINACTIVATION OF THYMIDINE PHOSPHORYLASE . However, 5-iodo-2'-deoxyuridine at 5.0mM only enhanced the photoinactivation of enzyme by factors of 83 (pH 5) and 21 (pH 7.4) . Neither 5-bromo-2'-deoxyuridine or 5-bromo-uracil was as potent in sensitizing the enzyme as the iodo analogs . Combinations of 5-iodouracil or 5-iodo-2'-deoxyuridine with thymine resulted in higher inactivation rates than the additive inactivation rates of individual compounds, whereas combinations of either iodo analog with thymidine resulted in lower inactivation rates . Increasing concentrations of phosphate or NaCl lessened the photoinactivation rate of thymidine phosphorylase alone and protected the enzyme from the sensitization caused by the different bases and nucleosides . No quantitative changes in the number of primary amino groups in thymidine phosphorylase was evident as a result of irradiation in the presence or absence of 5-iodouracil or 5-iodo-2'-deoxyuridine . Examination of the irradiated enzyme on Sephadex G-150 indicated that a larger protein species is formed and that 5-iodouracil promotes this process.

J Biol Chem, 1975 May 25, 250(10), 3874 - 7
The role of polyamines in the aminoacyl transfer ribonucleic acid synthetase reactions . Demonstration of the requirement for magnesium ion and a secondary stimulatory effect of spermine; Santi DV et al.; Spermine and related polyamines have been reported to substitute for Mg2+ in the aminoacylation of tRNA catalyzed by aminoacyl-tRNA synthetases, but not in the ATP-PP-i exchange reaction . Such observations have led some workers to propose that these reactions proceed via a concerted mechanism rather than the usual two-step mechanism involving an aminoacyladenylate intermediate . In an attempt to elucidate the mechanism of the spermine effect on acylation and exchange, both reactions were re-examined using isoleucyl-tRNA synthetase from Escherichia coli . In the absence of added Mg2+ untreated tRNA was acylated in the presence of spermine, but tRNA from which Mg2+ had been scrupulously removed was not . ATP-PP-i exchange was not observed when spermine was used in place of Mg2+; however, if tRNA possessing sequestered Mg2+ was added, the exchange reaction was observed . These data suggest that a primary effect of spermine is to displace bound Mg2+ from tRNA in quantities sufficient to promote both the ATP-PP-i exchange and esterification of tRNA . The previously reported stimulatory effects of polyamines on these reactions are believed to be artifacts due to Mg2+ contamination of tRNA . Providing trace levels of Mg2+ are present, spermine exerts a secondary stimulation of the rate of aminoacylation, the mechanism of which is unknown . The results presented refute arguments that these enzymes proceed by a concerted mechansim and support the intermediacy of aminoacyladenylates.

J Biol Chem, 1975 May 25, 250(10), 3866 - 73
Kinetic demonstration of the intermediate role of aminoacyl-adenylate-enzyme in the formation of valyl transfer ribonucleic acid; Midelfort CF et al.; The question whether aminoacyl-tRNA synthetases act in a stepwise or in a concerted mechanism has been investigated kinetically with the valine enzyme of Escherichia coli, which had been used in previous studies by others who concluded that the physiological mechanism is concerted . An exchange between aminoacyl-tRNA and tRNA, dependent upon AMP, was studied . PP-i inhibits this exchange completely in the presence of Mg2+ and AMP but in the absence of added Mg2+ or with dAMP as the nucleotide the inhibition by PP-i is only partial; this is compatible with a stepwise, not a concerted, reaction . Exchange of isotopically labeled substrates in a system at chemical equilibrium also shows effects of substrate concentrations on rates in agreement with the predictions of a stepwise mechanism.

J Biol Chem, 1975 May 25, 250(10), 3679 - 82
Differentiation between binding and transport of dansylgalactosides in Escherichia coli; Schuldiner S et al.; The results presented in this paper confirm and extend previous observations which indicate that fluorescent dansylgalactsodes bind to the beta-galactoside carrier protein but do not penetrate the cytoplasmic membrane . The conclusion is supported by the following observations . (a) Although 2'-(N-dansyl)aminoethyl-beta-D-thiogalactopyranoside and 2'-(N-dansyl)aminoethyl-beta-D-galactopyranoside are competitive inhibitors of lactose transport in intact cells of Escherichia coli and induce the in vitro synthesis of beta-galactosidase, they do not induce beta-galactosidase in vivo . (b) p-Chloromercuribenzenesulfonate does not cause efflux of lactose from the intravesicular pool, but causes rapid reversal of D-lactate-induced dansylgalactoside fluorescence . (c) Dansylgalactosides inhibit dilution-induced, carrier-mediated lactose efflux.

Biochemistry, 1975 May 20, 14(10), 2043 - 50
Sequence holology between mitochondrial DNAs of different eukaryotes; Jakovcic S et al.; The sequence divergence of mitochondrial DNAs (mtDNA) from rat, mouse, guinea pig, monkey, and chicken has been examined by DNA-DNA hybridization . mtDNAs, isolated as closed circular molecules by propidium iodide-CsCl centrifugation, were labeled in vitro by use of Escherichia coli DNA polymerase I, and renatured (Tm-35 degrees) in the presence of a 2500-fold excess of heterologous mtDNA . Single-stranded and duples DNA were separated by hydroxylapatite chromatography . The thermal stability of heteroduplexes was compared to the homoduplex by thermal elution chromatography on hydroxylapatite columns . Heteroduplex fromation between the tritiated myDNAs and a 2500-fole excess of rar mtDNA were 70, 59, 37, and 22%, respectively, for mouse, guinea pig, monkey, and chicken . Similar results were obrained in reciprocal hybridizations where one of the other mtDNAs was present in excess . Considerable mismatching of sequences in all the heterohybrids was indicated by a 18-24 degrees depression in the te50 of the heteroduplexes compared with the homoduplex . There was no apparent change in heteroduplex formation when the concentration ratio of driving DNA in excess to {3H}mtDNA was varied between 1250 and 7500 . Furthermore, a second renaturation with excess driving DNA after completion of the first reaction resulted in no detectable augmenting of heteroduplex formation . Similar sequences appear to be conserved preferentially in different organisms, since the presence of two of fouf different heterologous mtDNAs in excess resulted in only moderate and nonadditive increases in heteroduplex formation . Evolutionary divergence of mtDNA sequences appears to have occurred at rates similar to that for unique sequences nuclear DNA.

Biochemistry, 1975 May 20, 14(10), 2037 - 42
Sequence homology of the mitochondrial leucyl-tRNA cistron in different organisms; Jakovcic S et al.; Sequence divergence of the mitochondrial leucytl-tRNA cistron in several eukaryotes has been examined by RNA-DNA hybridized . Rat mitochondria Leucyl-tRNA was hybridized with rat, mouse, guinea pig, monkey, chicken, and yeast mitochondrial DNAs (mtDNA) immobilized onfilters . Hybridization was carried out in 50% formamide (Tm -12degrees) or in 20% fromamide (Tm -21degrees) . melting profiles of the hybrids were obtained for evaluation of the extent of base sequence micmatching . Under the more stringent hybridization conditions (50% formamide, Tm -12degrees), only mouse and quines pig mtDNAs hybridized with rat mitochondrial leucyl-tRNA . The Tm's of the heterohybrids were depressed by 2 and 9 degrees, respectively . Under less stringent hybridization conditions (Tm-21 degrees), monkey mtDNA also hybridized, and the Tm was depressed by about 15 degrees . Chicken and yeast mtDNAs did not form specific hybrids with rat mibochondrial leucyl-tRNA under these hybridization conditions . Mitochondrial leucyl-tRNA sequences in different eukaryotes appear to be conserved to a less extent than cytoplasmic rRNA, 5S RNA, or hemoglobin mRNA sequences.

Biochemistry, 1975 May 20, 14(10), 2219 - 24
Nuclear magnetic resonance study of ligand binding to Mn-aspartate transcarbamylase; Fan S et al.; Aspartate transcarbamylase from Escherichia coli has been prepared with up to four of zinc ions replaced by manganese, and the effect of this substitution on the proton nuclear magnetic resonance properties of succinate bound to the catalytic site and of cytidine 5'-triphosphate bound to the regulatory site has been determined, The specific activity and allosteric properties of the Mn-substituted enzyme are essentially identical with those of the native enzyme . The longitudinal relaxation time, T1, of the succinate protons is shortened by the native enzyme and is shortened further by the Mn-substituted enzyme at both 100 and 220 MHz in D2O solutions of 0.02 M immidazole chloride (pH 7.0), 10 minus 3 M beta-mercaptoethanol, 0;2 mM ethylenediamenetetraacetic acid, and 2.5 mM carbamyl phosphate over a temperature range of 5 to 35 degrees . Under the same conditions, the transverse relaxation time, T2, of the succinate protons at 90 MHz is shortened to the same extent by native and Mn-substituted enzyme . The temperature dependence of the relaxation times indicates that the shortening of the transverse relaxation time is determened by the lifetime of bound succinate, whereas the further shortening of the longitudinal relaxation time by the Mn-substituted enzyme is due to dipolar relaxation, i.e . to the interaction between Mn and the succinate protons . The distance between the Mn and the protons of succinate bound to the enzyme can be calculated from the relaxation time measurements and is 15,3 A . The dipolar interaction correlation time which is needed for the calculation of this distance, was found to be 3.5 X 10 minus 9 sec from the frequency dependence of T1 . The transverse relaxation time of the C-6 proton of CTP is shortened to the same extent by both the native and Mn-substituted enzyme in D2O solutions of 0.02 M imidazole chloride (pH 7.0), 10 MINUS 3 M beta-mercaptoethanol, 0.2 mM ethylenediaminetetraacetic acid, and 2.5 mM carbamyl phosphate over the temperature 5-30 degrees . Since the temperature depencece of the relaxation time indicates the relaxation is not exchange limited, the manganese must be too distant from the bound CTP for an appreciable interaction to occur . This requires that the manganese be greater than 20A from the CTP . These results are used together with other available structural data to construct a schematic model for aspartate transcarbamylase.

Biochemistry, 1975 May 20, 14(10), 2275 - 82
Zinc and magnesium content of alkaline phosphatase from Escherichia coli; Bosron WF et al.; Since alkaline phosphatase from Escherichia coli was first reported to contain 2.1 g-atoms of zinc and 0.8 g-aton of magnesium per molecular weight 80,000 (Plocke, D.J., Levinthal, D., and Vallee, B . L . (1962), Biochemistry 1, 373-378), the procedures for isolation and purification of the enzyme, as well as values for the protein molecular weight, specific absorptivity, and maximal activity, have changed repeatedly . Such variations have resulted in uncertainties concerning the molar metal content of this phosphatase . The present paper reviews the initial and recent results of metal analyses of alkaline phosphatase preparations in this laboratory and compares them with those obtained elsewhere, while simultaneously identifying some of the factors which have affected reports on the metal content of this enzyme . A purification procedure is described eliminating the features of all methods known to alter the metal content of phosphatase . In addition, the three isozymic forms, as well as preparations from four E . coli strains commonly employed for phosphatase isolation, were analyzed and compared.

Biochim Biophys Acta, 1975 May 16, 390(3), 312 - 8
Difference in the action of ethyl and methyl methane sulfonates on DNA template activity for RNA synthesis in vitro; Guertin M et al.; RNA produced in vitro from alkylated T7 DNA has been characterized by polyacrylamide gel electrophoresis . Methylation of T7 DNA by methyl methane sulfonate reduces RNA chain length . In contrast, ethylation of T7 DNA by ethyl methane sulfonate, while reducing RNA synthesis to the same extent, does not alter chain length.

N Engl J Med, 1975 May 15, 292(20), 1041 - 5
Enterotoxigenic Escherichia-coli-associated diarrheal disease in Apache children; Sack RB et al.; A search for intestinal enterotoxigenic Escherichia coli was made in 59 Apache children hospitalized with 64 episodes of acute diarrhea . Esch . coli isolates from acute-phase and convalescent-phase specimens of small-bowel fluid and stool were tested in three currently recognized models: the adult-rabbit ileal loop; infant rabbit; and the adrenal-cell assay . Enterotoxigenic strains were isolated from 10 children during acute diarrheal episodes (16 per cent); none were isolated from convalescent-phase specimens . None of 64 "enteropathogenic" serotypes of Esch . coli from 43 children with diarrhea, however, caused fluid production in the ileal-loop model . These results suggest that enterotoxigenic Esch . coli may be the cause of considerable diarrhea in this population and that the term "enteropathogenic" as applied to serotypes of Esch . coli needs to be redefined.

Cancer Chemother Rep, 1975 May-Jun, 59(3), 611 - 20
Interaction of Rhodium(II) carboxylates with molecules of biologic importance; Bear JL et al.; Rhodium(II) acetate, propionate, and butyrate showed a considerable variation in their antitumor activity against Ehrlich ascites tumor cells in mice, with the butyrate complex being the most active . The three complexes markedly inhibited DNA synthesis of Ehrlich ascites tumor cells in vivo . Rhodium (II) butyrate was the most potent inhibitor followed by the propionate complex . One hour after administration, rhodium(II) propionate and butyrate induce more uridine-5-3H incorporation into RNA than is seen in the controls . Equilibrium dialysis studied showed that rhodium(II) acetate-1-14C binds to single stranded DNA, poly-A, ribonuclease A, and bovine serum albumin but not to highly polymerized native calf thymus DNA, poly-G, or poly-C . In these cases binding occurred at the two axial positions of rhodium(II) acetate to a nitrogen donor in the ligands . The formation constants of the rhodium(II) acetate and propionate complexes with 5'-adenosine monophosphate were determined . The rhodium(II) propionate complex was more stable . Sedimentation and viscosity measurements of poly-A and poly-A/rhodium(II) acetate complexes indicate a high degree of intramolecular crosslinking in the rhodium(II) acetate/poly-A complex . The rhodium(II) carboxylate complexes were also found to be potent inhibitors of purified DNA polymerase I and RNA polymerase from Escherichia coli.

Klin Wochenschr, 1975 May 1, 53(9), 441 - 3
{Cholestatic jaundice following disseminated intravascular coagulation ("shock-liver") (author's transl)}; Kunzer W et al.; Recently we gained increasing evidence of the fact that cholostatic syndromes in young infants are frequently associated with preceeding shock and disseminated intravascular coagulation . This is illustrated by the case of a 4 weeks-old male infant with urosepticemia due to E . coli, which was successfully treated with additional streptokinase . Therefore, in the age-group of early infancy we have to pay attention to the liver as a potential shock-organ similar to the well known shock organs such as kidneys, lungs, skin and brain.

J Biol Chem, 1975 May 10, 250(9), 3352 - 8
Kinetic and equilibrium studies on the activation of Escherichia coli K12 tryptophanase by pyridoxal 5'-phosphate and monovalent cations; Hogberg-Raibaud A et al.; An improved purification of Escherichia coli K12 tryptophanase is presented . It is shown that the apoenzyme crystals, oxidized by exposure to air, can be reactivated by treatment with a reducing agent . The titration of sulfhydryl groups shows that four --SH groups are exposed and two are masked per protomer . The influence of two effectors, monovalent cations and the coenzyme pyridoxal 5'-phosphate, on the reactivity of --SH groups and the enzymatic activity was investigated . The --SH groups react more slowly in holo- than in apoenzyme in the presence of potassium ions . If these ions are replaced by sodium ions, the reactivity becomes the same . Potassium and ammonium ions, both activators, give sigmoidal activation curves . The sodium ion is a Michaelian inhibitor of potassium activation . The binding of pyridoxal 5'-phosphate was examined by kinetics and at equilibrium . The kinetics are shown to be very slow; the rate constants of the forward and reverse reactions have been measured . The binding equilibrium, examined with 3H-labeled pyridoxal 5'-phosphate, gives one site per protomer with a K-D value of (3.2 plus or minus 0.8) times 10-7 M . The K-m for pyridoxal-P was determined by activity measurements . The binding equilibrium is attained after several hours, giving a value of 4.2 times 10-7 M, being nearly identical with the dissociation constant and 5 times smaller than previously reported.

J Biol Chem, 1975 May 10, 250(9), 3545 - 51
Purification and properties of guanosine triphosphate cyclohydrolase II from Escherichia coli; Foor F et al.; An enzyme that uses GTP as substrate for the formation in stoichiometric quantities of formate, inorganic pyrophosphate, and 2,5-diamino-6-hydroxy-4-(ribosylamino)pyrimidine-5'-phosphate has been purified 2200-fold from extracts of Escherichia coli B . This enzyme is named GTP cyclohydrolase II to distinguish it from a previously studied E . coli enzyme, named GTP cyclohydrolase (and called GTP cyclohydrolase I in this paper), that catalyzes the first of a series of enzymatic reactions leading to the biosynthesis of the pteridine portion of folic acid (Burg, A . W., and Brown, G . M . (1968) J . Biol . Chem . 243, 2349-2358) . Some of the properties of GTP cyclohydrolase II are: (a) divalent cations are required for activity (Mg2+ is most effective); (b) its molecular weight, estimated by filtration on Sephadex G-200, is 44,000; (c) the K-m for GTP is 41 mum; (d) its pH optimum is 8.5; and (e) its activity is inhibited by inorganic pyrophosphate, one of the products of the reaction . Compounds not used as substrate are: GDP, GMP, guanosine, dGTP, ATP, ITP, and XTP . Properties a, b, c, and e (above), as well as the nature of the products, distinguish this enzyme from GTP cyclohydrolase I . Since GTP cyclohydrolase II apparently is not concerned with the biosynthesis of folic acid, the possible physiological role of this enzyme in the biosynthesis of riboflavin is considered in the light of the present investigations and the previously published work on riboflavin biosynthesis by other investigators.

J Biol Chem, 1975 May 10, 250(9), 3505 - 9
Use of the sodium borohydride reduction technique to identify a gamma-glutamyl phosphate intermediary in the Escherichia coli glutamine synthetase reaction; Todhunter JA et al.; Incubation of unadenylylated Escherichia coli glutamine synthetase with ATP, L-{14C}glutamate and metal ion results in the formation of gamma-glutamyl-P which can under appropriate conditions be reduced by sodium borohydride . The acyl-P compound is formed catalytically as judged by the quantity of radioactive alpha-amino-delta-hydroxyvalerate produced compared to the concentration of enzyme subunits . Formation of the glutamyl-P compound occurs in the presence of magnesium or manganous ions, and the relation of this apparent lack of metal ion specificity with regard to the highly specific Mg2+-supported biosynthetic activity of the unadenylylated form is discussed.

J Biol Chem, 1975 May 10, 250(9), 3261 - 6
Enhancement of the glutaminase activity of carbamyl phosphate synthetase by alterations in the interaction between the heavy and light subunits; Wellner VP et al.; Glutamine-dependent carbamyl phosphate synthetase (from Escherichia coli) was previously shown to be composed of a light subunit (molecular weight similar to 42,000) which has the binding site for glutamine and a heavy subunit (molecular weight similar to 130,000) which has binding sites for the other reactants and allosteric effectors . The subunits may be separated with retention of catalytic activities; only the separated light subunit exhibits glutaminase activity . The previous finding that storage of the native enzyme at pH 9 at 0 degrees increased its glutaminase activity by about 25-fold was further investigated; such storage markedly decreased the glutamine- and ammonia-dependent synthetase activities of the enzyme . Treatment of the enzyme with p-hydroxymercuribenzoate led to transient increase of glutaminase activity followed by inhibition . When the enzyme was treated with N-ethylmaleimide or with 5,5'-dithiobis-(2-nitrobenzoate), the glutaminase activity was increased by about 250-fold with concomitant loss of synthetase activities . The enhancement of glutaminase produced by storage of the enzyme at pH 9 was associated with intermolecular disulfide bond formation and aggregation of the enzyme . Aggregation also was observed after extensive treatment of the enzyme with 5,5'-dithiobis-(2-nitrobenzoate) or N-ethylmaleimide . However, a moderate increase of glutaminase activity (about 30-fold) was observed without aggregation under conditions in which one sulfhydryl group on the light subunit reacted with either reagent . The findings suggest that the increased glutaminase activities observed here are associated with structural changes in the enzyme in which the intersubunit relationship is altered so as to uncouple the catalytic functions of the enzyme and to facilitate access of water to the glutamine binding site on the light subunit.

J Biol Chem, 1975 May 10, 250(9), 3243 - 53
The nucleotide sequences of the two glutamine transfer ribonucleic acids from Escherichia coli; Yaniv M et al.; The nucleotide sequences of the two glutamine tRNA species in Escherichia coli K12 have been determined . Sufficient data was obtained to order unambiguously the products of complete RNase digestion of tRNA2Gln, and all but one oligonucleotide from tRNA1Gln . The sequence of tRNA1Gln was established by analogy with tRNA1Gln, as the two tRNAs are very similar, differing by only 7 residues out of 75 . tRNA1Gln has the anticodon NUG, where N is a modified nucleotide which is likely to be a derivative of 2-thiouridine, and is specific for the codon CAA . tRNA1Gln has the anticodon CUG, and is specific for the codon CAG (Folk, W . R., and Yaniv, M . (1972) Nature 237, 165) . The complete sequences of the tRNAGln species are: See journal for formula (Unique residues are enclosed in parentheses, with the residue in tRNA1Gln above that in tRNA2Gln.).

Eur J Biochem, 1975 May 6, 53(2), 605 - 13
Comparative effect of heparin on RNA synthesis of isolated rat-liver nucleoli and purified RNA polymerase A; Ferencz A et al.; The polyanion heparin has been employed to study the interaction of rat liver DNA-dependent RNA polymerase A and its template under various conditions . Heparin very efficiently inhibits polymerase molecules, which are not bound to DNA or are associated with the template in a loose, i.e . non-specific fashion . Purified nucleoli, isloated from rat liver nuclei, contain RNA polymerase A in abundant quantities of which only a portion is bound in a transcriptional complex . Excess enzyme, which is contained in the nucleolus in a quasi free form, can be transferred to an exogenously added template and can be completely inhibited by the prior addition of heparin . The enzyme contained in a transcriptional complex, however, initiated in vivo and completing these RNA chains in vitro, is fully resistant to heparin . In contrast to these results it has been found that RNA polymerase A extracted from nuclei and purified by various chromatographic steps does not form heparin-resistant complexes, even after the enzyme has been bound to the DNA template . Moreover it has been found that purified RNA polymerase A transcribes truly native DNA extremely poorly, indicating that the enzyme is highly deficient in the act of initiation on duplex DNA . It is therefore questionable whether the interaction of the purified enzyme and isolated DNA represents binding to true initiation complexes as is observed in the intact nucleolus.

Eur J Biochem, 1975 May 6, 53(2), 599 - 603
The effect of tRNA derivatives bound with natural or synthetic mRNA on the interaction of Escherichia coli ribosomes with colicin E3; Kaufmann Y et al.; Ribosomal binding complexes directed by poly(U) or T4 mRNA were formed with aminoacyl-tRNA or its derivatives bound to predominantly the P or A binding site . The defined binding complexes were reacted with colicin E3 and the reaction was assessed by the ability of the complexes to proceed with polypeptide synthesis . The results indicated that only one of the four complexes tested was completely resistant to colicin E3-induced inactivation: that of Phe-tRNA bound in the presence of poly(U) to the A-site . The poly(U) directed complex of AcPhe-tRNA and the T4-mRNA-directed complex at the A-site appeared slightly resistant, while the T4 mRNA initiation complex was inactivated by colicin E3 in a manner similar to non-complexed ribosomes . Colicin E3 added to ribosomes after protein synthesis had been initiated affected the subsequent polymerization in a manner corresponding to the response of the binding complexes . Thus, poly(U)-translating ribosomes were less affected than ribosomes translating the viral mRNA . The vulnerability of natural-mRNA-directed binding complexes to inactivation by colicin E3 is in accord with the mode of inactivation by the colicin in vivo.

Eur J Biochem, 1975 May 6, 53(2), 419 - 27
Glucose catabolite repression in Escherichia coli K12 mutants defective in methyl-alpha-d-glucoside transport; Bourd GI et al.; 1 . Two spontaneous Escherichia coli K12 mutants resistant to glucose catabolite repression were isolated using minimal agar plates with methyl alpha-D-glucoside . Mutants grow well on glucose and mannitol . 2 . Glucose does not inhibit the inducible enzyme synthesis in isolated mutants: mutant cell (in contrast to parent cells) produce high levels of beta-galactosidase and L-tryptophanase under the conditions of glucose catabolite repression . 3 . The isolated mutants are negative in methyl-alpha-D-glucoside transport; glucose uptake is not severely damaged . But the mutants (named tgl, transport of glucose) retained the ability to phosphorylate methyl alpha-D-glucoside in vitro at the expense of phosphoenolpyruvate . 4 . The tgl mutation is cotransduced with purB and pyrC markers, i.e . locates near 24 min of the E . coli chromosome map . 5 . It is thought that E . coli cells possess two glucose transport systems . The first one is represented by the glucose-specific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system . The second glucose transport system (coded for tgl gene) functions as permease and possesses high affinity to methyl alpha-D-glucoside . The integrity of glucose permease determine the sensitivity of the cell to glucose catabolite repression.

Biochim Biophys Acta, 1975 May 6, 389(2), 358 - 69
Lipid phase transitions in cytoplasmic and outer membranes of Escherichia coli; Overath P et al.; The cytoplasmic and outer membranes containing either trans-delta-9-octadecenoate, trans-delta-9-hexadecenoate or cis-delta-9-octadecenoate as predominant unsaturated fatty acid residues in the phospholipids were prepared from a fatty acid auxotroph, Escherichia coli strain K1062 . Order-disorder transitions of the phospholipids were revealed in both fractions of the cell envelope by fluorescent probing or wide angle X-ray diffraction . The mid-transition temperatures, Tt, and the range of the transition, delta-T, are similar in the outer and cytoplasmic membrane . Relative to the corresponding extracted lipids, 60-80% of the hydrocarbon chains take part in the transition in the cytoplasmic membrane whereas in the outer membrane only 25-40% of the chains become ordered . The results suggest that in the outer membrane part of the lipids form fluid domains in the form of mono- and/or bilayers.

Biochim Biophys Acta, 1975 May 6, 389(2), 236 - 50
Membrane reconstitution in chl-r mutants of Escherichia coli K 12 . IX . Part played by phospholipids in the complementation process; Azoulay E et al.; The supernatant extracts of the chl A and chl B mutants of Escherichia coli K 12, the phospholipids of which are labeled by growth in 32 P or {2- 3H}glycerol media, contain 20 times more radioactivity than the supernatant extract of the wild-type strain grown under the same conditions . We have observed that, after complementation, 80% of the radioactivity previously contained by Extracts A and B is incorporated into reconstituted particles . The chromatography of 3H-labeled Extract B on DEAE-cellulose and followed by gel filtration of radioactive fractions on Sephadex G-200 has shown that the phospholipids of Extract B are only bound to soluble proteins and not to fragments of membranes; it can be assumed that they have been solubilized in the form of a lipid-protein complex by cell breakage . When Extracts A and B are treated by phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) before being mixed together, an inhibition of the reconstitution of nitrate reductase activity which is proportional to the phospholipase C concentration and the length of treatment is observed . The analysis of lipids and phospholipids of particles (Peak I, Peak II and Peak III) formed during complementation and reconstituted nitrate reductase shows that their phospholipid contents (phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and phosphatidylserine) and especially that of Peak II (d equals 1.18) are closely related to that of native particles from the wild-type strain . These results allow one to propose a hypothesis explaining the mechanism involved in complementation.

Biochim Biophys Acta, 1975 May 6, 389(2), 219 - 35
Membrane reconstitution in chl-r mutants of Escherichia coli K 12 . VIII . Purification and properties of the FA factor, the product of the chl B gene; Riviere C et al.; The isolation and purification of the product of the chl B gene of Escherichia coli K 12 from the chl A mutant have been attempted . The purified protein gives a single band in 10% sodium dodecylsulfate/polyacrylamide gel electrophoresis . The molecular weight is estimated to be 35 000 . This protein, that we have named "FA factor", does not contain any lipid, has a strong tendency to lose its activity by polymerizing but can be kept in an active state when stored in buffer containing NaCl . The addition of purified FA protein to a soluble extract from the chl B mutant strain grown under anaerobiosis in the presence of nitrate initiates the "complementation reaction", i.e . the reconstitution of the nitrate reductase activity and the formation of particulate material similar to the native membrane with respect to the structure and enzymatic function . FA protein acts both on the rate of reconstitution and on the total amount of reconstituted enzyme . The complementation leads to the reconstitution of nonsedimentable nitrate reductase and to the formation of three types of particles of different buoyant densities (1.10, 1.18 and 1.23) the two lightest of which contain nitrate reductase . It is shown that FA factor is incorporated only into the particles of intermediate density . In vivo, this factor is located in the native membranes of chl A, chl C, chl D and wild-type strains, whatever the growth conditions, aerobiosis or anaerobiosis, and in the presence or absence of nitrate . Protein FA can be released from either of these membranes (native or reconstituted) by removing Mg-2+ or by subjecting Kaback's vesicles to mechanical treatments; in the case of 1.18-reconstituted particles and wild-type membranes, the release of FA protein does not exert any effect on the level of the nitrate reductase activity.

Biochemistry, 1975 May 6, 14(9), 1869 - 76
Conformations and structural transitions in polydeoxynucleotides; Pilet J et al.; Polydeoxynucleotides of different base sequence, the alternating poly{d(A-T)}-poly{d(A-T)}, crab satellite DNA, on the one hand, and double-stranded homopolymer complexes poly{d(A)}-poly{d(T)}, poly{d(I)}-poly{d(C)}, on the other, display significant differences in their conformation and conformational transitions . Infrared linear dichroism investigations indicate that the alternating poly{d(A-T)}-poly{d(A-T)}, enzymatically synthesized, adopts a lower humidity a well-expressed A* form in which stability is relatively small,i.e., restricted to limited relative humidity . This A form is characterized by the orientation of the bisector of the phosphate OPO group at 34 degrees with respect to the helical axis, which is slightly lower than that of DNA . In contrast, for the homopolynucleotide double-stranded complex poly(dA)-poly(dT) and also for poly(dI)-poly(dC), the B yields A conformational change is not observed . Instead poly(dA)-poly(dT) exists at lower humidity in a stable modified B form . Thus the present results indicate that homo(dA)-homo(dT) double-stranded sequences prevent the B yields A structural transition . All AT-containing polydeoxynucleotides and crab satellite DNA adopt a high humidity a modified B form characterized by the orientation of the bisector of the phosphate group OPO at 64 degrees with respect to the helical axis which is significantly lower than 68-74 degrees observed in DNAs . The base pairing geometry in poly(dA)-poly(dT), poly{d(A-T)}-poly{d(A-T)}, and also in poly(dI)-poly(dC) is apparently a Watson and Crick type . Thus the observed differences in conformation are not due to different base pairing scheme . It is suggested that in DNAs of high AT content the presence of homo(dT)-homo(dA) sequences and the relatively low stability of the A form of d(A-T) alternating sequences may inhibit the change to the A form . A possible role of these sequences in DNA recognition by protein is suggested.

Biochemistry, 1975 May 6, 14(9), 1859 - 66
Evidence for fidelity of chromatin reconstitution; Stein GS et al.; Several lines of evidence are presented which support the contention that chromatin may be dissociated, fractionated, and reconstituted without altering the compositional, structural, or transcriptional integrity of the genome . The similar compositions of native and reconstituted chromatins are suggested by the absence of significant differences in their protein/DNA ratios and in the polyacrylamide gel electrophoretic profiles of their histones and nonhistone chromosomal proteins . Criteria for fidelity of genome structure in reconstituted chromatin include binding of reporter molecules with specificity for the minor groove of DNA, binding of histones, number of sites available for addition of nucleotides, and circular dichroism spectra . When the transcriptional activities of native and reconstituted chromatins were compared under conditions where reinitiation is prohibited, significant changes were not observed . Taken together, the present results strongly suggest, but do not conclusively establish, fidelity of chromatin reconstitution.

Biochemistry, 1975 May 6, 14(9), 1805 - 14
A comparative study of the 50S ribosomal subunit and several 50S subparticles in EF-T-and EF-G-dependent activities; Sander G et al.; A series of ribosomal subparticles derived from the 50S subunit has been studied and compared in EF-T- and EF-G-dependent reactions . Three different 50S cores were prepared by CsC1 isophycnic centrifugation and one by NH(4)Cl-ethanol extractionm the 50S CsCl core a had lost proteins L1, L7, L8, L10, L12, L16, L25, L33, and some L6 and L11 . The 50S CsCl core b additionally lacked protein L6, and 50S CsCl core c also lacked protein L5, L15, L18, L27, L28, L30, and most of L9, L14, L19, and L21 . The 50S NH(4)Cl-ethanol core had lost up to 90 percent of proteins L7, L12 and 30-60 percent of proteins L8, L10, and L29 . The 50S CsCl core a had much reduced activity in EF-G and none in EF-T GTPase reactions while 50S CsCl cores b and c were inactive . Addition of proteins L7, L12 restored the activity for both the EF-T- and EF-G-dependent GTPase with all of the three 50S CsCl cores, increasing stepwise from core c to core a; The 50S NH(4)Cl-ethanol core was partially active in the EF-G GTPase over the 2-30 mM MG-2+ range tested, while EF-T only showed some activity inthe upper portion of this range...

Biochemistry, 1975 May 6, 14(9), 1980 - 9
Regulation of Escherichia coli glutamine synthetase . Evidence for the action of some feedback modifiers at the active site of the unadenylylated enzyme; Dahlquist FW et al.; The interaction of unadenylylated form of Escherichia coli glutamine synthetase with several substrates and effectors has been examined by magnetic resonance techniques . These studies show that two manganese ions bind per enzyme subunit . From the dramatic line broadening observed in the alanine spectra in the presence of manganese and enzyme, it is concluded that the binding of alanine occurs at a site nearer one of the two manganese sites . Electron spin resonance (ESR) titration experiments suggest apparent dissociation constants of 20 and 120 muM for manganese to these sites in the presence of 1.0 mM magnesium ion . The manganese concentration dependence of the broadening of alanine suggests an affinity of 30 muM for the manganese closest to the alanine binding site . This suggests that alanine binds closer to the more tightly bound manganese ion . Glutamate appears to displace the alanine and also appears to bind close to the strongly bound manganese ion . It is proposed that alanine and glutamine bind competitively and in the same site . The binding of alanine and ATP is shown to thermodynamically interact such that the presence of one ligand increases the affinity of the enzyme for the other ligand . The presence of ATP dramatically sharpens the alanine line width when manganese and glutamine synthetase are present . Addition of ADP or phosphate alone has little effect on the alanine line width but the addition of both ADP and phosphate shows the same dramatic sharpening as the addition of ATP alone, suggesting an induced fit conformational change in the enzyme induced by ATP or by both ADP and phosphate . A binding scheme is proposed in which all feedback inhibitors of the enzyme bind in a competitive fashion with substrates.

Biochim Biophys Acta, 1975 May 6, 389(2), 370 - 9
Requirement of heat and metabolic energy for the expression of inhibitory action of colicin K; Okamoto K; Escherichia coli B, induced for beta-galactoside permease, can accumulate thio-methyl-beta-galactoside in the cell even at 0 degrees D . At this temperature, cells adsorb colicin K but the adsorbed colicin does not inhibit thiomethyl-beta-galactoside uptake . Inhibition by colicin K is, however, seen at 0 degrees C after exposure of the colicin K-cell complex to a high temperature: a greater degree of inhibition occurs with increasing temperature or duration or exposure . There is a transition point at around 21 degrees C in Arrhenius plots of this colicin K activation reaction . If inhibitors of energy yielding reactions are present during the heat treatment, the inhibitory action of colicin K (as measured by thiomethyl-beta-galactoside uptake after returning the colicin K-cell complex to 0 degrees C and removal of the inhibitors) is prevented . These results indicate that adsorbed colicin K is converted into the active state only in the presence of metabolic energy and that cell surface fluidity appears to be concerned in this process.

Biochemistry, 1975 May 6, 14(9), 1956 - 64
2'-O-methylated oligonucleotides in ribosomal 18S and 28S RNA of a mouse hepatoma, MH 134; Hashimoto S et al.; Simple two-dimensional thin-layer chromatography was found to be useful for the separation of sugar methylated dinucleotides in RNA . Of the 16 possible sequences of the type Nm-Np, 15 were separated and all the sequences were determined . In a mouse hepatoma, MH 134, the levels of the sugar methylation in the 18S and 28S RNA molecules were 17-18 and 11-12 per 1000 nucleotides, respectively . Thus, 18s RNA contained approximately 35 2'-O-methylated dinucleotides and 28S RNA approximately 60 2'-O-methylated dinucleotides . The pattern of distribution was also distinct between these two molecules . Two 2'-O-methylated trinucleotides were identified in the 28S RNA with the sequences Um-Gm-Up and Um-Gm-psip . A unique 2'-O-methylated tetranucleotide was present also in the 28S RNA, the sequence of which was Am-Gm-Cm-Ap . The 5'-terminal nucleotides of both 18S and 28S RNA were obtained as nucleoside 3',5'-diphosphates (pNp) in the trinucleotide fraction of the RNase T2 digest . The 5'-termimi of 18S and 28S RNA were pUp and pCp, respectively, and found to be almost homogeneous.

Biochemistry, 1975 May 6, 14(9), 1866 - 8
The role of superoxide radical in the autoxidation of cytochrome c; Cassell RH et al.; The net rate of autoxidation of ferrocytochrome c was decreased by ferricytochrome c . Superoxide dismutase accelerated this autoxidation to a limit and overcame the inhibitory effect of ferricytochrome c . This was the case whether the autoxidationwas observed in the presence or in the absence of denaturants, such as alcohols orurea, and whether the superoxide dismutase used was the Cu-2+-Zn-2+ enzyme from bovine erythrocytes or the Mn-3+-enzyme from Escherichia coli . It can be deduced that the autoxidation of ferrocytochrome c, under a variety of conditions, geenerates O2 minus which can then dismute to H202 + O2 or can reduce ferricytochrome c back to ferrocytochrome c . Superoxide dismutase, by accelerating the dismutation of O2 minus, prevents the back reaction and thus exposes the true rate of reaction of ferrocytochrome c with molecular oxygen.

Biochemistry, 1975 May 6, 14(9), 1821 - 5
Different cyclic adenosine 3',5'-monophosphate requirements for induction of beta-galactosidase and tryptophanase . Effect of osmotic pressure on intracellular cyclic adenosine 3,5-monophosphate concentrations; Piovant M et al.; In this study we have tried to answer the following questions: (1) is it possible for different catabolite-repressible genes, although submitted to the same control, to be expressed selectively depending upon the growth conditions, and (2) what is the effect of increasing the osmolarity of the medium on the intracellular level of cAMP? Two conditions were found to cause a continuous variation of intracellular cAMP levels during growth . With different strains, higher cAMP levels are required for induction of the tryptophanase gene than one required for induction of the lactose operon . cAMP has been provided externally in adenyl cyclase minus cells of a mutant that has been made permeable by EDTA treatment . Although external cAMP concentrations, 10 times higher than the usual intracellular levels, are required for induction of beta-galactosidase and tryptophanase, the difference of requirements of cAMP is maintained . An increase in the osmolarity of the medium by sucrose addition causes a fourfold decrease in the intracellular cAMP level . As a consequence this prevents the induction of tryptophanase whereas beta-galactosidase is still inducible . After pulse induction, a difference in the kinetics of expression of the tryptophanase and beta-galactosidase genes was found . Its relationship with the previous results is discussed.

Biochim Biophys Acta, 1975 May 6, 389(2), 203 - 18
Membrane reconstitution in chl-r mutants of Escherichia coli K 12 . VII . Purification of the soluble ATPase of supernatant extracts and kinetics of incorporation into reconstituted particles; Giordano G et al.; Membrane-bound ATPase (EC 3.6.1.3) of Escherichia coli K 12 is released in a soluble form by the mechanical treatments applied to the cells in order to break them . The purification of the soluble enzyme is described . The purified protein gives a single band in 7.5% polyacrylamide gel electrophoresis . The molecular weight is estimated to be 350 000 . The enzyme is cold-labile, Mg-2+ dependent, insensitive to inhibition by N, N'-dicyclohexylcarbodiimide and specific for ATP and ADP . Membranes depleted of their ATPase activity by dilution in a buffer of low ionic strength and without Mg-2+ are able to incorporate the purified ATPase only in the presence of 2-6 mM Mg-2+ . ATPase binds to particles formed by complementation between supernatant extracts of chl A and chl B mutants . There are three kinds of particles of different buoyant densities (1.10, 1.18 and 1.23); ATPase binds only to the 1.10 and 1.18 particles . The kinetics of incorporation have been studied . ATPase begins to be incorporated into the 1.10 particles after 10 min of incubation up to a maximum at 20 min: from 30 min, ATPase is incorporated only into 1.18 particles and the amount of incorporated ATPase increased in proportion with the peak of 1.18 particles . These kinetics have a hyperbolic pattern . In order to explain the mechanism of assembly involved in complementation, two hypotheses are proposed.

Nucleic Acids Res, 1975 May 5, 2(5), 735 - 44
Stimulation of transcription of mouse kidney chromatin by sulfated polysaccharides; Warnick CT et al.; The sulfated polysaccharides polydextran sulfate (PDS) and heparin stimulate in vitro transcription of mouse kidney chromatin by E . coli RNA polymerase by about 100 and 40 fold respectively . Heparin which has been N-desulfated and N-acetylated stimulates only 13 fold . Chondroitin sulfate B and heparitin sulfate do not stimulate transcription under similar conditions . PDS inhibits transcription of deproteinized chromatin . Therefore, the stimulation with chromatin is due to interaction with the chromatin and not the polymerase . Polydextran sulfate has no effect on the size of the RNA that is made either under conditions in which the enzyme can reinitiate or under conditions in which reinitiation is blocked . If reinitiation of the enzyme is blocked, the time required to complete the synthesis of the RNA is the same whether or not the enzyme is stimulated by PDS . These observations indicate that sulfated polysaccharides stimulate transcription by making available new RNA polymerase binding sites on the chromatin.

Nucleic Acids Res, 1975 May 5, 2(5), 723 - 33
A study of the interaction of histones with DNA using isopycnic centrifugation in metrizamide gradients; Rickwood D et al.; Isopycnic sedimentation in metrizamide gradients has shown that mouse-liver histones bind co-operatively to both homologous and bacterial DNA's . However, at low input ratios of histone to DNA, two types of stable complex are formed, depending on the histone concentration . One complex contains half as much histone as DNA while the other contains approximately equal amounts of histone and DNA . At high input ratios of histone to DNA extra histone is bound giving complexes containing up to twice as much histone as DNA . Poly-L-lysine and protamine were also found to bind co-operatively to DNA.

Nucleic Acids Res, 1975 May 5, 2(5), 683 - 90
The preparation of 5-cyanouracil and 5-cyano-2'-deoxyuridine from the corresponding 5-iodo derivative and cuprous cyanide; Bleackley RC et al.; 5-Cyanouracil has been prepared in high yield from cuprous cyanide and 5-iodouracil . The deoxynucleoside has been similarly prepared form 5-iodo-2'-deoxyuridine and this has enabled these compounds to be labelled with (14-C) cyanide . Attempts have been made to incorporate 5-cyanouracil into Escherichia coli 15T and into Mycoplasma mycoides var . capri DNA under conditions in which several other 5-substituted uracils have been incorporated, but without success . Similarly 5-cyano-2'-deoxyuridine could not be incorporated into the DNA of T3 phage under conditions in which 5-bromo-2'-deoxyuridine is easily incorporated . These results suggest that the criteria for a 5-substituted uracil to be incorporated into DNA in vivo depends on some factor other than the size of the substituent.

Nucleic Acids Res, 1975 May 5, 2(5), 635 - 46
Enzymic in vitro repair and chemical nature of DNA chain breaks induced by incorporated phosphorus-32P decay; Fodor I et al.; In vitro repair of single strand breaks in T4 and phage DNA caused by 32p decay was studied . Zone centrifugation procedure showed that polynucleotide kinase, ligase enzyme system failed to repair 32P-damages . It was found that damaged DNA contained gaps and could be repaired by DNA-polymerase I, polynucleotide ligase treatment.

Arch Microbiol, 1975 May 5, 103(3), 251 - 7
On the structure of the peptidoglycan of cell walls from Myxobacter AL-1 (myxobacterales); Harcke E et al.; Basically the peptidoglycan of Myxobater AL-1 consists of alternating beta-1,4-linked N-acetylglucosamic-N-acetylmuramic acid chains . After splitting the aminosugar backbone with a specific algal enzyme three subunits arise: a monomer, a dimer and a timer . Investigation of the monomer with specific enzymes and comparison of the degradation products to standards derived from other bacterial peptidoglycans suggest the following structure of the monomer peptide: L-alanyl-D-glutamic-L-meso-diaminopimelic-D-alanine . A D-alanyl-D-meso-diaminopimelic acid bond is the bridgebond between the peptides of the subunits.

Nucleic Acids Res, 1975 May 5, 2(5), 613 - 24
Enzymatic synthesis of oligonucleotides of defined sequence . Addition of short blocks of nucleotide residues to oligonucleotide primers; Gillam S et al.; Polynucleotide phosphorylase from Escherichia coli can be used to catalyse the addition of short tracts of deoxyadenylate residues to the 3'-termini of deoxyribooligonucleotides of the type pdAn-dN (where dN = dC, dT or dG) using dADP as donor . Similarly, the enzyme can also be used to catalyse the addition of short tracts of adenylate residues to the 3'-termini of ribooligonucleotides of the type An-N (where N = C, U or G) using ADP as donor . In the ribooligonucleotide series, phosphorolytic cleavage of the primer oligonucleotides is significant and results in the concommitant production of oligoadenylates lacking the N residue . Oligomers of the same length, with and without the residue N, were readily separated by thermal elution from cellulose-pdT9 columns . This latter procedure therefore provides a simple method for purification of the oligoadenylates containing an internal base substitution and it also provides a convenient assay for oligonucleotide phosphorolysis.

Nucleic Acids Res, 1975 May 5, 2(5), 691 - 8
Maturation of a hypermodified nucleoside in transfer RNA; Agris PF et al.; E . coli C6 rel- met- cys- was cultured in a fully supplemented medium and in media lacking cysteine or methionine . tRNA isolated from the three cultures containted, respectively, a normal complement of modified nucleosides; a deficiency in thiolated nucleosides and a deficiency in methylated nucleosides . Both sulfur-deficient tRNA and methyl-deficient tRNA contained large amounts of N-6- (delta-2-isopentenyl) adenosine and small amounts of the 2-methylthio derivative . Methyl-deficient tRNA contained, in addition a large amount of a cytokinin active, differently modified nucleoside that is believed to be a sulfur derivative of N6-(delta-2-isopentenyl) adenosine . The structure of this compound is unknown . When methly-deficient tRNA and the precusor the tRNA-Tyr su3-+ A25 were enzymatically methylated in vitro, methyl groups were incorporated into derivatives of isopentenyladenosine . These results indicate that the biosynthesis of the 2-methylthio derivative of isopentenyladenosine may occur in a sequential manner, i.e., thiolation of isopentenyladenosine followed by methylation.

Mol Biol (Mosk), 1975 May-Jun, 9(3), 426 - 34
{Possibility of internal organization of RNA and proteins in ribosomal subparticles . A structural model of Escherichia coli 30S ribosomal subparticle}; Potapov AP; The hypothetic model of reciprocal spatial arrangement of 18 from 21 proteins in the E . coli 30S ribosomal subparticle is suggested . The model is based on conception of the 16S R-A molecule macrostrand which is the right superhelix in the subparticle composition . Macrohelix's biopolarity against single-stranded sites of RNA and its small width result in that proteins binding with single-stranded RNA organized in chain, one-number sequence . The double helixes uniting the corresponding one single-stranded sites of RNA play the role of rigid transmission between them . So, in the course of subparticles reconstruction from RNA and proteins the spatially uncoupled proteins can interact without its direct contact . The model takes into consideration the vast amount of information.

Am J Physiol, 1975 May, 228(5), 1479 - 82
Prostaglandin F and E levels during endotoxin-induced pulmonary hypertension in calves; Anderson FL et al.; Prostaglandin F and E (PGF and PGE) concentrations in sequential blood samples obtained simultaneously from the pulmonary artery (PA) and pulmonary vein (PV) during endotoxin-induced pulmonary hypertension in calves were determined by radioimmunoassay . Three groups of calves were studied . In nine control calves in which no endotoxin was given PA pressure and PGF and PGE concentrations in four pairs of samples taken at 0, 5, 15, and 45 min did not change . In 17 calves given 1 mg E . coli endotoxin, PGF concentrations were increased significantly in the PV and to a lesser degree in the PA in the 15-and 45-min samples . The increased PGF concentration in the 15-min sample corresponded to an increased PA pressure of 74 plus or minus 4 mmHg (mean plus or minus SE) . In three of the endotoxin-treated calves studied a second time and three separate calves indomethacin pretreatment completely blocked the hemodynamic effect of endotoxin as well as PGF release . PGE concentrations did not change in either group . These data suggest that endotoxin-induced pulmonary hypertension may be mediated by PGF, a known pulmonary pressor agent in the bovine, and that blockade of this effect by indomethacin may be due to inhibition of prostaglandin synthesis and/or release.

Proc Natl Acad Sci U S A, 1975 May, 72(5), 1734 - 8
Repetitive DNA replication of the incomplete genomes of phage T4 petite particles; Kozinski AW et al.; The genomes of petite T4 phage particles presumably cannot circularize because they are deficient for a significant terminal segment and hence not terminally redundant like normal T4 genomes . Combined density- and 32P-labeling shows that the majority of such deficient DNA molecules can nevertheless replicate their entire length . Furthermore, the density-shift technique shows that replicated parental strands can exchange their partners for new light strands, indicating that noncircularized T4 DNA molecules replicate repeatedly . When taken together with previously published data, these results indicate that T4 replication is bidirectional from multiple, genetically fixed points of origin . Rolling circle models can, therefore, not be considered as an essential mechanism for the early rounds of T4 replication.

J Biochem (Tokyo), 1975 May, 77(5), 1095 - 106
Conformational transitions of polypeptide chain elongation factor Tu . I . Studies with hydrophobic probes; Arai K et al.; The conformational difference between polypeptide chain elongation factor Tu (EF-Tu)-GTP and EF-Tu-GDP has been studied using hydrophobic and fluorescent probes . The interaction of EF-Tu-GDP with 1-anilino-8-naphthalenesulfonate (ANS) was measured in terms of the enhancement of the fluorescence intensity at the emission maximum of 475 nm . When EF-Tu-GDP-ANS complex was converted to EF-Tu-GTP-ANS complex by incubation with phosphoenolpyruvate and pyruvate kinase {EC 2.7.1.40}, there was a roughly 2-fold increase in fluorescence intensity and a blue shift of the emission maximum from 475 to 467 nm, indicating a conformational transition of the protein . The conformational change was found to be reversible and the spectrum promptly returned to that of EF-Tu-GDP-ANS complex upon addition of excess GDP . A similar change in the spectrum was also observed when aminoacyl-tRNA, but not deacylated tRNA, was added to EF-Tu-GDP-ANS complex . Measurement of the number of binding sites by gel filtration indicated that EF-Tu-GTP and EF-Tu-GDP bind 2.9 and 1.7 molecules of ANS, respectively . These results suggest that in EF-Tu-GTP the conformation was altered and one additional binding site for ANS was created at or near the site interacting with aminoacyl-tRNA . Another reagent, N-(1-anilinonaphthyl-4) maleimide (ANM) was covalently bound to the sulfhydryl group in EF-Tu-GDP which is essential for interaction with aminoacyl-tRNA . The binding could be determined spectrofluorometrically, since the reagent, which is nonfluorescent in aqueous solution, emitted a strong fluorescence upon binding with the sulfhydryl group, indicating a marked hydrophobicity of the local environment . Measurements of the kinetics of the binding revealed that ANM reacted rapidly with the sulfhydryl group in EF-Tu-GTP, while the reaction with that in EF-Tu-GDP proceeded more sluggishly . The difference in the reactivity of the sulfhydryl group essential for aminoacyl-tRNA binding between EF-Tu-GTP and EF-Tu-GDP probably reflects a conformational transition of the protein near the active site . These results, together with those on spin-label studies previously published (Arai, Kawakita, Kaziro, Maeda, & Onishi (1974) J . Biol . Chem . 249, 3311), demonstrate that reversible conformational transition does occur in EF-Tu on changing the ligand from GDP to GTP.

Biochim Biophys Acta, 1975 May 1, 390(2), 209 - 25
Photoinduced convalent crosslinkage, in situ, of Escherichia coli 50 S ribosomal proteins to rRNA; Gorelic L; Irradiation of aqueous solutions of Escherichia coli 50 S ribosomal subunits with 253.7 nm light results in the covalent crosslinkage of the rRNA and protein components . Neither peptide bond cleavage nor protein-protein crosslinkage accompanies the crosslinkage reactionmin addition, substantial photoinduced modifications in the primary structure of the ribosomal proteins, other than crosslinkage to rRNA, are not detected . The crosslinkage of the ribosomal proteins to the rRNA proroceeds in two discrete, dose-dependent steps . The first step, requires an input of up to 3 with 10-20 Quanta of 253.7nm radiation, and results in the crosslinkage of less than half of the ribosomal proteins to the rRNA . The second step requires an input of greater than 3 with 10-20 Quanta of 253.7 nm radiation, and results in the crosslinkage of the remaining ribosomal proteins to the rRNA . The possible relationship of the nature of the corsslinkage reaction to the spatial orientations of the rRNA and protein molecules in the intact 50 S ribosomal subunits is discussed.

Biochim Biophys Acta, 1975 May 1, 390(2), 141 - 54
Purification and properties of nucleic acids from an unusual cytoplasmic organelle in the flagellate protozoan Crithidia oncopelti; Spencer R et al.; A simple, rapid method for preparing bipolar bodies from sonicated (rithidia oncopelti cells, with a yield of 2-5%, is described . Apart from 2-4% contamination with unbroken cells the fraction was considered pure with respect to contamination by other nucleic acid-containing organelles, as judged by light and electron microscopy . A light satellite DNA, f bouyant density 1.695 g/ml in neutral CsCl, and derived from the bipolar body, had a Tm of 81.6 degrees C in0.15 M NaCl/0.015 M sodium citrate (pH 7.0) and a kinetic complexity of 2.7 with 109 . The bipolar body fraction also contained ribonucleoprotein particles with and s20,w of 67 S, in contrast to cytoplasmic ribosomes (87 S) . Bipolar body ribosomes contained rRNA components which migraged coincidentally with Escherichia coli rRNA (molecular weights 1.07 with 10-6 and 0.56 with 10-6) on polyacrylamide gel electrophoresis . Cytoplasmic ribosomes contained rRNAs of molecular weights 1.30 with 10-6 and 0.83 with 10-6 . Bipolar body rRNA accounted for up to 10% of the rRNA extracted from cells . The properties of these bipolar body nucleic acids provide good evidence for the bacterial nature of this subcellular component.

Biochem J, 1975 May, 148(2), 349 - 52
Synthesis of alternative membrane-bound redox carriers during aerobic growth of Escherichia coli in the presence of potassium cyanide; Ashcroft JR et al.; Aerobic growth of Escherichia coli with an oxidizable substrate as carbon source in the presence of low concentrations of KCN leads to the synthesis and integration into the membrane of menaquinone and cytochromes b558, a1 and d in addition to the redox carriers normally present under aerobic growth conditions, namely ubiquinone and cytochromes b562, b556 and o . The results are discussed with reference to other phenotypic and genotypic modifications to the electron-transport chains of E . coli.

Acta Pathol Microbiol Scand {A}, 1975 May, 83(3), 283 - 91
Pulmonary vascular lesions in chickens following intravenous injections of disintegrated cells of Escherichia coli; Nordstoga K et al.; Intravenous injections of disintegrated cells of Escherichia coli in chickens were almost constantly followed by extensive lesions in pulmonary arteries; the alterations consisted of mural fibrinoid necrosis, sometimes with slight intramural occurrence of mononuclear inflammatory cells and eosinophils . Massive perivascular accumulations of the same cell types were also very common findings . Affected arteries were frequently occluded by precipitates, predominantly consisting of the injected material, which were rapidly replaced by proliferating endothelial cells, resulting in obliterative lesions, where giant cells sometimes occurred . The conclusion was drawn that the arterial lesions could most adequately be categorized as hypersensitivity angiitis.

Proc Natl Acad Sci U S A, 1975 May, 72(5), 1725 - 8
gene mutations.GENE MUTATIONS; Richardson JP et al.; Rho factor has been purified from a strain of E . coli containing the Su78 mutation in the suA gene and assayed in another strain with an amber mutation in the suA gene . The rho from the Su78 mutant strain is present in normal amounts but has altered termination function; it does not terminate transcription at some sites that are recognized effectively by the rho factor from the isogenic wild-type strain . Rho in cells with an amber mutation in the suA gene has been assayed by its RNA-dependent ATPase activity . Extracts of cells of this strain have only 9% as much of this rho activity as extracts of cells of the isogenic wild-type strain . These results suggest that rho is the product of the suA gene . Since mutations in the suA gene are known to decrease polar effects of mutations in other genes, it is also suggested that rho factor is at least partially responsible for polar effects.

Poult Sci, 1975 May, 54(3), 674 - 81
Blood parameters of dwarf and normal pullets from growth selected lines before and after Escherichia coli challenge; Reddy PR et al.; Two experiments were conducted to study the hematological differences and the effect of challenge with Escherichia coli on the hemopoietic system among dwarfs and their normal sisters in lines selected for high and low juvenile body weight . Erythrocyte counts, packed cell volumes, and hemoglobin concentrations were significantly higher to dwarf than for normal pullets, while no such differences were noted for mean corpuscular volume, mean corpuscular hemoglobin and hemoglobin concentration . Dwarfs had a significantly lower erythrocyte sedimentation rate than normal pullets . The effect of challenge with E . coli was evident in all the groups . Body weight decreased and leucocyte counts increased significantly . Lymphopenia, accompanied by heterophilia and a consequent increase in H:L ratio was the conspicuous effect of the challenge with E . coli indicating a polymorphonuclear response . Forty-eight hours after challenge the differential response of lines and genotypes to heterophilia was uniform . The hematological data suggest that the dw gene may not contribute to resistance to bacterial diseases . Mortality and autopsy lesions data indicate that the dw gene had a deleterious effect in the low weight but not in the high weight line, suggesting that its influence on the traits measured is dependent upon the genetic background of the population.

J Med Chem, 1975 May, 18(5), 528 - 30
Inhibition of phenylalanyl-tRNA synthetase by aromatic guanidines and amidines; Danenberg PV et al.; Aromatic guanidines and amidines were investigated for their ability to inhibit phenylalanyl-tRNA synthetase from E . coli B . 2-Phenylacetamidine (1), benzylguanidine (2), and N-benzylbenzamidine (3) are competive inhibitors with respect to phenylalanine, binding nearly as well as the substrate, The remainder of the inhibitors was unexpectedly found to be noncompetitive, indication the presence of a secondary binding site on the enzyme . Inhibition by these compounds appears to be specific for phenylalanyl-tRNA synthetase and requires the presence of a phenyl ring as well as the amidine or guanidine moiety.

Eur J Biochem, 1975 May, 54(1), 93 - 6
No correlation between native and denatured forms of tRNA(Trp) form Escherichia coli and the resistant and sensitive molecules characterised by phosphorolysis . Two classes of conformation characterised by phosphorolysis in both native and denatured tRNA(Trp); Thang MN et al.; Some tRNA molecules in solution are sensitive to attack by polynucleotide phosphorylase while others are resistant, even with pure species of tRNA . Further analysis of this behaviour has revealed an underlying microheterogeneity in tRNA structure . In order to clarify the relation between the sensitive and resistant classes of tRNA, and the native and denatured forms with respect to amino acid acceptance, the phosphorolysis of tRNATrp from Escherichia coli has been investigated . Native tRNATrp is similar to species examined previously: resistant and sensitive classes are observed and the sensitive proportion increases with temperature . At 20 degrees C both native and denatured tRNATrp are stable under phosphorolysis conditions, and denaturated tRNATrp is found also to possess resistant and sensitive classes . About 10% of both native and denatured tRNATrp is rapidly phosphorolysed at 20 degrees C, but the rate of conversion of resistant denatured tRNATrp to the sensitive class is about twice as fact as for the native form . Thus it can be concluded that the sensitive molecules of tRNATrp attacked by polynucleotide phosphorylase are not due to denaturation.

Eur J Biochem, 1975 May, 54(1), 55 - 63
Metabolism of the phosphatidylglycerol molecular species in Escherichia coli; Kito M et al.; The fractionation, turnover and biosynthesis of the phosphatidylglycerol molecular species of Escherichia coli were studied . Monoacetyldiglycerides derived from phosphatidylglycerol were separated into five subfractions; cis-vaccenyl-palmitoleyl, cis-vaccenyl-cis-vaccenyl, palmityl-palmitoleyl, palmityl-cis-vaccenyl and the disaturated molecular species on a silica gel plate impregnated with silver nitrate . Individual molecular species had different turnover rates . The palmityl-cis-vaccenyl species was metabolized faster than the others . Disaturated species were rather stable . Various phosphatidylglycerol molecular species were synthesized in the presence of sn-glycerol 3-phosphate, palmityl-CoA, palmitoleyl-CoA, cis-vaccenyl-CoA and CTP by the E . coli membrane particulate fraction . When only the proportion of palmityl-CoA among the three acyl-CoAs was increased, the molecular species containing the palmityl residue were increased . Similar results were obtained with the other acyl-CoAs . However, a temperature-sensitive incorporation of unsaturated and saturated fatty acids into phosphatidylglycerol molecular species was observed with no change in the proportions of the three acyl-CoAs, completely reflecting the in vivo unsaturated/saturated ratio.

Eur J Biochem, 1975 May, 54(1), 45 - 53
Digestion with matrix-bound proteases as a possible probe for the topography of the DNA-dependent RNA polymerase from Escherichia coli; Lill HR et al.; DNA-dependent RNA polymerase lacking subunit sigma was digested with matrix-bound chymotrypsin or trypsin in the presence of 0.4 M NaCl in the monomeric form or at low ionic strength in the oligomeric form . Sigma-containing polymerase was digested in the same way . The course of proteolysis was followed by polyacrylamide gel electrophoresis after dissociation of the enzyme with detergent into subunits and the fragments produced by the hydrolysis . The following results were obtained . (a) The large subunits beta and beta' are cleaved with a much higher rate in the monomeric than in the oligomeric polymerase . (b) Both large subunits are hydrolysed with the same rate . (c) Subunit alpha is hydrolysed almost with the same rate in the monomeric and oligomeric form of polymerase . (d) The same was found for subunit sigma . (e) These effects were independent of the substrate specificity of the protease used . (f) Subunit sigma is much more susceptible to chymotrypsin than to trypsin . (g) Subunit sigma protects the large subunits beta and beta' against tryptic cleavage . These results can be explained in terms of a tentative model for the topography of the protomer-protomer interactions in RNA polymerase . According to this model subunits beta and beta' contain two sites for isologous interactions of protomers . One site can be blocked by attachment of subunit sigma . Subunits alpha and sigma do not participate directly in the association.

Eur J Biochem, 1975 May, 54(1), 293 - 9
Topology of binding sites for carbamyl phosphate in aspartate transcarbamylase from Escherichia coli . The use of pyridoxal phosphate as covalent probe; Suter P et al.; Pyridoxal phosphate, a competitive inhibitor of aspartate transcarbamylase, binds to six sites in the catalytic and to twelve sites in the regulatory subunits of this hexameric protein . The properties of its association to the active sites of the enzyme are very similar to those observed with one of its substrates, carbamyl phosphate . It tightly binds to one half of the sites in the absence of succinate, an analogue of the second substrate . Since pyridoxal phosphate can be linked covalently to the protein by reduction, the distribution of the high affinity binding sites on catalytic trimers was studied after dissociation of modified holoenzyme . Electrophoresis of isolated subunits under non-denaturing conditions revealed four distinct bands, corresponding to trimers containing 0 to 3 pyridoxal phosphate derivatives . The distribution among the four species as a function of ligand concentration in the absence of succinate indicates that in the native oligomer, pyridoxal phosphate (and by extrapolation, carbamyl phosphate) binds to both catalytic trimers, rather than to three sites on a single subunit.

Eur J Biochem, 1975 May, 54(1), 163 - 7
The "hidden ligand" of the galactose-binding protein; Silhavy TJ et al.; Following tryptophan fluorescence of the galactose-binding during dissociation of the ligand it has been found that glucose dissociates with a half life of less than 5 s . Similarly, fast dissociation was also observed by following release of radioactively labelled glucose from Sepharose-coupled galactose-binding protein upon dilution . Accordingly, a previous claim that the galactose-binding protein contains glucose as a non-dissociable "hidden ligand" {G . Richarme and A . Kepes (1974) Eur . J . Biochem . 45, 127-133} has to be reinterpreted

Appl Microbiol, 1975 May, 29(5), 685 - 91
Effect of dichlorodifluoromethane on the appearance, viability, and integrity of Escherichia coli; Prior BA et al.; Cultures of Escherichia coli H52 were treated with liquid dichlorodifluoromethane (fluorocarbon-12 {f-12}) for 2 h at 22 C and then examined microscopically . Treated cells tended to clump, and their cytoplasms were generally less dense and less uniform in appearance than those of control cells . E . coli ML30 was exposed to f-12 at a concentration of 1.25 X saturation for times up to 1,200 min at 22 C . Cells were examined for changes in viability (plate count), permeability (as measured by exit of alpha-{14-C}methylglucoside or uptake of omicron-nitrophenyl-beta-D-galactopyranoside), release of compounds absorbing at 260 nm, and lysis (changes in absorbance at 420 nm) . Large losses of alpha-methylglucoside and of percentage of viability occurred after brief exposure to f-12 . Release of compounds absorbing at 260 nm occurred more slowly than the aforementioned events, possibly because these molecules are larger than alpha-methylglucoside . During 1,200-min exposure to f-12, the number of survivors decreased from 10-9 to 10-4 organisms/ml, the loss of compounds absorbing at 260 nm amounted to 50 percent, and 32 percent lysis occurred . Most of these changes occurred during the first 300 min of treatment . Loss of alpha-methylglucoside was almost complete after 1-min exposure to f-12 . These results suggest that death of the cell involves several stages, with a change of permeability, occurring first, followed by leakage of compounds of increasing size and, finally, lysis.

Chem Biol Interact, 1975 May, 10(5), 363 - 75
Effect of 5-bromodeoxyuridine on the transcriptional properties of the genome in WI-38 human diploid fibroblasts; Hill BT et al.; Growth of WI-38 diploid fibroblasts in a medium containing 5-bromodeoxyuridine (BrdU) resulted in an increased GMP and a decreased AMP incorporation into the RNA synthesised in vitro on a chromatin template . This effect was similar to that previously reported using 3T6 mouse fibroblasts-1 . Substitution of thymidine by BrdU in DNA, also altered the characteristics of the DNA template itself, since the increased incorporation of guanine and decreased incorporation of adenine into RNA were evident also when purified, isolated DNA was used as template . The extent of replacement of AMP by GMP was proportional to the extent of replacement of thymidine by BrdU . Although there are variations in the base composition of RNA transcribed from BrdU-containing DNA templates, there are no significant difference in overall template activity or in the number of available chromatin binding sites for E . coli RNA polymerase . Confluent monolayers of BrdU-treated WI-38 fibroblasts are still able to respond with cell proliferation to a change of medium, as evidenced by an increased incorporation of (-3H)thymidine and an increase in chromatin template activity . The length of the prereplicative phase is similar in both BrdU-treated and untreated cells, although the magnitude of the increase of (-3H)thymidine incorporation is reduced by approximately 30% after BrdU treatment . The increase in chromatin template activity is associated with an increase in the number of chromatin binding sites for E . COLI RNA polymerase, suggesting that the presence of BrdU does not interfere with the availability of initiation sites or alter the actual rate of transcription.

Am J Vet Res, 1975 May, 36(5), 625 - 30
In utero immunization of calves against colisepticemia; Gay CC; A total of 21 bovine fetuses was inoculated in utero with Escherichia coli antigen to determine if nonserotype-specific resistance to colisepticemia could be induced . (Seven of these fetuses were inoculated through the intact flank of the dam.) After birth, the calves were deprived of colostrum and challenge exposed to a serologically distinct E coli which killed nonvaccinated controls . Of 21 calves vaccinated as fetuses, 10 survived challenge exposure, 8 died of colisepticemia, and 3 were stillborn . Premature birth precluded an adequate period of vaccination in 6 of the calves that died of colisepticemia . A relationship was not observed between E coli serum antibody and survival after challenge exposure . The results indicate that in utero vaccination with a single serotype of E coli can result in heterogenetic protection against neonatal colisepticemia . However, the occurrence of stillbirth and premature birth in calves vaccinated in utero indicates need for furthur research before field application of this technique.

Cell, 1975 May, 5(1), 69 - 74
Regulation of stable RNA synthesis and ppGpp levels in growing cells of Escherichia coli; Sokawa Y et al.; Under the balanced condition of growth of E . coli cells, no distinct difference is observed in stable RNA and protein synthesis between CP78 (rel+) and CP79 (rel minus), whereas a considerable difference is present in RNA accumulation between NF161 (rel+) and NF162 (rel minus), where NF161 smaller than NF162 . The RNA content of NF161 is lower than that of NF162 in four different cultures with different growth rates . These two sets of isogenic pairs of rel+ and rel minus strains are commonly used in the study of rel gene function; however, NF161 is a mutant in the spoT gene whose product may be responsible for the degradation of ppGpp . The basal levels of ppGpp in these four strains growing with three different growth rates were examined: NF161 (rel+ spoT minus) has a much higher content of ppGpp than do other strains . Furthermore, the contents of ppGpp tend to be lower when the above four strains are growing at a faster rate . Thus a close correlation seems to exist between the content of RNA and the basal level of ppGpp under the condition of balanced growth.

Am J Clin Pathol, 1975 May, 63(5), 735 - 47
Lymphocyte surface receptors and leukemia virus-induced immunosuppression; Friedman H et al.; The number and distribution patterns of lymphocytes in the spleens and lymph nodes of Balb/c mice which express immunoglobulin surface receptors were studied in terms of the effects of a murine leukemia virus on the immune-response mechanism . Friend leukemia virus induces a prompt, marked depression of the immune response of mice to antigens such as sheep erythrocytes and E . coli LPS . A functioning T- and B-lymphocyte system is necessary for the response to the SRBC's whereas E . coli LPS, a T cell-independent antigen, stimulates B cells alone . Although the responses to both classes of antigen were markedly depressed in FLV-infected mice, the major defect appeared to be impairment of B-cell function, at least early in the course of infection . In order to examine in more detail the mechanism of interaction between FLV and lymphoid cells with Ig surface receptors, presumably B cells, immmunofluorescent analyses were performed with spleen, and lymph node cells from FLV-infected mice . Within a few days after infection there was a marked decrease in the percentage of spleen cells with Ig surface molecules, although the absolute number of these cells was either unchanged or increased due to marked splenomegaly caused by the virus . A marked decrease in the percentage of splenocytes with theta antigen, considered a marker for mature T cells, also was evident in infected mice . The number of spleen cells showing evidence of FLV infection (i.e., positive for FLV-associated antigens) increased rapidly during the first few days after infection, and within 2 to 2 1/2 weeks nearly all of the nucleated splenocytes were positive for the tumor antigen . In contrast to the results for spleen cells, there were increases rather than decreases in the percentages of Ig-positive and theta-positive cells in the lymph nodes after infection . The number of lymph-node cells that showed the presence of FLV antigen was much lower than in the spleen, and their appearance was also much slower as the leukemic process progressed . Despite these differences between spleen and lymph-node cells in terms of relative percentages of Ig- and theta-positive lymphocytes, relatively similar depressions were evident for the percentages of lymphoid cells that could redistribute their surface Ig receptors into polar caps when incubated with anti-Ig serum at 37 C . Marked impairment of the Ig-capping responses for both spleen and lymph-node cells paralleled the course of infection and development of immunosuppression . These observations indicate that murine leukemia virus infection can both alter the responsiveness of immunocompetent cells to T-dependent and independent antigens and depress the number and normal functional activity of these cells, as reflected by altered surface Ig receptors and antigens.

Urology, 1975 May, 5(5), 626 - 31
Treatment of chronic prostatitis . Comparison of Minocycline and Doxycycline; Brannan W; The results of minocycline and doxycycline therapy in 41 patients with chronic prostatitis and minocycline therapy in 6 patients with acute prostatitis were evaluated . In the comparative study of chronic prostatitis, minocycline and doxycycline were given on the same dosage schedule, milligram for milligram: a loading dose of 200 mg . followed by 100 mg . twicd daily . Over-all clinical responses to therapy with either agent were generally satisfactory, and no statistically significant difference was demonstrable in this regard . In the group with chronic prostatitis treated wtih minocycline, however, all symptoms manifested before therapy had disappeared after therapy even where over-all results had been judged unsatisfactory . This was not true of the group with chronic prostatitis treated with doxycycline . Symptoms in 6 patients persisted after therapy had been terminated . Here the difference in results between the two groups was found to be statistically significant . A review of symptoms two to eight weeks after therapy revealed no significant difference between the two groups . After two years only 6 patients in the entire group with chronic prostatitis had returned with recurrent problems: 3 of these patients had been treated with minocycline and 3 had been treated with doxycycline . Results of therapy in the small series of patients with acute prostatitis treated with minocycline were generally satisfactory.

J Lipid Res, 1975 May, 16(3), 244 - 6
Lyophilized 7 alpha-hydroxysteroid dehydrogenase: a stable enzyme preparation for routine bile acid analysis; Macdonald IA et al.; Preparations of 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) from Escherichia coli strain 23 can be frozen and thawed without significant loss of activity . 7 alpha-HSDH may then be lyophilized into powder form, which is stable for more than 6 months (3% loss of activity) . The lyophilized 7 alpha-HSDH preparation has the additional advantage over previously described preparations of a low and stable fluorescence background when applied to the fluorometric estimation of bile acids, especially in combination with thin-layer chromatography . Analysis of duodenal aspirates from 18 normal subjects gave bile acid ratios identical with those reported earlier and obtained by using gas-liquid chromatography . A significant difference in the glycine: taurine ratio between males and females was observed.

J Bacteriol, 1975 May, 122(2), 791 - 3
Location of the Escherichia coli K-12 ruv gene affecting septum formation after inhibition of deoxyribonucleic acid synthesis; Iyehara H et al.; Escherichia coli ruv gene was located at 36.1 min on the chromosome by P1 transduction experiments and the gene order his - supD - uvrC, dar4 - ruv - eda - fadD - pps was proposed . Complementation analysis by an F' factor carrying genes in the his region indicated that ultraviolet light sensitivity genes, ruv and uvrC, consist of different cistrons and wild-type alleles of these genes are dominant over the mutant alleles.

J Bacteriol, 1975 May, 122(2), 749 - 63
Electron microscopic heteroduplex studies of sequence relations among plasmids of Escherichia coli: structure of F13 and related F-primes; Hu S et al.; The structure of F13, a plasmid containing lac, purE, and proC, has been determined by heteroduplex analysis . As expected for an F-prime formed by a type II excision event, it contains all the sequences of F plus a large segment of Escherichia coli chromosomal deoxyribonucleic acid . There is a sequence of F with coordinates 16.3-17.6F which has been shown in other studies to be the insertion sequence IS2 . This IS2 occurs twice on F13, once at each of the two junctions of F deoxyribonucleic acid with chromosomal deoxyribonucleic acid . The sequence alpha beta which occurs twice on F with coordinates 93.2-94.5/OF and 13.7-15.0F occurs an additional three times, twice in an inverted order relative to the alpha beta sequences of F, on the chromosomal sequences of F13 . The structures of the plasmids F13-4 and F210 have been determined . The common sequences of F13 with F152-1 (a derivative of F152, the classical F2gal) and with F13-4 and F210 have been mapped . These results partially map lac, proC, tsx, and purE on F13 . On the basis of all of these results, it is proposed that Hfr 13 (the parent of F13) was formed by recirpocal recombination between IS2 on F and an IS2 resident at a point between lac and proC on the chromosome of the F+ parent of Hfr 13 . It is proposed that this IS2 and the several alpha beta sequences on the chromosomal part of F13 are hot spots for recombination with F, i.e., for Hfr formation . The point of origin and direction of transfer of many Hfr's can be explained by this hypothesis . In particular, the sequence relations of F42-1 (Flac) and of F152-1 (F 2gal) with F13 are completely consistent with this model.

J Bacteriol, 1975 May, 122(2), 686 - 90
Lipophilic chelator inhibition of electron transport in Escherichia coli; Crane RT et al.; The lipophilic chelator bathophenanthroline inhibits electron transport in membranes from Escherichia coli . The less lipophilic 1,10-phenanthroline, bathophenanthroline sulfonate, and alpha,alpha-dipyridyl have little effect . Reduced nicotinamide adenine dinucleotide oxidase is more sensitive to bathophenanthroline inhibition than lactate oxidase activity . Evidence for two sites of inhibition comes from the fact that both reduced nicotinamide adenine dinucleotide menadione reductase and duroquinol oxidase activities are inhibited . Addition of uncouplers of phosphorylation before bathophenanthroline protects against inhibition.

J Bacteriol, 1975 May, 122(2), 623 - 8
Reinitiation of chromosome replication in the presence of chloramphenicol under an integratively suppressed state by R6K; Sotomura M et al.; The autonomous replication of an R plasmid, R6K (amp, str) was shown not to be affected by chloramphenicol . It provoked integrative suppression and gave rise to Hfr strains when integrated into the chromosome of a strain of Escherichia coli K-12 with a temperature-sensitive mutation in the gene, dnaA . An Hfr strain designated as Hfr(R6K) no . 1 was thus obtained and characterized . It was not completely stable as shown by a plating efficiency of 0.6 at 42 C relative to that at 30 C . The density labeling and the ultracentrifugation analysis suggested that the deoxyribonucleic acid replication in this Hfr strain did not stop immediately after completion of the round already started before temperature shift-up and the addition of chloramphenicol . These observations are discussed in relation to a possibility that the chromosome replication of this Hfr strain is under the control of the integrated plasmid at a nonpermissive temperature.

J Bacteriol, 1975 May, 122(2), 592 - 8
Effect of ribonuclease on the association of deoxyribonucleic acid with the membrane in Escherichia coli; McIntosh MA et al.; The Mg-2+-Sarkosyl crystals (M band) procedure was used to study the effect of ribonuclease (RNase) A on the association of Escherichia coli deoxyribonucleic acid (DNA) with membrane . Incubation of gently prepared cell extracts with RNase results in the release of DNA from membrane . This effect appears to result from the activation, by RNase, of endonuclease I and subsequent limited activity of this deoxyribonuclease . In support of this explanation, it is demonstrated (i) that the extent of the RNase-induced loss of DNA from membrane is directly correlated with the endogenous level of endonuclease I, and (ii) that endonucleolytic activity occurs when gently lysed cell preparations are incubated in the presence of RNase.

J Bacteriol, 1975 May, 122(2), 570 - 4
Mapping of sul, the suppressor of lon in Escherichia coli; Johnson BF et al.; The suppressor sul, which is allele specific for the ultraviolet sensitivity gene lon, has been mapped by conjugation and transductional crosses in Escherichia coli K-12 and B/r . Previously, sul was reported to lie in the azi region of the Escherichia coli chromosome . Evidence is presented which positions sul close to and clockwise of fabA on the Escherichia coli map . Cotransductional frequencies of 31.3% were obtained between sul and pyrD, and frequencies of 82% were obtained between sul and fabA . Also, the mucoid phenotype of K-12 lon strains grown on minimal glucose agar plates at 37 C was not significantly effected in sul derivatives . No differences between the sul of Escherichia coli B/r and that of K-12 derivatives with regard to map location or effect on mucoid production were observed.

J Bacteriol, 1975 May, 122(2), 565 - 9
Rapid cessation of phospholipid synthesis in fructose-1,6-diphosphate aldolase mutants of Escherichia coli; Su CH et al.; Escherichia coli GH352, which was originally described as a temperature-sensitive strain containing a thermolabile acyl coenzyme A:monoacylglycerol 3-phosphate acyltransferase, does not now contain a thermolabile form of this enzyme . It has a defect in fructose-1,6-diphosphate aldolase and at least one additional temperature-sensitive lesion . Both strains GH352 and NP315, a temperature-sensitive aldolase mutant, show rapid cessation of 32-P1 incorporation into nucleic acids and phospholipids at 42 C . These characteristics of strain GH352 are therefore no longer attributed to thermolabile phospholipid synthesis, but can be attributed to the fructose-1,6-diphophate aldolase lesion.

J Bacteriol, 1975 May, 122(2), 538 - 48
Formation of chromatographically unique species of transfer ribonucleic acid during amino acid starvation of relaxed-control Escherichia coli; Fournier MJ et al.; Examination of the transfer ribonucleic acid (tRNA) produced by starving, relaxed-control (rel minus) strains of Escherichia coli for required amino acids revealed the occurrence of a number of chromatographically unique subspecies . Leucine starvation results in the formation of new isoacceptor species of leucine-, histidine-, arginine-, valine-, and phenylalanine-specific tRNA and quantitative changes in the column profiles of serine, glycine, and isoleucine tRNA . Evidence that the unique tRNA species are synthesized de novo during amino acid starvation comes from the findings that the major unique leucine isoacceptor species is not formed in stringent control cells or in rel minus cells starved for uracil or treated with rifampin . Furthermore, heat treatment of the unique leucine tRNA does not alter its chromatographic behavior, indicating that the species is not an aggregate or nuclease-damaged form of a normal isoacceptor tRNA . The methyl acceptor activities of tRNA from leucine-starved and nonstarved rel+ or rel minus cells were found to be essentially the same . This result and the finding that the chromatographic behavior of the unique leucine-specific tRNA was not altered after treatment with tRNA methylase suggests that gross methyl deficiency is probably not the biochemical basis for the occurrence of the unique species.

J Bacteriol, 1975 May, 122(2), 518 - 25
Five control systems preventing transfer of Escherichia coli K-12 sex factor F; Gasson MJ et al.; The transfer inhibition systems of 28 Fin+ plasmids have been characterized, using Flac mutants insensitive to inhibition by R100 or R62 . All F-like plasmids (except R455) and one N group plasmid determined systems analogous to that of R100; this is designated the FinOP system . None of these plasmids could supply a FinP component of the transfer inhibitor able to replace that of F itself . In addition to the FinOP and R62 transfer inhibition systems described previously, new systems were encoded by the F-like plasmid R455, the I-like plasmid JR66a, and the group X plasmid R485 . Besides inhibiting F transfer, JR66a also inhibited F pilus formation and surface exclusion, whereas R485 inhibited only pilus formation and R455 inhibited neither . All three R factors inhibited transfer of J-independent Flac elements, indicating that they act directly on one or more genes (or products) of the transfer operon, rather than directly via traJ . The tral products and transfer origin sequences of two Fin+ F-like plasmids, ColB2 and R124, appear to have similar specificities to those of F itself.

J Bacteriol, 1975 May, 122(2), 492 - 501
Multiple gene loci for a single species of glycine transfer ribonucleic acid; Fleck EW et al.; The study of suppressors of tryptophan synthase A protein missense mutations in Escherichia coli has led to the establishment of two nonadjacent genetic loci (gly V and gly W) specifying identical nucleotide sequences for a single isoaccepting species of glycine transfer ribonucleic acid (tRNA GLY 3 GGU/C) . In one case, suppression of the missense mutation trpA78 was due to a mutation in a structural gene (gly W) for tRNA Gly 3 GGU/C . This mutation resulted in a base change in the anticodon and modification of an A residue adjacent to the 3' side of the anticodon, leading to the production of a tRNA Gly 3 UGU/C species . The resulting glyW51 (SU UGU/C) allele was mapped by interrupted mating and was located at approximately 37 min on the Escherichia coli genetic map . Other suppressor mutations affecting the primary sequence of tRNA Gly GGU/C and giving rise to the Ins and SU+A58 phenotypes were positioned at 86 min (glyV) . Several independently arising missense suppressor mutations resulting in the SU+A78 phenotypes were isolated and mapped at these two genetic loci (glyV and glyW) . The ratio of appearance of suppressor mutations at glyV and glyW suggests that there are three of four tRNAGly3 GGU/C structural gene copies at the glyV locus to one copy at the glyW locus . Structural genes for tRNA ly isoacceptors are now known at four distinct locations on the Escherichia coli chromosome: glyT (77 MIN), TRNA Gly 2 GGA/G; gly U (55 min), tRNAGly-1 minus; and gly V (86 MIN) AND GLYW (37 min), tRNAGly 3 GGU/C.

J Bacteriol, 1975 May, 122(2), 474 - 84
Isolation and characterization of mutator strains of Escherichia coli K-12; Hoess RH et al.; A selection procedure was devised to select for mutants of Escherichia coli K-12 with enhanced rates of spontaneous frameshift mutation . Three types of mutants were isolated . Two of the mutations apparently represent alleles of previously isolated mutL13 and mutS3 . The third type of mutation, represented by two alleles, lies between lysA and thyA, and has been designated mutR . mutR increases the rate of spontaneous frameshift mutation and also the rate of base substitution mutations . The mutator phenotype is recessive . Reversion of a lac amber mutation located on an episome is increased in the presence of the mutator, indicating that mutR can act in trans . No change in sensitivity to ultraviolet irradiation or mitomycin C could be found when mutR34 was compared to the isogenic mutR+ strain . The mutator's activity was little affected by the type of medium in which the strain was grown . Deoxyribonucleoside triphosphate pools were normal in mutR34 . Intergenic recombination frequencies were the same in mutR and mutR and mutR+ strains, but a two- to threefold increase in intragenic recombination was observed in Hfr times Fminus crosses when the recipeint was mutR34 as compared with mutR+ . This increase appeared independent of the distance between the two markers within the gene in which the crossover took place.

J Bacteriol, 1975 May, 122(2), 437 - 42
Events following prophage Mu induction; Razzaki T et al.; Escherichia coli strains lysogenic for a thermoinducible Mu prophage (Mu cts62) undergo rapid lysis about 50 min after heat induction . Induction of Mu cts62 apparently causes damage to the host sequences in which Mu is inserted . The normal expression of A, BU, and X genes of Mu is needed for this specific deleterious effect on the prophage-containing host sequences . Mu deoxyribonucleic acid can be shown to reintegrate extensively at different sites on the host genome during the lytic cycle after prophage induction or after infection of sensitive cells by clear-plaque mutants of Mu . We estimate that approximately 10 copies of Mu deoxyribonucleic acid are inserted per chromosome during vegetative growth . The episome rescue method for detecting vegetative Mu deoxyribonucleic acid insertion, in which an episome is transferred from the lytically infected cells to F- receipient cells, can be applied to study Mu integration without requiring the host cells to survive . It also provides an easy system to isolate Mu insertions in transmissible episomes and plasmids.

J Bacteriol, 1975 May, 122(2), 352 - 8
Thermosensitive mutants of Escherichia coli K-12 altered in the catalytic Subunit and in a Regulatory factor of the glutamy-transfer ribonucleic acid synthetase; Lapointe J et al.; The glutamyl-transfer ribonucleic acid synthetase (GluRS) of a partial revertants (ts plus or minus) of the thermosensitive (ts) mutant strain JP1449 (LOcus gltx) and of a ts mutant strain EM111-ts1 with a lesion in or near the locus gltx have been studied to find the relation between these two genetic loci known to influence the GluRS activity in vitro and the presence of a catalytic subunit and of a regulatory subunit in the GluRS purified from Escherichia coli K-12 . The ts character of strain JP1449-18ts plus or minus is co-transduced with the marker dsdA at the same frequency as is the ts character of strain JP1449 . Its purified GluRS is very thermolabile and its Km for glutamate is higher than that of a wild-type GluRS . These results indicate that the locus gltX is in the structural gene for the catalytic subunit of this enzyme . The location of the mutation causing the partial ts reversion in strain JP1449-18ts plus or minus is discussed . The GluRS purified from the ts mutant strain EM111-ts1 has the same stability as the wild-type enzyme, but its Km forglutamate increases with the temperature, suggesting that the locus gltE codes for a regulatory factor, possibly for the polypeptide chain that is co-purified with the catalytic subunit.

J Immunol, 1975 May, 114(5), 1523 - 31
Chemotaxis of basophils by lymphocyte-dependent and lymphocyte-independent mechanisms; Ward PA et al.; Guine pigs basophils obtained from blood or bone marrow have been studied for their chemotactic responsiveness . Chemotactic factors for basophils include a substance (lymphokine) present in culture fluids from antigen-stimulated lymphocytes, a material generated in zymosan-activated guinea pig serum, a C5 cleavage factor, and a bacterial factor . When compared with homologous neutrophils and monocytes, basophils respond most rapidly to a chemotactic stimulus . The lymphokine basophil chemotactic factor is physicochemically similar to the previously described monocyte chemotactic factor but appears to be distinct from it as well as MIF and neutrophil chemotactic factor present in the same fluids, Part of the evidence for this is the ability to detect basophil chemotactic factor in the absence of other lymphokine activities under appropriate experimental conditions . More evidence, specifically relating to the monocyte factor, is that monocytes can adsorb basophil chemotactic activity but not vice versa . This latter observation may have implications for the mechanism whereby the accumulation of basophils is controlled and limited in vivo . In addition, it was noted that specific antigen could also suppress basophil chemotaxis . Although the mechanism of this phenomenon is unclear, it could serve as a second means by which basophil accumulation may be controlled in the intact animal . Taken together, these observations provide further definition of the chemotactic behavior of basophils in general, and underscore some of the ways in which lymphocytes can influence basophils through lymphokine-dependent mechanisms.

Infect Immun, 1975 May, 11(5), 1010 - 3
Significane of intravascular coagulation in canine endotoxin shock; From AH et al.; The contribution of disseminated fibrin clot formation to the pathogenesis of canine endotoxin shock was explored in control dogs and in those defibrinated with a purified fraction of Malayan pit viper venom . The hemodynamic and humoral responses after the administration of an intravenous challenge dose of Escherichia coli endotoxin were comparable as was mortality . It is concluded that, although the role of the coagulation sequence in canine endotoxin shock is unclear, it does not appear to be determinative.

J Med Chem, 1975 May, 18(5), 524 - 6
Substituted 1-{(5-nitrofurfurylidene)amino}-4-imidazolin-2-ones; Snyder HR Jr et al.; A series of 1-{(5-nitrofurfurylidene)amino}-4-imidazolin-2-ones has been prepared . A new synthesis of 4-alkyl-1-{(5-nitrofurfurylidene)amino}-4-imidazolin-2-ones involving the oxidative ring closure of 5-nitro-2-furaldehyde 2-(2-hydroxyethylalkyl)semicarbazones is described . The in vitro testing of the compounds against a variety of bacteria is reported.

Eur J Biochem, 1975 May, 54(1), 267 - 77
Interactions of heteroaromatic compounds with nucleic acids . 1 . The influence of heteroatoms and polarizability on the base specificity of intercalating ligands; Muller W et al.; We have examined the origins of base specificity in intercalating ligands by studying the interaction with DNA of a series of proflavine and acridine orange analogs differing in the heteroatoms present in the chromophore . Base specificity was determined by differential dialysis of the dye against DNA samples of differing G-C content . We find that G-C specificity increases as the visible absorbance band of the chromophore moves to longer wavelength, implying a relation between specificity and polarizability of the chromophore . This can be rationalized by recognizing that the G-C pair is more polar than A-T, and should therefore interact more favorably with an easily polarized ring system . We find in addition that dimethylation of the chromophore amino groups increases specificity which we discuss in terms of steric and coupled steric-electronic contributions . Our results also bear on the origin of G-C specificity in binding actinomycin to DNA . Some of the compounds studied are as G-C specific as actinomycin, yet they lack hydrogen-bonding functions as plausible determinants of specificity . This observation gives new life to the hypothesis that the specificity of actinomycin is determined primarily by preferential interaction of the chromophore with a G-C pair.

J Bacteriol, 1975 May, 122(2), 425 - 32
Stability of ribosomal and transfer ribonucleic acid in Escherichia coli B/r after treatment with ethylenedinitrilotetraacetic acid and rifampicin; Yuan D et al.; A short treatment with ethylenedinitrilotetraacetic acid to permeabilize bacteria for various antibiotics or treatment with the ribonucleic acid (RNA) synthesis inhibitor rifampin causes a slow degradation of 50S and 30S ribosomal particles and of the corresponding 23S and 16S ribosomal RNA species (about 25 percent in 1 h) . The effects are additive such that the decay is about 50 percent/h if rifampin is employed after permeabilization by ethylenedinitrilotetraacetic acid . The 5S ribosomal RNA and transfer RNA are essentially stable under these conditions.

Mol Biol (Mosk), 1975 May-Jun, 9(3), 459 - 66
{Phage T4 partial diploidy obtained with the method of DNA interrupted injection . I . Analysis of the genetic structure and phage progeny reproduction process}; Mekshenkov MI et al.; Phage T4 chromosome fragmentation is shown to take place when DNA injection is interrupted, a fragment length being strictly controlled by the interval from the moment of adsorbtion till the moment of an interruption . Populations of the bacteria cells infected by the phage T4 partial diploids are produced with the method of DNA interrupted injection . In the population a merodiploid involves some phage T4 amber mutant and a phage "wild" type chromosome fragment of the size controlled . To construct merodiploids the amber mutant in gene 43 and the mutant in gene 32 with the higher and the lower recombination frequency, accordingly, are used . Every merodiploid which is the heterozygote by one of these genes or which is the heterozygote by the late genes is determined to reproduce mixed phage progeny . Both the mean of the burst and the parent genotypes ratio in progeny either in the E . coli CR-63 cells or in the E . coli B depend on neither the heterozygote genetic structure nor the diploid region size . The results obtained conclude that phage genes express their function in the small fragments and the fragment recombination with the mutant partner whole chromosome follows their autonomous replication.

Mol Biol (Mosk), 1975 May-Jun, 9(3), 459 - 66
{Phage T4 partial diploidy obtained with the method of DNA interrupted injection . I . Analysis of the genetic structure and phage progeny reproduction process}; Mekshenkov MI et al.; Phage T4 chromosome fragmentation is shown to take place when DNA injection is interrupted, a fragment length being strictly controlled by the interval from the moment of adsorbtion till the moment of an interruption . Populations of the bacteria cells infected by the phage T4 partial diploids are produced with the method of DNA interrupted injection . In the population a merodiploid involves some phage T4 amber mutant and a phage "wild" type chromosome fragment of the size controlled . To construct merodiploids the amber mutant in gene 43 and the mutant in gene 32 with the higher and the lower recombination frequency, accordingly, are used . Every merodiploid which is the heterozygote by one of these genes or which is the heterozygote by the late genes is determined to reproduce mixed phage progeny . Both the mean of the burst and the parent genotypes ratio in progeny either in the E . coli CR-63 cells or in the E . coli B depend on neither the heterozygote genetic structure nor the diploid region size . The results obtained conclude that phage genes express their function in the small fragments and the fragment recombination with the mutant partner whole chromosome follows their autonomous replication.

Mol Biol (Mosk), 1975 May-Jun, 9(3), 426 - 34
{Possibility of internal organization of RNA and proteins in ribosomal subparticles . A structural model of Escherichia coli 30S ribosomal subparticle}; Potapov AP; The hypothetic model of reciprocal spatial arrangement of 18 from 21 proteins in the E . coli 30S ribosomal subparticle is suggested . The model is based on conception of the 16S R-A molecule macrostrand which is the right superhelix in the subparticle composition . Macrohelix's biopolarity against single-stranded sites of RNA and its small width result in that proteins binding with single-stranded RNA organized in chain, one-number sequence . The double helixes uniting the corresponding one single-stranded sites of RNA play the role of rigid transmission between them . So, in the course of subparticles reconstruction from RNA and proteins the spatially uncoupled proteins can interact without its direct contact . The model takes into consideration the vast amount of information.

Biochim Biophys Acta, 1975 May 1, 390(2), 226 - 30
ATPase and GTPase activities associated with the 5-S RNA-protein complex of Escherichia coli ribosomes; Gaunt-Klopfer M et al.; Specific 5-S RNA-protein complexes were reconstituted from Escherichia coli 5-S RNA and 50-S ribosomal proteins . These complexes consist of 5-S RNA and two major proteins, namely E-L18 and E-L25 . Analysis for enzymatic activities shows that ATP and GTP are hydrolyzed and that this hydrolysis is independent of elongation factors.

Biochim Biophys Acta, 1975 May 1, 390(2), 192 - 208
Affinity labeling of the ribonucleic acid component adjacent to the peptidyl recognition center of peptidyl transferase in Escherichia coli ribosomes; Yukioka M et al.; N-Iodacetylphenylalanyl-tRNA was used as an affinity label for localizing the RNA components intimately related to the peptidyl transferase activity of Escherichia coli ribosomesmthis analogue could specifically alkylate a unique nucleotide chain of 23-S RNA . The alkylation was strongly enhanced by poly(U), and was dependent on the presence of both 50- and 30-S subunits; Chloramphenicol inhibited the reaction, wheras blasticidin S stimulated it . The alkylated RNA base was found to be adenine . The nucleotide chain attacked by N-iodoacetylphenylalanyl-tRNA seemed to be localized at or near to the peptidyl recognition center of peptidyl transferase.

Acta Virol, 1975 May, 19(3), 177 - 81
Excision of bromodeoxyuridine from T4-DNA by an antimutator polymerase of T4 phage; Presber W; With gene-43 (DNA polymerase)-ts-mutants of T4 phage, L98 (mutator) and CB121 (antimutator), and the T4 wild type, double labelling of DNA was carried out with (H3)-bromodeoxyuridine (BUdR) and (C14)-thymidine (TdR) . Experiments on (C14) TdR for DNA synthesis measurement in the presence of BUdR offered evidence of the ability of the CB121 mutant to excise BUdR from the DNA . This effect took place only at increased temperature . As distinct from DNA synthesis of the host, all T4 phages used preferred TdR rather than BUdR for net synthesis.

Eur J Biochem, 1975 May, 54(1), 239 - 45
Studies on the de novo biosynthesis of NAD in Escherichia coli . The separation of the nadB gene product from the nadA gene product and its purification; Griffith GR et al.; Quinolinic acid (pyridine 2,3-dicarboxylic acid) which is an immediate precursor of the pyridine nucleotides, is synthesised from L-asparate and dihydroxyacetone phosphate in Escherichia coli . Extracts from certain nadB mutants complement the extracts prepared from all nadA mutants for the enzymic synthesis of quinolinate . Using the complementation assay, the quinolinate synthetase B protein has been purified more than 300-fold . The quinolinate synthetase B protein exists in all nadA and nadC mutants examined . The quinolinate synthetase A protein was present in all nadC mutants and most (but not all) nadB mutants . The facile separation of the wild-type quinolinate synthetase A and B proteins out of a nadC mutant suggests that quinolinate synthetase does not exists as a tightly bound complex . The partially purified quinolinate synthetase is inhibited by physiological concetrations of NAD and NADH but not by NADP or NADPH.

Eur J Biochem, 1975 May, 54(1), 169 - 73
The effects of spermine and Mg2+ on the catalytic mechanism of isoleucine: tRNA ligase; Carr AC et al.; Isoleucyl-tRNA formation catalysed by isoleucine: tRNA ligase is stimulated by both Mg2+ and spermine in the pH-range 7.0 to 8.0 at 310 K . At low {Mg2+} the acceleration caused by both cations together exceeds the sum of their individual effects . 2 . The spermine-stimulated reaction has a steeper temperature-dependence than reaction in the presence of Mg2+ . Two phases in the kinetics of isoleucyl-tRNA formation are detected in the presence of Mg2+ plus or minus spermine, but only a single step is observed in the presence of spermine alone . Thus the rate-limiting steps under normal assay conditions are different for the two cations . 3 . Enzyme-bound isoleucyl-AMP can be formed in the absence of Mg-2+ and plus or minus spermine . 4 . It is concluded that there is no evidence for cation-dependent differences in the reaction mechanism of isoleucine: tRNA ligase, though there are certainly differences in the relative rates of some of the individual steps.

J Med Microbiol, 1975 May, 8(2), 375 - 84
Standardisation of the nitroblue-tetrazolium test; Merzbach D et al.; Optimal conditions for the NBT-reduction test have been sought . Increasing heparin concentrations up to 100 units per ml and a delay in performance of the test, especially when blood specimens are kept at room temperature, resulted in higher values for the NBT index, which then sometimes exceeded the upper limit of normal in healthy people and in uninfected patients . The effect of pH, composition of the buffer, and dye concentration was also investigated . Phosphate-buffered saline pH 7-2 containing 0-1% NBT dye, without glucose, gave the most reliable results . In endotoxin-stimulated NBT tests, the following procedure is recommended: incubation of 0-1 ml whole blood with lyophilised endotoxin 20 mug per ml . for 15 min . in a 37 degree C water bath, followed by the standard test with a 0-2% NBT solution . By this technique, the leucocyte reaction to various types of lipopolysaccharides was of the same order of magnitude . Drug therapy having an effect on blood components lowered this reaction, whatever the source of endotoxin used as stimulant . The importance of NBT-reduction tests is discussed . Standard conditions of test performance are strictly requisite if comparable results are to be obtained and if data not corresponding with the apparent clinical and other laboratory findings are to be evaluated correctly . The stimulated NBT test, performed in parallel with the standard test, is useful in the interpretation of abnormal results and in the detection of factors with a temporary or permanent effect on the phagocytic activity of pmn leucocytes.

Clin Chim Acta, 1975 May 1, 60(3), 315 - 22
The radiometric assay of alkaline phosphatase activity with 125-I-labelled phenolphthalein monophosphate; Aw SE; The coupling of 125-I to phenolphthalein monophosphate is described . Hydrolytic cleavage of the phosphate group by alkaline phosphatases (EC 3.1.-3.1) releases labelled phenolphthalein which is extracted into ethyl acetate at pH 10 . This forms the basis of a sensitive assay for the enzyme with the advantages of sensitivity and rapidity from the use of a gamma-emitter . Results with Escherichia coli and calf intestine alkaline phosphatases are presented.

J Bacteriol, 1975 May, 122(2), 407 - 11
Thiolases of Escherichia coli: purification and chain length specificities; Feigenbaum J et al.; The presence of only one thiolase (EC 2.3.1.9) in wild-type Escherichia coli induced for enzymes of beta oxidation was demonstrated . A different thiolase was shown to be present in a mutant constitutive for the enzymes of butyrate degradation . The two thiolases were purified to near homogeneity by a simple two-step procedure and were found to be associated with different proteins as shown by gel electrophoresis . The thiolase isolated from induced wild-type Escherichia coli cell was active on beta-ketoacyl-coenzyme A derivatives containing 4 to 16 carbons, but exhibited optimal activity with medium-chain substrates . In contrast, the thiolase isolated from the constitutive mutant was shown to be specific for acetoacetyl-coenzyme A.

Biochem J, 1975 May, 148(2), 329 - 33
Synthesis and sideedness of membrane-bound respiratory nitrate reductase (EC1.7.99.4) in Escherichia coli lacking cytochromes; Bile composition and bile cast formation after transplantation of the liver in man; Transplantation of the liver in man is frequently complicated by biliary fistula and obstruction of the biliary tree by casts, which suggests that the composition of the bile may be abnormal . In part of the present study, bile composition was investigated in three recipients during the first few weeks after transplantation, when a T tube was in place . Supersaturation of bile with cholesterol was found in two patients immediately after surgery and during episodes of acute rejection, but bile was never lithogenic in the third . There was no evidence of bile stasis in the absence of acute rejection and bile viscosity was normal in all three patients throughout the study . Free bile acids and free bilirubin, which are relatively insoluble products of bacterial metabolism, were not present in the bile of any patient . However, chemical analysis of casts found at autopsy in a fourth recipient showed that the major component was free bilirubin . Escherichia coli was grown in cultures of the casts and the organisms were shown to possess glucuronidase activity, thus providing an explanation for the high bilirubin content . There was also some evidence in one case that damage to the bile duct mucosa has led to the precipitation of material upon it, and it is concluded that a number of factors, including infection, supersaturation with cholesterol, and mucosal damage, may be involved in bile case formation after transplantation of the liver.

Proc Natl Acad Sci U S A, 1975 May, 72(5), 1960 - 4
Interaction of partially purified simian virus 40 T antigen with circular viral DNA molecules; Jessel D et al.; Mixing chromatographic fractions containing simian virus 40 (SV40) T antigen with SV40 {3H}-DNA I (double-stranded, circular, supercoiled) results in the conversion of the nucleic acid to a form that will bind to a nitrocellulose filter . Unlabeled SV40 DNA I successfully competes with this reaction . Under the conditions employed, the antigen-containing fractions bind a variety of circular, viral DNA molecules . Chromatography of the antigen in three systems reveals that the T immunoreactivity migrates with DNA binding activity . In a kinetic heat inactivation experiment, the antigenic reactivity disappears simultaneously with the DNA binding activity . The data indicate the presence of a discernible DNA binding activity in fractions containing T antigen and suggest that the T antigen is the DNA binding protein being measured.

Proc Natl Acad Sci U S A, 1975 May, 72(5), 1922 - 6
Simian virus 40 DNA directs synthesis of authentic viral polypeptides in a linked transcription-translation cell-free system; Roberts BE et al.; A linked cell-free system has been developed which is capable of transcribing and translating mamalian viral DNA, and its characteristics and requirements are outlined . In this system, simian virus 40 (SV40) DNA Form I (supercoiled) directed the synthesis of discrete polypeptides up to 85,000 daltons in size . One of these products was indistingusihable from authentic major virus capsid protein VPI, as judged by mobility on sodium dodecyl sulfate/polyacrylamide gels, antibody predipitation, and peptide analyses . The cell-free products larger than VPI comprised a number of polypeptides ranging in molecular weight from 50,000 to 85,000 . These polypeptides demonstrated no immunological relationship whatsoever to the structural protein VPI . However, two of these products, along with one of approximately 25,000 dlatons, were precipitated with antiserum to SV40 tumor antigen . Linear SV40 DNA generated by the cleavage of Form I DNA with the restriction endonuclease EcoR1 was an efficient template in this system and also directed the synthesis of a polypeptide migrating with VPI on polyacrylamide gels . The potential of this system for defining a functional map of a DNA genome is discussed.

Proc Natl Acad Sci U S A, 1975 May, 72(5), 1843 - 7
Folding of the DNA double helix in chromatin-like structures from simian virus 40; Germond JE et al.; Relaxed circular, covalently closed simian virus 40 DNA molecules were associated with the four histones that are present in virions . In electron micrographs the resulting complexes appear twisted, with globular structures (nucleosomes) along the DNA . Incubation with an untwisting extract converts the twisted complexes to relaxed structures . Extraction of the DNA from the relaxed complexes yields supercoiled molecules . The number of superhelical turns in these molecules corresponds to the number of nucleosomes per DNA molecule in the complexes.

Proc Natl Acad Sci U S A, 1975 May, 72(5), 1768 - 72
Anatomy of herpes simplex virus DNA: strain differences and heterogeneity in the locations of restriction endonuclease cleavage sites; Hayward GS et al.; Digestion of herpes simplex virus DNA by the HinIII or Eco RI restriction endonucleases yielded 11 to 15 fragments with molecular weights between 1 x 10(6) and 28 x10(6) . The electrophoretic profiles obtained in 0.3% agarose gels with DNA fragments from none different strains of herpes simplex virus type 1 could be readily differentiated from the patterns exhibited by the corresponding fragments from four separate strains of type 2 virus; however, with each serotype, the laboratory strains differed significantly among themselves and also from isolates passaged a minimum number of times outside the human host . Digestion of all DNAs of herpes simples virus with either enzyme reproducibly generated two classes of fragments (major and minor) which differed in molar ocncentration . Moreover, although the molecular weight of an intact herpes simplex 1(F1) DNA molecule is approximately 98 x 10(6), the summed molecular weights of all major and minor HinIII fragments totalled 160 x 10(6), and the seven major fragments alone accounted for only 60 x 10(6) . These unusual features indicate the existence of limited heterogeneity in the positions of cleavage sitet along individual molecules . We have eliminated the possibility that minor fragments arose from contamination with the defective DNA of high byoyant density which appears on serial undiluted passage of the virus . In fact, this latter type of DNA was resistant to cleavage by HinIII and gave large amounts of only two species of EcoRI fragments; suggesting that the defective molecules consist of many tandem repeats of a small segment of viral DNA . The heterogeneity in the viral DNA of normal density appears to be related to the structural organization of the molecules and does not necessarily imply differences in genetic content.

J Med Chem, 1975 May, 18(5), 473 - 6
Synthesis and biological activity of 4-beta-Iribofuranosyl-1,3-dihydroxybenzene ("1,3-dideazauridine"); Sharma RA et al.; In view of the marked antitumor activity of 3-deazauridine, the synthesis of 4-(beta-D-ribofuranosyl)-1,3-dihydroxybenzene (1,3-dideazauridine) and its dibenzyl derivative was carried out . 4-Bromo-1,3-dihydroxybenzene was converted to its dibenzyl derivative, which, upon reaction with n-butyllithium followed by treatment with anhydrous cadmium chloride, gave bis(1,3-dibenzyloxyphenyl-4)cadmium . Condensation of this intermediate with 2,3,5-tri-O-benzoyl-D-ribofuranosyl chloride in refluxing toluene, and subsequent removal of the protecting benzoyl groups, afforded 4-(beta-D-ribofuranosyl)-1,3-dibenzyloxybenzene which, upon catalytic hydrogenation over Pd/C, furnished the desired 4-(beta-D-ribofuranosyl)-1,3-dihydroxybenzene . The beta configuration at the anomeric center was established by NMR and hydrogen bonding studies . 4-(Beta-D-ribofuranosyl)-1,3-dibenzyloxybenzene inhibited the growth of leukemia L1210 cells by 50% at 7 x 10(-6) M, and that of mammary carcinoma TA3 cells at 5 x 10(-5) M . Dideazauridine itself was less active, inhibiting the leukemia L1210 but not the TA3 cells at 1 x 10(-4) M, but the compound was significantly active against herpes simplex (type I) virus in vitro.

Eur J Biochem, 1975 May, 54(1), 279 - 91
Interactions of heteroaromatic compounds with nucleic acids . 2 . Influence of substituents on the base and sequence specificity of intercalating ligands; Muller W et al.; This paper presents the results of a systematic study on the effects of substituents on the base and sequence specificity of tricyclic heteroaromatic compounds interacting with DNA by intercalation . All the compounds tested are derived from proflavine and acridine orange analogs with different heteroatoms in the middle ring . Their base and sequence specificities were determined by differential dialysis of the ligand against DNA samples of differing G-C content . The main results indicate that (a) the introduction of a phenyl substituent into one of the two available positions of the middle ring increases or decreases the G-C specificity of the ligand depending on the position where the substitution takes place; (b) compounds of the substitution type of neutral red (2-methyl-3-amino-7-dimethyl-amino-phenazine) show unexpectedly high G-C specificities and (c) DNA ligands of pronounced sequence specificity for adjacent G-C pairs can be constructed by combining the structural elements of neutral red with an additional phenyl residue in the same molecule . The further study of compounds related to the phenylated neutral red revealed that the G-C specificity can be improved or destroyed by additional substituents . The comparison of the G-C specificity and the DNA-affinity data of the compounds studied leads to the suggestion that the specificity arises mainly from electronic factors which are strongly controlled through steric constraints on possible ocmplex geometries . As a basis for the discussion a possible structure for the DNA complex of the phenylated neutral red is considered in which the extra phenyl ring at N-5 of the phenazinium system, protrudes into the large groove of the DNA helix while the tricyclic part of the ligand is inserted between the DNA base-pairs.

J Bacteriol, 1975 May, 122(2), 669 - 75
Effect of glucose and its analogues on the accumulation and release of cyclic adenosine 3',5'-monophosphate in a membrane fraction of Escherichia coli: relation to beta-galactosidase synthesis; Seto H et al.; Correlation between beta-galactosidase synthesis and cyclic adenosine 3',5'-monophosphate (cAMP) levels in a membrane fraction obtained from disrupted spheroplasts of Escherichia coli was investigated . Repression of beta-galactosidase synthesis in the membrane fraction by glucose-6-phosphate and by 2-deoxyglucose differed in sensitivity to reversal by cAMP . The difference between the two repressions could be due to the fact that glucose-6-phosphate inhibited severely the accumulation of exogenous {3-H}cAMP by the membrane fraction, whereas 2-deoxyglucose had little effect on the accumulation of the nucleotide . On the other hand, a quick decrease in the level of {3-H}cAMP preaccumulated in the membrane fraction resulted from addition of either glucose-6-phosphate or 2-deoxyglucose . Results reported here suggest that repression of beta-galactosidase synthesis is associated with anabrupt decrease in cAMP levels at the intramembranal sites where beta-galactosidase is synthesized, and the major, if not sole, mechanism which leads to instantaneous drop of cAMP level is via the release of cAMP, but not by degradation of the nucleotide since the membrane fraction retained less than 10 percent of cellular cyclic phosphodiesterase and the activity of the enzyme was not affected by repressing sugars.

J Bacteriol, 1975 May, 122(2), 660 - 8
Two types of glucose effects on beta-galactosidase synthesis in a membrane fraction of Escherichia coli: correlation with repression observed in intact cells; Seto H et al.; A membrane fraction obtained from an osmotic lysate of Escherichia coli spheroplasts retains capability to synthesize beta-galactosidase . The system also retains cellular regulatory functions, one of which is known as catabolite repression . Two types of repression of beta-galactosidase synthesis were observed in this membrane system: one was caused by the addition of 2-deoxyglucose or glucose at a low concentration (3 times 10- minus 4 M), and the other was caused by glucose-6-phosphate or glucose at a high concentration (3 times 10- minus 2 M) . In the presence of cyclic adenosine 3',5'-monophosphate (10 mM), repression caused by the former was completely reversed, whereas repression by the latter was only partially reversed . Conditions in intact cells causing transient and permanent repression were also investigated . Upon addition of 2-deoxyglucose or glucose at a low concentration to intact cells, only transient repression of beta-galactosidase synthesis was observed . Glucose at a high concentration caused both transient and subsequent permanent repression, and intensity of permanent repression depended upon glucose concentration, whereas duration and intensity of transient repression were independent of glucose concentration . Mutants deficient in phosphoenolpyruvate-phosphotransferase system (Hpr minus and enzyme I minus) showed transient repression but failed to show permanent repression . In mutants deficient in glucose catabolism beyond glucose-6-phosphate, both transient and permanent repression were observed . Correlation between the observations in the membrane system and in intact cells is discussed . The results obtained here strongly suggest that transient repression is caused by glucose itself, and that permanent repression is caused by glucose-6-phosphate of high intracellular levels of glucose.

Z Immunitatsforsch Exp Klin Immunol, 1975 May, 148(5), 414 - 24
Cellular- and permeability-changes in the peritoneal cavity of mice after injection of endotoxon; Jokay I et al.; Injection of endotoxin into the peritoneal cavity of mice causes an early emigration of PMN-leucocytes and therafter a decrease in macrophage count . The number of lymphocytes shows only a transient increase . One and 2 injections of endotoxin rise the fibrinogen and protein level of the peritoneal fluid . A simple test for measuring the change in permeability (and lymph circulation) was described ("congo red elimination's test") . Changes in permeability occur after a latent period of 2 hours following endotoxin administration, culminate during the 1st day and last about 3-4 days . The possible mechanisms of the above phenomena are discussed.

Z Naturforsch {C}, 1975 May-Jun, 30(3), 406 - 11
Inhibition of excision repair without influence upon UV-sensitivity and UV-mutability in Escherichia coli B/r Hcr+; Balgavy P et al.; Pre-irradiation starvation of exponentially growing Escherichia coli B/r Hcr+ thy-try- strain for thymine and tryptophan causes inhibition of pyrimidine dimer excision from ultraviolet damaged cells DNA . This inhibition of excision repair has not resulted in increasing ultraviolet sensitivity nor in increasing frequency of ultraviolet induced tryptophan revertants . The possible mechanisms of the non-excision repair in the prestarved cells, which is at least as accurate and effective as the whole dark repair in exponentially growing cells, are discussed.

J Periodontal Res, 1975 May, 10(2), 65 - 72
Elevation of a serum component in periodontal disease capable of modulating chemotactic infiltration; Gothier DE et al.; Serum from dental patients was examined for its capacity to affect migration of polymorphonuclear leukocytes toward a complement-independent chemotactic factor, bacterial filtrate of Escherichia coli, (BF) . Seven subjects with advanced periodontitis, documented with clinical measurements of pocket depth, loss of attachment, indices for gingival inflammation and oral hygiene, were paired with normal subjects of similar age and sex . This study shows that patients whose only apparent clinical symptom was severe periodontal inflammation harbor a heat-stable, serum component which neutralizes factors chemotactic for polymorphonuclear leukocytes . A role for this component in modulation of inflammatory infiltration is discussed.

J Biol Chem, 1975 Apr 25, 250(8), 3174 - 8
Decay of ribosomal ribonucleic acid in Escherichia coli cells starved for various nutrients; Kaplan R et al.; Decay of pre-existing ribonucleic acid was studied in Escherichia coli cells subjected to high temperature or to starvation for nitrogen, phosphate, amino acids, or a carbon source . In these studies a series of mutants affected in ribonucleic I(RNase I, EC 3.1.4.22) polynucleotide phosphorylase (EC 2.7.7.8) or ribonuclease II (RNase II, EC 3.1.4.23) were used . Degradation of total RNA and the disappearance of 23 S and 16 S rRNA were followed . The results obtained indicated that, by and large, decay of 23 S and 16 S RNA parallels that of total RNA . Decay of RNA depended on the nuclease content of the cells as well as on the treatment of applied . It was most pronounced during carbon starvation and least in cells deprived of phosphate ions . It was most effective in strains containing all three nucleases and least in the strain defective in all three . The exonucleases polynucleotide phosphorylase and RNase II did not seem to affect the extent of 23 S and 16 S RNA disappearance . Strains with modified exonucleases did accumulate low molecular weight RNA species during treatments which induced considerable degradation of 23 S and 16 S RNA . Based on the above date and previous observations, we suggest that during various starvations a similar mechanism is operative . The 23 S and 16 S RNAs are degraded endonucleolytically, and this is the rate-limiting step during starvation . The exonucleases polynucleotide phosphorylase and RNase II seem to participate primarily in the decay of the low molecular weight RNA species formed by the endonuclease(s), not as yet identified.

J Biol Chem, 1975 Apr 25, 250(8), 3057 - 61
Synthesis and turnover of ribosomal ribonucleic acid in guanine-starved cells of Escherichia coli; Erlich H et al.; The relationship between the rate of RNA accumulation and the level of guanosine triphosphate was examined . Cells auxotrophic for guanine show a 6-fold drop in the intracellular level of GTP in response to the exhaustion of the exogenous guanosine supply . Contraction of the GTP pool results in a 2.5-fold reduction in the rate of RNA synthesis and the cessation of RNA accumulation . The decrease in the rate of RNA synthesis is seen to occur at the level of chain elongation . Analysis of RNA made in guanine-starved cells by competition-hybridization and sucrose gradient sedimentation suggests that the turnover of newly synthesized ribosomal RNA accounts for the observed failure of RNA to accumulate.

J Biol Chem, 1975 Apr 25, 250(8), 2872 - 7
Transcription of Azotobacter phage deoxyribonucleic acid . Salt-dependent equilibrium between steps in initiation; Domingo E et al.; The transcription of Azotobacter phage A21 DNA by Escherichia coli or Azotobacter vinelandii RNA polymerase differs from that of some other DNAs in its inhibition by moderate concentrations of KCl . This characteristic results in an apparent low template activity for this DNA as compared with T4 DNA under standard assay conditions . From an analysis of the dependence of the various steps in initiation on KCl it is concluded that the effect is exerted on an equilibrium between an inactive polymerase-DNA complex and an active preintitiation complex . This salt-sensitive equilibrium favors the inactive complex at a lower KCl concentration than with other templates . It can be approached from other low or high salt concentrations at a measurably slow rate.

J Biol Chem, 1975 Apr 25, 250(8), 2855 - 65
Coupling of alanine racemase and D-alanine dehydrogenase to active transport of amino acids in Escherichia coli B membrane vesicles; Kaczorowski G et al.; Isolated membrane vesicles from Escherichia coli B grown on DL-alanine-glycerol carry out amino acid active transport coupled to D-alanine oxidation by a membrane-bound dehydrogenase . Several other D-amino acids are substrates for this D-alanine dehydrogenase and also drive concentrative uptake of solutes . Additionally, L-alanine and L-serine can energize solute transport by virtue of conversion to oxidizable D isomers by a membrane-bound alanine racemase . No other physiological L-amino acids were effective . Both membrane enzymes and consequent solute transport are markedly reduced in vesicles from glucose-grown cells . Respiratory chain uncouplers abolish the racemase-dehydrogenase-supported transport activity . When amino-oxyacetate at 10-4 M is added to the vesicles, the racemase activity and transport driven by L-alanine and L-serine is specifically and reversibly inhibited . D-Alanine-driven transport is unaffected . Similarly beta-chloro-L-alanine is an irreversible inactivator of the bound racemase but not the D-alanine dehydrogenase . Both the D and L isomers of beta-chloroalanine support oxygen uptake by the vesicles and initially stimulate L-(14C)proline active transport . However, oxidation of the beta-chloro-D-alanine rapidly uncouples active transport from substrate oxidation . This transport inactivation can be protected partially by dithiothreitol, putatively scavenging a reactive product of chloroalanine oxidation . Authentic beta-chloropyruvate produces the same transport uncoupling . When beta-chloro-L-alanine is employed as a substrate, no such transport inactivation is observed . This difference may stem from the possibility that the alanine racemase eliminates HCl from beta-chloro-L-alanine producing pyruvate, not the beta-chloropyruvate that would arise from racemization and then dehydrogenation . We have shown that exogenous pyruvate is oxidized by the vesicles and will also stimulate active transport of amino acids.

J Biol Chem, 1975 Apr 25, 250(8), 3179 - 84
Escherichia coli 30 S ribosomal proteins uniquely required for assembly; Held WA et al.; Two 30 S ribosomal proteins from Escherichia coli have been found to be uniquely required for assembly of 30 S ribosomal subunits . 30 S ribosomes were reconstituted in vitro from 16 S RNA and a mixture of purified 30 S ribosomal proteins (sigma Si) . In the absence of S16, sigmaSi-S16 particles were slowly assembled wich had physical and functional properties similar to complete particles (sigmaSi) . The results indicate that S16 affects the rate of 30 S ribosome assembly, but does not appear to be directly involved in any known ribosomal function . Particles assembled in the absence of S18 (sigmaSi-S18) had high activity in poly(U)-directed polyphenylalanine synthesis but lost considerable activity upon isolation or purification . The loss of activity could be attributed primarily to the loss of proteins S11 and S21 . S18 appears to have a major role in the stabilization of ribosome structure, especially the binding of proteins S11 and S21, and does not appear to be directly required for activity in poly(U)-directed polyphenylalanine synthesis . However, it is possible that S18 has some functional role which is not required for polyphenylalanine synthesis.

J Biol Chem, 1975 Apr 25, 250(8), 2911 - 19
Fractions of lipopolysaccharide from Escherichia coli O111:B4 prepared by two extraction procedures; Morrison DC et al.; Lipopolysaccharides have been extracted from Escherichia coli O111:B4 by phenol extraction and by a new method employing aqueous butanol . Both methods yield very similar lipopolysaccharide preparations . Gel filtration chromatography of either preparation yields two physically and chemically distinct lipopolysaccharide fractions . One fraction contains lipopolysaccharide molecules with long antigenic side chains . It acts like a highly asymmetric unit with an apparent weight of 1.5 times 10-6 and is not dissociated by detergents or deacylation . The second fraction has a short antigenic side chain and can be dissociated by sodium dodecyl sulfate and Triton X-100 into units of approximately 90,000 . Some properties of the lipopolysaccharide fractions vary with the method of extraction.

J Biol Chem, 1975 Apr 25, 250(8), 2778 - 82
Thioredoxin reductase-mediated hydrogen transfer from Escherichia coli thioredoxin-(SH)2 to phage T4 thioredoxin-S2; Berglund O et al.; Thioredoxin from Escherichia coli B and phage T4-infected E . coli B are small hydrogen carrier proteins which in their reduced forms are specific hydrogen donors to E . coli and T4-induced ribonucleotide reductase, respectively . The oxidation-reduction active group of both thioredoxins consists of a single cystine residue which is reduced to sulfhydryl form by NADPH in the presence of E . coli thioredoxin reductase . Reduction of T4 thioredoxin-S2 to thioredoxin-(SH)2 led to a 3-fold increase in the quantum yield of tyrosine fluorescence . By using the spectrofluorimetric properties of T4 thioredoxin and E . coli thioredoxin as markers for their oxidized and reduced forms we have shown that E . coli thioredoxin reductase catalyzed the reaction: (see article) whose equilibrium constant favors formation of E . coli thioredoxin-S2 and T4 thioredoxin-(SH)2 . This finding suggests that in the T4-infected cell most of the deoxyribonucleotides required for the viral DNA might be synthesized by the T4-induced ribonucleotide reductase while the host ribonucleotide reductase is inactive due to the shortage of reduced E . coli thioredoxin.

J Biol Chem, 1975 Apr 25, 250(8), 3101 - 7
Guanosine monophosphate synthetase from Escherichia coli B-96 . Inhibition by nucleosides; Spector T et al.; The mechanism of inhibition of GMP synthetase by purine and purine-analog nucleosides was investigated . It was found that in addition to allowing the nucleoside to bind to the enzyme (Udaka, S., and Moyed, H . S . (1963) J . Biol . Chem . 238, 2797)PPi was also a competitive inhibitor with respect to ATP . A rate equation was derived to describe this inhibitory model for two competitive inhibitors where the binding of one inhibitor is contingent upon the binding of the other . The inhibition constants for a large number of nucleosides were then determined . It was found that the initial enzyme-inhibitor complex (of all nucleoside inhibitors) was slowly (0.2 min-1) transformed into a secondary (nondissociating) complex . The two inhibitory complexes appeared to exist in equilibrium . While decoyenine, N6-allyladenosine, and adenosine had similar inhibition constants for the initial complex (0.7 to 1.0 muM), their apparent inhibition constants for the secondary complex were 0.004, 0.06, and 0.5 muM respectively . These differences in the apparent dissociation constants from the secondary complexes are due to different equilibria between the initial and the secondary complexes . The ratios of the secondary complex to the initial complex at equilibrium were 3,250, 290, and 11 for decovenine, N6-allyladenosine, and adenosine, respectively.

Biochemistry, 1975 Apr 22, 14(8), 1648 - 60
Structural transitions of deoxyribonucleic acid in aqueous electrolyte solutions . I . Reference spectra of conformational limits; Hanlon S et al.; The circular dichroism properties of calf thymus DNA have been examined at 27 degrees over the wavelength range of 215-300 nm in aqueous solutions of NaCl, KCl, LiCl, CsCl, and NH4Cl at pH 7 . The concentrations of these electrolytes were varied from 0.01 to ca . 5-10 m . The spectral changes induced by changes in concentration of NaCl and KCl and all but the highest concentrations of NH4Cl as well as lower concentrations of Cstcl and LiCl could be represented by a common two-state transition involving the conversion of the typical conservative spectrum commonly seen in dilute solutions of these salts to a nonconservative spectrum similar to that obtained by Tunis-Schneider and Maestre ((1970), J . Mol . Biol . 52, 521) for the C form of DNA . At higher concentrations of CsCl, LiCl, and NH4Cl, an additional component, resembling an A type spectrum, was required to account for the observed CD changes with changing concentration of electrolyte . Relying on the published spectra of the B, the C, and the A forms of DNA by Tunis-Schneider and Maestre for identification and approximate values of the molecular ellipticities of these forms, we have analyzed these spectral transitions by two least mean squares methods in order to obtain accurate reference spectra of aqueous "B", C, and "A" conformations of calf thymus DNA . The results obtained suggest that although the C form in solution is identical with that obtained in film, the aqueous B conformational limit is not identical with the crystallographic Watson-Crick structure . In addition, the A form generated in solution under our experimental conditions appears to be more similar to that assumed by low molecular weight Escherichia coli DNA at 75% relative humidity rather than calf thymus DNA at the same relative humidity.

Biochemistry, 1975 Apr 22, 14(8), 1553 - 9
Ribosomal subunit interaction as studied by light scattering . Evidence of different classes of ribosome preparations; Debey P et al.; The Mg2+-dependent equilibrium of ribosomal subunits was studied by light scattering in the absence of any other factor of protein synthesis . The difference of intensity in the light scattered by dissociated 30S-50S couples and by 70S particles was used as a method to measure the percentage of association as a function of Mg2+ under conditions of nonperturbation of the equilibrium . We found that pressure-resistant (type A) and non-pressure-resistant (type B) ribosomes may be characterized by the different behavior of their association equilibrium curves . The thermodynamic parameters of this equilibrium and their comparison for the two classes of ribosomes were calculated from experiments at different temperatures.

Biochemistry, 1975 Apr 22, 14(8), 1700 - 12
Interaction of effecting ligands with lac repressor and repressor-operator complex; Barkley MD et al.; The equilibrium association constants for the binding of a wide variety of effecting ligands of the lac repressor were measured by equilibrium dialysis . Also, detailed investigations of the apparent rate of dissociation of repressor-operator comples as a function of ligand concentration were carried out for several inducers and anti-inducers . The affinity of repressor-ligand comples for operator DNA was evaluated from the specific rate constants at saturating concentrations of effecting ligand . By fitting the experimental data depicting the functional dependence of the rate of dissociation upon ligand concentrations to calculated curves, assuming simple models of the induction mechanism, the equilibrium association constant for the binding of effecting ligand to repressor-operator comples was determined . Inducers reduce the affinity of lac repressor for operator DNA by a factor of approximately 1000 under standard conditions; the extent of destabilization depends on Mg2+ ion concentration . Anti-inducers increase the affinity of repressor for operator at most a factor of five . Only one neutral ligand, which binds to repressor without altering the stability of repressor-operator comples, was found . No homotropic or heterotropic interactions in the binding of effecting ligands either to repressor or to repressor-operator complex are evident.

Biochim Biophys Acta, 1975 Apr 18, 388(1), 29 - 37
Substrate activity of phosphonic acid analogues of CDPdiglyceride in the synthesis of phosphoglycerides in Escherichia coli; Tyhach RJ et al.; Two phosphonic acid analogues of CDPdiglyceride, D L-2-hexadecoxy-3-octadecoxypropylphosphonyl-O-(cytidine 5'-phosphate) (analogue (I), and D L-3,4-dioctadecoxybutylphosphonyl-O-(cytidine 5'-phosphate) (analogue (II), have been synthesized and examined as substrates for the enzymes involved in the synthesis of phosphoglycerides in Escherichia coli . Both compounds were substrates for CDPdiglyceride:sn-glycerol-3-phosphate phosphatidyl transferase . The analogues had similar Km values (Km of 0.060 mM for analogue (II): Km of 0.080 mM for analogue (I) and a V identical to that of CDPdipalmitin (Km of 0.044 mM) . In contrast, the analogues were poor substrates for CDPdiglyceride:L-serine phosphatidyl transferase . The analogues had lower Km values (Km of 0.40 mM for analogue (II); Km of 0.80 mM for analogue (I) than CDPdipalmitin (Km of 1.4 mM) . The V, although identical for both analogues, was ten-fold lower than that observed with the natural substrate . An analysis of the products of these enzymatic reactions suggests that phosphatidylglycerophosphate phosphatase and phosphatidylserine decarboxylase may also possess a certain degree of substrate specificity.

Biochim Biophys Acta, 1975 Apr 16, 390(1), 69 - 77
The effect of double-stranded cowpea mosaic viral RNA on protein synthesis; Reijnders L et al.; The effect of double-stranded cowpea mosaic viral RNA on several in vitro protein synthesizing systems was studied . No inhibitory effect of this RNA at concentrations up to 16 mug/ml was found in wheat germ protein synthesis programmed with cowpea mosaic viral-, alfalfa mosaic viral- or globin 9 S-RNA . At high concentrations of this double-stranded RNA, inhibition of Escherichia coli in vitro protein synthesis programmed with MS-2- and cowpea mosaic viral-RNA was found, whereas at low concentrations its inhibitory effect on rabbit reticulocyte in vitro protein synthesis was strong . The implications of these observations for the suggestions of other authors, based on the study of mammalian protein synthesizing systems, are discussed.

Biochim Biophys Acta, 1975 Apr 16, 390(1), 56 - 68
Gene activation in human diploid cells . Age-dependent modifications in the stability of messenger RNAs for nonhistone chromosomal proteins; Stein GS et al.; Serum stimulation of early as well as late passages of nondividing WI-38 human diploid fibroblasts to proliferate, results in DNA synthesis beginning at 12 hours and MITOSIS AT 20 HOURS . A 2-fold increase in the transcriptional activity of chromatin isolated from early and late passage W2-38 cells is evident one hour following serum stimulation . An increased synthesis and association with the genome of two defined molecular weight classes of nonhistone chromosomal proteins one hour following serum stimulation of early and late passage cells is also observed . The increased chromatin template activity and nonhistone chromosomal protein synthesis occur in early passage cells stimulated to proliferate in the presence of actinomycin D . However, when late passage WI-38 cells are stimulated in the presence of antinomycin D, increases in chromatin template activity and nonhistone chromosomal protein synthesis are not observed one hour following serum stimulation . The possibility that nonhistone chromosomal protein synthesis and activation of transcription early during the prereplicative phase of the cell cycle in early passage human diploid fibroblasts may be regulated at the translational level is discussed . Consideration is also given to the possibility that there may be an age-dependent modification in such a regulatory mechanism.

Biochim Biophys Acta, 1975 Apr 16, 390(1), 14 - 23
Synthesis of heteropolyribonucleotides by toluene-treated Escherichia coli cells; Rebrov LB; Escherichia coli cells, made permeable to ribonucleoside-5'-diphosphates by treatment with toluene, efficiently promote the synthesis of homo- and heteropolynucleotides . This synthesis is catalyzed by polynucleotide phosphorylase because, among other things, it is inhibited by orthophosphate, and E . coli Q13, a mutant having a Mn-2+-dependent polynucleotide phosphorylase, promotes polynucleotide synthesis in the presence of Mn-2+ but not of Mg-2+ . Cells of E . coli B and E . coli MRE 600 (A Mutant lacking ribonuclease I) are about equally active in promoting poly(A, U, G, C) synthesis . Sucrose density gradient and agarose gel electrophoretic analysis of the product show that it is polydisperse with sedimentation coefficients ranging between 4 S and 27 S . The synthesized polynucleotides can be translated by the toluene-treated cells.

Tijdschr Diergeneeskd, 1975 Apr 15, 100(8), 421 - 5
Leptospirae of serotype lora of the serogroup Australis isolated for the first time from swine in the Netherlands; Hartman EG et al.; Leptospirosis in swine, caused by serotype lora (serogroup Australis) was detected both serologically and by culture . The most important symptoms consisted in abortion during the final month of gestation and the birth of dead or not viable piglets.

Biochim Biophys Acta, 1975 Apr 14, 387(1), 12 - 22
Energy requirement for the initiation of colicin action in Escherichia coli; Jetten AM et al.; 1 . Starved cells of a strain of Escherichia coli and its mutant uncA, treated with colicin K, E2 or E3, remained fully rescuable upon trypsin treatment (stage I in colicin action) . The transition to stage II in colicin action (cells no longer rescuable by trypsin) was promoted by the addition of either glucose or D-lactate . 2 . Aerobically glucose-grown cells of the normal strain were irreversibly killed by colicin K, E2 or E3 under anerobic conditions, while similarly treated cells ot its mutant uncA remained fully rescuable . The stage I-stage II transition in colicin action was blocked in normal cells under anaerobic conditions when succinate was the sole carbon source . 3 . Arsenate alone had little effect on the progression of the stage I-stage II transition in normal cells, treated with colicin K . However, this transition was abolished in the presence of both arsenate and anaerobic conditions . 4 . The initiation of colicin action could be coupled to the anaerobic electron transfer systems formate dehydrogenase-nitrate reductase and alpha-glycerophosphate dehydrogenase-fumarate reductase . 5 . These results indicate that an energized state of the cytoplasmic membrane is required for the initiation of colicin action and that no high-energy phosphorylated compounds are necessary.

Biochim Biophys Acta, 1975 Apr 14, 387(1), 23 - 36
The maintenance of the energized membrane state and its relation to active transport in Escherichia coli; Rosen BP et al.; 1 . An ATPase mutant of Escherichia coli and two partial revertants of that mutant were examined for the ability to generate a high energy membrane state with D-lactate or ATP, as measured by the quenching of the fluorescent dye quinacrine . 2 . All three strains showed reductions in the aerobically-driven quenching of fluorescence compared to the wild type, but the reduction could be reversed by the addition of eitherN,N'-dicyclohexylcarbodiimide or the crude soluble ATPase of the wild type . 3 . The mutant exhibited a decreased ability to accumulate sugars and amino acids and showed an increased permeability to protons . 4 . One partial revertant showed a slight increase in active transport and a slight decrease in proton permeability . 5 . The other partial revertant showed a large increase in transport ability and a large decrease in proton permeability . 6 . A model is proposed in which the conformation of the Mg-2+-ATPase is important in the utilization of energy derived from the electron transport chain and this function is independent of the catalytic activity of the Mg-2+-ATPase.

J Biol Chem, 1975 Apr 10, 250(7), 2626 - 9
Enzymatic arginylation of beta-melanocyte-stimulating hormone and of angiotensin II; Soffer RL; Porcine beta-melanocyte-stimulating hormone and angiotensin II were examined as acceptors in the reaction catalyzed by arginyl-tRNA-protein transferase . Both inhibited enzymatic transfer of {14C}arginine from tRNA to bovine albumin . Inhibition was competitive with albumin and the K-i values were, respectively, 15 and 0.8 muM . The expected arginylated compounds were isolated and characterized . Beta-melanocyte-stimulating hormone and its arginylated product had identical activities in the frog epithelium bioassay . In contrast, the biological activity of angiotensin II was diminished by enzymatic arginylation . The pressor effect of the arginylated derivative on anesthetized rats and its activity on the isolated rat uterus were, respectively, approximately 60% and 20% of those found for the unmodified peptide.

J Biol Chem, 1975 Apr 10, 250(7), 2620 - 5
Purification and properties of rabbit reticulocyte protein synthesis elongation factor 2; Merrick WC et al.; A homogeneous preparation of elongation factor 2 (EF-2) has been obtained from rabbit reticulocytes . EF-2, purified 1,960-fold, appears to be active as a single polypeptide chain with a molecular weight of approximately 100,000 based upon the following determinations: sodium dodecyl sulfate gel electrophoresis (95,000); sedimentation equilibrium centrifugation (112,000); gel filtration (97,000); ADP-ribosylation (103,000) . The amino acid composition of rabbit reticulocyte EF-2 is almost identical with that of rat liver EF-2 . The unknown amino acid in rat liver EF-2 which can be ADP-ribosylated appears also to be present in rabbit reticulocyte EF-2 . A comparison of the amino acid composition of rabbit reticulocyte and rat liver EF-2 with Escherichia coli EF-G shows a high degree of similarity with only four amino acids differing by more than 10% (alanine, lysine, cysteine, and leucine).

J Biol Chem, 1975 Apr 10, 250(7), 2587 - 92
Amino acid sequence of beta-galactosidase . IV . Sequence of an alpha-complementing cyanogen bromide peptide, residues 3 to 92; Langley KE et al.; Intracistronic alpha-complementation between a cyanogen bromide digest of beta-galactosidase and an extract of the lac Zminus operator-proximal deletion mutant M15 was used to monitor the purification of a cyanogen bromide peptide (CB2) responsible for the complementation . Key steps in the purification were ion exchange chromatography on carboxymethylcellulose and sulfopropyl-Sephadex in the presence of urea, and Sephadex gel filtration . CB2 contains residues 3 to 92 of beta-galactosidase . Its sequence is: Ile-Thr-Asp-Ser-Leu-Ala-Val-Val-Leu-Gln-Arg-Arg-Asp-Trp-Glu-Asn-Pro-Gly-Val-Thr-Gln-Leu-Asn-Arg-Leu-Ala-Ala-His-Pro-Pro-Phe-Ala-Ser-Trp-Arg-Asn-Ser-Glu-Glu-Ala-Arg-Thr-Asp-Arg-Pro-Ser-Gln-Gln-Leu-Arg-Ser-Leu-Asn-Gly-Glu-Trp-Arg-Phe-Ala-Trp-Phe-Pro-Ala-Pro-Glu-Ala-Val-Pro-Glu-Ser-Trp-Leu-Glu-Cys-Asp-Leu-Pro-Glu-Ala-Asp-Thr-Val-Val-Val-Pro-Ser-Asn-Trp-Gln-Met . Thus no more than 1/13 of the beta-galactosidase polypeptide chain, starting 2 residues from the NH2 terminus, is necessary for alpha-complementation with M15 as alpha-acceptor.

J Biol Chem, 1975 Apr 10, 250(7), 2581 - 6
Interactions of a glutamate-aspartate binding protein with the glutamate transport system of Escherichia coli; Willis RC et al.; Escherichia coli cells cultured with succinate as the carbon source display apparent K-m values for the uptake of L-glutamate of 10 muM in the absence of added sodium ion and 0.7 muM in the presence of an optimal level of sodium ion (15 to 50 mM) . The glutamate transport system of the succinate cultured cells is noncompetitively inhibited by L-aspartate . A protein which binds glutamate and aspartate with K-D values of 0.7 and 1.2 muM, respectively, is released from the succinate cultured cells by osmotic shock or with the formation of spheroplasts during the preparation of membrane vesicles . The membrane vesicles of succinate cultured cells do not retain the whole cell capacity for L-glutamate uptake, but do retain much of the whole cell capacity for L-aspartate uptake . Culture of E . coli cells with glucose as carbon source causes a 2- to 3-fold repression of glutamate-aspartate binding protein but does not affect the velocity component of glutamate transport . As shown by other workers, the glutamate transport system of glucose cultured cells displays a sodium affected K-m value (FRANK, L., AND HOPKINS, I . (1969) J . Bacteriol . 100, 329-336) and is noncompetitively inhibited by L-aspartate (HALPERN, Y . S., AND EVEN-SHOSHAN, A . (1967) J . Bacteriol . 93, 1009-1016) . Membrane vesicles prepared from glucose cultured cells retain the whole cell capacity for the uptake of glutamate (LOMBARDI, J . F., AND KABACK, H . R . (1972) J . Biol . Chem . 247, 7844-7857) . The glutamate transport system of E . coli strain W appears to be conditionally dependent on the presence of the osmotic shock-releasable glutamate-aspartate binding protein . The results are interpreted to suggest that the binding protein-ligand complex acts as a substrate which is competitive with unbound substrate(s) for a sodium affected translocation process; the organization and specificity of which are dependent on the carbon source of the culture.

J Biol Chem, 1975 Apr 10, 250(7), 2457 - 62
Effects of colicin Ia on transport and respiration in Escherichia coli; Gilchrist MJ et al.; Treatment of Escherichia coli with colicin Ia leads to an inhibition in the active transport of exogenously supplied proline, thiomethyl-beta-D-galactoside and potassium ion . Furthermore, the addition of colicin Ia to cells preloaded with these substances leads to their almost immediate efflux . In contrast, colicin tia treatment enhances by as much as 10-fold the level of accumulation of alpha-methyl-D-glucoside . The colicin Ia-induced stimulation of glucoside accumulation is mediated by the phosphotransferase system . Cells treated with colicin Ia exhibit an increased rate of respiration when glucose is the substrate and a decreased rate when glycerol or succinate is the substrate; The decreased rate of succinate-dependent respiration is probably due to the failure of Ia-treated cells to accumulate succinate.

J Biol Chem, 1975 Apr 10, 250(7), 2609 - 13
Xanthosine-5'-phosphate amidotransferase from Escherichia coli; Patel N et al.; The purified enzyme xanthosine-5'-monophosphate (XMP) aminase from Escherichia coli strain B-96 is shown to possess catalytic activity with either glutamine or ammonia as a substrate . This enzyme, which possesses identical subunits, has the following properties: (a) a pH optimum of 8.3 for both aminase and amidotransferase; (b) an apparent K-m for both glutamine and NH3 of 1 mM; (c) an amidotransferase that is approximately 2 times more active than the aminase; (d) a linear relationship between velocity and enzyme concentrationfor both activities; (e) inhibition of both activities by the glutamine analogue 6-diazo-5-oxo-L-norleucine, but the amidotransferase is more sensitive than the aminase; and (f) inhbiition of both activities by the adenosine analogue, psicofuranine, but again the amidotransferase activity is more sensitive than the aminase . The so-called XMP aminase from the E . coli mutant B-24-1 also has been examined in both crude extracts nad ammonium sulfate fractions and the following data have been obtained: (a) both preparations of enzyme contain aminase and amidotransferase activity; (b) both activities have the same substrate requirements; (c) the pH optima for both activities in the crude extract are identical with those found with the purified enzyme preparation; and (d) the amidotransferase activity in the crude extract and the ammonium sulfate fractions is 2- to 3-fold more active than the aminase . These data demonstrate that this enzyme from E . coli is not strictly a XMP aminase but is, in fact, an amidotransferase capable of utilizing either glutamine or NH3 as a substrate.

J Biol Chem, 1975 Apr 10, 250(7), 2698 - 702
The compatibility of netropsin and actinomycin binding to natural deoxyribonucleic acid; Wartell RM et al.; The simultaneous binding of netropsin and actinomycin to four natural DNAs was studied to determine the influence of one ligand on the binding of the other . Actinomycin binds specifically to GC sites, whereas netropsin binds specifically to AT sites . Spectral titrations, thermal denaturation, and analytical buoyant density centrifugation were employed to measure the binding interference of these drugs . The binding of actinomycin to DNA was decreased by the presence of netropsin . Increasing the GC content of the DNA resulted in a decreased effect of netropsin on actinomycin binding . Quantitative analysis of the binding parameters indicated that netropsin and actinomycin can bind in close proximity along the DNA chain . Supercoiled DNA gave the same result as linear DNA . These results imply that DNA can absorb alterations in conformation within a short distance.

Biochemistry, 1975 Apr 8, 14(7), 1464 - 70
Kinetic studies on coenzyme binding and coenzyme dissociation in tryptophanase immobilized on sepharose; Ikeda S et al.; The binding rate of pyridoxal '5-phosphate (Pxa-P) to apotryptophanase and the dissociation rate of the coenzyme from holotryptophanase were able to be determined by following the enzyme activity in continuous flow reactions on a column of immobilized tryptophanase . When the enzyme activity was assayed continuously in the flow system in the absence of coenzyme added to the reaction mixture, immobilized holotryptophanase lost gradually its initial activity owing to dissociation of coenzyme . The coenzyme dissociation at a given concentration of substrate (tryptophan) followed first-order kineticsmin a low substrate concentration range below the Km value, a more decreased rate constant was obtained for the coenzyme dissociation . This indicates that the coenzyme is more dissociable from the apoenzyme-coenzyme-substrate complex (ECS complex) rather than from the apoenzyme-coenzymecomplex (holoenzyme) . Immobilized tryptophanase freed of coenzyme restored rapidly its original activity, when the assay mixture containing a given concentration of substrate and Pxa-P was passed through the immobilized enzyme column . The coenzyme binding at a given coenzyme concentration followed first-order kinetics, but the rate was not first order in regard to the coenzyme concentration . A plot of the reciprocal of the first-order rate constant obtained vs . the reciprocal of the coenzyme binding occurs in a two-step fashion; the first step is rapid and the second step is rate determining . Both the dissociation constant for the first step and the rate constant for the second step were shown to be independent of the substrate concentration . This means that Schiff base formation between Pxa-P and tryptophan in the assay mixture has no effect on the binding of Pxa-P to apoenzyme . The coenzyme dissociation constant at a given substrate concentration was calculated from both the rate constant of the coenzyme binding and the rate constant of the coenzyme dissociation . The values obtained by this method at different substrate concentrations were almost identical with those measured at the corresponding substrate concentrations directly by an ordinary method.

Biochemistry, 1975 Apr 8, 14(7), 1436 - 44
Europium as a fluorescent probe of transfer RNA structure; Wolfson JM et al.; The binding of europium(III) to Escherichia coli tRNA-fMet,Glu and to unfractionated E . coli tRNA has been investigated by using the 4-thiouridine sensitization of europium 5-Do yields 7-F1 emission and changes in the lifetime of the 5-Do state of europium reported earlier (J . M . Wolfson and D . R . Kearns (1974), J . Am . Chem . Soc . 96, 3653) . Binding of the first 3-4 europium ions is independent and sequential, approximately 600 times stronger than the magnesium binding, and the binding sites are located near the 4-thiouridine residue found at position 8 in a number of E . coli tRNA . Competition experiments suggest the strong binding sites are the same for magnesium and europium . The europium binding properties of both unfractionated E . coli tRNA and purified tRNA-fMet are quite similar, indicating that the location of the strong binding sites and their binding constants are nearly the same for a large group of tRNA . The europium binding properties of native and denatured tRNA are quite different, however.

Biochemistry, 1975 Apr 8, 14(7), 1373 - 8
The nature of protein association with chromatin; Doenecke D et al.; The reality of the nonrandom distribution of histones along the chromatin strand was investigated in several ways . It does not appear to derive from histone exchange during shearing and is evident in chromatin fixed with formaldehyde prior to shearing . Endogenous or Escherichia coli polymerase are preferentially associated with regions of chromatin with a low protein/DNAratio . Although RNA polymerase and histones are fixed to chromatin after formaldehyde treatment with high efficiency, only a minor fraction of non-histone protein is fixed under similar conditions . Even after washing in high salt to minimize adventitious association, most remaining non-histone protein fails to be fixed . The utility of this approach for defining chromosomal proteins is discussed.

Biochemistry, 1975 Apr 8, 14(7), 1454 - 61
Magnetic circular dichroic spectra of cobalt(II) substituted metalloenzymes; Holmquist B et al.; The magnetic circular dichroic (MCD) spectra of cobalt(II) sugstituted metalloenzymes have been studied and compared to a series of four-, five-, and six-coordinate cobalt(II) model complexes previously examined (T . A . Kaden et al . (1974), Inorg . Chem . 13, 2582) . The MCD spectra of cobalt substituted carboxypeptidase A, procarboxypeptidase ta, and thermolysin are consistent with earlier deductions of tetrahedral coordination from absorption spectra and also with X-ray structure analysis . Inhibitors fail to alter their MCD spectra significantly . The MCD spectra of cobalt alkaline phosphatase and carbonic anhydrase are more complex and their pH dependence and alteration by inhibitors are discussed in terms of known cobalt(II) models.

Biochemistry . 1975 Apr 8;14(7):1503.
Partial reconstitution of active ribosomes and 50S subunits; Hernandez F et al.; Escherichia coli ribosomes and their 50S subunits disassembled by LiCl treatment can be reconstituted into structurally completed but inactive particles . However, peptidyltransferase and polyphenylalanine synthesizing activity can be partly recovered by the addition of methanol to the reconstitution system . Furthermore, entirely active ribosomes and 50S subunits are reconstituted when methanol is present during the initial treatment with LiCl to disassemble the ribosomal components . The presence of methanol (10% v/v) during this treatment diminished the release of some proteins but does not affect the separation of the 5S RNA.

Arch Microbiol, 1975 Apr 7, 103(2), 155 - 62
The control by respiration of the uptake of alpha-methyl glucoside in Escherichia coli K12; Hernandez-Asensio M et al.; The uptake of methyl alpha-D-glucopyranoside (alpha-MG) by Escherichia coli K12 was decreased by the addition of substrates which stimulated the rate of oxygen consumption by the cells . The inhibition, which occurred only at non-saturating concentrations of alpha-MG, was not the result of a stimulation of the rate of exit of intracellular alpha-MG, and was abolished by the presence of carbonyl cyanide m-chlorophenylhydrazone or sodium azide . Since those drugs inhibit energy conservation at the respiratory chain and did not alter significantly the rate of oxygen consumption under the conditions for the assay of alpha-MG uptake, it appears that the inhibition of the transport system by respirable substrates is mediated by some form of energy derived from respiration.

Biochim Biophys Acta, 1975 Apr 7, 385(2), 305 - 11
Structure of the glycopeptide storage material in GM 1 gangliosidosis . Sequence determination with specific endo- and exoglycosidases; Tsay GC et al.; 1 . An endo-beta-galactosidase from Escherichia freundii, specific for the hydrolysis of desulfated keratan sulfate, quantitatively liberated a trisaccharide (Gal-GlcNAc-Gal) from a glycopeptide (Mr 1800) isolated from the liver of a patient with GM 1 (generalized) gangliosidosis . 2 . The remaining glycopeptide was susceptible to sequential digestion with purified beta-N-acetylhexosaminidase and exo-beta-galactosidase from Jack Bean meal . 3 . These and other studies established the structure of the stored glycopeptide to be:(see article) which probably represents the desulfated linkage region of skeletal keratan sulfate.

Biochim Biophys Acta, 1975 Apr 2, 383(4), 457 - 63
A simple method for the preparation of large quantities of pure plasmid DNA; Humphreys GO et al.; Polyethylene glycol quantitatively precipitates plasmid DNA of molecular weight 6-123-10-6, from cleared lysates of plasmid-carrying bacterial strains, After resuspension and density-gradient centrifugation of the precipitated DNA, it is unchanged in length and in transformation efficiency for Escherichia coli K12 . Plasmid DNA can be easily prepared in large quantities by including a polyethylene glycol precipitation step in standard plasmid isolation procedures.

Biochim Biophys Acta, 1975 Apr 2, 383(4), 435 - 40
Ineffectiveness of rifampicin in inhibiting RNA synthesis in Escherichia coli and T(4)-infected Escherichia coli cells after exposure to ultraviolet radiation; Prakash RK et al.; Resdiual RNA synthesis in Escherichia coli cells and T(4)-infected E . coli cells exposed to ultraviolet radiation is insensitive to rifampicin . On the other hand, residual RNA synthesis in these cells after gamma irradiation is further inhibited by rifampicin . The specific effect exerted by ultraviolet irradiation on transcription seems to arise from alterations in the DNA structure rather than in bacterial RNA polymerase since RNA synthesis in ultraviolet-irradiated E . coli cells infected with unirradiated T(4) is sensitive to rifampicin.

Biochim Biophys Acta, 1975 Apr 2, 383(4), 359 - 69
Mitochondrial DNA of Tetrahymena pyriformis strain ST contains a long terminal duplication-inversion; Arnberg AC et al.; 1 . We have studied denatured Tetrahymena mtDNA by electron microscopy using the formamide technique . 2 . After denaturation all DNA is single stranded, but within a few minutes single-stranded circles with a duplex tail are formed . 3 . The duplex tail is 1.3 mum long, i.e . 8 percent of the length of native mtDNA, and it often contains a small single-stranded eye . 4 . Digestion of the duplex DNA with exonuclease III of Escherichia coli abolishes its ability to form circles and duplex tails after denaturation . 5 . Renaturation of denatured mtDNA leads to the formation of duplex circles with single-stranded section and/or duplex tails . In addition, a minority of duplex circles without apparent tails is formed, but these circles contain a small ambiguous section . 6 . We conclude that this mtDNA contains a long terminal duplication-inversion, that could be involved in the replication of this linear mtDNA.

Biochim Biophys Acta, 1975 Apr 2, 383(4), 345 - 50
Cyclic adenosine monophosphate receptor: effect of cyclic AMP analogues on DNA binding and proteolytic inactivation; Krakow JS; The cyclic AMP receptor protein of Escherichia coli in the presence of cyclic AMP undergoes a conformational change resulting in an increased affinity for DNA and an increased susceptibility to attack by proteolytic enzymes resulting in loss of DNA binding capacity . Of several cyclic nucleotides tested only cyclic AMP and cyclic tubercidin monophosphate are able to effect the conformational transition in cyclic AMP receptor protein, prerequisite to proteolytic inactivation or DNA binding . Other analogues such as cyclic GMP or cyclic IMP which are competitive inhibitors of cyclic AMP do not support DNA binding or proteolytic inactivation.

Eur J Biochem, 1975 Apr 1, 52(3), 445 - 9
The specificity of a neutral deoxyribonuclease from Cancer pagurus; Sabeur G et al.; The recently isolated neutral deoxyribonuclease from crab (Cancer pagurus) testes has been characterized in its mode of action and its specificity . The enzyme is a typical endonuclease, forming 5'-phosphate oligonucleotides of large average size; after extensive digestion of calf thymus DNA over 75% of the fragments have a size larger than pentanucleotides and mononucleotides are absent . As far as specificity is concerned, thymidine is very abundant in the 5'-penultimate position (approximately 50%) and in the 3'-terminal position (37%) and almost absent in the 5'-terminal position (approximately 1%), the values quoted concerning Escherichia coli digests of average size (Pn) between 50 and 10.

Cell, 1975 Apr, 4(4), 329 - 35
Effect of reduced temperatures on protein synthesis in mouse L cells; Craig N; The rate of incorporation of leucine into protein, the rate of polypeptide elongation and termination, and the relative quantity and size of polysomes were analyzed in mouse L cells grown in suspension culture at various temperatures between 0 degrees C and 36 degrees C . Between 10 degrees C and 36 degrees C protein synthesis exhibited two different apparent activation energies (39 kcal/mole, 10-25 degrees C; 14 kcal/mole, 25-36 degrees C), whereas elongation and termination had only one (16 kcal/mole) . Below 36 degrees C, the polysome level and size decreased, reaching a minimum of 30% of the control 36 degrees C values at 10 degrees C; below 10 degrees C the level increased again back to control values at 0 degrees C . The polysome decline was time dependent, requiring about 5 hr to reach the equilibrium value . This decline is completely reversible within 60 min, even in the presence of 4 mug/ml of actinomycin D, and even after 15 hr of incubation at the lower temperature . The results suggest that polypeptide initiation is rate limiting, particularly below 25 degrees C; whereas above this temperature, elongation or perhaps some other process may be limiting . These results are quite different from those obtained for E . coli and rabbit reticulocyte protein synthesis.

South Med J, 1975 Apr, 68(4), 479 - 80
Monilial esophagitis; Mann NS et al.; A case of monilial esophagitis, developing in a diabetic patient treated with gentamicin for Escherichia coli septicemia, is described . The esophagogram was normal and the diagnosis was confirmed by fiberoptic esophagoscopy and biopsy examination . The patient responded to oral nystatin . The literature on esophageal moniliasis is reviewed.

Clin Exp Immunol, 1975 Apr, 20(1), 161 - 77
Ultrastructural and serological studies on the resistance of activated B cells to the cytotoxic effects of anti-immunoglobulin serum . Patch and cap formation of surface immunoglobulin on mitotic B lymphocytes; Kerbel RS et al.; Previous studies have shown that rabbit anti-mouse immunoglobulin sera (anti-Ig) which kill non-activated B lymphocytes in the presence of complement, are incapable of doing so when the cells are activated by antigen or mitogen into mitosis . Results reported here indicate that the resistance is not dependent on either the source of antiserum or complement, or on the presence of a mitotic inhibitor, colcemid . Immunoperoxidase staining-electron microscopy techniques were applied to assess whether there was any conspicuous difference between unstimulated versus mitogen-stimulated, mitotic cells with respect to density or distribution of cell surface Ig . No such differences were found; furthermore, mitotic cells showed rapid classical 'patch and cap' formation of cell surface Ig when incubated with anti-Ig at room temperature, indicating the retention of fluid membrane dynamics by lymphocytes in this stage of the cell cycle . In contrast to this cytotoxic resistance, T or B lymphocytes in mitosis were found to be as sensitive, or more so, to lysis by various other antisera when compared to non-mitotic cells . Thus the resistance of mitotic B cells to the cytotoxic effects of anti-Ig serum seems unique and appears independent of any conspicuous quantitative or qualitative change in cell surface Ig.

Acta Pathol Microbiol Scand {B}, 1975 Apr, 83(2), 121 - 4
Two new Escherichia coli o antigens, o162 and o163, and one new h antigen, h56 . withdrawal of h antigen h50; orskov I et al.; Three E . coli strains representing strains from faeces of normal persons were established as antigenic test strains of two new O antigens, O162 and O163, and one new H antigen, H56.

Z Klin Chem Klin Biochem, 1975 Apr, 13(4), 149 - 55
Determination of serum nucleotidase with cytidine monophosphate as substrate, (I); van der Kooij PJ et al.; This paper deals with a new method for the determination of serum nucleotidase (EC 3.1.3.5) . The assay is performed with cytidine-5'-monophosphate as substrate, followed by deamination of the generated cytidine . The principle of the method and the determination of the liberated ammonia by the Berthelot indophenol-reaction are comparable to the Persijn--van der Slik method in which adenosine-5'-monophosphate is used as substrate . The correlation between the results obtained with these two methods was found to be good; the new method has the advantage of higher sensitivity.

J Biochem (Tokyo), 1975 Apr, 77(4), 753 - 9
Effects on ribosomes of the substitution of spermidine or divalent cations for magnesium ions; Igarashi K et al.; The inactivation of rat liver ribosomes (polysomes) resulting from the replacement of bound magnesium by spermidine has been studied using a dialysis method . Inactivation of the ribosomes (polysomes) occurred when more than 40% of the Mg2+ originally bound to ribosomes in the presence of 2 mM Mg2+ was replaced by spermidine . The polypeptide-synthesizing activity of polysomes was retained partially when Mg2+ was replaced by Mn2+ by dialysis . E . coli 30S ribosomal subunits were more resistant to inactivation due to the replacement of Mg2+ by spermidine than the 50S subunits.

J Biochem (Tokyo), 1975 Apr, 77(4), 719 - 28
Binding of aminoacyl-tRNA to ribosomes promoted by elongation factor Tu . Studies on the role of GTP hydrolysis; Yokosawa H et al.; The role of guanosine triphosphate (GTP) in the elongation factor Tu(EF-Tu)promoted binding of aminoacyl-tRNA to ribosomes has been investigated . In the presence of EF-Tu and GTP, phenylalanyl-tRNA (Phe-tRNA) was bound to the A site of the poly(U).ribosome complex having N-acetylphenylalanyl-tRNA on its P site . EF-Tu could be utilized repeatedly in this binding reaction in the presence of GTP but was used only once and remained bound to ribosomes when GTP was replaced by 5'guanylyl methylenediphosphonate (Gpp(CH2)p), a nonhydrolyzable analog of GTP . The binylalanyl-tRNA, while little dipeptide was formed in the latter case . However, when EF-Tu and Gpp(CH2)p bound to the ribosomal complex were released by centrifugation through 10% sucrose containing 0.2 mM GDP, the yield of the dipeptide was correspondingly increased . It was concluded that the role of GTP in this reaction is to facilitate the transfer of Phe-tRNA to ribosomes by virtue of the high affinity of EF-Tu.GTP for ribosomes . The subsequent conversion of EF-Tu.GTP to EF-Tu.GDP, a form of EF-Tu with low affinity for ribosomes as well as for Phe-tRNA, resulted in the detachment of EF-Tu . Thus, the hydrolysis of GTP seems to be required for the release of EF-Tu from ribosomes, which is necessary for peptidyl transfer and the reutilization of EF-Tu . A freely reversible interaction between ribosome and Phe-tRNA.EF-Tu.Gpp(CH2)p complex was also demonstrated.

J Biochem (Tokyo), 1975 Apr, 77(4), 689 - 94
Preferential production of IgG2 anti-hapten antibody by immunization with hapten-conjugated Escherichia coli; Furuichi K et al.; It is well-known that immunization of guinea pigs with hapten-protein conjugates induces concomitant production of IgG1 and IgG2 anti-hapten antibodies . However, the synthesis of antibody to 2, 4-dinitrophenyl groups (DNP) was found to be restricted to one of the IgG antibodies (IgG2 antibody) when guinea pigs were immunized through repeated intraperitoneal injections of 0.1 mg of 2, 4-dinitrophenylated Escherichia coli (DNP-E coli) . This selective induction of IgG2 anti-DNP antibody formation occurred both in the presence and absence of Freund's adjuvants, whereas a trace of IgG1 anti-DNP antibody was produced concomitantly on increasing the immunizing dose of DNP-E . coli (3.0 mg) in Freund's complete adjuvant (FAC).

Nippon Yakurigaku Zasshi, 1975 Apr, 71(3), 263 - 71
{Mechanisms of release of leucocytic pyrogen}; Ogawa Y et al.; Mechanisms of the production and release of leucocytic pyrogen (LP) from peritoneal polymorphonuclear leucocytes (PMN) from rabbits were studied in vitro . The following results were obtained . 1) The fever curves of the microsomal and the lysosomal fractions had a later onset and were longer lasting than those from the extracellular fluid . The fever curves of the supernatant of 105,000 g showed the typical response of endogenous pyrogen characterized by a rapid onset and short lasting fever, as shown by the extracellular fluid . 2) The pyrogenicity of LP was the most potent at 60 min while the 105,000 g supernatant was most potent at 30 min after PMN incubation at 37 degrees C . 3) The extracellular protein attained the maximum level at 30 min after incubation of PMN, while the protein content in the supernatant of 105,000 g of PMN decreased gradually to the constant level at 30 min . 4) It was observed that the lysosomal degradation was stimulated with bacterial pyrogen (LPS) at 37 degrees C but LPS did not directly affect the lysosomal fraction of PMN.

Surgery, 1975 Apr, 77(4), 520 - 9
Effect of antithymocyte globulin and other immune reactants on human platelets; Rosenberg JC et al.; Human platelets suspended in autologous plasma do not respond to nonspecific immune complexes as do platelet suspensions from rabbits and dogs . However, platelets of all three species do undergo aggregation and the release reaction when exposed to antibodies directed against platelets . Antithymocyte globulin (ATG) contains such antibodies, apparently because of antigens common to both thymocytes and platelets . ATG-induced platelet aggregation and release is thus a specific reaction which may be responsible for the thrombocytopenia and thrombotic complications occasionally seen following the administration of ATG . However, if ATG is given properly, its effect on platelets should not constitute a contraindication to the use of this immunosuppressive drug . Since nonspecific immune complexes do not affect human platelets in the presence of plasma, it would appear that platelet aggregates seen in hyperacute and acute rejection result from endothelial damage rather than an effect of immune complexes on platelets.

Mutat Res, 1975 Apr, 28(1), 1 - 7
Induction and mutagenesis of prophage lambda in Escherichia coli K12 by metabolites of aflatoxin B1; Goze A et al.; Like most carcinogens, aflatoxin B1 must be activated by mammalian microsomal enzymes to give rise to coupounds active on bacteria . These compounds act as inducers of E . coli K12 (lambda) at a high efficiency, whereas unmodified aflatoxin B1 has no effect . Moreover, metabolites of aflatoxin B1 have a mutagenic action on phage lambda, as shown by the appearance of clear plaque mutants . We propose the hypothesis that the same derivative is responsible for carcinogenesis of liver cells by aflatoxin B1 . Therefore, our system provides a simple way of measuring in vitro, in the same assay, the mutagenic and inducing activities of compounds to which the cells are permeable, thereby detecting potentially carcinogenic agents.

Equine Vet J, 1975 Apr, 7(2), 97 - 101
Maintenance of fertility in the horse including artificial insemination; Frhr J et al.; A high fertility rate depends on many different factors and is always related to inheritance and enviorment . The successful feritly control system in the German Thoroughbred breeding industry shows that fertility can be increased by good management and veterinary supervision . The insemination of horses with frozen semen is discussed . Replacement of natural service by A.I . with frozen semen is not generally accepted in horsebreeding, as the conditions are entirely different from cattle breeding . However, there are several ways in which A.I . can be assistance in stud management.

Equine Vet J, 1975 Apr, 7(2), 86 - 90
Surgical repair of cleft palate in the horse; Jones RS; Surgical repair of a cleft palate was carried out in three horses . Mandibular symphisotomy allowed adequate exposure of the defect . The first subject, a young foal died from inhalation pneumonia but the other two made satisfactory recoveries . The problems of closure of the lip and symphysis are discussed.

Nucleic Acids Res, 1975 Apr, 2(4), 537 - 44
The activation of RNA polymerase by alkylammonium ions; Malcom AD et al.; Tetramethylammonium and tetraethylammonium ions both lower the melting temperature of DNA . We have shown that these ions increase the activity of E . Coli RNA polymerase . When the base composition of the DNA template was varied, there was a correlation between the decrease in melting temperature and increase in RNA polymerase activity . No stimulation was observed when heat denatured DNA was used as template . It was shown that initiation of RNA synthesis was stimulated but to a smaller degree than was total RNA synthesis . The lag observed at low temperatures before RNA synthesis commences was found to be shorter in the presence of these alkyl ammonium ions . All these results are consistent with an unwinding of the double helix being an important step in transcription.

Nucleic Acids Res, 1975 Apr, 2(4), 447 - 58
Detection of cation-specific conformational changes in ribosomal RNA by gel electrophoresis; Morris DR et al.; Electrophoresis of ribosomal RNA in polyacrylamide-agrose composite gels separates 16S and 23S species into multiple bands . These bands of RNA represent multiple conformational forms of the molecules as judged by oligonucleotide analysis of the 16S RNA . Gel elctrophoresis was used to test for cation-specific conformational changes in ribosomal RNA . Relative to magnesium-equilibrated RNA, barium ion and putrescine induced alterations in the electrophoretic behavior of ribosomal RNA while calcium ion produced no change . Exchange of a critical level of bound magnesium ion for barium or putrescine was necessary for these changes to take place . The alterations in electrophoretic behavior were unaffected by simply restoring magnesium ion, but in addition required heating for reversal . We suggest that these conformational changes are a result of interaction at a specific class of cation binding sites previously observed with intact ribosomes.

Proc Natl Acad Sci U S A, 1975 Apr, 72(4), 1533 - 7
A ribonucleoprotein precursor of both the 30S and 50S ribosomal sunbunits of Escherichia coli; Duncan MJ et al.; A ribonucleoprotein particle (46S) has been isolated from {3H}uridine pulse-labeled cultures of E . Coli AB301/105 . Evidence from pulse chase experiments and from protein analysis suggested that this particle may give rise to both the 30S and 50S ribosomal subunits . Direct deproteinization of the particle yielded 30S RNA, while deproteinization after treatment with a crude RNase III preparation yielded products similar to 23S and 16S RNA . This result is consistent with the idea that the 46S ribonucleoprotein is the in vivo counterpart of 30S RNA, which is the in vitro product obtained after phenol extraction.

Proc Natl Acad Sci U S A, 1975 Apr, 72(4), 1505 - 9
Affinity labeling of the ribosomal decoding site with an AUG-substrate analog; Pongs O et al.; The trinucleotide AUG was condensed at the 5'-end with N-bromoacetyl-p-aminophenylphosphate . This bromoactylated AUG analog reacted irreversibly with the mRNA binding site of Escherichia coli 70S ribosomes . After reaction of 70S ribosomes with the AUG analog, labeled 30S subunits could be isolated that were programmed for initiation-factor-dependent binding of fMet-tRNAfMet . This shows that this AUG-affinity label reacted in the decoding site for fMet-tRNAfMet . By combination of sodium dodecyl sulfate-, Sarkosyl-, and ureapolyacrylamide gel electrophoresis the AUG-affinity label was found to be irreversibly bound to ribosomal proteins S4, the ram gene product, and S18.

Proc Natl Acad Sci U S A, 1975 Apr, 72(4), 1354 - 8
Proposal concerning mechanism of evolution of the genome of Escherichia coli; Zipkas D et al.; Many pairs of genes whose gene products are functionally related lie either 90 degrees or 180 degrees apart on the circular map of the E . coli chromosome . A mechanism of evolution is proposed that involves two sequential duplications of an ancestral genome, followed by mutation and divergence of function of replicate genes.

Proc Natl Acad Sci U S A, 1975 Apr, 72(4), 1344 - 8
Mapping of late adenovirus genes by cell-free translation of RNA selected by hybridization to specific DNA fragments; Lewis JB et al.; Cytoplasmic RNA, isolated from cells late after infection by adenovirus type 2 and fractionated by hybridization to specific fragments of adenovirus DNA produced by cleavage with the endonuclease R-EcoRI, was used as template for protein synthesis in cell-free mammalian extracts . Each of the R-EcoRI fragments of DNA selects RNA that encodes specific subsets of the viral polypeptides . From the known order of the R-EcoRI fragments, the following partial map is deduced: (III, IIIa, IVa2, V, P-VII, IX), (II, P-VI), 100K, IV-where the relative order of the components enclosed in parentheses has not yet been determined.

Proc Natl Acad Sci U S A, 1975 Apr, 72(4), 1254 - 7
Molecular basis of beta-galactosidase alpha-complementation; Langley KE et al.; In previous studies, a cyanogen bromide peptide derived from amino-acid residues 3-92 of beta-galactosidase (EC 3.2.1.23; beta-D-galactoside galactohydrolase) was shown to have alpha-donor activity in intracistronic alpha-complementation . We have now isolated the defective beta-galactosidase alpha-acceptor protein from the deletion mutant strain M15 of Escherichia coli and find that it lacks residues 11-41 of betal-galactosidase . This is demonstrated by the isolation and sequence determination of a cyanogen bromide peptide from the M15 protein, which is identical to the corresponding peptide from beta-galactosidase except for the missing amino acids . We conclude that the alpha-donor peptide restores the region missing in the M15 protein.

Lab Invest, 1975 Apr, 32(4), 561 - 9
Pathophysiologic responses of the subhuman primate in experimental septic shock; Coalson JJ et al.; The grave clinical aspects of septic shock have stimulated the search for an experimental animal model which more closely relates to human pathophysiology . This study of the cardiovascular-pulmonary-morphologic responses of the baboon to slow infusions of live Escherichia coli organisms was designed to approximate more closely the human clinical entity . Anesthetized young adult baboons received 3-hour intravenous infusions of organisms at an average dosage of 8 times 10-9 organisms per kg . body weight . Responses of animals were followed during a period of 6 hours in the anesthetized state . There was progressive systemic hypotension and steadily decreasing cardiac output . Total peripheral resistance was uniformly depressed during the infusion, but was variable during the post-infusion survival period . Increases in heart rate, alveolar-arterial oxygen tension gradient, and oxygen uptake were uniformly present . These alterations bear close resemblance to those seen in other subhuman primates administered short term doses of live organisms . There were extensive morphologic changes in pulmonary, cardiac, and renal beds . Glomeruli contained multiple fibrin thrombi and disrupted platelets, and the glomerular capillary endothelium was focally edematous and disrupted . The myocardium exhibited capillary endothelial edema and fluid accumulation in interfiber and intrafiber spaces . There were sequestration, degranulation, and fragmentation of polymorphonuclear leukocytes and platelets, and characteristic endothelial lesions within the pulmonary vascular bed . Findings demonstrate both cardiovascular-pulmonary dysfunction and renal, cardiac, and pulmonary morphologic lesions . The baboon shock model appears to be well suited for studies of experimental septic shock and bears close resemblance to the human clinical entity.

J Exp Med, 1975 Apr 1, 141(4), 753 - 60
Genetical control of B-cell responses . III . Requirement for functional mitogenicity of the antigen in thymus-independent specific responses; Coutinho A et al.; Spleen cells from C3H/HeJ mice fail to respond with polyclonal antibody synthesis to mitogenic concentrations of lipopolysaccharide (LPS) which are optimal for activating spleen cells from a high-responder strain (B10.5M) . This unresponsiveness is selective for LPS, since C3H/HeJ cells respond as normals to another B-cell mitogen, purified protein derivative of tuberculin . Spleen cells from low-responder mice also fail to mount a specific anti-NNP plaque-forming cell (PFC) response, when challenged in vitro by NNP-LPS . However, C3H/HeJ cells develop normal responses to another thymus-independent hapten conjugate, DNP-AECM-Ficoll . C3H/HeJ mice fail to mount a specific anti-LPS antibody response, when challenged in vivo with doses of soluble LPS which are fully immunogenic for the high-responder strain . However, C3H/HeJ mice develop normal direct and indirect PFC responses to LPS, when challenged with a thymus-dependent form of the immunogen . These results are interpreted as indicating as absolute requirement for functional mitogenicity of the antigen, in the induction of specific thymus-independent antibody responses.

Bioinorg Chem, 1975 Apr, 4(3), 245 - 55
Preparation, properties and biological activities of succinyl derivatives of vitamin B12; Toraya T et al.; Three new derivatives of vitamin B12,0-2'-succinyl-, 0-5'-succinyl-, and 0-2', 0-5'-disuccinyl-vitamin B12, whose alpha-ribose moieties of the nucleotide ligand are succinylated, were prepared by reaction of the vitamin with succinic anhydride . The first succinylation took place rapidly and almost predominantly on 5'-OH of alpha-ribose, and the second succinylation much more slowly on 2'-OH . From the behaviors in paper electrophoresis and the lability to CH- of cobalt-base bond of 0-2'-succinylated vitamin B12 derivatives, it was suggested that the terminal COOH of the 0-2'-succinyl group forms an inner salt with the imidazole nucleus of 5,6-dimethylbenzimidazole . Monosuccinyland disuccinyl-vitamin B12 by mild acid or base hydrolysis . Heating at 130 degrees for 5 min also led to the complete severance of the succinyl group of 0-5'-succinyl-vitamin B12 . None of the three succinly derivatives inhibited the diol dehydrase reaction when added with coenzyme B12, SUggesting that the ability to bind to the apoenzyme is strongly diminished or almost lost by succinylation on 2'- or 5'-OH of alpha-ribose . None of the succinyl vitamin B12 compounds showed either biological activity or anti-vitamin B12 activity when tested with Escherichia coli 215, a methionine-B12 auxotroph . None of them significantly inhibited {3-H} vitamin B12 uptake by E . coli 215 cells . This observation implies that succinyl derivatives of vitamin B12 are hardly incorporated into the cells of E . coli.

Appl Microbiol, 1975 Apr, 29(4), 537 - 9
Violet red bile 2 agar for stressed coliforms; Hartman PA et al.; Counts on a new, autoclave-sterilizable violet red bile (VRB-2) agar were compared with counts on freshly boiled VRB agar . Yields on VRB-2 agar averaged 217, 180, 130, and 112% of counts obtained on the control medium for samples of water, cottage cheese, frozen vegetables, and raw milk, respectively . The general principle used for the development of VRB-2 agar could be applied to many other kinds of selective plating media.

Am J Vet Res, 1975 Apr, 36(4 Pt 2), 568 - 71
Immune response to Escherichia coli; Gross WB et al.; Chickens from a line artifically selected for lew level of plasma corticosterone (LPC) produced higher antibody titers, produced antibody earlier, continued the production of antibody longer, responded to lower dosages of antigen, and had greater body weight gain than those from a line slected for a high level of plasma corticosterone (hpc) . the HPC line resisted Escherichia coli challenge and antigen more effectively than the LPC line . It appears that, although the HPC line is more effective in its defense against bacterial infections, the LPC line can be aided more by vaccines.

J Med Chem, 1975 Apr, 18(4), 399 - 403
Cyclopenta{f}isoquinoline derivatives designed to bind specifically to native deoxyribonucleic acid . 2 . Synthesis of 6-carbamylmethyl-8-methyl-7(5)H-cyclopenta{f}isoquinolin-3(2H)-one and its interaction with deoxyribonucleic acids and poly(deoxyribonucleotides); Kundu NG et al.; 3-Ethoxy-8-methyl-5,6-dihydro-7H-cyclopenta{f}isoquinolin-5-one (2) was converted to 6-carbethoxymethyl-3-ethoxy-8-methyl-5,6-dihydro-7H-cyclopenta{f}isoquinolin-5-one (6) through an oxalyl derivative . Treatment of 6 with ammonia gave the corresponding amide 7 which on sodium borohydride reduction and subsequent dehydration yielded 6-carbamylmethyl-3-ethoxy-8-methyl-7(5)H-cyclopenta{f}isoquinoline (9) . The analogous ester 10 was similarly obtained from 6.Numerous attempts to dealkylate the 3-ethoxy group of 9 or 10 failed . However, 6 coould easily be dealkylated on heating with 25% hydrochloric acid in a sealed tube.The ester, 6-carbethoxymethyl-8-methyl-5,6-dihydro-7H-cyclopenta{f}isoquinoline-3(2H),5-dione (11), so obtained was converted to the corresponding amide 12 which on reduction with sodium borohydride and subsequent dehydration afforded the desired compound, 6-car-bamylmethyl-8-methyl-7(5)H-cyclopental{f}isoquinolin-3-(2H)-one (1) . 1 was found to be mildly cytotoxic againstL5178Y mouse leukemia cells in culture.1 was also found to bind to native calf thymus DNA . 1 inhibited RNA synthesis by a DNA-dependent RNA polymerase and a higher inhibition of RNA synthesis was observed when poly(dG-dC) was used as a template than when poly(dA-dT) was used . A significant increase of thermal transition temperature of calf thymus DNA and poly(dG)-poly(dC) was observed in the presence of 1 . The accumulated evidence demonstrates that 1 interacts weakly with calf thymus DNA and interacts preferentially with poly(deoxyribonucleotides)-containing GC pairs.

J Bacteriol, 1975 Apr, 122(1), 73 - 9
Indirect selection for plasmid mutants: isolation of ColVBtrp mutants defective in self-maintenance in Escherichia coli; Koyama AH et al.; An efficient method for isolation of a large number of plasmid mutants is described . It is based on the fact that N-methyl-N'-nitro-N-nitrosoguanidine induces a number of closely linked mutations within a short segment of the bacterial chromosome . Thus, selection for reversions of an auxotrophic marker located on the ColVBtrp plasmid yielded a large fraction (more than 50 percent) of mutants defective in some plasmid functions, including its own maintenance in the host bacteria . The results of preliminary characterization of strains carrying these mutated plasmids are presented.

J Bacteriol, 1975 Apr, 122(1), 66 - 72
Properties of alpha-dehydrobiotin-resistant mutants of Escherichia coli K-12; Eisenburg MA et al.; We have isolated four classes of mutants resistant to alpha-dehydrobiotin, a biotin analogue . One mutant group, referred to as bioR shows high excretion levels of biotin vitamers, derepressed levels of the biotin biosynthetic enzymes, and resistance to repression by biotin . The mutation has been mapped between argC and bfe at min 79 . A second class of mutants, with lesions in the bioA operon at min 17.5, shows derepressed levels of the dethiobiotin synthetase enzyme and has been tentatively designated as bioO mutants . The other two mutant groups show alterations in permeability: biotin uptake is markedly reduced in one, whereas in the other proline uptake is also affected . The former mutation lies near metE at min 75 and has been designated as bioP . The permeability mutants in the second group also show poor growth on minimal media, suggesting a generalized permeability effect . This mutation, designated as P, has not been mapped.

J Bacteriol, 1975 Apr, 122(1), 59 - 65
Detection of a protein, similar to the sex pilus subunit, in the outer membrane of Escherichia coli cells carrying a derepressed F-like R factor; Beard JP et al.; The outer membranes of Escherichia coli K-12 cells carrying a derepressed F-like R factor contain about 7 times 10-4 molecules per cell of a protein similar to the subunit of the sex pili specified by the R factor . This protein pool is absent in cells carrying the repressed variant of the R factor . The size of the pool is about one-half of the amount of protein incorporated into mature sex pili at the peak of production and is independent of the phase of growth of the culture . The molecular weight of the protein in the pool and of the subunit of the sex pili specified by the cells is 12,500 plus or minus 600.

J Bacteriol, 1975 Apr, 122(1), 257 - 65
Isolation and partial characterization of three Escherichia coli mutants with altered transfer ribonucleic acid methylases; Marinus MG et al.; Seven transfer ribonucleic acid (tRNA) methylase mutants were isolated from Escherichia coli K-12 by examining the ability of RNA prepared from clones of unselected mutagenized cells to accept methyl groups from S-adenosylmethionine catalyzed by crude enzymes from wild-type cells . Five of the mutants had an altered uracil-tRNA methylase; consequently their tRNA's lacked ribothymidine . One mutant had tRNA deficient in 7-methylguanosine, and one mutant contained tRNA lacking 2-thio-5-methylaminomethyluridine . The genetic loci of the three tRNA methylase mutants were distributed over the E . coli genome . The mutant strain deficient in 7-methylguanosine biosynthesis showed a reduced efficiency in the suppression of amber mutations carried by T4 or lambda phages.

J Bacteriol, 1975 Apr, 122(1), 19 - 24
Characterization of mucoid mutants of Escherichia coli K-12 isolated after exposure to ozone; Hamelin C et al.; Seventy mucoid mutants retaining the phenotypic properties of the ultraviolet-induced lon strains of Escherichia coli K-12 were isolated after treatment of strain MQ259 (lon+) with ozone . They produce mucoid colonies at 37 C on minimal agar, are abnormally sensitive to radiation, and tend to grow in long aseptate filaments after irradiation with ultraviolet light . Results indicate also that ozone sensitivity, radiosensitivity, and mucoidy are pleiotropic properties of the lon gene.

J Bacteriol, 1975 Apr, 122(1), 159 - 70
Stimulation of adenosine 5'-triphosphate-dependent in vitro deoxyribonucleic acid replication by factors from the periplasmic space of Escherichia coli; Smith DW et al.; In vitro deoxyribonucleic acid (DNA) synthesis systems based on an earlier system using pencillin have been developed which use osmotic lysis of lysozyme-formed spheroplasts of Escherichia coli cells embedded in an agarose matrix . An adenosine 5'-triphosphate (ATP)-dependent semiconservative mode, or replicative mode, of in vitro DNA synthesis is exhibited which is sensitivie to nalidixic acid . These systems require growth of the agar-embedded cells in a preincubation medium before spheroplast formation and osmotic lysis . Inhibitor studies suggest that one or more required macromolecular species are synthesized during this preincubation growth period . Osmotic shock fluid from E . coli contains macromolecular factors which preferentially stimulate the ATP- dependent semiconservative mode of in vitro DNA synthesis . In some cases, the ATP independent mode of synthesis is inhibited by shock fluid . Evidence is presented that the stimulating factors found in the osmotic shock fluid come from the E . coli periplasmic space . This stimulation is observed using either toluene-treated cells or lysed agar-embedded ethylene glycol-bis-(beta-aminoethyl ether) N,N'-tetraacetate-lysozyme spheroplasts, and is thus independent of the in vitro DNA synthesis system used . Shock fluid obtained from a given E . coli dna mutant does not stimulate in vitro DNA synthesis by that mutant . However, in some cases, shock fluid from one class of dna mutants does stimulate ATP dependent in vitro DNA synthesis by another class of dna mutants, in a thermosensitive reacaction . Gently prepared cell extracts also stimulate ATP-dependent in vitro DNA synthesis, whereas cell extracts prepared by more severe procedures inhibit this in vitro synthesis . Severl stimulating DNA replication factors may be present in the osmotic shock fluid, including products of E . coli dna genes.

J Bacteriol, 1975 Apr, 122(1), 129 - 38
Deoxyribonucleic acid-cytosine methylation by host- and plasmid-controlled enzymes; May MS et al.; Deoxyribonucleic acid (DNA)-cytosine methylation specified by the wild-type Escherichia coli K 12 mec+ gene and by the N-3 drug resistance (R) factor was studied in vivo and in vitro . Phage lambda and fd were propagated in the presence of L-{methyl-3H}methionine in various host bacteria . The in vivo labeled DNA was isolated from purified phage and depurinated by formic acid-diphenylamine treatment . The resulting pyrimidine oligonucleotide tracts were separated according to size and base composition by chromatography on diethylaminoethyl-cellulose in 7 M urea at pH 5.5 and 3.5, respectively . The distribution of labeled 5-methylcytosine in DNA pyrimidine tracts was identical for phage grown in mec+ and mec minus (N-3) cells . For phage lambda the major 5-methylcytosine containing tract was the tripyrimidine, C2T; for both fd-mec minus (N-3) DNA and fd-mec+DNA, C2T was the sole 5-methylcytosine-containing tract . When various lambda DNAs were methylated to saturation in vitro by crude extracts from mec+ and mec minus (N-3) cells, the extent of cytosine methylation was the same . This is in contrast to in vivo methylation where lambda-mec minus (N-3) DNA contains twice as many 5-methylcytosines per genome as lambda-mec+ DNA . Therefore, we suggest that the K12 met+ cytosine methylase and the N-3 plasmid modification methylase are capable of recognizing the same nucleotide sequences, but that the in vivo methylation rate is lower in mec+ cells.

J Bacteriol, 1975 Apr, 122(1), 110 - 9
Regulation of methionine transport activity in Escherichia coli; Kadner RJ; Methionine transport activity in cells of Escherichia coli K-12 is regulated by the level of the internal methionine pool . Transport activity is depressed either in cells grown in the presence of methionine or in cells exposed to methionine immediately prior to harvest . Alpha-Keto-gamma-methiol-butyrate, D-methionine, or methionine sulfoxide have little effect on the initial rate of uptake of L-methionine when they are added simultaneously with the substrate . However, methionine transport is markedly reduced in cells exposed to these sources of L-methionine before the addition of substrate . This reduction is prevented if the cells are treated with amino oxyacetic acid . The initial rate of uptake into L-methionine-loaded cells was lower than that into unloaded cells . This inhibition affected both methionine transport systems and the inhibition by the internal pool appeared to be non-competitive with the external methionine concentration . Two classes of mutants with increased methionine pools have decreased rates of uptake . Conversely, starvation for methionine in a methionine auxotroph with high rates of methionine degradation resulted in a substantial increase in the rate of methionine transport . Thus, these transport systems are subject to regulation by the internal pool size and possibly by repression.

Chem Biol Interact, 1975 Apr, 10(4), 265 - 8
The effects of unsubstituted polycyclic aromatic hydrocarbons on the growth of Escherichia coli; Hass BS et al.; The effect on growth of E . coli of a series of 3-, 4-, and 5-ring unsubstituted polycyclic aromatic hydrocarbons (PAH) is determined at concentrations of 10-5M, 10-6M, and 10-7M . The angular hydrocarbon configurations of 1,2-benzanthracene, 1,2,5,6-dibenzanthracene (DIBA), and 3,4-benzpyrene (BP) promote growth; tetracene and pyrene have little or no effect on growth while at most concentrations anthracene, phenanthrene chrysene, 1,2,3,4-dibenzanthracene, and pentacene inhibit bacterial growth . No universal pattern of concentration effect is manifest, but based on the results an angular acene configuration is a necessary condition for growth stimulation.

J Virol, 1975 Apr, 15(4), 720 - 5
Growth studies of three phi chi 174 mutants in tsDNA mutants Escherichia coli; Haworth SR et al.; Three mutants of phi chi 174 were examined for their abilities to grow in temperature-sensitive dna,A, dnaC, dnaE, or dnaG mutants of Escherichia coli . The results indicate that the phage mutants have acquired the ability to grow in some tsDNA mutants that normally block the replication of wild-type phi chi 174 . Evidence is presented indicating that the phage mutants contain one or more altered structural proteins . Several models are presented to explain how altered phage structural proteins could affect phi chi 174 replication.

J Immunol, 1975 Apr, 114(4), 1430 - 3
Interference of anti-allotype antisera with antigen-antibody binding; Acuto O et al.; Rabbit anti-b4 antisera are capable of inhibiting the antigen-binding ability of antibodies carrying b4 allotype . The data support the hypothesis of the localization of at least one b group allotypic determinant in the V region.

J Immunol, 1975 Apr, 114(4), 1296 - 301
The role of macrophages in the production of lymphokines by T and B lymphocytes; Wahl SM et al.; The role of macrophages in the production of two lymphokines, monocyte chemotactic factor and macrophage activating factor, was investigated . Lymphokine production by guinea pig lymph node and spleen cells required macrophages for thymus-dependent antigens and mitogens . In contrast, B cell stimulants which also induce the synthesis of lymphokines were macrophage independent . When populations of relatively pure B or T lymphocytes were isolated, it was found that T cells required viable macrophage cooperation to produce these two lymphokines and to undergo propliferation in response to specific antigens, whereas B cells could be directly activated in the absence of macrophages . These findings suggest that T and B cells have different requirements for activation and for macrophage cooperation . Futhermore, since lymphokine synthesis is evident within the first 4 hr of stimulant presentation, these observations demonstrate that macrophages play an essential role in the earliest events of lymphocyte activation.

J Immunol, 1975 Apr, 114(4), 1226 - 9
Electrophoretic fractionation of guniea pig lymphocytes: evidence for different subsets of T and B cells in spleen and lymph node; Anderson LC et al.; Guinea pig lymph node and blood lymphocytes have been physically fractionated in preparative cell electrophoresis into two functionally viable populations, the high mobility cell population (HMC) and the low mobility cell population (LMC) . By using cell surface markers and functional tests known to be specific for T and B lymphocytes, respectively, it is shown that the T lymphocytes localize in the HMC population and the B lymphocytes in the LMC population . The spleen lymphocytes do not separate into the two populations . They move into one single broad peak containing both T and B cells . This finding indicates the presence of electrokinetically different subsets of T and B lymphocytes in the spleen on one hand and in the lymph node and blood on the other hand.

Blood, 1975 Apr, 45(4), 569 - 75
Inhibition of intravascular fibrin deposition by dipyridamole in experimental animals; Gurewich V et al.; Intravascular fibrin deposition was induced in rabbits by endotoxin, the infusion of fibrin monomer (FM), and by the infusion of thrombin and EACA . A previously developed radioisotope technique was used to measure the fibrin deposits in various organs . Dipyridamole treatment of rabbits caused significant inhibition of fibrin deposition in all three experimental models . The drug also inhibited platelet consumption and, in the thrombin- and EACA-infused animals, fibrinogen consumption as well . The results obtained with dipyridamole were compared with the effect of thorotrast . It was concluded from this comparison that the effect of dipyridamole could not be attributed to inhibition of the reticuloendothelial system . It is postulated that dipyridamole inhibits the final step at which soluble FM is precipitated as fibrin in vivo.

Int J Radiat Biol Relat Stud Phys Chem Med, 1975 Apr, 27(4), 355 - 62
Prophage inductive efficiency of alkylating agents and radiations; Hussain S et al.; The prophage inducing efficiency in E . coli K-12 (lambda) of a number of agents--alkylating agents and radiations--has been compared at a high survival of bacteria . The inducing effectiveness (per alkylation in DNA) and efficiency (compared with mutation frequency in E . coli Sd-4) was found to decrease in the order 2-hydroxyethylating agents (2-hydroxyethyl methanesulphonate and ethylene oxide) greater than isopropyl methanesulphonate approximately equal to methyl methanesulphonate greater than ethylating agents (diethyl sulphate, ethyl methanesulphonate) . The low inducing activity of the ethylating agents could not be explained with respect to their reactivity towards targets of differing nucleophilicity, nor could the effectiveness in mutagenicity or the ability to break the chromosomes in the presence of metal ions be invoked . The high inducing efficiency of hydroxyalkylating agents may be related to their ability to break DNA strands.

J Pathol, 1975 Apr, 115(4), 241 - 4
Ulcerative colitis in apes: A comparison with the human disease; Scott GB et al.; The pathological changes in the colons of two young gorillas and an adult orang-utan which developed diarrhoea and died, are described . Since no causative agents could be identified and the changes were indistinguishable from the active phase of ulcerative colitis in humans, these cases were considered examples of this disease in apes . Evidence of early healing was found in one case and the suitability of apes and monkeys as possible animal models of the human disease is discussed.

Proc Natl Acad Sci U S A, 1975 Apr, 72(4), 1364 - 7
Metabolites influence control of lysine transfer ribonucleic acid synthetase formation in Escherichia coli K-12; Hirshfield IN et al.; A mutant of E . coli K-12 has been isolated which has only 1-3% of the wild-type lysyl-tRNA synthetase activity {L-lysine:tRNA ligase (AMP forming), EC 6.1.1.6} . Additions of 20 mM L-alanine or 6 mM leucine dipeptides to the culture medium can restore the activity of lysyl-tRNA synthetase in the mutant strain to the wild-type level . Experiments on the in vivo charging of lysine tRNA in the mutant show that in the absence of the metabolites lysine tRNA is charged 15-23% . Upon the addition of 3 mM L-leucyl-L-alanine to the medium the lysyl tRNA synthetase activity increases 25-fold and the in vivo charging of lysine tRNA returns to the wild-type level . Experiments with antibody against lysyl-tRNA synthetase show that the stimulation of lysyl-tRNA synthetase activity by the metabolites is the result of new protein synthesis.

J Bacteriol, 1975 Apr, 122(1), 80 - 8
Plasmid mutations affecting self-maintenance and host growth in Escherichia coli; Koyama AH et al.; As reported in the accompanying paper, a number of mutants of the ColVBtrp plasmid that can not be maintained stably in the host cell of Escherichia coli have been isolated . Each of the mutated plasmids has been transferred to an isogenic Col minus strain, and the resulting Col+ strains were studied to examine the effects of plasmid mutations on some properties of the host bacteria . Many of the strains harboring a mutated plasmid were thus found to be temperature sensitive; they failed to grow and divide normally at high temperatures . Some of them formed "filaments" under these conditions . These abnormal growth characteristics were accompanied by an increased susceptibility to sodium deoxycholate and methylene blue, suggesting that the cytoplasmic membrane has been altered . Moreover, studies of temperature-independent revertants obtained from two of these temperature-sensitive Col+ strains suggested that a single mutation on the plasmid is responsible for the pleiotropic effects exerted on the host cell . The bearing of these findings on the mode of replication and segregated of stringent-type plasmids such as ColVBtrp in the host bacteria is discussed.

J Nucl Med, 1975 Apr, 16(4), 280 - 3
Distribution of 67-Ga following intravenous administration: effects of disodium edetate therapy; Hurwitz SR et al.; Following intravenous administration of 67Ga-citrate to normal and abscessed rats, the colon content of 67Ga decreased with increasing oral doses of Na2EDTA . The effects observed, however, were not thought to be of potential clinical value.

Eur J Biochem, 1975 Apr 1, 52(3), 469 - 74
Transcriptional units for ribosomal proteins of Escherichia coli; Hirsch-Kauffmann M et al.; Transcriptional units for ribosomal proteins in Escherichia coli were measured using the ultraviolet sensitivities of the rates of synthesis of individual ribosomal proteins . The ultraviolet sensitivities of gene transcriptions are proportional to the distances from the promoters . The longest transcriptional units for ribosomal proteins are 3.6 x 10(6) of DNA molecular weight corresponding to 1.8 x 10(6) of RNA or to 180 000 of protein . The length would cover 10--12 genes of ribosomal proteins (of an average Mr of 15000-18000).

Eur J Biochem, 1975 Apr 1, 52(3), 459 - 68
RNA-protein interactions in the ribosome . Binding of 50-S-subunit proteins to 5' and 3' terminal segments of the 23-S RNA; Spierer P et al.; Limited digestion of the Escherichia coli 50-S subunit permits the isolation of two large fragments of the 23-S RNA . One arises from the 5' end of the 23-S RNA and contains about 1200 nucleotides . The other arises from the 3' end of the 23-S RNA and contains about 2000 nucleotides . Each of the ten 50-S proteins known to bind specifically and independently to the 23-S RNA were tested for their ability to interact with these two RNA fragments . It was determined that the 5' terminal segment contains the specific binding sites for proteins L4, L20 and L24, whereas the 3' terminal segment contains the specific binding sites for proteins L1, L2, L3, L6, L13, L13 and L23.

Eur J Biochem, 1975 Apr 1, 52(3), 451 - 7
The specificity of five DNAases as studied by the analysis of 5'-terminal doublets; Bernardi A et al.; The 5'-terminal dinucleotides released by five deoxyribonucleases (spleen acid DNase, snail acid DNase, pancreatic DNase, Escherichia coli endonuclease I and crab DNase) have been determined on E . coli DNA (51% dG & dC) digests having different average sizes (Pn) in the range 50 to 10 . It has been shown that the composition of the 5'-terminal dinucleotide (a) is independent upon the degradation level, at least in the range explored; (b) is strongly different from the composition of E . coli DNA doublets, these differences being characteristic for each enzyme; (c) is very significantly different from the statistical composition of 5'-terminal dinucleotides, as calculated from the composition of 5'-terminal and penultimate nucleotides . A calculation of the statistical composition of the trinucleotides split by each enzyme, using the 3'-terminal nucleotide data in conjunction with the 5'-terminal dinucleotide results provided a qualitative "specificity spectrum" for each enzyme.

Genetika, 1975 Apr, 11(4), 97 - 105
{A further study of the nature of phenotypical reversions in thymidine phosphorylase deletion mutants of Escherichia coli K-12}; Mironov AS et al.; Thymidine dependent (thy) strains of Escherichia coli carrying deletions for thymidine phosphorylase (tpp gene) formed phenotypical reversions for the ability of growth at the medium containing thymine as a source of thymidilate . The ability of the thy tpp strains to utilyze thymine for growth is due to mutations of the regulatory genes cytR and udpR, which control the uridine phosphorylase activity . This enzyme in constitutive amounts catalyzes conversion of thymine to thymidine in bacterial cells with a block of deoxyribose catabolism (deoxyribomutase-negative strains) . The udpR mutant by contrast to cytR mutants reported by Munch-Petersen et al . (1972) are shown to contain low, inducible levels of cytidine deaminase and deo-enzymes . The udpR mutation is recessive with respect to wild type allele . It is supposed that the product of udpR gene is a specific repressor of uridine phosphorylase (udp) gene . The udpR mutation is closely linked (90% contransduction) to uridine phosphorylase (udp) gene, and is located approximately at 75 min, on the E . coli chromosome.

Zh Mikrobiol Epidemiol Immunobiol, 1975 Apr, (4), 47 - 53
{New methods of determination of the functional peculiarities of autoantibodies in various types of autoimmune reactions}; Klemparskaia NN; Anderson's method (with our modification of Jerne's method) was applied in experiments on 574 albino female mice; a study was made of the affinity of autoantibodies in three groups of animals: intact, gamma (Co60) irradiated in a dose of 600 r, and with stimulation of auto-immune reaction by injection of a complete adjuvant (0.05 ml subcutaneously) or injection of living E . coli cells (subcutaneously 300 million) or hemostimulation (subcutaneously 0.1--0.3 or 0.5 ml of homologous blood) . The maximal autoantibody indices were obtained in the irradiated mice, the least -- in the intact animals . Apart from the plaque-formation inhibition phenomenon, there were observed two more phenomena; these were an immune erythrocyte adherence to the immunocompetent cells and a marked increase in the formation of plaques of autoimmune hemolysis in the preparation with addition of diluted (1:10) lysate of erythrocytes obtained from mice with the administration of an adjuvant or homologous blood.

Nucleic Acids Res, 1975 Apr, 2(4), 603 - 11
A rapid assay technique for RNA ribose methylases; Svensson I et al.; A rapid technique for quantitative separation of ribose-methylated nucleosides from base-methylated and non-methylated nucleosides by chromatography on DEAE-cellulose paper in the presence of borate is described . The method has been used as an assay for tRNA ribose methylases from yeast, using under methylated Escherichia coli tRNA as substrate . The main product formed with a partly purified yeast enzyme was characterized as 2'-O-methylcytidine.

Chem Phys Lipids, 1975 Apr, 14(2), 113 - 22
Infrared and Raman spectra of phosphatidyl-ethanolamine and related compounds; Akutsu H et al.; Infrared and Raman spectra of phosphatidylethanolamine from Escherichia coli, L-alpha-glycero-phosphorylethanolamine and 0-phosphorylethanolamine were obtained . Most of the bands were assigned to each vibrational mode based on the N deuteration effect, comparison of the intensity in the infrared and Raman spectra and the depolarization degree measurement in the Raman spectra . The spectra of phosphatidylethanolamine can be interpreted by assuming that the molecule takes the dipolar ionic structure both in non-polar solvent and in solid.

Can J Biochem, 1975 Apr, 53(4), 444 - 54
The control of pyruvate kinases of Escherichia coli . II . Effectors and regulatory properties of the enzyme activated by ribose 5-phosphate; Waygood EB et al.; The pyruvate kinases of Escherichia coli activated by ribose 5-phosphate (RP) has been partially purified . The active form of the enzyme has a molecular weight of about 180 000 as judged by sucrose density gradient centrifugations and Sephadex G-150 chromatography . On dissociation in the absence of sulfhydryl reagents such as dithiothreitol, the enzyme is inactivated and it has a molecular weight of about 110 000 . Various substrates and effectors of the enzyme, with the exception of phosphate, do not influence the association-dissociation equilibrium of the enzyme . The enzyme, unlike pyruvate kinases from many other sources, is not activated by potassium ions . Sulfate and phosphate ions are inhibitory to the enzyme . Phosphate seems to be an allosteric inhibitor and its effect is completely antagonized by activators . The enzyme is activated in an allosteric manner by two classes of compounds, nucleoside monophosphates and sugar phosphates of the hexose monophosphate pathway . Amongst the nucleotides, guanosine 5'-phosphate and adenosine 5'-phosphate are the most effective activators . Amongst the hexose monophosphate pathway intermediates, RP is the most powerful activator, with apparent activation constants as low as 1 Mu . Sugar phosphates esterified at C-1 or both terminal positions are entirely ineffective in activation . The effectors act by changing the Michaelis constant for the substrates . Both of the substrates of the enzyme, adenosine diphosphate and phosphoenolpyruvate, yield cooperative-concentration plots in the presence of unsaturating concentrations of the fixed changing substrate . The initial velocity plots for both substrates become hyperbolic in the presence of saturating concentrations of RP.

Infect Immun, 1975 Apr, 11(4), 622 - 9
T lymphocyte function as the principal target of lymphocytic choriomeningitis virus-induced immunosuppression; Bro-Jorgensen K et al.; Plaque-forming cell responses against sheep erythrocytes, Escherichia coli lipopolysaccharide, pneumococcal polysaccharide, and polyvinylpyrrolidone were examined in mice infected with lymphocytic choriomeningitis virus . A 92 to 96 percent reduction of the thymus-dependent anti-sheep erythrocyte responses was observed 2 to 4 weeks after infection . However, the thymus-independent responses against the three other antigens were close to normal at all stages of the infetion . Studies on allograft immunity of infected C3H mice against DBA/2 mastocytoma cells revealed a severe suppression of the T cell-mediated cytotoxic response which was temporally related to the impaired humoral responsiveness against sheep erythrocytes . The capacity of spleen cells from infected mice to restore immune responsiveness of lethally irradiated recipients against sheep erythrocytes was significantly reduced . The adoptive responses, however, were clearly improved when normal thymus cells were added to the inferior spleen cells . Moreover, it appeared that the spleen cells from immunosuppressed donor mice could not confer suppression to normal lymphoid cells . The presented findings are consistent with the assumption that a numeric deficiency of T cells, or cells belonging to some T cell subpopulation, is the primary cause of lymphocytic choriomeningitis virus-induced immunosuppression.

Mutat Res, 1975 Apr, 28(1), 15 - 26
Pleiotropic effects of a DNA adenine methylation mutation (dam-3) in Escherichia coli K12; Marinus MG et al.; The dam-3 mutation results in a five-fold reduction in the number of 6-methyl-adenine (6-meA) residues in the DNA of E . coli K12 or phage lambda . The DNA of phage fd appears to be devoid of 6-meA when propagated on dam-3 bacteria . The phenotypic differences between dam-3 and dam+ bacteria include: (i) increased free phage in lysogenic dam-3 cultures, (2) increased sensitivity to methyl methanesulfonate (MMS), (3) inviability of dam-3 lex-I strains, (4) lower molecular weight of DNA in dam-3 bacteria in the absence of DNA ligase and (5) increased rate of DNA degradation in dam-3 recA strains.

Proc Natl Acad Sci U S A, 1975 Apr, 72(4), 1605 - 9
T antigen binds to simian virus 40 DNA at the origin of replication; Reed SI et al.; A technique employing ferritin-conjugated antibody has been developed to visualize specific protein-DNA complexes in the electron microscope and has been used to demonstrate the preferential binding of simian virus 40 (SV40) T antigen at or near the origin of replication of SV40 DNA, 0.67 fractional length clockwise from the EcoRI restriction endonuclease cleavage site . urified covalently closed supercoiled circles of SV40DNA were treated with partially purified T antigen and the complex was stabilized by crosslinking with glutaraldehyde . Hamster antiT antigen gamma-globulin, ferritin-labeled goat anti-hamster gamma-globulin, and glutaraldehyde were then added sequentially . The location of the bound ferritin cores was measured with respect to the EcoRI cleavage site and the orientation of the cores relative to the ends of the DNA was determined with respect to the locations of Escherichia coli DNA unwinding protein, which binds to covalently closed supercoiled SV40 DNA at either of two preferred sites, 0.46 or 0.90 fractional length clockwide from the EcoRI cleavage site.

Proc Natl Acad Sci U S A, 1975 Apr, 72(4), 1501 - 4
On the question of translocation of heart cAMP-dependent protein kinase; Keely SL Jr et al.; Rat hearts were perfused with epinephrine and/or 1-methyl-3-isobutylxanthine for 2 min . These agents raised the concentration of cAMP and increased the fraction of cAMP-dependent protein kinase (EC 2.7.1.70) in the active form . However, the content of cAMP-dependent protein kinase in the soluble fraction of homogenates of these hearts was reduced and the amount in the particulate fraction was increased . A similar redistribution was obtained by adding cAMP to homogenates of control hearts . The reduction in soluble protein kinase content was due to apparent binding of the free catalytic subunit of the enzyme to particulate material (12,000 times g pellet) in media of low ionic strength (smaller than 100 mM KCl) . The amount bound was, therefore, proportional to the dissociation of the holoenzyme . The binding was not altered by prior boiling or trypsin treatment of the particulate material, but it was prevented or reversed by the addition of 150 mM KCl . The catalytic subunit of the protein kinase from heart also bound to particulate fractions from liver or Escherichia coli and to various denatured proteins . These findings suggest that the protein kinase activity of membranes and particulate fractions has frequently been overestimated, since isolation of particulate materials has usually been carried out at low ionic strength . The data also imply that intracellular translocation of the protein kinase catalytic subunit, at least in heart tissue, is of questionable physiological significance.

Cell, 1975 Apr, 4(4), 301 - 10
The interaction of estradiol-receptor protein with the genome: an argument for the existence of undetected specific sites; Yamamoto K et al.; In extracts from rat and calf uterus, the steroid hormone 17 beta-estradiol stimulates the binding of its specific receptor protein to DNA . This interaction appears to be of low affinity (half of the estradiol-activated, 5S receptor bound at 300-400 mug/ml DNA) and nonspecific with respect to DNA base sequence . No binding to double-stranded RNA is observed . These findings are consistent with several in vivo observations . In particular, when the cytoplasmic receptor protein binds hormone, it migrates to the cell nucleus to an extent consistent with its affinity for DNA in vitro, and this in vivo nuclear binding is uniform and nonsaturable in the testable range (to greater than 3 times 10-4 sites per cell) . The level of biological response appears to parallel the hormone dose up to these high levels of receptor binding . How are these observations to be reconciled with the prevalent view of steroid receptors as gene control proteins regulating transcription at specific loci on the genome? Our model is based on an analogy with the DNA binding properties of the E . coli lac repressor protein . We believe that the estradiol receptor exerts its effect by binding to a small number of high affinity sites on the genome, while also having a finite low affinity for nonspecific DNA sequences . These nonspecific loci, because of their vast number, completely mask the presence of the high affinity sites . We estimate that up to 10-3 specific sites, with affinities in the range 10- minus 8 minus 10- minus 10 M, could exist without being detected by bulk binding assays currently in use . However, alternative approaches should allow detection of these sites, and some of these are suggested.

J Biochem (Tokyo), 1975 Apr, 77(4), 705 - 18
Cytoplasmic membrane vesicles of Escherichia coli . A simple method for preparing the cytoplasmic and outer membranes; Yamato I et al.; A simple preparative method is described for isolation of the cytoplasmic and outer membranes from E . coli . The characteristics of both membrane fractions were studied chemically, biologically, and morphologically . Spheroplasts of E . coli K-12 strain W3092, prepared by treating cells with EDTA-lysozyme {EC 3.2.1.17}, were disrupted in a French press . The crude membrane fraction was washed with 3 mM EDTA-10% (w/v) sucrose, pH 7.2, and the cytoplasmic membranes and outer membranes were separated by sucrose isopycnic density gradient centrifugation . The crude membrane fraction contained approximately 10% of the protein of the whole cells, 0.3% of the DNA, 0.7% of the RNA, 0.3% of the peptidoglycan, and about 30% of the lipopolysaccharide . The cytoplasmic membrane fraction was rich in phospholipid, while the outer membrane fraction contained much lipopolysaccharide and carbohydrate; the relative contents of lipopolysaccharide and carbohydrate per mg protein in the cytoplasmic membrane fraction were 12 and 40%, respectively, of the contents in the outer membrane fraction . Cytochrome b1, NADH oxidase, D-lactate dehydrogenase {EC 1.1.1.28}, succinate dehydrogenase {EC 1.3.99.1}, ATPase {EC 3.5.1.3}, and activity for concentrative uptake of proline were found to be localized mainly in the cytoplasmic membranes; their specific activities in the outer membrane fraction were 1.5 to 3% of those in the cytoplasmic membrane fraction . In contrast, a phospholipase A appeared to be localized mainly in the outer membranes and its specific activity in the cytoplasmic membrane fraction was only 5% of that in the outer membrane fraction . The cytoplasmic and outer membrane fractions both appeared homogeneous in size and shape and show vesicular structures by electron microscopy . The advantages of this method for large scale preparation of the cytoplasmic and outer membrane fractions are discussed.

J Immunol, 1975 Apr, 114(4), 1345 - 7
Functional heterogeneity among the T-derived lymphocytes of the mouse . IV . Nature of spontaneously induced suppressor cells; Burns FD et al.; When spleen cells are cultured for 4 days in the presence of fetal bovine serum, T-cells are generated which nonspecifically suppress the humoral responses of non-precultured cells to a variety of antigens . These T cells can be generated from both short-lived, sessile T1 cells and long-lived, recirculating T2 cells, and thus appear similar to suppressor T cells activated by concanavalin A.

Transplantation, 1975 Apr, 19(4), 326 - 34
Allogeneic radiation chimeras . Long-term studies; Tyan ML; Lethally irradiated mice protected with allogeneic fetal liver cells or with syngeneic or allogeneic marrow and spleen cells treated with antisera to mouse immunoglobulins or to the T cell-associated theta antigen and their controls were observed for up to 750 days . The best survival rates were found in the large groups given syngeneic marrow and spleen or allogeneic fetal liver cells (70-85% 700-day survival); in contrast, 43% of the group injected with allogeneic cells treated with anti-theta serum and 19% of those given antiimmunoglobulin-treated cells were alive 700 days postradiation . Pulmonary infection was the most frequent cause of death of long-term survivors in all groups . Tumor incidence was increased in recipients of allogeneic cells (13% versus 4% among syngeneic chimeras), but the renal pathology seen in these groups was no greater than that noted in the syngeneic controls . Beginning 600 days after irradiation, mice from experimental and control groups were killed and their spleens were cultured with thymus-dependent antigens and the mitogens concanavalin A and lipopolysaccharide, Escherichia coli . The most frequent finding in all groups was mild to moderate impairment of T cell-dependent responses.

Proc Natl Acad Sci U S A, 1975 Apr, 72(4), 1268 - 71
Evolution of biosynthetic pathways: immunological approach; Truffa-Bachi P et al.; Through the use of specific immunoadsorbent columns, it is shown that Escherichia coli aspartokinase I-homoserine dehydrogenase I, aspartokinase II-homoserine dehydrogenase II, aspartokinase III, and homoserine kinase, enzymes involved in the same complex biosynthetic pathway, share antigenic determinants . This raises the question of a common origin for the four cibtenoirart kinases . (Aspartate kinase or ATP:L aspartate 4-phosphotransferase, EC 2.7.2.4; homoserine dehydrogenase or Lhomoserine:NADP oxidoreductase, EC 1.1.1.3; homoserine kinase or ATP:L-homoserine O-phosphotransferase, EC 2.7.1.39.)

J Cell Physiol, 1975 Apr, 85(2 Pt 2 Suppl 1), 449 - 58
Control of transcription of the globin gene; Gilmour RS et al.; This report provides a more rigorous proof of previous findings that the RNA transcribed in vitro from the chromatins of different organs shows different sequence specificities . Here the particular case of the globin gene is considered for a comparison of embryonic mouse liver chromatin and mouse brain chromatin using the reverse transcriptase cDNA copy of globin 9S m RNA as a definitive probe . It can be shown that globin sequences are transcribed in vitro from embryonic liver chromatin and not brain chromatin . This specificity in liver chromatin can be reconstituted after dissociation of the structural elements of the chromatin . It can be shown that the non-histone protein fraction of liver chromatin can confer specificity for the transcription of globin sequences from brain chromatin which otherwise lacks this ability . Preliminary results are described with the Friend cell system, in which haemoglobin synthesis can be induced in vitro in the presence of dimethylsulphoxide.

Biochim Biophys Acta, 1975 Mar 28, 386(1), 99 - 106
The primary structure of the ribosomal protein L29 from Escherichia coli; Bitar KG; The amino acid sequence of ribosomal protein L29 was determined utilizing an improved Beckman sequenator, a solid phase peptide sequenator, and more conventional techniques . L29 is composed of 63 amino acid residues and has a molecular weight of 7262 . It lacks proline, cysteine, isoleucine, tyrosine and tryptophan . The N-terminal and C-terminal regions (residues 1-9 and 50-63) are basic, while the rest of the molecule is hydrophobic and neutral . Calculated P-a values show two helical regions: one from residues 1 through 32 and the other from residues 37 through 47 . No beta-sheet regions are predicted . Short identical regions occur within the sequence of L29 as well as between L29 and other ribosomal proteins.

Biochim Biophys Acta, 1975 Mar 28, 386(1), 168 - 80
Purification and properties of glutamate binding protein from the periplasmic space of Escherichia coli K-12; Barash H et al.; Glutamate binding protein released from the periplasmic space of Escherichia coli K-12 by lysozyme-EDTA treatment was purified to homogeneity and its physical and chemical properties were studied . It is a basic protein with a pI of 9.1 . Its molecular weight, determined in an analytical ultracentrifuge, and by gel filtration on Sephadex G-100 and dodecylsulphate acrylamide is 29 700, 27 800 and 32 000, respectively . The KD value for glutamate was 6.7 - 10- minus 6 M . L-Aspartate, reduced glutathione, G-glutamate-gamma-benzylester and L-glutamate-gamma-ethylester competitively inhibited glutamate binding with K-i; values of 7.8 - 10- minus 5, 1.1 - 10- minus 5, 1.0 - 10- minus 5 and 1.0 - 10- minus 5 M, respectively . Spheroplasts retained 40% of glutamate transport as compared to intact cells . The glutamate binding activity of a glutamate-utilizing strain (CS7), was 1.6 times as high as that of the glutamate non-utilizing parent strain (CS101) . Similarly, the glutamate binding activity of a temperature conditional glutamate-utilizing mutant (CS2-TC) was 1.9 times higher when grown at the permissive temperature (42 degrees C) than when grown at the restrictive temperature (30 degrees C).

Biochemistry, 1975 Mar 25, 14(6), 1235 - 44
Endonuclease II of Escherichia coli: degradation of gammairradiated DNA; Kirtikar DM et al.; Irradiation of DNA in a nitrogen atmosphere with 60Co gamma-radiation produces at least two types of damage . The first type leads to single strand breaks in the DNA observed after exposure to alkali . This type of alkali-labile bond will be designated a spontaneous break . The second type of damage to DNA is an alteration which makes the DNA susceptible to phosphodiester bond hydrolysis by a 1600-fold purified preparation of endonuclease II of Escherichia coli and is designated an enzyme-sensitive site . This site is not alkali-labile . After irradiation, preincubation of the DNA either for days at 0 degrees or for 4 hr at 37 degrees increases both the spontaneous breaks and the enzyme sensitive sites . There is a greater increase of spontaneous breaks when the preincubation is in O2 compared to N2 . The increase of enzyme sensitive sites due to the preincubation is not altered significantly by O2 . The increase of spontaneous breaks during the preincubation is almost completely prevented by addition of either NaBH4 or NH2OH after the irradiation . The treatment can be before or after the preincubation . This effect indicates that these breaks are due to alkali-labile bonds possibly produced by depurination or depyrimidination reactions . That the spontaneous breaks are due primarily to alkali-labile bonds is supported by an experiment in which formamide gradients were used . Neither NaBH4 nor NH2OH has any effect on the enzyme sensitive sites . Addition of beta-mercaptoethanol (0.5 M) at the start of the preincubation prevents in part the appearance of both spontaneous breaks and enzyme-sensitive sites . It has no effect when added at the end of the preincubation . Catalase added before the preincubation has no effect on either type of damage . It is postulated that the spontaneous breaks occur because purine or pyrimidine radicals are formed (possibly hydroxyl radicals) which can then interact with oxygen to produce unstable intermediates . The intermediates then undergo either depurination or depyrimidination . The subsequent alkali catalyzed beta-elimination reaction of depurinated or depyriminidinated DNA is prevented by NaBH4 or NH2OH . An alternative hypothesis would involve damage to the sugar rather than to bases . The enzyme-sensitive sites represent another form of base damage which is not oxygen dependent . The chemical nature of either form of primary damage is not known.

J Biol Chem, 1975 Mar 25, 250(6), 2407 - 9
Escherichia coli ribosomal ribonucleic acids are not cut from an intact precursor molecule; Gegenheimer P et al.; Ribonuclease III-deficient strains of Escherichia coli accumulate a "30 S" RNA species . Kinetic analysis of label incorporated into rRNA species shows, however, that 30 S RNA is not the major precursor to the 16 S and 23 S RNA species is in 7- to 10-fold excess over that into 30 S RNA . The 30 S RNA species turns over with a half-life of about 2.5 min, which could account for no more than one-tenth of the incorporation into 16 S plus 23 S RNA . Thus, under these conditions RNase III-strains of E . coli do not cut rRNAs from an intact tandem precursor molecule but rather from the elongating nascent transcript, possibly by an alternate pathway not involving RNase III.

J Biol Chem, 1975 Mar 25, 250(6), 2383 - 7
Evidence for the allosteric regulation of glycogen synthesis in the intact Escherichia coli cell . Agreement of the values of the parameters of the Hill equation fitted to data for glycogen synthesis in vivo with the abailable values obtained in vitro with adenosine diphosphoglucose synthetase; Dietzler DN et al.; In various nutrient-limited cultures of either Escherichia coli W4597(K) or G34 a 10-fold range of rates of glycogen synthesis is observed while the energy charge values (0.86 plus or minus 0.01) and glucose 6-phosphate levels are essentially the same in each condition . The steady state level of fructose 1,6-diphosphate in these cultures varies from experiment to experiment as a function of the observed rate of glycogen synthesis . These data were fitted to the Hill equation by a nonlinear regression analysis and the statistically most probable values obtained for the Hill coefficient (n), A0.5, and V were, respectively, 2.08, 0.82mM, and 1030 mumol/g of protein per hour . The values of the first two parameters agree well with values available at energy charge 0.85 for the in vitro synthesis of ADPG by the ADPG synthetase of E . coli . When the difference in the glucose 1-phosphate concentration used in the studies in vitro from the apparent glucose 1-phosphate concentration in vivo (estimated from the glucose 6-phosphate levels) is considered, the in vitro value of V (1140 mumol of ADPG synthesized per g of protein per hour) is quite similar to the value of V (1030 mumol of glucose incorporated into glycogen per g of protein per hour) for glycogen synthesis in vivo . The close agreement of the values of the parameters of the Hill equation for glycogen synthesis in vivo to the values obtained for ADPG synthesis in vitro provides the most quantitative evidence yet obtained that allosteric regulation of bacterial glycogen synthesis functions in vivo.

J Biol Chem, 1975 Mar 25, 250(6), 2056 - 61
The activity of oligonucleotides containing guanosine 5'-triphosphate in protein synthesis . I . The interaction of protein synthesis elongation factor I with cytidylyl (5'-3')-guanosine 5'-triphosphate; Allende JE et al.; The interaction of protein synthesis elongation factor 1 (EF-1) from wheat embryos and elongation factor Tu from Escherichia coli with cytidylyl(5'-3')guanosine 5'-triphosphate(pppGpC) has been studied . The dinucleotide 5'-triphosphate interacts strongly with EF-1 as evidenced by its capacity to inhibit the binding of {3H}GTP to the factor . The analogs pGpC and GpC do not interfere with GTP binding to EF-1 but guanosine 5'-triphosphate cyclic 2',3'-monophosphate and ppGpC are also potent inhibitors . The binding of the dinucleotide 5'-triphosphate to EF-1 was also demonstrated directly by the nitrocellulose retention method and by Sephadex G-50 fractionation using a radioactive analog iodinated with 125I in the 5 position of the cytosine of pppGpC . The dinucleotide triphosphate can replace GTP in the formation of a ternary complex EF-1-aminoacyl-tRNA-GTP and in its requirement for the binding of aminoacyl-tRNA to ribosomes catalyzed by EF-1 . The absolute requirement for GTP in an in vitro polypeptide-synthesizing system can also be met by pppGpC and by guanosine 5'-triphosphate cyclic 5',3'-monophosphate . The bacterial factor EF-Tu differs drastically from eukaryotic EF-1 in its nucleotide specificity since EF-Tu only interacts slightly (if at all) with pppGpC . The low inhibition of {3H}GTP binding to EF-Tu by pppGpC could be due to a slight contamination in the latter compound.

J Biol Chem, 1975 Mar 25, 250(6), 1972 - 80
The deoxyribonucleic acid unwinding protein of Escherichia coli . Properties and functions in replication; Weiner JH et al.; The DNA unwinding protein of Escherichia coli (Sigal, N., Delius, H., Kornberg, T., Gefter, M., and Alberts, B . (1972) Proc . Nat . Acad . Sci . U.S.A . 69, 3537-3541) has been purified to homogeneity by a simple procedure which utilizes its stability to heating . The protein is an asymmetric tetramer of 18,500 dalton subunits which binds preferentially to single-stranded DNA at a ratio of one protein molecule per 32 nucleotides . Binding to DNA is complete in less than 10 s at 0 degrees while release of the protein from single-stranded DNA is relatively slow even at 37 degrees . A simple functional assay for unwinding protein depends on its essential role in the conversion of phage G4 single-stranded DNA to the replicative form . Unwinding protein stimulates initiation of replication of all single-stranded phage DNAs . Approximately 300 copies of unwinding protein are present per cell, as estimated by antibody titration, an amount sufficient to cover substantial lengths of DNA in several replicating forks.

J Biol Chem, 1975 Mar 25, 250(6), 2339 - 50
Modification of Escherichia coli membranes in the prereplicative phase of phage T4 infection . Specificity of association and quantitation of bound phage proteins; Takacs BJ et al.; Reinfection of Escherichia coli with the bacterial virus T4 causes modifications of the properties of the host cell envelope during the preeplicative phase of the lytic cycle . These changes include altered densities cell enveloped and their subfractions, morphological modifications of membrane vesicles, and association of newly synthesized proteins with the host cell envelope . Polypeptide analysis by high resolution electrophoresis on polyacrylamide slab gels in dodecyl sulfate revealed that most of some 30 prereplicative phage-coded polypeptides are attached to this structure . Different means of cell disruption and selective extraction procedures, such as variations of ionic strength, removal of divalent cations, and the addition of chaotropic agents or detergents were used to study the characteristics of these attachments . Many proteins appeared to be artifactually absorbed or weakly bound to the envelope, Separation of cell walls from plasma membranes showed that all of the tightly bound proteins were associated withthe cell membrane fraction . The partitioning of phage proteins between the different fractions was monitored using 12 polypeptides which were identified as products of distinct phage genes . Of these, 8 were eliminated as potential membrane markers . Four polypeptides, the products of genes rIIA, rIIB, 39, and 52 were operationally defined as membrane proteins . The number of molecules of the 12 identified phage gene products, synthesized during a single lytic cycle, was determined . The results allowed the estimation of the concentration in the membrane of those proteins which were found to be quantitatively associated with that structure . Association of phage proteins with the cell envelope was found to be unaffected by mutations in any of the identified phage polypeptides.

Biochemistry, 1975 Mar 25, 14(6), 1221 - 5
Kinetics and specificity of T4 polynucleotide kinase; Lillehaug JR et al.; The kinetics of T4 polynucleotide kinase has been investigated at pH 8.0 and 37 degrees . Double reciprocal plots of initial rates vs . substrate concentrations as well as product inhibition studies have indicated that the enzyme reacts according to the ordered sequential mechanism shown in eq 2 in the text for phosphorylation of a DNA molecule . Based on this mechanism the rate equation for the overall reaction was deduced and the various kinetic constants estimated . Hill plots indicated little or no interaction between active sites in the enzyme . The apparent Michaelis constants and V-max were determined at a fixed ATP concentration, 66 muM, for a number of different substrates varying in chain length, base composition, and nature of the sugar, and a wide variation was found . For the nucleoside 3'-monophosphates tested both the apparent Michaelis constant and V-max values were from approximately 2 to 5 times larger than for the corresponding oligonucleotide . The following orders were obtained with regard to apparent Michaelis constants and V-max for the nucleoside 3'-monophosphates investigated: Michaelis constant, rGP greater than rUp greater than rCp greater than rAp greater than dTp; V-max, rGp greater than rCp greater than rAp greater than dTp greater than rUp . Somewhat similar results were also obtained with the deoxyoligonucleotides tested.

J Biol Chem, 1975 Mar 25, 250(6), 2311 - 4
Ketopantoic acid and ketopantoyl lactone reductases . Stereospecificity of transfer of hydrogen from reduced nicotinamide adenine dinucleotide phosphate; Wilken DR et al.; The stereospecificity of hydrogen transfer from NADPH to the appropriate carbonyl substrate catalyzed by ketopantoic acid and ketopantoyl acid and ketopantoyl lactone reductases of yeast (Saccharomyces cerevisiae) and Escherichia coli has been determined . Yeast and E . coli ketopantoic acid reductases are B-specific enzymes which transfer hydrogen from {4B-3H}-NADPH to ketopantoic acid to form {3H}pantoic acid . In contrast to the usual observations on the stereospecificity of functionally similar dehydrogenases from different species, yeast and E . coli ketopantoyl lactone reductases exhibit opposite stereospecificities . Both of two forms of yeast ketopantoyl lactone reductases are A-specific enzymes which form {3H}pantoyl lactone from ketopantoyl lactone and {4A-3H}NADPH, whereas, two forms of E . coli ketopantoyl lactone reductases are B-specific enzymes.

J Biol Chem, 1975 Mar 25, 250(6), 2299 - 304
On the structure-function relationship of acyl carrier protein of Escherichia coli; Schulz H; The conformations of Escherichia coli acyl carrier protein (ACP) and acetylated ACP have been studied as a function of pH and salt concentration by circular dichroism measurements . The results show that the amino groups of ACP in their protonated form are important for maintaining the native conformation of the protein at physiological pH . However, externally added cations (divalent more effectively than monovalent ones) can substitute for the ammonium groups in maintaining the ordered structure pf ACP . It is suggested that both the ammonium groups of ACP and externally added cations reduce the repulsion between carboxylate groups of ACP and thereby prevent the unfolding of the protein . A reduction of the number of negatively charged carboxylate groups by either protonation or chemical modification abolished the requirement for either ammonium groups or other cations . A qualitative agreement between the effect of salt on the conformation and on the biological activity of acetylated ACP has been observed . The single arginine residue of acetylated ACP has been modified by treatment with a trimer of 2,3-butanedione with the resulting derivative of ACP retaining most of its biological activity.

Biochemistry, 1975 Mar 25, 14(6), 1225 - 9
Effect of salts and polyamines on T4 polynucleotide kinase; Lillehaug JR et al.; The activity of T4 polynucleotide kinase (EC 2.7.1.78) was found to be greatly stimulated by salts, such as NaCl and KCl, and polyamines such as spermine and spermidine . Up to a sixfold increase in initial rates was observed with a variety of different single-stranded DNAs and mono- and oligonucleotides . The optimal concentrations of salts were 0.125 M, corresponding to a total ionic strength of mu equals 0.19 . For polyamines the optimal concentrations were found to be at approximately 2 mM . With low enzyme concentration and in the absence of activators complete phosphorylation was not achieved for a number of substrates . In the presence of salts or polyamines or high concentration of enzyme the phosphorylation proceeded to completion . Addition of salt led to an increase in both the apparent V-max and the Michaelis constant for the DNA substrate whereas the Michaelis constant of ATP remained unchanged . Polyamines had a similar influence on the kinetic constants for the DNA substrate whereas a decrease was found for the apparent Michaelis constant for ATP . The overall mechanism in the presence of activators was found to be sequential but probably of a rapid equilibrium random type . Of the inorganic anions tested both P-i and PP-i inhibited the enzyme in a competitive manner with both substrates.

J Biol Chem, 1975 Mar 25, 250(6), 2189 - 95
Activation of transcription by guanosine 5'-diphosphate,3'-diphosphate, transfer ribonucleic acid, and novel protein from Escherichia coli; Aboud M et al.; A protein factor TFms) that is required for ppGpp to stimulate RNA synthesis has been purified from an eluate of crude ribosomes . TFms also has the capacity to stimulate RNA synthesis without ppGpp present . Under standard conditions the action TFms and ppGpp requires uncharged tRNA . TFms and ppGpp act at inhibition to promote the formation of rifampicin-resistant or polytrI)-resistant preinitiation complexes . In the presence of rifampicin or poly(rI), tRNA is no longer required . With lambdah80dlacPs DNA as template, ppGpp together with TFms stimulated gal RNA synthesis to a much greater extent than total RNA synthesis . The stimulation of both lac and gel RNA synthesis was increased in the presence of cyclic AMP receptor and cyclic AMP.

Biochim Biophys Acta, 1975 Mar 24, 380(3), 414 - 20
Polyglycerophosphatide metabolism in Escherichia coli; Audet A et al.; When Escherichia coli B cells were labelled with {14-C} glycerol and chased, there was a marked sparing of the phosphatidyl moiety compared to the nonacylated glycerol moiety of phosphatidylglycerol . When energy-depleted cells were restored to an energy-rich medium there resulted a conversion of 32-P-labelled cardiolipin to phosphatidylglycerol, a lack of phosphatidic acid accumulation and no loss in total polyglycerophosphatide counts . In cell-free extracts, phosphatidic acid produced from 32-P-labelled cardiolipin by the action of Escherichia coli phosphalipase D, was readily recycled to form poly-glycerophosphatide . In the presence of glycerol, such extracts displayed traansphosphatidylase activity by degrading cardiolipin to phosphatidyglycerol mainly . The results as a whole indicate that the enzyme synthesizing cardiolipin together with cardiolipin-hydrolyzing phospholipase D constitute a cycle which is normally involved in the turnover of polyglycerophosphatides in Escherichia coli.

Biochim Biophys Acta, 1975 Mar 24, 380(3), 403 - 13
The inhibition of phospholipid synthesis in escherichia coli by phenethyl alcohol; Nunn WD; The kinetics of lipid metabolism during phenethyl alcohol treatment of Escherichia coli were examined . Phenethyl alcohol at a non-bacteriostatic concentration reduces the accumulation of {32-P} phosphate into phospholipids and alters the phospholipid composition of the cell membrane . The changes in phospholipid composition are a result of the inhibitory effect of phenethyl alcohol on the rates of synthesis of the individual phospholipids . The inhibition in the rate of phosphatidylethanolamine synthesis by phenethyl alcohol was twice the inhibition in the rate of phosphatidyglycerol synthesis . The de novo rate of cardiolipin synthesis was only slightly inhibited . However, net cardiolipin accumulation increased during phenethyl alcohol treatment due to a more rapid turnover of phosphatidylglycerol to cardiolipin . Phenethyl alcohol also altered the fatty acid composition of the cell as a result of its inhibitory effect on the rate of individual fatty acid synthesis . However, the inhibition of phospholipid synthesis was not reversed by fatty acid supplementation of phenethyl alcohol treated cells . This result indicates that phenethyl alcohol does not inhibit phospholipid synthesis solely at the level of fatty acid synthesis.

Biochim Biophys Acta, 1975 Mar 21, 383(3), 290 - 304
Regulation of RNA synthesis in Escherichia coli . III . Degradation of guanosine 5'-diphosphate 3'-diphosphate in cold-shocked cells; Raue HA et al.; Cold-shocked cells of Escherichia coli can degrade intracellularly accumulated guanosine 5'-diphosphate 3'-diphosphate (ppGpp) . The rate of ppGpp degradation is governed, as in whole cells, by the spoT gene; a rapid breakdown reaction is associated with the presence of the spoT+ allele and at least a five-fold slower decay occurs in spoT-minus mutants . The two degradation reactions in shocked cells display the following similarities: (i) the rates of degradation are equivalent to whole cell estimates, (ii) both require a full complement of activated amino acids, (iii) both are dependent upon supplements in the reaction mixture which stimulate the availability of energy-rich compounds and (iv) neither is inhibited by concentrations of ribosomal antibiotics which severely restrict protein synthesis . Apart from characteristic rate differences, decay of ppGpp in shocked cells derived from spoT-minus strains is discerned from spoT+ mediated decay in shocked cells by sensitivity to high concentrations of tetracycline and by manganese ion dependence.

Biochim Biophys Acta, 1975 Mar 21, 383(3), 236 - 41
Polynucleotides . XXVIII . Stimulation of the binding of aminoacyl-tRNA to ribosomes by tri- and polynucleotide analogs; Otsuka E et al.; Messenger activity of synthetic tri- and polynucleotide analogs was studied by binding of 14C-labeled aminoacyl-tRNAs to ribosomes in the presence of the analogs . Synthetic messengers used were: poly(A) analogs in which adenosine was replaced by tubercidine (I), 3-deazaadenosine (II), 1-deazaadenosine (III) and 2-methyladenosine (IV); copolymers of adenosine and aristeromycin (V); cyclic triadenylate (VI); the heptanucleotide of 6,2'-O-cyclouridine (VII); the pentanucleotide of 8,2'-S-cycloadenosine (VIIIa); A-U-G analogs in which adenosine was replaced by 8,2'-O- and S-cycloadenosine (VIII), 8,5'-O- and S-cycloadenosine (IX); 8-oxyadenosine (x); 8-bromoadenosine (XI) and formycine (XII) . Among these oligo- and polynucleotides, analogs which contained nucleotides of anti conformation having appropriate bases for Watson-Crick type hydrogen bonding stimulated the binding of corresponding tRNAs to ribosomes.

Biochim Biophys Acta, 1975 Mar 20, 376(3), 485 - 91
Energy requirements for biosynthesis of DNA in Escherichia coli . Role of membrane-bound energy-transducing ATPase (coupling factor); Mevel-Ninio MT et al.; A mutant of Escherichia coli missing energy-transducing ATPase and known to be defective in a variety of membrane functions from earlier studies (Yamamoto, T . H., Mevel-Ninio, M . and Valentine, R . C . (1973) Biochim . Biophys . Acta 314, 267-275; Thipayathasana, P . and Valentine, R . C . (1974) Biochim . Biophys . Acta 347, 464-468; Mevel-Ninio, M . and Yamamoto, T . (1974) Biochim . Biophys . Acta 357, 63-66) has been found to be blocked for anaerobic DNA synthesis . The rate of anaerobic DNA synthesis in the mutant, measured as radioactive adenine incorporation into the alkali-resistant fraction of whole cells, is about 1/6 the rate of DNA synthesis in the wild type culture under similar conditions . Addition of NO-3- or O-2 restores DNA biosynthesis in the mutant . The entry of radioactive adenine is not appreciably affected in the mutant by anaerobiosis . It is concluded that coupling factor plays a role in some step(s) of DNA biosynthesis.

Eur J Biochem, 1975 Mar 17, 52(2), 385 - 9
Molecular morphology of ribosomes . Iodination of Escherichia coli ribosomal proteins with solid-state lactoperoxidase; Michalski CJ et al.; Using either soluble or solid-state lactoperoxidase, a comparison was made between the enzymic iodination of ribosomal proteins iodinated as 30-S and 50-S subunits or as 70-S monosomes . Proteins S7, S11 and S12 of the 30-S subunit and proteins L2, L11, L26 and L28 of the 50-S subunit were labelled to a greater extent in isolated particles than in the 70-S ribosome . In contrast, proteins S4, S19 and S20 were labelled to a lesser extent in the isolated subunit . No significant differences were observed in the iodination patterns of ribosomes iodinated in the presence of soluble lactoperoxidase and those iodinated in the presence of lactoperoxidase bound to Sepharose 4B . It is suggested that the 30-S subunit undergoes a conformational change during its association with the 50-S subunit to form a 70-S monosome . Implications from results obtained with solid-state lactoperoxidase-catalyzed iodination of ribosomal proteins are also discussed.

Eur J Biochem, 1975 Mar 17, 52(2), 291 - 9
The mechanism of template activation by exonuclease V; Ferdinant FJ et al.; In crude extracts from Escherichia coli cells the ATP-dependent exonuclease V was found to be most active in converting double-stranded DNA into a suitable template for DNA polymerase . This phenomenon was studied in some detail with isolated exonuclease V and T7 DNA polymerase . We found that, at ATP concentrations arount 1 mM, the exonuclease produces a broad spectrum of DNA fragments . One class of fragments is largely single stranded with hydrogen-bonded small primer sequences . These structures allow the synthesis of remarkably homogeneous polynucleotide strands by T7 DNA polymerase.

Eur J Biochem, 1975 Mar 17, 52(2), 391 - 400
Mechanism of action of rifamazine, a member of a new class of (dimeric) rifamycins; Fietta AM et al.; 1 . Rifamazine (AF/RP) a dimeric rifamycin, is active against bacterial DNA-dependent RNA polymerase and against viral RNA-dependent DNA polymerase . 2 . Rifamazine is active also against DNA-dependent RNA polymerase extracted from rifampicin-resistant mutants of Escherichia coli . It does not interfere with enzyme-template interaction or with RNA elongation . It blocks initiation . 3 . A comparison is made between the mechanism of action of rifamazine and that of rifampicin, and of AF/013 (octyloxime of 3-formylrifamycin SV), a C-class rifamycin . Our results show that the mechanism of action of rifamazine is more similar to that of rifampicin than to that of the octyloxime derivative . 4 . Activity of rifamazine against RNA polymerase from rifampicin-resistant mutants is thought to be due to binding of the dimer to both the rifamycin-specific binding site and to a second weak site.

Proc Natl Acad Sci U S A, 1975 Mar, 72(3), 1082 - 6
Chromatin directed transcription of 5S and tRNA genes; Marzluff WF Jr et al.; Chromatin prepared by gentle methods from mouse myeloma cells retained its ability to synthesize RNA using bound endogenous RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) . The transcription resembles that observed in vivo in several respects . The low-molecular-weight RNA species 5S RNA and the 4.5S precursor to 4S RNA, are transcribed accurately and transcription is reinitiated continually in vitro . Their synthesis was not inhibited by alpha-amanitin (1 mug/ml) as was found previously for these species in isolated nuclei.

Am J Pathol, 1975 Mar, 78(3), 525 - 36
Effects of intravascular clotting on the activation of the complement system: The role of the platelet; Kalowski S et al.; Total hemolytic complement activity and the third component of complement were found to be significantly depressed in vivo in rabbits following the induction of disseminated intravascular coagulation by both thrombin and thromboplastin . Production of severe thrombocytopenia by the administration of platelet antiserum prior to the infusion of thrombin or thromboplastin partially prevented complement activation . The data show that, when clotting is triggered, complement activation takes place and that platelets are required to some extent for this reaction.

J Bacteriol, 1975 Mar, 121(3), 785 - 93
Regulation of branched-chain aminoacyl-transfer ribonucleic acid synthetases in an ilvDAC deletion strain of Escherichia coli K-12; Coleman W Jr et al.; Valyl-, isoleucyl-, and leucyl-transfer ribonucleic acid synthetase formation was compared in isogenic strains of Escherichia coli K-12 that differed only in that one strain carried a deletion of three genes of the ilv gene cluster, ilvD, -A, and -C . It was found that: (i) the activities of these synthetases in the deletion strain were less than those in the normal strain during growth in minimal medium supplemented with excess isoleucine, valine, and leucine, and (ii) their stability was reduced in the deletion strain during specific branched-chain amino acid limitations . The results of density-labeling experiments suggest that the in vivo stability of valyl-, isoleucyl-, and leucyl-transfer ribonucleic acid synthetases requires some product missing in the ilvDAC deletion strain.

J Virol, 1975 Mar, 15(3), 479 - 83
Biological activity of T4 DNA synthesized in toluene-treated Escherichia coli cells; McNicol LA et al.; T4 DNA synthesized in a toluene-treated cell system can act as the genetic donor in a DNA transformation assay . This material transforms a variety of markers at high efficiency . We present evidence that the genetic activity is due to newly synthesized, double-stranded DNA.

Biochemistry, 1975 Mar 11, 14(5), 970 - 3
Nucleotide specificity of stringent factor and the synthesis of analogs of guanosine 5'-diphosphate 3'-diphosphate and guanosine 5'-triphosphate 3'-diphosphate; Sy J; The ribosome-dependent stringent factor reaction was found to be nonspecific with regard to the number of phosphate groups linked to the 5' position of guanosine nucleotides . Both GMP and guanosine 5'-tetraphosphate could accept a pyrophosphoryl group from ATP although at a much lower rate than GDP or GTP . Guanosine 5'-monophosphate 3'-diphosphate and guanosine 5'-tetraphosphate 3'-diphosphate were the products of these reactions . Furthermore, 3'-linked analogs of guanosine 5'-diphosphate 3'-diphosphate and guanosine 5'-triphosphate 3'-diphosphate were synthesized from the corresponding ATP analog, adenosine 5'-O-(3-thiotriphosphate) . The stringent factor catalyzed reverse reaction was found to be specific for guanosine 5'-diphosphate 3'-diphosphate, and was essentially inactive to the isomeric form, guanosine 5'-diphosphate 2'-diphosphate.

Biochemistry, 1975 Mar 11, 14(5), 1079 - 87
Physical properties of some ribosomal proteins in solution and evidence for molecular interactions between isolated ribosomal proteins; Rohde MF et al.; Many previous studies have been directed toward obtaining a physical visualization of the relationship between the protein and RNA in the ribosomal subunits isolated from Escherichia coli . The current study is the first report where an attempt has been made to directly assess interactions between a pair of isolated ribosomal proteins separate from the intact system by means of sedimentation equilibrium analysis . The molecular weights of the proteins S3, S4, S5, S6, S7, S8, and S20 from the 30S subunit of the E . coli ribosome were determined under conditions of assembly of the subunit by sedimentation equilibrium . All of the proteins exhibited molecular weights consistent with monomeric behavior (i.e., in agreement with the measurement of the ultimate molecular weight in denaturing solvents as reported in other studies as well as in the current study) except S8 which indicates a tendency to self-associate . Hydrodynamic measurements on the proteins indicate that these proteins are not completely disorganized in solution such as a random coil, although not as compact as globular proteins . The frictional coefficient ratios found for these ribosomal proteins range from 1.4 to 1.9 . The hydrodynamic data are discussed as containing some evidence that stable interaction sites could exist in the proteins . The molecular weight data are considered pertinent to a sedimentation equilibrium study of protein-protein interactions that may be occurring in the ribosomal subunits . Two proteins, S3 and S5, considered in this investigation were found to exhibit no tendency to self-associate under conditions of reassembly . When the two proteins are mixed under those same conditions, however, a species with a molecular weight greater than that of either S3 or S5 is observed to be formed . The interpretation is presented that a molecular interaction between S3 and S5 is the cause . The system is described as containing S3, S5, and a complex between S3 and S5 with a stoichiometry of 1:1 and an association equilibrium constant of 5.7 times 10-5 l./mol (delta G-o equals minus 7.25 kcal/mol) . Since the association appears to be specific and of moderate strength, it is concluded that the interaction could have some pertinence with respect to conferring a structural arrangement in the ribosomal subunit . Moreover, it is concluded that protein-protein interactions, in general, must be considered in addition to the well documented significant RNA-protein relationships when models for ribosome structure and assembly are formulated.

Biochemistry, 1975 Mar 11, 14(5), 1060 - 7
Binding of Met-tRNA-f to native and derived 40S ribosomal subunits; Smith KE et al.; Our previous work has shown that the native 40S ribosomal subunits (those found free in the cell sap) but not polyribosomal 40S subunits have additional associated proteins that are removed by 0.5 M KCl . In this communication we present evidence that in the Ehrlich cell one of the native subunit associated proteins is the mammalian initiation factor that forms a Met-tRNA-f-factor-GTP complex, and is required for the binding of Met-tRNA-f to the 40S subunit . Initial examination of the KCl wash of the Ehrlich cell total ribosomal pellet revealed a factor which (1) shifted the elution of Met-tRNA-f and of GTP from the included to the excluded volume on Sephadex G-100 chromatography, (2) stimulated the binding of Met-tRNA-f to Millipore filters, and (3) stimulated the binding of Met-tRNA-f to salt-washed 40S subunits . These activities were dependent upon or enhanced by GTP; were inhibited by GDP; were much greater for Met-tRNA-f than for Met-tRNA-m or for lysyl-tRNA; and were concentrated in the KCl ribosomal wash and were not detected in the cell soluble fraction . Met-tRNA-f bound in conjunction with a specific amount of KCl wash protein, to form a distinctive particle of bouyant density 1.40 g cm minus 3 in CsCl, identical in density to one form of the native 40S subunit . Native 40S subunits, but no other subunits, contained a factor which was eluted by 0.5 M KCl and which (1) stimulated the binding of Met-tRNA-f to Millipore filters, and (2) stimulated the binding of Met-tRNA-f to salt-washed 40S subunits . The factor appeared to be localized on the native 40S subunit of density 1.40 g cm minus 3.

Biochemistry, 1975 Mar 11, 14(5), 1057 - 9
The predicted secondary structure of the N-terminal sequence of the lac repressor and proposed models for its complexation to the lac operator; Patel DJ; Rules for the prediction of protein conformation (Chou, P . Y., and Fasman, G . D . (1974), Biochemistry 13, 211, 222) have been applied to the N-terminal sequence 1-60 of the lac repressor . This analysis predicts beta structure at sequences 4-9 and 15-20, helices at 26-32, 38-45, and 52-57, and beta turns at 48-51 and 14-17 . Repressor mutants lacking operator binding capacity in which Pro replaces Ser-16 and Ala replaces Thr-19 (Weber, K., Platt, T., Ganem, D., and Miller, J . H . (1972), Proc . Natl . Acad . Sci . U.S.A . 69, 3624) have no effect on the prediction of beta structure at residues 15 to 20, which suggests that the polar side chains of Ser-16, Tyr-17, Gln-18, and Thr-19 participate in intermolecular hydrogen bonding with conplementary polar groups on the lac operator . The loss of operator binding capacity on replacement of Ala by Val at position 53 in the repressor results from a predicted secondary structural change from helix to beta structure for residues 52-57 which can be transmitted to the N-terminal sequence via a beta turn at residues 48-51 . The basic residues at positions 33, 35, and 37 between the helical regions 26-32 and 38-45 probably bind to the phosphate groups on the operator on complexation . It is proposed that complex formation involves the interaction of either a beta structure (residues 15-20) or a right-hand twisted antiparallel beta-pleated sheet (residues 4-9 and 12-20) with operator DNA.

Biochemistry, 1975 Mar 11, 14(5), 1047 - 51
The irreversible step in formation of initiation complexes of Escherichia coli; Gottlieb M et al.; At some stage during initiation the ribosomal subunits of Escherichia coli must become irreversibly coupled, since polysomal ribosomes, in contrast to free ribosomes, are not dissociated by initiation factor IF-3 . To determine when irreversibility develops we have compared the response to IF-3 of mature, puromycin-reactive initiation complexes, made with GTP, and of intermediate, puromycin-unreactive complexes, made with GMPPCP . The latter complexes initially appeared to be dissociated by the factor but this effect was found to be due to artificial loss of the ligands at the Mg2+ concentration customary in the test for dissociation . At a slightly higher Mg2+ concentration (4 mM), sufficient to retain the ligands, the GMPPCP complexes were not significantly dissociated by IF-3, at concentrations that caused complex dissociation of free ribosomes . It thus appears that the intermediate 70S initiation complex, though less stable to ionic dissociation than the mature complex, is in effect irreversible under physiological conditions.

Biochim Biophys Acta, 1975 Mar 10, 383(2), 188 - 94
Excision of pyrimidine dimers in normal and T4-infected Escherichia coli: effect of polA and other mutations; Katsuki M et al.; Strains carrying both polA1 and recBts1 mutations, which are defective in DNA polymerase I and have thermolabile exonuclease V (the recBC enzyme), are viable at 30 degrees C but not at 42 degrees C . These mutants exhibit almost normal rate of dimer excision in vivo even at the restrictive temperature . Similar results were obtained with other polA minus strains . We have also investigated effect of host and phage mutations on excision of dimers in T4-infected cells . Only a small amount of dimer is excised in T4v-1-infected cells whereas an extensive and selective release of dimers takes place in T4D-infected cells . Other phage mutations, including mutations in gene 43 and gene 30, do not affect excision of dimers in infected cells.

Biochim Biophys Acta, 1975 Mar 10, 383(2), 178 - 87
Action of exonuclease V (the recBC enzyme) on ultraviolet-irradiated DNA; Tanaka JI et al.; Exonuclease V (the recBC enzyme) of Escherichia coli can release pyrimidine dimers from ultraviolet-irradiated linear duplex DNA though it acts more slowly on irradiated DNA than on non-irradiated DAN . However, close circular lambda-dv DNA or phi X174 replicative form I DNA is not attacked by exonuclease V even though the DNA has been irradiated and treated with T4 endonuclease V to produce single-stranded breaks at the 5'-side of pyrimidine dimers . When irradiated circular DNA, previously nicked by T4 endonuclease V, is briefly exposed to elevated temperature, the DAN becomes susceptible to the action of exonuclease V, and pyrimidine dimers are selectively released . The increased susceptibility to exonuclease V may be resulted from locarized denaturation, or "fraying" of the 5'-termini at the nicks . The preferential release of pyrimidine dimers was observed when irradiated DNA, treated with T4 endonuclease V, was incubated with crude extracts of Escherichia coli . The activity was found in various strains defective in exonuclease V and/or DNA polymerase I.

J Biol Chem, 1975 Mar 10, 250(5), 1854 - 63
The fate of ribosomes in Escherichia coli cells starved for a carbon source; Kaplan R et al.; The disappearance of ribosomes in Escherichia coli cells starved for a carbon source was studied . We used a series of mutants, some of them lacking in ribonuclease I(RNase I, EC 2.7.7.17), and other containing various combinations of modified polynucleotide phosphorylase (PNPase, EC 2.7.7.8) and modified ribonuclease II (RNase II, EC 3.1.4.1) . RNA was prepared from the starved mutant cells and separated on polyacrylamide gels . The results obtained indicate that 23 S RNA degradation is similar in all strains that lack RNase I, and is slightly increased in the strain that contains this enzyme . The extent of 16 S RNA degradation is identical in all strains tested . RNA species in the size of 4 S and smaller accumulate in mutants containing modified forms of PNPase and RNase II . The appearance of an RNA species 10% smaller than 16 S RNA (d16 S RNA) was observed in all strains that contain unmodified RNase II . Analysis of ribosomes and polysomes and their RNA content indicated that polysomes are converted to monosomes and these, in turn, to ribosomal subunits . No RNA degradation products were found in polysomes, 70 S, OR 50 C particle; 30 S subunits contained 16 S RNA as well as the d16 S RNA species . Subunits are degraded to a similar extent in all strains lacking RNase I, and at a slightly faster rate in the strain that contains RNase I . The RNA to protein ratio in subunits prepared from starved cells is similar to that of unstarved cultures . Very little degradation of ribosomal proteins occurs in these mutants during carbon starvation . The proteins released from degraded ribosomes are found in the fast sedimenting (20,000 times g) pellet . Cell viability studies indicated a direct correlation between the capacity of the mutants to recovery from starvation and their capacity to degrade RNA . Thus a biological necessity for degradation of ribosomes during starvation is implied . Based on these data we propose that the endonucleolytic degradation of ribosomal RNA is the primary event in starvation degradation . It takes place in ribosomal subunits, which fall apart after the endonucleoltic attack . The RNA pieces produced by this cleavage are degraded to nucleotide by RNase II and PNPase . The ribosomal proteins attach to the cell membrane.

J Biol Chem, 1975 Mar 10, 250(5), 1690 - 3
N-Acetylglutamate synthase of Escherichia coli regulation of synthesis and activity by arginine; Leisinger T et al.; N-Acetylglutamate synthase, the first enzyme of arginine biosynthesis, was stabilized in crude extracts from Escherichia coli . At 4 degrees the enzyme lost less than 5% of activity per day . L-Arginine repressed the formation of N-acetylglutamate synthase . Under conditions of genetic or physiological derepression, a specific activity of approximately 50 nmol per min per mg of protein was measured . No activity (i.e . less than 0.2 nmol per min per mg of protein) could be detected in extracts from cells grown under conditions of repression, whereas an intermediate level was found in cell cultivated on minimal medium . In a 6-fold purified preparation L-arginine inhibited the enzyme . Of 11 precursors and analogues of arginine tested only O-{L-norvalyl-5}-isourea inhibited N-acetylglutamate synthase as strongly as L-agrinine.

J Biol Chem, 1975 Mar 10, 250(5), 1661 - 7
Studies on tyrosine phenol lyase . Modification of essential histidyl residues by diethylpyrocarbonate; Kumagai H et al.; Tyrosine phenol-lyase of Escherichia intermedia is inactivated by treatment with diethylpyrocarbonate at pH 6.0 AND 4 degrees . Spectrophotometric studies show that the inactivation is stoichiometric, with a modification of 2 histidyl residues per molecule of the enzyme . Finding that this inactivation is largely reversed by treatment with hydroxylamine indicates that the inactivation is mainly due to modification of the histidyl residues . No changes in the sulfhydryl content or in the aromatic amino acids are observed as a result of this modification . The modified tyrosine phenol-lyase retains most of its ability to form a nearly normal complex with its coenzyme, pyridoxal phosphate . This has been shown by studies of its absorption, by the determination of pyridoxal phosphate, and by reduction of the holoenzyme with tritiated sodium borohydride . The modified enzyme also appears to form a Schiff base intermediate with L-alanine . The modified holoenzyme fails to catalyze the exchange of the alpha-hydrogen of L-alanine with tritium from tritiated water . This is consistent with a catalytic role for modified histidyl residues at the active site of the enzyme; this role is the removal of the alpha-hydrogen of substrates.

J Biol Chem, 1975 Mar 10, 250(5), 1633 - 9
Investigations concerning the mode of action of 3,4-dihydroxybutyl-1-phosphonate on Escherichia coli; Cheng PJ et al.; Experiments were performed to evaluate the ability of the enzymes of Escherichia coli involved in glycerol 3-phosphate metabolism to recognize phosphonic acid analogues of the natural substrate . Neither the catabolic membrane-bound glycerol-3-phosphate dehydrogenase nor the acyl coenzyme A: glycerol-3-phosphate acyltransferase can use 3,4-dihydroxybutyl-1-phosphnate or 2,3-dihydroxypropyl-1-phosphonate are inhibitors of the reduction of dihydroxyactone phosphate as substrates . The 4-carbon phosphonic acid analogue does not exhibit inhibitory activity for either of these enzymes . While the 3-carbon phosphonic acid analogue has no inhibitory effect upon the catabolic dehydrogenase, it does appear to have a slight but reproducible inhibitory effect on the acyltransferase . Glycerol 3-phosphate and 3,4-dihydroxybutyl-1-phosphonate by glycerol 3-phosphate:NAD (P) oxidoreductase . rac-2,3-Dihydroxypropyl-1-phosphonate does not appear to be recognized by this enzyme . The apparent K-i for snglycerol 3-phosphate is 19 muM and for D-3,4-dihydroxybutyl-1-phosphonate it is 42 muM . In addition the glycerol 3-phosphate:NAD(P) oxidoreductase catalyzes the reduction of 4-hydroxy-3-oxobutyl-1-phosphonate (apparent K-m of 182 muM), a phosphonic acid analogue of dihydroxyacetone phosphate . 3,4-Dihydroxybutyl-1-phosphonate is both a competitive inhibitor (apparent Ki of 740 muM) and a substrate (apparent K-m of 450 muM) for the CDP-diglyceride: glycerol 3 phosphate phosphatidyltransferase but it has no effect upon CDP-diglyceride:L-serine phosphatidyltransferase . The relationship ofthese in vitro studies to in vivo investigations is discussed.

J Biol Chem, 1975 Mar 10, 250(5), 1640 - 7
The mechanism of end product inhibition of serine biosynthesis . V . Mechanism of serim inhibition of phosphoglycerate dehydrogenases; Winicov I; The reduction of enzyme-bound DPN constitutes a half-reaction of phosphoglycerate dehydrogenase and has been investigated fluorometrically . Serine was found to inhibit the half-reaction to the same extent and with the same degree of cooperation as the steady state reaction . This finding identifies the ternary complex conversion as the point in the reaction sequence at which serine inhibition occurs . Delta H determinations for the half-reaction showed no difference whether serine was or was not present and led to the conclusion that the inhibitory effect of serine could only manifest itself through the delta S term in the expression for the formation of the activated transition state complex . DL-3-P{2-2H}glyceric acid showed no primary isotope effect in the half-reaction . This result excludes hydrogen transfer as the rate-limiting step in the half-reaction and confirms that an isomerization step, affected by serine, exists in the ternary complex conversion scheme . The deuterated 3-P-glyceric acid shows an isotope effect of 2 in the steady state reaction.

Eur J Biochem, 1975 Mar 3, 52(1), 93 - 8
The mode of action of pleuromutilin derivatives . Location and properties of the pleuromutilin binding site on Escherichia coli ribosomes; Hogenauer G; Using equilibrium dialysis techniques it could be demonstrated that (a) the pleuromutilin derivative 14-deoxy-14{(2-diethylaminoethyl)-mercaptoacetoxy} dihydromutilin HCl binds to one site per ribosome specifically, (b) the binding constant is 1.3 times 10(7) M(-1) and (c) chloramphenicol and puromycin compete with binding of the pleuromutilin derivative . Similarly the nucleotides CpA and CpCpA also displace the unsaturated derivative of the above-mentioned pleuromutilin compound . These findings suggest that the ribosomal binding site for pleuromutilin overlaps with that for chloramphenicol and analogs of the 3'-terminus of a tRNA, like puromycin or the nucleotides CpA and CpCpA.

Eur J Biochem, 1975 Mar 3, 52(1), 197 - 202
A study of codon-dependent binding of aminoacyl-tRNA with the ribosomal 30-S subparticle of Escherichia coli . Determination of the active-particle fraction and binding constants in different media; Glukhova MA et al.; Titration of isolated Escherichia coli ribosomal 30-S particles with {14C}phenylalanyl-tRNA in the presence of poly(uridylic acid) was used for a quantitative assay of codon-dependent binding of aminoacyl-tRNA with the small ribosomal subparticle . The technique has allowed the estimation both of the fraction of "active" 30-S subparticles capable of forming the 30-S - poly(U) - phenylalanyl-tRNA complexes and the equilibrium constants of phenylalanyl-tRNA binding in different media . Heterogeneity of the ternary complexes formed has been revealed: at least two classes of complexes differing in stability have been observed . The stability of the 30-S - poly(U) - phenylalanyl-tRNA complexes has been shown to decrease with the lowering of the Mg2+ concentration, the increase of K+ concentration and the addition of urea . The stability of the complexes increases with the increase of Mg2+ concentration, with the addition of ethanol and decrease of temperature . It is demonstrated that the fraction of actively binding 30-S particles also varies in different medium conditions; it decreases with the increase of ionic strength (K+) and with the addition of urea, and increases with the increase of Mg2+ concentration and addition of ethanol.

C R Acad Sci Hebd Seances Acad Sci D, 1975 Mar 3, 280(9), 1197 - 200
{Adjuvant and mitogenic effects of detoxified lipopolysaccharides}; Chedid ML et al.; McIntire and al . have observed that a lipopolysaccharide (LPS) extracted from E . coli could be detoxified by succinylation or phtalylation and remained capable of enhancing the immune respose to serum albumins . The data reported here confirm that several LPS preparations after treatment by phtalylation retained their adjuvant activity when injected with bovine serum albumin or influenza vaccine . Yet their toxicity (as measured in adrenalectomized mice) was at least 10 000-fold smaller . Furthermore it was observed that after phtalylation LPS could still induce blastic transformation of murine B-derived lymphocytes . Thus it was possible to dissociate the toxicity of LPS from both its adjuvant and mitogentic activities.

C R Acad Sci Hebd Seances Acad Sci D, 1975 Mar 3, 280(9), 1189 - 92
{RNA fragments rich in G and A nucleotides obligatory for in vitro DNA replication of phages phi-X 174 and lambda}; Beljanski MM; Evidence was presented that in vitro conversion of single-stranded DNA of phage phi X 174 to the double-stranded replicative form by partially purified DNA-dependent DNA polymerase I requires a specific RNA fragment acting as primer (25-50 nucleotides) . RNA fragments highly rich in nucleotides A and G were obtained by partial degradation of E . coli M 500 Sho-R ribosomal RNA with pancreatic ribonuclease . They become covalently bound to the newly synthesized DNA chain of the replicative form of phage phi X 174 . These RNA fragments are also required for in vitro replication of lambda phage DNA.

Genetics, 1975 Mar, 79(3), 349 - 60
Studies of mutations in T4 control genes 33 and 55; Horvitz HR; Available mutations in transcriptional control genes 33 and 55 of coliphage T4 have been examined . By complementation analysis and map position, 15 mutants (13 in T4D, 2 in T4B) have been shown to lie in gene 33 and 6 (5 in T4D, 1 in T4B) in gene 55 . According to patterns of suppression and recombination, these mutants define three distinct amber sites in gene 33 and also three distinct amber sites in gene 55 . All of these mutations are true amber mutations, in apparent contrast to some traditional T4 "amber" mutants which grow in su+ E . coli CR63 but not in su minus E . coli B because of a strain difference other than the su+ determinant . Evidence is presented that, contrary to previous suggestions (BOLLE et al . 1968; pulitzer and geiduschek 1970), the gene 33 product is absolutely essential for T4 development.

Biochem J, 1975 Mar, 146(3), 697 - 703
The effect of spermine on transcription of mammalian chromatin by mammalian deoxyribonucleic acid-dependent ribonucleic acid polymerase; Moruzzi G et al.; Isolated rat liver nuclei demonstrate an increased ability to synthesize RNA in the presence of either spermine or spermidine . Spermidine has more effect on the low-salt alpha-amanitin-insensitive reaction, and spermine has more effect on the high-salt alpha-amanitin-sensitive reaction . Spermine is effective at concentrations of 0.1 mM and 1 muM, showing a biphasic effect . The RNA polymerase activity associated with nuclear chromatin is increased in the presence of spermine only at a concentration of 0.1 mM . Aso the transcription of deproteinized liver DNA by liver form-B polymerase or Escherichia coli enzyme is more efficient in the presence of 0.1 mM-spermine . Only when liver chromatin is transcribed by its homologous enzyme (and not by E . coli enzyme) is spermine active at both 0.1mM and 1 muM as in purified nuclei . The lower concentration of spermine (1 muM) is able to affect chromatin transcription by increasing the affinity of chromatin for the enzyme . Our findings suggest a regulatory role of spermine at the level of genome transcription.

Bol Med Hosp Infant Mex, 1975 Mar-Apr, 32(2), 307 - 20
{Solute charge in milk feeding of babies with diarrhea}; Vega-Franco L et al.; In each one of the samples, investigation of osmolarity, concentration of sodium chloride and proteins and bacteria was carried out . With these data, concentration of solutes in milks was estimated, together with a calculation of renal charge they represented . The most outstanding results showed that mothers offer whole and half skimmed milks at concentrations less than normal dilutions; thus, the solute charge was found in most cases, far below figures reported as average . In a good number of milks, osmolarity showed to very high, specially in those added with corn syrup . Only in 9 of the formulas germs were not identified; in the rest, E . coli predominated and there was high index of contamination.

Antibiotiki, 1975 Mar, 20(3), 253 - 7
{Caffeine as an inhibitor of the conjugation transfer of R-factors . A study of certain aspects of the mechanism of action of caffeine on the conjugation transfer of R-factors}; Tiagunenko IuV et al.; Some aspects of the inhibitory effect of caffeine on conjugation transfer of R-factors described by the authors earlier were studied . The effect of the above substance on the donor and recipient competence was tested in experiments with cultivation of the parent strains for 18 hours in the presence of caffeine . For this purpose the effect of caffeine on reduction of the donor and recipient cell competence after starvation in a physiological solution was investigated . It was shown that caffeine markedly decreased the donor competence of strain 15-3Mdrd of E . coli in the experiments of both types . Caffeine also inhibited reduction of the recipient competence of strain C600 of E . coli after starvation without its changing on 18-hour treatment . For the study of the caffeine effect on formation of the conjugation pairs experiments were carried out with dilution of the conjugation mixture after definite intervals which practically stopped formation of new conjugation pairs and eliminated further effect of caffeine on conjugation . Under such conditions transfer of R-factors may occur in the conjugation pairs after elimination of caffeine by dilution, if they were formed in the presence of caffeine before the mixture dilution . The experiments showed that caffeine inhibited not the formation of the conjugation pairs but the genetic transfer of R-factors . In addition, it was found that the substance insignificantly inhibited the process of phenotypic manifestation of the resistance markers . Inhibition of conjugation R-transfer by caffeine was associated with its eliminating effect, since the concentrations used did not induce elimination of the resistance markers in R+ strains.

Lloydia, 1975 Mar-Apr, 38(2), 153 - 61
Quantitation of amanitins in Amanita verna with calf thymus RNA polymerase B; Preston JF et al.; A procedure utilizing the specific inhibition of calf thymus DNA-directed RNA polymerase B has been applied to the quantitation of amanitins . This procedure has permitted the accurate quantitation of alpha-amanitin in amounts as low as 0.05 nanogram, a sensitivity 2000-fold greater than chemical detection methods used following tlc . Analysis of extracts of specimens of Amanita verna identified by morphological criteria has demonstrated that while toxin concentration is variable, some specimens are practically devoid of amanitins and may represent a variety of A . verna or a distinct species.

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