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| Naturwissenschaften, 1979 Apr, 66(4), 182 - 9 Gentic engineering for practical application; Klingmuller W; Genetic engineering has ushered in a new era in biology . Although many problems are still to be solved, there are examples that point to a possible later application for the benefit of mankind: Bacteria can be manipulated to degrade crude-oil spillages, to produce human insulin and to bind nitrogen from the air . If all the bacteria that are indigenous to agricultural soils could be made to bind nitrogen, an increase in soil fertility might well result. J Bacteriol, 1979 Apr, 138(1), 40 - 7 Modulation of gene expression by drugs affecting deoxyribonucleic acid gyrase; Sanzey B; Nalidixic acid (Nal), a drug which affects deoxyribonucleic acid gyrase activity, inhibits the expression of catabolite-sensitive genes: the three maltose operons, the lactose and galactose operons, and the tryptophanase gene . A correlation between the degree of sensitivity to Nal and that to catabolite repression has been observed . The expression of the threonine and tryptophan operons, insensitive to catabolite repression, is insensitive to Nal . The expression of the lacZ gene under the control of the IQ promoter is activated by Nal . Strains carrying a mutation in the nalA locus are resistant to these effects . Novobiocin, which inhibits the negative supercoiling activity of deoxyribonucleic acid gyrase, affects expression of the operons similarly to Nal . The involvement of promoters in Nal and novobiocin action, as well as a possible role of in vivo negative supercoiling in the selectivity of gene expression, are discussed. Nucleic Acids Res, 1979 Apr, 6(4), 1521 - 34 Pyrophosphate-condensing activity linked to nucleic acid synthesis; Volloch VZ et al.; In some preparations of DNA dependent RNA polymerase a new enzymatic activity has been found which catalyzes the condensation of two pyrophosphate molecules, liberated in the process of RNA synthesis, to one molecule of orthophosphate and one molecule of Mg (or Mn) - chelate complex with trimetaphosphate . This activity can also cooperate with DNA-polymerase, on condition that both enzymes originate from the same cells . These results point to two general conclusions . First, energy is conserved in the overall process of nucleic acid synthesis and turnover, so that the process does not require an energy influx from the cell's general resources . Second, the synthesis of nucleic acids is catalyzed by a complex enzyme system which contains at least two separate enzymes, one responsible for nucleic acid polymerization and the other for energy conservation via pyrophosphate condensation. Nucleic Acids Res, 1979 Apr, 6(4), 1309 - 21 A new method for the size estimation of the RNA genome segments of influenza virus; Sleigh MJ et al.; Previous estimates of the size of the RNA genome segments of influenza virus have been unreliable because of a lack of suitable RNA species as size markers . We have attempted to overcome this problem by utilising the ability of AMV reverse transcriptase to synthesise full length DNA copies of RNA molecules in the presence of a suitable primer . By comparing such DNA copies of the RNA segments of the influenza virus genome with sequenced restriction fragments from the E . coli plasmid pBR322, we have made more reliable estimates of the sizes of the eight genome segments from influenza virus A/NT/60/68. Nucleic Acids Res, 1979 Apr, 6(4), 1221 - 39 Polyadenylation and reverse transcription of influenza viral RNA; Emtage JS et al.; The polyadenylation of Fowl Plague Viral RNA and of Influenza A/Victoria Viral RNA using E . coli poly (A) polymerase and the subsequent reverse transcription of the polyadenylated species is reported . We have shown that all 8 genome fragments are adenylated and that an average of 25--30 adenylic acid residues per molecule is sufficient for maximal transcription with reverse transcriptase . The cDNA product is 95% sensitive to Sl-nuclease and hybridisation analysis against viral RNA reveals it to be a faithful copy of the RNA . Amongst the transcription products are long, discrete copies of genes 1--8, the lengths of which are comparable with those of the vRNA determined by electrophoresis on formamide acrylamide gels . These single-stranded cDNAs have been further transcribed to form double-stranded products with hair-pin structures at one end . Analysis of this material on native acrylamide gels revealed some DNA bands corresponding to the predicted sizes for genes 4--8. Biochim Biophys Acta, 1979 Mar 29, 572(3), 472 - 82 Glycerol 3-phosphate analogues as metabolic inhibitors in Escherichia coli, 3-hydroxy-4-oxobutyl-1-phosphonate, a drug that interferes with normal phosphoglyceride metabolism; Tang CT et al.; 3-Hydroxy-4-oxobutyl-1-phosphonate, the phoshonic acid analogue of glyceraldehyde 3-phosphate, enters Escherichia coli via the glycerol 3-phosphate transport system . There is no differential effect upon the accumulation of deoxyribonucleic acid, ribonucleic acid, or phosphoglycerides, although the accumulation of proteins was less effected . Examination of the phospholipids revealed that phosphatidylglycerol accumulation was most severely inhibited and cardiolipin accumulation was least affected . Concentrations of glyceraldehyde 3-phosphate and its phosphonic acid analogue that markedly inhibit macromolecular and phosphoglyceride biosynthesis have no effect upon the intracellular nucleoside triphosphate pool size . The phosphonate is a competitive inhibitor of sn-glycerol 3-phosphate in reactions catalyzed by acyl coenzyme A:sn-glycerol-3-phosphate acyltransferase and CDP-diacylglycerol:sn-glycerol-3-phosphate phosphatidyltransferase . A Km mutant for the former enzyme was susceptible to the phosphansferase activity . Studies with mutant strains ruled out the aerobic glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate synthase, and fructose-1,6-biphosphate aldolase as the primary sites of action. Biochim Biophys Acta, 1979 Mar 28, 562(1), 51 - 61 DNA synthesis with methylated poly(dC-dG) templates . Evidence for a competitive nature to miscoding by O(6)-methylguanine; Abbott PJ et al.; The alternating copolymer poly(dC-dG) has been methylated with either dimethyl sulphate or N-methyl-N-nitrosourea and the levels of the various methylation products determined . In addition to the 3-methylcytosine, 3-methylguanine and 7-methylguanine (produced by both agents) reaction with N-methyl-N-nitrosourea also yielded easily detectable amounts of O(6)-methylguanine and phosphotriesters . These methylated polymers were then used as templates in an in vitro assay with Escherichia coli DNA polymerase I measuring the incorporation of complementary (dCMP and dGMP) and noncomplementary (dAMP and dTMP) nucleotides . When the dimethyl sulphate-methylated polymer was used as template there was virtually no detectable incorporation of non-complementary nucleotides indicating that no miscoding could be attributed to the presence of 3-methylcytosine, 3-methylguanine or 7-methylguanine . However, when the N-methyl-N-nitrosourea-methylated polymer was used as template there was a specific incorporation of dTMP but not of dAMP . The amount of dTMP incorporated was always less than the level of O(6)-methylguanine in the template and was found to vary with the relative concentrations of the deoxynucleoside 5'-triphosphates in the assay . As the amount of dCTP present in the assay was decreased the wrong incorporation of dTMP increased and approached the level that would have been expected for a one-to-one miscoding by O(6)-methylguanine as the concentration of dCTP approached zero . The results indicate that O(6)-methylguanine is capable of miscoding with a DNA polymerase but the miscoding is competitive with the normal incorporation of dCMP: when the 5'-triphosphate precursors are present in equal amounts approximately one O(6)-methylguanine in three miscodes leading to the incorporation of dTMP. Biochim Biophys Acta, 1979 Mar 28, 562(1), 112 - 30 Specificity of chromatin transcription in vitro . Anomalies due to RNA-dependent RNA synthesis; Pays E et al.; In the presence of Mn2+, globin mRNA can be transcribed into a partial RNA copy by Escherichia coli RNA polymerase . This process also occurs when the mRNA is transcribed together with chromatin . A fraction, at least, of the newly synthesized RNA copy (anti-globin RNA) can serve as a template for the synthesis of globin sequences of the same polarity as the original mRNA . This process is sufficient to explain the specific synthesis of a subset of the globin RNA on mouse foetal liver chromatin . It also accounts for the synthesis of double-stranded RNA sequence by E . coli RNA polymerase, on chromatin as well as on pure mRNA . Results are presented suggesting that the poly(A) tract of the mRNA could be preferentially transcribed . In the presence of Mg2+, the RNA-dependent transcription is strongly inhibited, as well as the synthesis of double-stranded RNA . Under these conditions, the transcription on chromatin appears to be largely DNA dependent, and the synthesis of globin sequences is completely asymmetric . Spermine (0.3 mM) seems to improve the specificity of transcription . The transcription of chromatin in vitro is thus largely dependent on the nature of the divalent cation present in the in the incubation mixture. Mol Gen Genet, 1979 Mar 27, 171(3), 261 - 75 Threonine deaminase: autogenous regulator of the ilv genes in Escherichia coli K-12; Abrescia P et al.; In this paper we analyze the effect of mutations in three genes, ilvO, ilvA and rho, on the expression of the ilvEJGDA gene cluster of Escherichia coli K-12 . The ilvO603 mutation causes a cis-dominant derepression of the ilvEJGDA genes . In particular, the ilvG gene, not expressed in the wild type, becomes expressed in the ilvO603 strain . We have introduced ilvA mutations (ilvA454 or ilvA628) in the ilvO603 strain and we show that ilvG expression requires the presence in cis of both an ilvO603 mutation and of an ilvA+ allele . The ilvG gene is not expressed when in trans is present an ilvO+, ilvA+ genotype . However, it is expressed when the chromosome in trans is ilvO603, ilvA+ (ilvG-) . We suggest that ilvO603 is part of ilvA, the structural gene for threonine deaminase, and that threonine deaminase from the ilvO603 mutant binds the ilvO603 site and not the ilvO+ site . Therefore, the ilvA gene product would be a cis-acting protein . Mutations in the rho gene cause derepression of the ilvEJGDA gene cluster without a concomitant expression of the ilvG gene . We show that introduction of either a rho-218 or a rho-115 mutation into the ilvO603, ilvA454 double mutant causes expression of ilvG . We therefore suggest that the ilvA gene product, threonine deaminase, is involved in termination of transcription as an antagonist of the rho gene product . Introduction of ilvA454 into an ilvO603 strain causes also a decrease in expression of the ilvE, ilvJ and ilvD genes . This effect is maximum in the case of the ilvD gene and we studied it in detail in isogenic strains containing also the rho-218 mutation. Mol Gen Genet, 1979 Mar 27, 171(3), 251 - 6 Pathways for repair of DNA damaged by alkylating agent in Escherichia coli; Yamamoto Y et al.; A strain with both the polA12 and the alk-1 mutation is only slightly more sensitive to methyl methane sulfonate (MMS) than isogenic strains with only one of the mutations . On the other hand, alk-1 recA1 double mutant is much more sensitive to MMS than are strains carrying either one of alk or recA mutation . It was suggested that the alk and the polA gene products are involved in the same DNA repair process whereas the recA function is independent from the process . The yield of MMS-induced mutation (Arg- (argE) to Arg+ reversion) in alk mutant is considerably higher than that in wild type strain . Thus, the repair process in which the alk gene product is involved is relatively accurate . When MMS-treated lambda phages were plated on MMS-treated bacteria, there were considerable increases in survival of treated phage even in recA alk double mutant . It seems that a new repair pathway, which is specific for alkylating agent-induced damages and is not dependent on the RecA function, may be induced on exposure of bacteria to the alkylating agent. J Biol Chem, 1979 Mar 25, 254(6), 1902 - 12 Error induction and correction by mutant and wild type T4 DNA polymerases . Kinetic error discrimination mechanisms; Clayton LK et al.; The fidelity of DNA synthesis as determined by the misincorporation of the base analogue 2-aminopurine in competition with adenine has been measured as a function of deoxynucleoside triphosphate substrate concentrations using purified mutator (L56), antimutator (L141), and wild type (T4D) T4 DNA polymerases . Although the rates of both incorporation and turnover of aminopurine and adenine decrease as substrate concentrations are decreased, the ratio of turnover/polymerase activity is increased . Thus, the nuclease/polymerase ratio of each of these three DNA polymerases can be controlled . The misincorporation of aminopurine decreases with decreasing substrate concentrations such that all three enzymes approach nearly identical misincorporation frequencies at the lowest substrate concentration . The increased accuracy of DNA synthesis corresponds to conditions producing a high nuclease/polymerase ratio . The misinsertion frequency for aminopurine is independent of substrate concentrations and enzyme phenotype; therefore, the increased accuracy of DNA synthesis with decreasing substrate concentrations is shown to be a result of increased nuclease activity and not increased polymerase or nuclease specificity . The data are analyzed in terms of a kinetic model of DNA polymerase accuracy which proposes that discrimination in nucleotide insertion and removal is based on the free energy difference between matched and mismatched base pairs . A value of 1.1 kcal/mol free energy difference, delta G, between adenine: thymine and aminopurine:thymine base pairs is predicted by model analysis of the cocentration dependence of aminopurine misincorporation and removal frequencies . An independent estimate of this free energy difference based on the 6-fold higher apparent Km of T4 DNA polymerase for aminopurine compared to adenine also gives a value of 1.1 kcal/mol . It is shown that the aminopurine misinsertion frequency for an enzyme having either extremely low 3'-exonuclease activity, Escherichia coli DNA polymerase I, or no measurable exonuclease activity, calf thymus DNA polymerase alpha, is 12 to 15%, which is similar to that for the T4 polymerases and consistent with delta G approximately 1.1 kcal/mol. J Biol Chem, 1979 Mar 25, 254(6), 1761 - 4 Methanol formation in vivo from methylated chemotaxis proteins in Escherichia coli; Toews ML et al.; Chemotactically wild type Escherichia coli were incubated with L-{methyl-3H}methionine to label the methyl groups of their methyl-accepting chemotaxis proteins . Cells were then treated to specifically demethylate these proteins . We have identified the end product of this demethylation as {3H}methanol in the cell-free medium from treated cells. J Biol Chem, 1979 Mar 25, 254(6), 1951 - 6 Identification of a novel RNA molecule in a new RNA processing mutant of Escherichia coli which contains 5 S rRNA sequences; Ghora BK et al.; A temperature-sensitive mutant of Escherichia coli at the nonpermissive temperature fails to produce normal levels of 5 S rRNA . Instead, a number of larger RNA molecules are accumulated . One of these molecules, a 9 S RNA, contains 5 S rRNA sequences . When the strain is shifted from a nonpermissive to a permissive temperature, radioactive label is lost from the 9 S RNA and appears in 5 S rRNA . The identification of this 5 S rRNA-containing molecule indicates the participation of a new processing ribonuclease (RNase E) in the maturation of rRNA in E . coli . The 9 S RNA was not detected in a wild type strain, indicating that the processing step(s) involved in the formation of 5 S rRNA might be performed before the growing rRNA transcript is terminated. J Biol Chem, 1979 Mar 25, 254(6), 1778 - 80 31P NMR of alkaline phosphatase . Saturation transfer and metal-phosphorus coupling; Otvos JD et al.; The rate constants which characterize the formation and breakdown of the noncovalent (E.P) and covalent (E-P) enzyme-phosphate intermediates on the alkaline phosphatase reaction pathway are known to be sensitive to the nature of the metal ion bound to the enzyme . 31P NMR saturation transfer has been demonstrated to provide a simple and sensitive method for measuring the metal ion dependence of these rates under equilibrium conditions . When the native Zn2+ was replaced by Cd2+, the 31P NMR spectrum at high pH revealed a new resonance at 12.6 ppm which has been assigned to the noncovalent enzyme.phosphate complex . Reconstituting the enzyme with enriched 113Cd2+ caused this unusually downfield-shifted resonance to appear as a doublet due to 113Cd-31P spin coupling (2J31P-O-113Cd = 30 Hz) . This result provides the first unequivocal evidence for direct metal-phosphate interaction in alkaline phosphatase. Biochim Biophys Acta, 1979 Mar 23, 552(1), 89 - 102 Small angle x-ray scattering from the inner and outer membranes from Escherichia coli; Harder ME et al.; Small angle X-ray data from purified forms of inner or cytoplasmic and outer membranes from Escherichia coli have been obtained and appear to be qualitatively similar . Transitory changes are apparent in the circularly averaged X-ray profiles from inner membranes . Such results could be due to the loss or denaturation of peripheral membrane proteins . Some partially dried forms of outer membrane are partly ordered and produce diffraction patterns which support an underlying bilayer structure . An extra light membrane fraction which results from membrane preparations utilizing a French pressure cell for spheroplast disruption has been characterized and shown to be similar to inner membrane . The purified membranes produce small angle X-ray diffraction patterns which are much different from those of lipid dispersions and the differences are attributable to the high protein content of the intact membranes . While the small angle X-ray region may be useful for characterizing the membrane preparations, the paucity of detail in the diffraction pattern suggest that it will be of little value in describing the complex underlying membrane structure. Mol Gen Genet, 1979 Mar 20, 171(2), 215 - 8 Study of a suppressor of mutT in Escherichia coli; Ray U; A suppressor mutation of E . coli mut T has been isolated and mapped in the lue--ace(E/F) region. Mol Gen Genet, 1979 Mar 20, 171(2), 193 - 203 Transposon-insertion mutants of Escherichia coli K12 defective in a component common to galactose and ribose chemotaxis; Harayama S et al.; From a collection of 8,000 transposon-insertion mutants of Escherichia coli K12 we identified two mutations, trg-1::Tn5 and trg-2::Tn10, that simultaneously eliminate chemotactic response to ribose and galactose, two attractants recognized by independent receptors . We show that these transposon-insertions confer a Trg phenotype, indicating that this specific pattern of tactic defects is a null phenotype . The two mutation sites are cotransductionally linked to an extend consistent with placement in the same gene . The Trg phenotype of a family of deletion mutants produced by curing trg-2::Tn10 implies that trg is a single gene . Experiments with appropriate F-primes and Hfr's locate the trg locus at approximately 31 min on the linkage map, with a marker order: pyrF-rac-(P.O . 43)-trg-man . We also found one trg mutant whose Trg phenotype was not linked to a transposon-insertion but is probably the result of a mutator activity in the parent strain . Selection of transposon-insertions near, but not in trg allowed demonstration of a very close linkage between the spontaneous trg-3 and the transposon-generated trg's, indicating all three mutations are probably in the same gene . In our manipulations of transposon-insertions we found that Tn5 had a tendency to translocate from its initial site of insertion while Tn10 was relatively stable . The trg-product is probably a chemotactic signal transducer, which interacts directly with two independent receptor proteins and transmits information to the central chemotactic machinery. Mol Gen Genet, 1979 Mar 20, 171(2), 135 - 43 Genetic evidence for the nature, and excision repair, of DNA lesions resulting from incorporation of 5-bromouracil; Krych M et al.; Escherichia coli mutants defective in DNA uracil N-glycosidase (ung-) or endonuclease VI active against apurinic/apyrimidinic sites in DNA (xthA-) exhibit enhanced sensitivity towards 5-bromodeoxyuridine relative to the wild type strain, pointing to involvement of these enzymes in repair of bromouracil-induced lesions in DNA . Mutants defective in DNA polymerase I, either in polymerizing activity (polAl-) or (5' leads to 3')-exonuclease activity (polA107-) exhibit unusually high sensitivity (including marked lethality) in the presence of 5-bromodeoxyuridine . The results indicate that DNA polymerase I, and its associated (5'--3')-exonuclease activity, are involved in repair of bromouracil-induced lesions and are not readily replaced, if at all, by DNA polymerases II and III . Thermosensitive mutant in DNA ligase gene (lig ts7) shows high sensitivity towards 5-bromodeoxyuridine at 42 degrees C indicating the role of the enzyme in repair of bromouracil-induced lesions in DNA . Involvement of DNA uracil N-glycosidase, and endonuclease active against apurinic/apyrimidinic sites in recognition and repair of 5-bromouracil-induced damage permits of some inferences regarding the nature of this damage (lesions), in particular dehalogenation of incorporated bromouracil to uracil residues. Biochemistry, 1979 Mar 20, 18(6), 979 - 83 Subunits of RNA polymerase in function and structure . 8 . Catalytic properties of self-reactivated core enzyme; Saitoh T et al.; As an attempt to identify the maturation pathway of Escherichia coli RNA polymerase, the catalytic properties of core enzyme reactivated in the absence of maturation-promoting factors (sigma subunit or DNA) (that is, of self-reactivated core enzyme) were compared with those of native core enzyme . Differences have been found in the intrinsic activities such as in the template specificity, Km value of DNA template for the polymerase, activation energy for RNA synthesis, and increment of enzyme activity by sigma subunit . These observations imply that the transcription initiation by self-reactivated core enzyme is inaccurate and, therefore, more strict conditions including the presence of maturation-promoting factors are required for premature core be activated to the genuine function with the transcription specificity of native core enzyme. Biochemistry, 1979 Mar 20, 18(6), 972 - 8 Subunits of RNA polymerase in function and structure . 7 . Structure of premature core enzyme; Ishihama A et al.; The structure of premature core enzyme, an obligatory intermediate in both in vivo and in vitro assembly of Escherichia coli DNA-dependent RNA polymerase, was compared with that of native core enzyme . Though this assembled but inactive form of core enzyme harbors the gross conformation similar to that of native enzyme, minor and presumably local differences exist, which were identified by near-ultraviolet circular dichroism spectra, tritium-hydrogen exchange rate, protease sensitivity, intersubunit cross-linking rate by bifunctional reagents, sedimentation behavior, and elution profile from phosphocellulose . Taken together these results indicate that the core enzyme subunits are loosely associated in the premature core . The temperature-dependent maturation is required for the core subunits to be tightly associated, leading to the formation of structurally stable and functionally active RNA polymerase. Biochemistry, 1979 Mar 20, 18(6), 1063 - 7 Lac UV5 transcription in vitro . Rate limitation subsequent to formation of an RNA polymerase-DNA complex; Stefano JE et al.; The kinetics of transcription of lac UV5 mRNA using purified DNA restriction fragment as template has been studied . This template, which contains only 203 base pairs, directs the formation of a 67-base lac mRNA with high specificity . The half-time for formation of a DNA-RNA polymerase complex is approximately 0.2 min . However, upon addition of 200 micron nucleoside triphosphates to this complex, RNA production proceeds with a half-time of approximately 1 min . Therefore, it is suggested that the rate-limiting step for lac UV5 mRNA production, under typical in vitro conditions, occurs subsequent to the formation of a promoter-specific complex. Eur J Biochem, 1979 Mar 15, 95(1), 39 - 49 Degradation of the DNA-binding domain of wild-type and i-d lac repressors in Escherichia coli; Schlotmann M et al.; It has been shown that 28 transdominant mutant lac repressors which have lost operator DNA-binding ability in vivo and in vitro, but still bind inducer and are able to form tetramers (i-d repressors), could be divided into two groups by their capacity or incapacity to bind non-specifically to the phosphate groups of the DNA backbone . All but one of 15 analysed i-d repressors with amino acid substitutions to the C-terminal of residue 52 showed uneffected non-specific DNA binding . All 13 tested i-d repressors with amino acid substitutions to the N-terminal of residue 53 did not bind to double-stranded DNA, and 11 of these repressors derived from missense mutations in the lacI gene were endogenously degraded . The degradation in vivo only affects the amino-terminal 50-60 residues producing a mutant-specific pattern of stable repressor fragments . These fragments are tetrameric and capable of binding inducer in vivo and in vitro . The proteolytic attack presumably takes place during synthesis of the i-d repressors, since the resulting fragments are stable, both in vivo (as shown by a pulse-chase experiment) and in vitro . The proteolysis in vivo depends on the growth conditions of the bacteria and is higher in cells grown in minimal media than in rich media . Wild-type repressor is only susceptible to limited proteolysis in cells grown in minimal media but not in cells grown in rich media . The results suggest that the majority of the sequence alterations before residue 53 in missense mutant i-d lac repressor proteins affect the three-dimensional structure of the amino-terminal DNA-binding domain of the repressor protein, making it susceptible to proteolytic attack by one or several intracellular proteases. J Mol Evol, 1979 Mar 15, 12(3), 189 - 95 A unique pattern of toxic synthesis in pentitol catabolism: implications for evolution; Scangos GA et al.; All of our Escherichia coli C mutants blocked in the first step of D-arabitol catabolism (D-arabitol dehydrogenase) became unable to grow in the presence of D-arabitol . We have shown that this sensitivity is eliminated by a defect in the second enzyme of the pathway (D-xylulokinase), leading to a pattern of toxicity and its relief which has not been previously reported . We have found a similar pattern of toxicity and its relief in the closely related ribitol pathway . The evolutionary significance of these findings is discussed. Int J Cancer, 1979 Mar 15, 23(3), 344 - 52 Characteristics of a low-molecular-weight factor extracted from mouse tumors that affects in vitro properties of macrophages; Cheung HT et al.; A dialysable low-molecular-weight factor capable of affecting in vitro properties of macrophages was extracted from four different mouse tumors . This factor not only modulates closely related properties of peritoneal macrophages such as spreading and migration but also inhibits lipopolysaccharde-induced tumoricidal activity of these cells . It can be extracted not only from tumor tissues but also from tumor cells grown in vitro . The appearance of this factor is unique to tumors and it is not present in detectable quantities in normal tissues . The factor from one of the tumors, Lewis lung carcinoma, was purified extensively and the partially purified factor retains all the above effects on macrophages . It is not sensitive to pronase or a mixture of bovine spleen phosphodiesterase II, E . coli alkaline phosphatase and pancreatic ribonuclease . The factor is lipid-like in character and it is soluble in both organic solvents and aqueous media . It has ionizable group(s) and is anionic at neutral pH but non-ionic under acidic conditions. Chromosoma, 1979 Mar 12, 71(3), 263 - 81 The organization of the ribosomal RNA genes of Chironomus tentans and some closely related species; Degelmann A et al.; Southern gel analysis of total DNA from Chironomus tentans showed that the rRNA genes (rDNA) are homogeneous in structure . After cloning in Escherichia coli plasmid pBR313, the rDNA organisation was further studied by restriction fragment analysis and R-loop mapping . No heterogeneity could be detected by heteroduplex analysis of six different cloned rRNA cistrons . R-loop sizes of 1.69 and 3.63 kilobases (kb) were measured for the 18S and 28S rRNA coding sequences . The two spacers are 0.75 and 1.77 kb long . Southern gel analysis showed also a homogeneous rDNA structure for a Canadian population of C . tentans and C . pallidivittatus . The same technique indicated, however, that the rDNA of two other closely related species of C . thummi and C . melanotus is heterogeneous in structure . A possible correlation between this heterogeneity and the presence of heterochromatin in these species is discussed. Med J Aust, 1979 Mar 10, 1(5), 154 - 5 Coliform status of domestic swimming pools; Gluer J et al.; An investigation of maintenance, usage, and coliform status of water in 104 domestic swimming pools in 13 Brisbane suburbs was made in January, 1978 . The results showed that domestic pools are often imperfectly serviced, and are potential infection sources . The need for health education of pool owners, and for more reliable methods of pool sanitization is emphasized. J Biol Chem, 1979 Mar 10, 254(5), 1748 - 53 DNA polymerase III of Escherichia coli . Purification and identification of subunits; McHenry CS et al.; DNA polymerase III, the core of the DNA polymerase III holoenzyme, has been purified 28,000-fold to 97% homogeneity from Escherichia coli HMS-83 . The enzyme contains subunits: alpha, epsilon, and theta of 140,000, 25,000, and 10,000 daltons, respectively . The alpha subunit has been previously shown to be a component of both DNA polymerase III and the more complex DNA polymerase III holoenzyme (Livingston, D.M., Hinkle, D., and Richardson, C . (1975) J . Biol . Chem . 250, 461-469; McHenry, C., and Kornberg, A . (1977) J . Biol . Chem . 252, 6478-6484) . It is demonstrated here that the epsilon and theta subunits are also subunits of the DNA polymerase III holoenzyme . Thus, the DNA polymerase III holoenzyme contains at least six different subunits . Our preparation has both the 3' leads to 5' and 5' leads to 3' exonuclease activities previously assigned to DNA polymerase III (Livingston, D., and Richardson, C . (1975) J . Biol . Chem . 250, 470-478). J Biol Chem, 1979 Mar 10, 254(5), 1699 - 706 The structure of cytochrome b562 from Escherichia coli at 2.5 A resolution; Mathews FS et al.; The structure of cytochrome b562 from Escherichia coli has been determined at 2.5 A resolution by x-ray diffraction methods . Protein phases were computed by the single isomorphous replacement method with anomalous scattering measurements from the native and uranyl acetate-substituted crystals . The electron density was averaged about the noncrystallographic 2-fold axis relating 2 molecules in the triclinic unit cell . The protein consists of four nearly parallel alpha helices and represents a new class of cytochrome structure . The heme group is inserted between the helices near one end of the molecule with one heme face partially exposed to solvent . The two heme ligands are histidine and methionine . The 2 phenylalanines are packed internally near the heme group, and the 2 tyrosines are on the surface, also near the heme group . The folding of the protein resembles that of hemerythrin and tobacco mosaic virus protein and shows a different topology from that of cytochrome b5, cytochrome c, or the globins. J Biol Chem, 1979 Mar 10, 254(5), 1457 - 61 Subunit structure of functional porin oligomers that form permeability channels in the other membrane of Escherichia coli; Nakae T et al.; Oligomers of a protein, porin, form permeability channels in the outer membrane of Escherichia coli B . A functional porin oligomer was identified and was purified to homogeneity by gel filtration in the presence of salts and sodium dodecyl sulfate . Molecular weights of purified porin oligomer and heat-dissociated monomer appeared to be 102,900 and 32,600, respectively, when determined by sedimentation equilibrium in the presence of sodium dodecyl sulfate . We concluded that the porin oligomer thus consists of three identical subunits . These data and results from other laboratories suggest porin trimers exist also in the outer membrane of intact cells, and participate in the formation of permeability channels . It was found that porin trimer bound less sodium dodecyl sulfate than the porin monomer. Mol Gen Genet, 1979 Mar 9, 171(1), 75 - 8 Molecular cloning of the 4.2 Md EcoRI fragment of Aspergillus nidulans mitochondrial DNA; Bartnik E et al.; The 4.2 Md EcoRI fragment of Aspergillus nidulans mitochondrial DNA was cloned using the plasmid pBR 332 as vector and E . coli as host . Hitherto unknown sequence of HindIII sites within this region of mitochondrial genome was established. Mol Gen Genet, 1979 Mar 9, 171(1), 7 - 13 Transposition of DNA inserted into deletions of the Tn5 kanamycin resistance element; Meyer R et al.; Tn5-trp hybrid transposons have been constructed by insertion of a trpPOED Hind III fragment into an in vivo Tn5 internal deletion mutant or by substitution of trp for the internal Tn5 Hind III fragment . These hybrids are called, respectively, Tn409 and Tn410 . Both Tn409 and Tn410 will transpose into lambda in the presence of a complementing Tn5 element . In the absence of a wild Tn5, lysogens carrying R1162::Tn409 and R1162::Tn410 plasmids will yield lambdatrp phages at less than six per cent of the complemented frequency . This reduction indicates that Tn409 and Tn410 lack a diffusible transposition function provided by wild Tn5 elements . However, the formation of lambdatrp phages without complementation is real . Most of these transducing particles contain Tn409 and Tn410 still linked to the carrier R1162 plasmid . This observation suggests that uncomplemented Tn409 and Tn410 elements mediate the formation of lambda-transposon-plasmid cointegrate structures . Thus, the missing transposition function may be involved in resolving these cointegrate structures to the final lambda::Tn409 or lambda::Tn410 product. Mol Gen Genet, 1979 Mar 9, 171(1), 53 - 8 DNA synthesis in UV-irradiated Escherichia coli K-12 strains carrying dnaA mutations; Jonczyk P et al.; It has been found that UV irradiation of dnaA mutants of E . coli K-12 enables the initiation of DNA synthesis at a temperature restrictive to these mutants . The UV-induced DNA synthesis is dependent on protein synthesis and on a transcriptional event at a time when protein synthesis is no longer required . In contrast to dnaA mutants UV irradiation fails to induce DNA synthesis in the two other initiation mutants dnaC2 and dnaB252 . DNA synthesis at the restrictive temperature is initiated also when the tif-1 phenotype is expressed in the dnaA46 tif-1 double mutant . Possible mechanisms of the observed capability of dnaA mutants to synthesize DNA at the restrictive temperature after UV irradiation or under conditions of tif-1 expression are discussed. Mol Gen Genet, 1979 Mar 9, 171(1), 1 - 6 Spontaneous degradation of pRD1 DNA into unique size classes is recA dependent; Puhler A et al.; The his and nif genes of the P1 type plasmid pRD1 were lost at a high frequency in a recA+ but not in a recA- Escherichia coli host during growth in a non-selective medium . 92% of the His- Nif- segregants after 6 subcultures retained the genetic markers of the precursor plasmid RP4, while the remainder lost all of the pRD1 markers with the concomitant loss of ccc-DNA . Plasmids purified from the His- Nif- segregants resembled RP4 in the physical and genetic properties examined . The contour length of pRD1 DNA molecules from a recA- strain was 49 micrometer corresponding to a molecular weight of 101 Mdals and the buoyant density was 1.715 g/cm3 . In contrast, the contour lengths of plasmid molecules isolated from a recA+ host carrying pRD1 fell into 3 size classes of 49, 19 and 2 micrometer corresponding to molecular weights of 101, 39 and 4 Mdals respectively and two DNA species of buoyant density 1.715 and 1.719 g/cm3 were observed. Science, 1979 Mar 9, 203(4384), 1014 - 6 Binding of cis- and trans-dichlorodiammineplatinum(II) to DNA: evidence for unwinding and shortening of the double helix; Cohen GL et al.; The antitumor drug cis-dichlorodiammineplatinum(II) (cis-DDP) and the inactive trans isomer bind and produce cooperative changes in closed and nicked circular duplex DNA's . Covalent binding of both platinum complexes to the closed circular DNA alters the degree of supercoiling, presumably by disrupting and unwinding the double helix . Electron micrographs show the platinated DNA's to be shortened by up to 50 percent of their original length . At similar ratios of bound platinum per nucleotide, the electrophoretic mobilities of the DNA's in gels containing the dye ethidium bromide are the same for both isomers . The only detectable difference in the binding of the two platinum isomers is an increase in the electrophoretic mobility in nondye gels of closed circular DNA having small amounts of bound cis-DDP that is not apparent for the trans complex. Mol Gen Genet, 1979 Mar 9, 171(1), 15 - 22 A missense mutation in the gene coding for ribosomal protein S17 (rpsQ) leading to ribosomal assembly defectivity in Escherichia coli; Herzog A et al.; The conditionally lethal mutation, 2861 mis, has been mapped inside the ribosomal protein gene cluster at 72 minutes on the Escherichia coli chromosome and was found to cotransduce at 97% with rpsE (S5) . The 2861 mis mutation leads to thermosensitivity and impaired assembly in vivo of 30S ribosomal particles at 42 degrees C . The strain carrying the mutation has an altered S 17 ribosomal protein; the mutational alteration involves a replacement of serine by phenylalanine in protein S 17 . Spontaneous reversion to temperature independence can restore the normal assembly in vivo of 30 S ribosomal subunits at 42 degrees C and the normal chromatographical behaviour of the S 17 ribosomal protein in vitro . We conclude therefore that the 2861 mis mutation affects the structural gene for protein S 17 (rpsQ). Science, 1979 Mar 9, 203(4384), 1012 - 4 beta-Galactosidase and selective neutrality; Holmquist R; Three hypotheses to explain the amino acid composition of proteins are inconsistent (P congruent to 10(-9) with the experimental data for beta-galactosidase from Escherichia coli . The exceptional length of this protein, 1021 residues, permits rigorous tests of these hypotheses without complication from statistical artifacts . Either this protein is not at compositional equilibrium, which is unlikely from knowledge about other proteins, or the evolution of this protein and its coding gene have not been selectively neutral . However, the composition of approximately 60 percent of the molecule is consistent with either a selectively neutral or nonneutral evolutionary process. MMW Munch Med Wochenschr, 1979 Mar 9, 121(10), 345 - 8 {Pathogenesis of coli enteritis (author's transl)}; Guggenbichler JP; The pathogenic mechanisms of E . coli in diarrheal diseases were largely obscure up to now . The discovery of an enterotoxin which corresponded with the cholera enterotoxin in its mode of action and is also largely identical with this molecule immunologically provided essentially new aspects of the pathogenic importance of E . coli in diarrheal diseases . The detection of enterotoxin formation depends on the biological test model and presently it is still expensive and unsuitable for routine laboratory use . The serological identification of coli bacteria is meaningless for the detection of enterotoxin formation . Enterotoxic coli are more frequently found (12%) in the large group of hemolytic organisms . In 1978 we isolated enterotoxic coli from the stools of 12 infants under one year old with stubborn diarrhea. Biochim Biophys Acta, 1979 Mar 8, 551(2), 238 - 47 Ionic selectivity of pores formed by the matrix protein (porin) of Escherichia coli; Benz R et al.; Incorporation of the matrix protein (porin) from the outer membrane of Escherichia coli into black lipid films results in the formation of ion-permeable pores with a single-pore conductance of the order of 2 nS (in 1 M KCl) . Information on the structure of this pore has been obtained by determining the selectivity for various species differing in charge and size . From the permeability of the pore for large organic ions (Tris+, glucosamine+, Hepes-) a minimum pore diameter of 0.8 nm is estimated . At neutral pH the pore is two to four times more permeable for alkali ions than for chloride . On the basis of the observed pH dependence of permeability, this cationic selectivity is explained by the assumption that the pore contains fixed negative charges. Mol Gen Genet, 1979 Mar 5, 170(3), 319 - 25 Studies of a plasmid coding for tetracycline resistance and hydrogen sulfide production incompatible with the prophage P1; Briaux S et al.; The plasmid pIP231, determining tetracycline resistance and hydrogen sulfide production is shown to belong to incompatibility group Y and to code for a restriction and modification system . Unlike the IncY plasmids, P7 and P15B, plasmid pIP231 shows only little genetic and physical homology with P1 prophage. Science, 1979 Mar 2, 203(4383), 887 - 92 Molecular cloning of polyoma virus DNA in Escherichia coli: lambda phage vector system; Chan HW et al.; The biological activity of recombinant phage and recombinant phage DNA containing monomeric or dimeric polyoma DNA inserts was examined in mice and cultured mouse cells . Recombinant preparations containing a single copy of viral DNA were invariably noninfectious; molecules containing a dimeric polyoma DNA insert were at least seven orders of magnitude less infectious than polyoma virions after parenteral inoculation . No infection was detected with any recombinant preparation after oral administration. Science, 1979 Mar 2, 203(4383), 883 - 7 Molecular cloning of polyoma virus DNA in Escherichia coli: plasmid vector system; Israel MA et al.; A series of recombinant plasmids containing polyoma virus (PY) DNA were constructed, and their biological activity was evaluated in mice and in cultured mouse cells . While all of the recombinants studied contain the complete, potentially infectious viral DNA, in no case was the intact recombinant PY-plasmid DNA, or live Escherichia coli containing the recombinant plasmids, capable of inducing PY infection of mice, either by feeding or by parenteral injection. Cell, 1979 Mar, 16(3), 535 - 49 Vitellogenin in Xenopus laevis is encoded in a small family of genes; Wahli W et al.; Vitellogenin, the yolk protein precursor, is produced in X . laevis liver from a 6.3 kilobase (kb) mRNA . Sequences of this mRNA have been transcribed into cDNA and cloned in E . coli . Some properties of 21 of these cloned DNAs, ranging in size from 1 to 3.7 kb, have been reported by Wahli et al . (1978b) . This paper reports restriction endonuclease mapping, cross hybridization, heteroduplex mapping in the electron microscope and heteroduplex melting experiments with these DNAs . We conclude that the cloned DNAs fall into two main groups of sequences which differ from each other in approximately 20% of their nucleotides . Each main group contains two subgroups which differ from each other by about 5% sequence divergence . By hybridizing cloned DNAs with restricted genomic DNA, we showed that sequences corresponding to all four sequence groups are present in a single animal . Furthermore, we have obtained tentative evidence for the presence of large intervening sequences in genomic vitellogenin DNA . Analysis of R loop molecules demonstrated that all four sequences are present in the vitellogenin mRNA population purified from individual animals . While some alternate explanations are not entirely excluded, we suggest that vitellogenin is encoded by a small family of related genes in Xenopus. J Lab Clin Med, 1979 Mar, 93(3), 437 - 48 Platelet and fibrinogen production: relative sensitivities to endotoxin; Alving BM et al.; The relative sensitivities of platelet and fibrinogen production to endotoxin were determined in male New Zealand rabbits . E . coli endotoxin was administered in single intravenous doses of 0.1 to 50.0 mu/g/kg body mass . 75SeM was injected 5, 12, or 18 hr after endotoxin, and the percent incorporation into platelets and fibrinogen was used to measure thrombopoiesis and fibrinogen synthesis . Leukopenia occurred after the infusion of endotoxin at all dose levels; the lowest dose that caused thrombocytopenia was 0.5 microgram/kg . Endotoxin was detected with the Limulus test in plasma of animals that received 5 to 50 microgram/kg . The stimulation of fibrinogen and platelet production, as well as the postinfusion decrease in platelet count, correlated directly with the log of the dose of endotoxin infused . The lowest dose of endotoxin that stimulated platelet and fibrinogen production was 0.5 microgram/kg . In animals that received this dose of endotoxin, increased fibrinogen production was detected only when 75SeM was injected 5 hr later . In animals injected 5, 12, or 18 hr later . Increased platelet production was detected at these two doses only when 75SeM was injected 18 hr after endotoxin . The data indicate that fibrinogen and platelet production have similar sensitivities to endotoxin, although the time course of stimulation is different for these two blood components. Immunopharmacology, 1979 Mar, 1(2), 125 - 35 Role of complement in endotoxin initiated lethality in mice; Curry BJ et al.; We have examined the role of complement in eliciting a lethal response in C3H/HeJ and C3H/St mice . The results reported here indicate that endotoxin-initiated complement activation, leading to significant drops in circulating C3 levels, is not sufficient to cause lethality . The complement system in both strains was demonstrated to be responsive in vitro to activation both by E . coli 0111:B4 LPS I, an alternative pathway activator in other systems, as well as S . minnesota R595 LPS, which activates almost exclusively the classical pathway . In vivo injection of high (lethal) doses of E . coli 0111:B4 LPS I and S . minnesota R595 LPS causes a significant decrease in the circulating C3 levels of both strains after 4 hr . In contrast, circulating C3 levels were not significantly different from normal values in either strain following injection with minimal (lethal) amounts of E . coli 0111:B4 LPS II, a weakly anticomplementary LPS preparation . In all cases, lethality was observed in only the C3H/St mice, indicating that neither complement activation, nor the lack of it, is responsible for lethality in mice. Neurosci Lett, 1979 Mar, 11(3), 279 - 82 Hypothalamic monoamine contents in endotoxin fever of new-born guinea pigs and kittens; Hahn Z et al.; In new-born guinea pigs and kittens Escherichia coli endotoxin did not significantly modify the hypothalamic level of either norepinephrine (NE) or 5-hydroxytryptamine (5-HT) . The hypothalamic 5-hydroxyindoleacetic acid (5-HIAA) content was significantly elevated in endotoxin-treated animals, but this elevation was confined to a period following the endotoxin injections by about 60 min in guinea pigs and 75-90 min in kittens. Ann Sclavo, 1979 Mar-Apr, 21(2), 235 - 41 {Comparative study of the different media of enrichment and isolation for the identification of enteric pathogens (author's transl)}; Mauro A et al.; In the present study, were compared several media in use in diagnostic laboratory for the isolation of enteric pathogens . The AA . have remarked the efficiency of Bacto-Selenite F and of Bacto-TT Broth Base Hajna, for the enrichment; it is suggested the use of Bacto Agar and of Bacto-Hektoen Enteric Agar for the isolation. Can J Genet Cytol, 1979 Mar, 21(1), 95 - 100 Reduction of sensitivity to ultraviolet light in excision-deficient Escherichia coli strain WP2 uvrA; Nestmann ER; Mutants with increased resistance to UV light were isolated from Escherichia coli WP2 uvrA . Most of these isolates were designated as true or partial revertants of the uvrA- mutation . However, one class of mutants showed only a moderate increase in resistance to UV . The gene responsible for this phenotypic change is closely linked to the lac locus and has been designated the suuA gene (suppressor of uvrA). Pediatr Res, 1979 Mar, 13(3), 148 - 51 Characteristics of impaired chemotactic function in cord blood leukocytes; Tono-Oka T et al.; Mobilities of cord blood granulocytes were studied using the agarose plate method and Boyden's chamber method . In the agarose plate, granulocytes of cord blood were shown to have moderately decreased responses in chemotaxis and chemokinesis induced by Escherichia coli-derived chemotactic factor and/or zymosan-activated serum, whereas they were shown to have a normal capacity of random mobility . Although their distance and index of chemotaxis or chemokinesis in the agarose plate were significantly less than those of adult granulocytes, response rate in both types of mobility were evidently higher compared with those in patients with chemotactic defect . Furthermore, there is a difference between chemotactic responses of cord blood granulocytes to E . coli-derived chemotactic factor and to zymosan-activated serum in the agarose plate method . Using the latter, a more distinguishable difference between chemotactic responses of cord blood granulocytes and adult granulocytes was shown . The Boyden's chamber method tended to show a more significant difference between chemotactic responses of granulocytes of cord blood and adults than in the agarose plate method. Gene, 1979 Mar, 5(3), 233 - 53 High frequency of yeast transformation by plasmids carrying part or entire 2-micron yeast plasmid; Gerbaud C et al.; By using two chimeric plasmids containing yeast ura3 gene and 2-micron yeast DNA linked to the bacterial plasmid pCR1, yeast transformation of a high frequency has been achieved . The first plasmid is such that the 2-micron DNA part, in which the ura3 gene is incorporated, can be removed in one step and thus the 2-micron-ura3 sequence can be considered as a "transposable" block . In contrast, the second one bears the entire 2-micron plasmid and the ura3 gene is inserted in the bacterial plasmid part . As shown through hybridization experiments and genetic studies, the ura3 gene was maintained as a cytoplasmic element . Plasmids recovered from the yeast transformants were used to transform Escherichia coli . Their analysis by EcoRI showed that in many cases the vector had recombined with the endogenous 2-micron DNA of the recipient strain . The specific activity of orotidine 5'-monophosphate decarboxylase (coded by ura3) in yeast transformants was 10- to 30-fold higher than in the wild type. Gene, 1979 Mar, 5(3), 207 - 31 Cloning and endonuclease restriction analysis of argF and of the control region of the argECBH bipolar operon in Escherichia coli; Crabeel M et al.; A 1.8 kb DNA fragment, liberated by endonuclease HindIII, contains the control region of the argECBH bipolar operon near one end and the weak secondary promoter of argH at the other extremity; it has been cloned in plasmid pBR322 . The same plasmid vector has been used to clone the argF gene liberated from the chromosome by endonuclease BamHI . Restriction patterns for the two hybrid plasmids have been determined, using enzymes AluI, BglI, EcoRI, HaeIII, HincII, HindIII, HpaI and II, PstI and SalI . Two AluI sites situated on either side of and close to a HincII target delineate two short fragments covering the whole of the argECBH control region . The argF control elements are located in a region accessible to further dissection by BamHI, EcoRI, PstI and HindIII . Carriers of the argF plasmid produce extremely high amounts of ornithine carbamoyltransferase, a feature useful for purification of this enzyme. J Gen Microbiol, 1979 Mar, 111(1), 193 - 200 Mutants of Escherichia coli K12 accumulating porphobilinogen: a new locus, hemC; McConville ML et al.; Mutants of Escherichia coli K12 which accumulated the haem precursor porphobilinogen are described . The mutants grew very slowly on carbon and energy sources which K12 uses only oxidatively, and they had low catalase activities, suggesting that they were deficient in haem . Extracts had one-tenth of the parental activity of the enzyme porphobilinogen deaminase . In transduction, the mutation mapped close to genes ilvD and metE at minute 84 . The gene was tentatively identified as hemC, coding for porphobilinogen deaminase . The gene symbol hemC replaces the earlier and temporary symbol popE. Infect Immun, 1979 Mar, 23(3), 690 - 9 Hemagglutination and adhesiveness of toxigenic Escherichia coli isolated from humans; Thorne GM et al.; Toxigenic strains of Escherichia coli isolated from humans were studied for adherence to human buccal mucosal epithelial cells . The E . coli strains were labeled with 3H-amino acids or fluorescein isothiocyanate . Toxigenic E . coli strains varied in their ability to adhere in the presence of mannose . Of 32 toxigenic strains examined, 52% bound to the buccal cells, whereas none of 8 control strains did so (Mann-Whitney U test, P =0.007) . The control strains were nontoxigenic E . coli isolates from humans, enterotoxigenic E . coli isolates from animals, and E . coli K-12 containing the K88 or K99 plasmid; these strains exhibited only background-level adherence in this assay . Among the toxigenic E . coli strains that bound to human buccal mucosal cells, there was no correlation with mannose-resistant hemagglutination (MR-HA) of guinea pig and human erythrocytes . Screening 32 strains, we found the following phenotypes: (i) MR-HA+, buccal adherent; (ii) MR-HA+, buccal nonadherent; (iii) MR-HA-, buccal adherent . Presumably the third group represents strains with another type(s) of surface attachment components not involved in the MR-HA reaction . Our findings indicate that a number of bacterial surface structures can function in MR-HA and buccal adherence. Appl Environ Microbiol, 1979 Mar, 37(3), 369 - 72 Induction of mutation in Escherichia coli by freeze-drying; Tanaka Y et al.; The effect of freeze-drying on phenotypic reversion of amino acid auxotrophy to prototrophy was studied in Escherichia coli . In a radioresistant strain, E . coli H/r 30 (uvr+ exr+), which can repair the deoxyribonucleic acid damaged due to freeze-drying, an increased mutation frequency from auxotrophy to prototrophy was observed with increased time of freeze-drying of the cells . On the other hand, in a radiosensitive strain, E . coli NG 30 (recA), which cannot repair the damaged deoxyribonucleic acid due to a lack of repair enzyme system, no significant reversion occurred, although the survival rate was very low . The rate of phenotypic reversion dut to freeze-drying in both E . coli RIMD 0509109 (uvr+ exr+) and RIMD 0509115 (uvr exr+) was almost the same, indicating that the phenomenon is independent of the uvr character . From these results it is concluded that mutation was induced in E . coli cells during the rehydration when the damaged deoxyribonucleic acid was repaired by exr character of the cells . Thus, we propose that a serious consideration should be paid to the freeze-drying technique to preserve bacterial cells. Lymphology, 1979 Mar, 12(1), 20 - 2 Qualitative and quantitative changes in thoracic duct lymph during canine experimental shock; Hayashi S et al.; Changes of the body fluid exchange and of the composition of metabolites in the hepatosplanchnic area in canine hemorrhagic and endotoxin or septic shock models were studied by investigating the qualitative and quantitative changes in thoracic duct lymph draining from abdominal organs . In the present study, it might be summarized that the changes in the flow rate and composition of thoracic duct lymph were put forward to much more directly and apparently indicate the degree of hepatosplanchnic cellular impairment in canine experimental shock than in the circulating blood. J Virol, 1979 Mar, 29(3), 1229 - 31 Development of Escherichia coli virus T1: escape from host restriction; Wagner EF et al.; The bacterial virus T1 grows interchangeably on different Escherichia coli strains (C, B, and K) . This implies that T1 has an efficient mechanism to overcome the host restriction barrier . The DNA of T1 was found to be methylated independently of the hosts . The percentage of N6-methyladenine varied from 1.6 to 1.8, and the 5-methylcytosine content varied from 0.1 to 0.4% . In contrast, the range in percentage of N6-methyladenine and 5-methylcytosine found in the hosts was 0.7 to 2.4 and 0.0 to 1.1, respectively. J Immunol, 1979 Mar, 122(3), 932 - 5 Lack of responsiveness of C3H/HeJ macrophages to lipopolysaccharide: the cellular basis of LPS-stimulated metabolism; Ryan JL et al.; The rate of glucose utilization has been used as a measure of LPS-induced activation of cultures of C3H/HeN and C3H/HeJ spleen cells, peritoneal cells, and purified peritoneal adherent cells . Peritoneal cells utilized 40 to 60 times more glucose than did spleen cells and purified adherent monolayers were more active than mixed peritoneal cells, suggesting that only macrophage metabolism was being measured . The cell preparations for C3H/HeJ mice were not activated by Escherichia coli K235 LPS prepared by extensive phenol extraction, whereas C3H/HeN cells were activated by the LPS . Cells from both strains were activated by a commercially obtained E . coli 0111:B4 LPS and butanol-extracted K235 LPS . The addition of 10% C3H/HeN spleen cells to C3H/HeJ peritoneal cells resulted in a marked enhancement of glucose utilization . These findings suggest that LPS-induced enhancement of macrophage metabolism occurs both by direct action of LPS on macrophages as well as indirectly through activated lymphocytes. Urology, 1979 Mar, 13(3), 321 - 3 Bilateral renal malakoplakia; Ho KL et al.; A case of malakoplakia is presented which was confined to the renal parenchyma of both kidneys in a thirty-five-year-old woman in whom fatal renal failure developed in three weeks . Bilateral renal malakoplakia is rare, and unlike malakoplakia of the bladder, it behaves as a progressive, destructive, and fatal disease. Proc Natl Acad Sci U S A, 1979 Mar, 76(3), 1363 - 7 Strand exchange in site-specific recombination; Enquist LW et al.; The site-specific recombination system of phage lambda promotes crossovers at its attachment site (att) . In this report we show that when phage are crossed in conditions where only the site-specific recombination system is active, a low frequency of crossovers can also be detected in a region that is close to but does not contain att . These crossovers require the phage int gene, the host hip gene, and the integrity of att . They are not detected if one of the parents carries a substitution of a heterologous attachment site (attB instead of attP) . To explain these findings we suggest that site-specific recombination can proceed by exchange of single strands between the participating chromosomes at att and migration of the resulting junction outside of att. Proc Natl Acad Sci U S A, 1979 Mar, 76(3), 1213 - 7 Requirements of acetyl phosphate for the binding protein-dependent transport systems in Escherichia coli; Hong JS et al.; In Escherichia coli, acetyl phosphate can be formed from acetyl-CoA via the phosphotransacetylase (phosphate acetyltransferase; acetyl-CoA:orthophosphate acetyltransferase, EC 2.3.1.8) reaction and from acetate (plus ATP) via the acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) reaction . By restricting acetyl phosphate formation to the phosphotransacetylase reaction alone, through the use of metabolic inhibitors, we were able to show that, with pyruvate as a source of energy, mutants defective in phosphotransacetylase are unable to transport glutamine, histidine, and methionine . However, with the same energy source, mutants defective in acetate kinase are normal in the transport of these amino acids . The inability of the phosphotransacetylase mutants to transport is due to their presumed inability to form acetyl phosphate, because pyruvate is found to be metabolized to acetyl-CoA in these mutants . Thus acetyl phosphate has been implicated in active transport . Evidence is also presented that neither the protonmotive force nor the ecf gene product is required for the shock-sensitive transport systems. Proc Natl Acad Sci U S A, 1979 Mar, 76(3), 1122 - 5 Mutations reducing the activity of c17, a promoter of phage lambda formed by a tandem duplication; Mozola MA et al.; We report the isolation of four independently selected mutations (scs) in the c17 promoter of phage lambda that reduce or eliminate the promoter activity . The c17 promoter is not normally present in lambda, and has been shown to be generated by a tandem duplication which creates a "Pribnow Box," a heptamer sequence implicated in promoter activity . This sequence is located upstream from the site of transcription initiation and is present, with some variation, in all promoters whose sequences have been determined . Analysis of the c17 duplications carrying the scs mutations reveals that three of these mutants carry single base-pair changes in the most highly conserved base pairs of the Pribnow Box and that the other mutation is a reversion to the wild type sequence in this region (i.e., a loss of the duplicated base pairs). Proc Natl Acad Sci U S A, 1979 Mar, 76(3), 1089 - 93 Enzymatic insertion of purine bases into depurinated DNA in vitro; Livneh Z et al.; An enzymatic activity that inserts purines into depurinated DNA was found in a soluble enzyme extract of Escherichia coli . This activity brings about the insertion of adenine and guanine into the appropriate apurinic sites in double-stranded DNA by using the corresponding deoxyribonucleoside triphosphates as the purine donors . Magnesium ions are required for this activity, it is inhibited by caffeine, and it does not act on depurinated single-stranded DNA . The insertion activity described here may represent a step in a repair mechanism, "base-insertion repair," whereby apurinic sites (which may occur in double-stranded DNA either due to the removal of damaged purines with specific glycosylases or by spontaneous depurination) are directly filled with the correct missing purine base. Proc Natl Acad Sci U S A, 1979 Mar, 76(3), 1084 - 8 Biochemical assay designed to detect formation of recombination intermediates in vitro; Potter H et al.; A biochemical assay that is designed to detect recombination intermediates formed in vitro is described . The assay measures the fusion of two essentially homologous plasmids, one of which is radioactively labeled and the other of which carries several copies of the lac operator . The fusion product is radioactive and can be bound to a nitrocellulose filter by lac repressor . This assay for genome fusion is rapid and readily applicable to the many fractions that result during enzyme purification . The fused product is not destroyed in the assay and may be recovered from the filter for further analysis by electron microscopy . The product is then seen to consist of figure 8 structures that can be cleaved by the restriction enzyme EcoRI to give chi forms, structures similar to those recovered from recombination-proficient cells . It is expected that this assay will be useful in the purification of the "recombinase-type" activity detected in crude cell lysates . To demonstrate this point, the assay was applied to the protein fractions recovered from a molecular sieve column . The results indicate that the fusion activity has an apparent molecular weight of 50,000--100,000. Proc Natl Acad Sci U S A, 1979 Mar, 76(3), 1054 - 8 Localization of the decoding region on the 30S Escherichia coli ribosomal subunit by affinity immunoelectron microscopy; Keren-Zur M et al.; The decoding region of the Escherichia coli ribosome has been localized by affinity immunoelectron microscopy . Valyl-tRNA1Val, derivatized at the alpha-amino end with the dinitrophenyl group, was bound to the ribosomal P site of 70S ribosomes and crosslinked specifically to 16S RNA by 310- to 325-nm irradiation . Previous work has shown that crosslink occurs between the 5' anticodon base of the tRNA and a pyrimidine in the 3' one-third of the 16S RNA . By reaction of the dinitrophenyl group with antibody, dimers of the 30S tRNA covalent complexes were prepared containing one covalently attached tRNA per 30S subunit . Electron microscopic visualization of the antibody attached to the dinitrophenyl group located the position of the 3' end of the tRNA . Several sites, with a strong preference for the large protrusion or cleft region in the upper one-third of the subunit, were found . The multiplicity of sites is likely due to the freedom of orientation of the 3' end of the tRNA which is approximately 80 A from the site of crosslinking . By extrapolating this distance from each of the antigenic sites, we concluded that the anticodon of tRNA when in the P site is probably located in the cleft region of the 30S subunit. Proc Natl Acad Sci U S A, 1979 Mar, 76(3), 1040 - 4 Interaction of Escherichia coli ribosomal protein S1 with ribosomes; Draper DE et al.; The binding affinity of Escherichia coli ribosomal protein S1 for 30S ribosomal particles has been determined by a sucrose gradient band sedimentation technique; the association constant (K) for the binding of one S1 protein per active 30S ribosomal subunit is approximately 2 X 10(8) M-1 . The involvement of the two polynucleotide binding sites of S1 protein (site I binding single-stranded DNA or RNA, and site II binding single-stranded RNA only) in the S1--ribosomal interaction have been examined by competition experiments with polynucleotides of known affinity for the two sites . We find that site I does not contribute to the interaction; site II binding appears to provide a major part of the binding free energy, presumably by interaction of S1 with the 16S rRNA of the 30S particle . The remaining binding free energy is probably derived from the interaction of S1 protein with other proteins of the 30S subunit . The affinity of S1 for 70S ribosomes is about the same as that for the 30S subunit; the affinity of S1 for 50S subunits is much less . Binding affinities and stoichiometries of S1 protein with "inactive" 30S ribosomal subunits have also been examined. Proc Natl Acad Sci U S A, 1979 Mar, 76(3), 1035 - 9 High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules; Struhl K et al.; A set of vector DNAs (Y vectors) useful for the cloning of DNA fragments in Saccharomyces cerevisiae (yeast) and in Escherichia coli are characterized . With these vectors, three modes of yeast transformation are defined . (i) Vectors containing yeast chromosomal DNA sequences (YIp1, YIp5) transform yeast cells at low frequency (1--10 colonies per microgram) and integrate into the genome by homologous recombination; this recombination is reversible . (ii) Hybrids containing endogenous yeast plasmid DNA sequences (YEp2, YEp6) transform yeast cells at much higher frequency (5000--20,000 colonies per microgram) . Such molecules replicate autonomously with an average copy number of 5--10 covalently closed circles per yeast cell and also replicate as a chromosomally integrated structure . This DNA may be physically isolated in intact form from either yeast or E . coli and used to transform either organism at high frequency . (iii) Vectors containing a 1.4-kilobase yeast DNA fragment that includes the centromere linked trp1 gene (YRp7) transform yeast with an efficiency of 500--5000 colonies per microgram; such molecules behave as minichromosomes because they replicate autonomously but do not integrate into the genome . The uses of Y vectors for the following genetic manipulations in yeast are discussed: isolation of genes; construction of haploid strains that are merodiploid for a particular DNA sequence; and directed alterations of the yeast genome . General methods for the selection and the analysis of these events are presented. Nucleic Acids Res, 1979 Mar, 6(3), 915 - 30 Construction and analysis of recombinant DNAs containing a structural gene for rat prolactin; Gubbins EJ et al.; Poly A-containing RNA enriched in prolactin-coding sequences was isolated from female rate pituitaries after induction with diethylstilbesterol . Double stranded cDNA was synthesized from this RNA and inserted into plasmid pBR322 at the Pst I site via the poly(dG):polyy(dC) tailing method . E . coli was transformed with this DNA and the recombinant plasmid in one of the transformants characterized in detail . About half of its 900 base pair cDNA insert was sequenced . The DNA sequence is consistent with most of the reported amino acid sequence of rat preprolactin . In addition, the recombinant plasmids in two of the other transformants appear to contain growth hormone coding sequences. Nucleic Acids Res, 1979 Mar, 6(3), 899 - 914 Chemical modification study of aminoacyl-tRNA conformation; Negishi K et al.; Chemical reactivity of cytosines in 32P-labeled E . coli tRNA1Leu, E . coli tRNAPhe and yeast tRNAPhe before and after aminoacylation was examined by use of a cytosine-specific reagent, semicarbazide-bisulfite mixture . In all the three tRNA species examined, the cytosine residues that were susceptible to the modification were the same in the aminoacylated tRNA and the unacylated tRNA . Only a limited number of the cytosine residues were modifiable: those that occur in the anticodon, the 3'-CCA terminus, the D-loop, and the extra loop . The sites accessible by the reagent are in good agreement with the general three-dimensional structure of tRNA proposed in literature . These results indicate that the gross conformation of these tRNAs does not change on aminoacylation, and consequently favor the view that the T psi C(G) sequence could become exposed in later steps of protein synthesis in order to achieve the binding of aminoacyl tRNA to ribosomes. Nucleic Acids Res, 1979 Mar, 6(3), 849 - 65 Cloning, mapping and expression of the genetic determinant that encodes for the K88ab antigen; Mooi FR et al.; The K88 antigen, a plasmid-specified virulence factor of E . coli involved in porcine neonatal diarrhoea, is often found to be associated with the ability to metabolize raffinose (Raf) . Plasmid pRI8801 (51 megadalton) was used to clone the determinants of K88 and Raf with the vector pBR322 . K88 was found to be encoded by a 7.7 megadalton HindIII fragment . The expression was highly dependent on the orientation of the HindIII fragment within pBR322 . By in vitro generation of deletions, the HindIII fragment was reduced in size to 4.3 megadalton . The expression of K88 by pRI8801 and the recombinant plasmids was studied using an enzyme-linked immunosorbent assay . Raf was found to be located on a 4.0 megadalton SalI fragment . A physical map of pRI8801 was constructed . The K88 antigen and Raf genes are not closely linked but separated by a stretch of DNA of about 20 megadalton. Nucleic Acids Res, 1979 Mar, 6(3), 1177 - 87 Photosensitized formation of thymine dimers in DNA by tyramine, tyrosine and tyrosine-containing peptides; Kaneko M et al.; The formation of Thy-Thy in DNA in the presence of tyramine, tyrosine and tyrosine-containing peptides such as Lys-Tyr and Lys-Tyr-Lys was studied with monochromatic UV irradiation . The formation of Thy-Thy by UV irradiation was enhanced in the presence of these compounds . The action spectrum of the photosensitization has a peak near 280 nm corresponding to the absorption spectrum of tyrosine . The triplet quencher reduced the sensitization substantially . The sensitization in native DNA was more than six times larger than that in denatured DNA . increasing the concentration of salts suppressed the sensitization . The nature of the interaction between DNA and the sensitizer is discussed. Nucleic Acids Res, 1979 Mar, 6(3), 1123 - 33 In vitro methylation of yeast tRNAAsp by rat brain cortical tRNA-(adenine-1) methyltransferase; Salas CE et al.; Rat brain cortices from young animals contain large amounts of tRNA (adenine-1)methyltransferase(s) . The enzyme(s) can methylate E . coli tRNA and to a lower degree yeast tRNA . Among yeast tRNA species which can be methylated we have selected tRNAAsp as a substrate for the brain enzyme . The digestions of in vitro methylated {Me-3H}-tRNAAsp with pancreatic and/or T1 ribonucleases followed by chromatographies on DEAE-cellulose, 7 M urea, suggested that the methylation of tRNAAsp occurred at a single position within the D-loop . Further digestion of the radioactive oligonucleotide recovered after DEAE-cellulose chromatography by phosphomonoesterase and snake venom phosphodiesterase enzymes followed by bidimensional thin layer chromatography enabled us to determine the location of the adenine residue which becomes methylated by the brain enzyme . This one resulted to be the adenine 14 in the D-loop of yeast tRNAAsp. Nucleic Acids Res, 1979 Mar, 6(3), 1111 - 22 Nucleotide sequence of the transposable DNA-element IS2; Ghosal D et al.; The complete sequence of the transposable DNA element IS2 in gal OP-308:: IS2 (I) has been determined . This element is 1.327 bp long . The integrated element is flanked by a five base pair long sequence duplication . The termini of IS2 are not perfect inverted repeats, but a close approximation. Mol Biol (Mosk), 1979 Mar-Apr, 13(2), 388 - 401 {Modification of the RNA-polymerase of Escherichia coli by diethylpyrocarbonate}; Rozovskaia TA et al.; E . coli DNA dependent RNA polymerase was modified by diethylpyrocarbonate . Optical and kinetic properties of the reaction were studied . More than 90% of RNA polymerase activity is inhibited by introduction of 9--11 ethoxyformyl groups per enzyme molecule without loss of its ability to bind DNA template . Furthermore the modified enzyme is able to form tight complexes with DNA and to compete with native enzyme for the formation of rifampicin-resistant complex . The ratio of the complex formation constants for the native and modified enzyme was determined to be equal to 10 . The enzyme modified to such extent loses the activity in DNA dependent RNA as well as pppApU synthesis . Vmax value rather than Km value for both ATP and UTP decreases following the modification reaction . Incubation of the enzyme modified to the 10% of residual activity with 0.2 M hydroxylamine for 2 hours results in restoration of RNA polymerase activity . Most but not all of the modified histidyl residues restore their native structure . Two of 13 histidyl residues were modified irreversibly due to Bamberger's cleavage reaction, but these two residues were found to be unessential for RNA polymerase activity . Reaction with higher concentration of the diethylpyrocarbonate induces modification of more than 15--20 histidyl residues and leads to irreversible inactivation of the enzyme . Nevertheless the modification of the additional histidyl redidues was reversible as well as the modification of the first 11 residues . RNA polymerase modified to such extent loses the ability to bind DNA . Preformation of the initiated ternary complex of RNA polymerase with template and product fails to protect the enzyme from reversible inactivation at a low reagent concentration, but markedly decreases the rate of the irreversible and unspecific modification of sulfhydryl or amino groups of the enzyme . Reaction with the ternary complex results in reversible inactivation of the enzyme with respect to elongation of RNA chains as well as the pyrophosphate exchange reaction . The complex itself was, however, completely stable under the reaction conditions and the enzyme subunit structure was also conserved after the reaction . Evidently, the mild modification of the histidyl residues with diethylpyrocarbonate selectively inhibits RNA chain elongation. Mol Biol (Mosk), 1979 Mar-Apr, 13(2), 281 - 91 {Isolation and properties of DNA-cytosine methyltransferase from Escherichia coli C}; Bogdarina IG et al.; The method of isolation and partial purification of DNA-cytosine-methyltransferase (DC-methylase) from E . coli C is described . The enzyme underwent approximately 100-fold purification . The obtained preparation of DC-methylase can be additionally considerably purified by sedimentation in sucrose gradient . Native molecular weight of DC-methylase from E . coli C . is 70,000 . The activity of enzyme does not depend on the Mg2+ ions . DC-methylase E . coli C provides DNA of lambda phage in vitro with full resistance against restriction endonuclease EcoRII . In DNA methylated by DC-methylase the modified cytosine, mainly in C-MC and C-MC-T sequences, corresponds to the pyrimidine sequences of specific site EcoRII . DNA of lambda.B phage contains approximately 80 sites for modification by DC-methylase E . coli C . The results obtained point to the same specificity in vitro of DNA-cytosine-methylase E . coli C and DNA-methylase EcoRII. Mikrobiologiia, 1979 Mar-Apr, 48(2), 307 - 10 {Morphological changes in the cells of Escherichia coli damaged by nitrous yperite}; Lobanok EV et al.; Nitrous yperite supressed division of E . coli cells; as a result, filaments and giant abnormal cells appeared . Apparently, giant forms were caused by defects in the cell wall induced by nitrous ypertie due to a mutation in the mon gene of the strain . These cells gave rise to normal cells as well as to small bacteria resembling mini-cell. J Bacteriol, 1979 Mar, 137(3), 1439 - 42 Enhancement of deoxyribonucleic acid polymerase I-directed repair synthesis in toluene-treated Escherichia coli after growth in the presence of low levels of N-methyl-N'-nitro-N-nitrosoguanidine; Billen D et al.; Deoxyribonucleic acid polymerase I-directed repair synthesis can be selectively measured in toluene-treated Escherichia coli cells exposed to alkylating chemicals such as N-methyl-N'-nitro-N-nitrosoguanidine . Prior growth of the cells in the presence of a low dose of N-methyl-N'-nitro-N-nitrosoguanidine results in an enhanced deoxyribonucleic acid polymerase I-directed repair synthesis and an increase in single-strand breaks. J Bacteriol, 1979 Mar, 137(3), 1421 - 4 Enumeration and identification of IS3 elements in Escherichia coli strains; Deonier RC et al.; Escherichia coli K-12 strains ordinarily contain five IS3 elements . Three of these correspond to previously mapped IS3 elements (R . C . Deonier, G . R . Oh, and M . Hu, J . Bacteriol . 129:1129--1140, 1977; S . Hu, E . Ohtsubo, and N . Davidson, J . Bacteriol . 122:749--763, 1975), and two additional IS3 elements are identified . The distribution of IS3 elements among deoxyribonucleic acid fragments generated by digestion with EcoRI indicates a basic pattern from which deviation is detected. J Bacteriol, 1979 Mar, 137(3), 1243 - 52 Enzymatic degradation of uracil-containing deoxyribonucleic acid . V . Survival of Escherichia coli and coliphages treated with sodium bisulfite; Simmons RR et al.; A number of mutants of Escherichia coli defective in the ung gene (structural gene for uracil-deoxyribonucleic acid {ura-DNA} glycosylase) are shown to be abnormally sensitive to treatment with sodium bisulfite when compared with congenic ung+ strains . These results provide further evidence that sodium bisulfite causes the deamination of cytosine to uracil in DNA and that ura-DNA glycosylase is required for the repair of U-G mispairs . The effect of the chemical is apparently selective with respect to base damage; coliphages containing cytosine in their DNA are inactivated by treatment with sodium bisulfite, whereas those containing hydroxymethylcytosine are not . ura-DNA glycosylase and the major apurinic-apyrimidinic endonuclease of E . coli may function in the same repair pathway, since the extent of inactivation of a congenic set of strains which are ung xth (structural gene for the major apurinic-apyrimidinic endonuclease of E . coli) or ung xth+ is the same. J Bacteriol, 1979 Mar, 137(3), 1151 - 7 Chromosome replication and cell division in plasmid-containing Escherichia coli B/r; Weinberger M et al.; The kinetics of chromosome replication and cell division have been examined in recA mutants of Escherichia coli B/r containing F' plasmids of various sizes . Plasmid-mediated alterations in growth properties were detected only with the presence of the larger F' plasmids, and were reflected in decreased mean cell sizes and growth rates . The lengths of C and D in all plasmid-containing strains were in accord with the values for plasmid-free parental strains growing with similar generations times . The findings were consistent with an absence of competition between the chromosomal and extrachromosomal replicons for rate-limiting components involved in the initiation of deoxyribonucleic acid synthesis or in the elongation of deoxyribonucleic acid chains. J Bacteriol, 1979 Mar, 137(3), 1111 - 8 Isolation and properties of Escherichia coli K-12 mutants impaired in the utilization of gamma-aminobutyrate; Metzer E et al.; We have isolated mutants of Escherichia coli K-12 CS101B that have lost the ability to utilize gamma-aminobutyrate as a source of nitrogen . One class of mutants, which were not affected in the utilization of other nitrogen sources (proline, arginine, glycine), included many isolates with lesions in gamma-aminobutyrate transport or in its transamination and one mutant completely devoid of succinic semialdehyde dehydrogenase activity and exhibiting low gamma-aminobutyrate transport and transamination . gamma-Aminobutyrate-utilizing revertants of the latter recovered full transport and transamination capacities but remained dehydrogenaseless . Another class of mutants showed pleiotropic defects in nitrogen metabolism . One such mutant was lacking glutamate synthase activity . The genes specifying the synthesis of gamma-aminobutyrate permease, gabP, gamma-aminobutyrate transaminase, gabT, and succinic semialdehyde dehydrogenase, gabD, and the control gene, gabC, that coordinately regulates their expression all form a cluster on the E . coli chromosome, linked to the srl and recA loci (at 57.5 min) . The mutations with pleiotropic effects on the metabolism of nitrogenous compounds are not linked to the gab cluster. J Bacteriol, 1979 Mar, 137(3), 1088 - 94 Identification of the ftsA gene product; Lutkenhaus JF et al.; A nonsense mutation was identified in the essential cell division gene ftsA of Escherichia coli . A gamma-transducing phage was isolated which complemented this mutation . This phage programmed the synthesis of four bacterial proteins in UV-irradiated cells . By substituting the nonsense mutation for the ftsA+ allele in this transducing phage and comparing the proteins programmed by it in UV-treated Su+ and Su- cells, the product of the ftsA gene was identified as a protein with a molecular weight of 50,000. J Bacteriol, 1979 Mar, 137(3), 1084 - 7 Occurrence of 1-methyladenosine and absence of ribothymidine in transfer ribonucleic acid of Mycobacterium smegmatis; Vani BR et al.; The minor base composition of Mycobacterium smegmatis tRNA has been studied . Thin-layer chromatographic patterns of a ribonuclease T2 digest of mycobacterial tRNA indicated the presence of appreciable amounts of 1-methyladenosine (which is commonly present only in eucaryotic tRNA), dihydrouridine, and 7-methylguanosine . Ribothymidine was absent . The S-adenosylmethionine-dependent tRNA methylases of M . smegmatis catalyzed the formation of 1-methyladenosine when Escherichia coli tRNA was used as acceptor . Similarly, E . coli extracts methylated the tRNA of M . smegmatis, forming ribothymidine. Mutat Res, 1979 Mar, 60(1), 13 - 23 Mutation induction by thymidine deprivation in Escherichia coli B/r . I . Influence of caffeine; Mennigmann HD et al.; The addition of caffeine to the plating medium after thymine deprivation of E . coli WP2 uvr+ thyA or WP2 uvrA thyA had no influence on survival . Caffeine, however, reduced the frequency of mutants . The hypothesis is presented that the reduced mutagenesis is due to the sensitivity to caffeine of an inducible error-prone repair mechanism operating during thymine deprivation and after the re-addition of thymine. J Exp Med, 1979 Mar 1, 149(3), 669 - 85 Form variation in Escherichia coli K1: determined by O-acetylation of the capsular polysaccharide; Orskov F et al.; The chemical basis for the alternating antigenic change called form variation noted for the Escherichia coli K1-capsular polysaccharide has been shown by 13C nuclear magnetic resonance to be a result of random O-acetylation of C7 and C9 carbons of the alpha-2-8-linked sialic acid homopolymer . A serologic method (antiserum agar) was developed to identify and isolate the form variants . The O-acetyl positive and O-acetyl negative K1 polysaccharides had unique biochemical and immunologic properties . The O-acetyl-positive variants resisted neuraminidase hydrolysis in contrast to the susceptibility of the O-acetyl negative variant to this enzyme . In addition, O-acetylation altered the antigenicity of the O-acetyl polysaccharides . When injected as whole organisms, O-acetyl positive organisms produced anti-K1 -antibodies in rabbits specific for this polysaccharide variant . O-acetyl negative organisms were comparatively less immunogenic; however, antibodies induced by these organisms reacted with both K1 polysaccharide variants . Burros, injected with either variant, produced antibodies reactive with both K1 polysaccharides. Eur J Biochem, 1979 Mar, 94(2), 477 - 84 Translational fidelity and specificity of ribosomes cleaved by cloacin DF13; Twilt JC et al.; The effect of cloacin DF13 cleavage on several functional properties of the ribosome has been studied in a translational system in vitro . Cleaved ribosomes synthesize relatively shorter polypeptide chains on synthetic and natural templates . Their translational specificity is, however, unchanged as judged by the read-out of MS2 RNA . Here, cleaved as well as control ribosomes start translation only on the coat cistron of the phage RNA . Cloacin cleavage of ribosomes increases their fidelity of translation . Differential inhibition of translation of synthetic and natural template was not observed. Surg Gynecol Obstet, 1979 Mar, 148(3), 361 - 6 Methylprednisolone in the prevention of cerebral hemodynamic and metabolic disorders during endotoxin shock in the dog; Emerson TE Jr et al.; These results provide evidence that steroid pretreatment and subsequent post-treatment prevent cerebral hemodynamic and metabolic alterations during four hours of Escherichia coli endotoxin shock in the dog . However, in this study, no data are provided on how the steroid prevents an increase in cerebral vascular resistance, and no clear answer is available in the literature . While active vasodilation or alpha-adrenergic blocking properties, or both, have been attributed to glucocorticoids, recent evidence does not support these findings during normal conditions or circulatory shock . If the increase in cerebral vascular resistance is passive, steroids may help by preventing platelet aggregation, cell disruption and subsequent microvascular plugging . Intravenously administered fluids, dextran-saline solution, while in themselves are probably not important to survival, may augment cerebral blood flow during shock through a blood dilutional effect . Finally, it is possible that steroids act to permit normal, long term cerebral auto-regulation, which is apparently impaired during endotoxin shock in the dog. Proc Natl Acad Sci U S A, 1979 Mar, 76(3), 1194 - 8 Subcellular localization of acyl carrier protein in leaf protoplasts of Spinacia oleracea; Ohlrogge JB et al.; This communication demonstrates that all de novo fatty acid biosynthesis in spinach leaf cells requires acyl carrier protein (ACP) and occurs specifically in the chloroplasts . Antibodies raised to purified spinach ACP inhibited at least 98% of malonyl CoA-dependent fatty acid synthesis by spinach leaf homogenates . Therefore, the presence of ACP in a compartment of the spinach leaf cell would serve as a marker for de novo fatty acid biosynthesis . A radioimmunoassay capable of detecting 10(15) mol (10(-11) g) of spinach ACP was developed to measure the levels of ACP in leaf cell components isolated by sucrose gradient centrifugation of a gentle lysate of spinach leaf protoplasts . All of the ACP of the leaf cell could be attributed to the chloroplast . Less than 1% of the ACP associated with chloroplasts resulted from binding of free ACP to chloroplasts . Of interest, ACP from Escherichia coli, soybean, and sunflower showed only partial crossreactivity with spinach ACP by the radioimmunoassay . These results strongly suggest that, in the leaf cell, chloroplasts are the sole site for the de novo synthesis of C16 and C18 fatty acids . These fatty acids are then transported into the cytoplasm for further modification and are either inserted into extrachloroplastic membrane lipids or returned to the chloroplast for insertion into lamellar membrane lipids. Rev Infect Dis, 1979 Mar-Apr, 1(2), 357 - 69 Superoxide, hydrogen peroxide, and oxygen tolerance of oxygen-sensitive mutants of Escherichia coli; Hassan HM et al.; Oxygen-intolerant mutants of Escherichia coli K12 were selected by a replica plating technique after treatment with the mutagen, N-methyl-N'-nitro-N-nitrosoguanidine, to a lethality of 99.5% . One group of mutants had lost the ability to induce both peroxidase and catalase when exposed to oxygen but retained the ability to induce the manganese-superoxide dismutase . The second group of mutants had lost the ability to induce the activity of all these enzymes . Failure to induce peroxidase and catalase was associated with enhanced susceptibility of the bacteria to the lethal effect of oxygen . When a member of the first group of mutants was prevented from producing the manganese-superoxide dismutase by the presence of puromycin, its susceptibility to the lethal effects of oxygen was greatly increased . Two types of revertants were seen . In one group the ability to induce enzyme activity was recovered and was accompanied by the return of oxygen tolerance . Members of the other group lost the ability to respire and, therefore, no longer produced O2- AND H2O2 . These results indicated that enzymic scavenging of both H2O2 and O2- provides an important defense against oxygen toxicity . The parallel loss of peroxidase and catalase, which was seen in all mutants, suggests that these enzymes constitute a precursor-product pair in E . coli . The parallel loss in two of these mutants of peroxidase, catalase, and the manganese-superoxide dismutase suggests a control linkage for these enzymes, the basis of which remains to be explored. Eur J Biochem, 1979 Mar, 94(2), 445 - 50 Replication of M13 duplex DNA in soluble extracts of Escherichia coli . Effect of helix-destabilising proteins; van Dorp B et al.; Soluble extracts of M13-am5-infected Escherichia coli cells can carry out multiple rounds of M13 duplex DNA replication when supplemented with helix-destabilising protein of E . coli . Similarly addition of the helix-destabilising M13 gene 5 protein in low concentrations (up to 30 micrograms/ml) stimulates the replication of double-stranded M13 DNA . In contrast, higher concentrations of gene 5 protein (but not of E . coli helix-destabilising protein) cause a preferential inhibition of complementary strand synthesis resulting in a switch from double-strand replication to single-strand synthesis . Depending on the addition of the appropriate amounts of these two helix-destabilising proteins either stage of M13 DNA replication can now be studied with cell-free preparations. Z Naturforsch {C}, 1979 Mar-Apr, 34(3-4), 248 - 52 On the dynamic model of tRNA: recent experimental findings; Gurel O; Recent experimental findings are shown to support various aspects of the dynamic model of tRNA proposed earlier and the interaction between tRNA, mRNA and the ribosomal RNA's . Extra loop of tRNA molecule is suggested to play a role in recognizing the corresponding amino acids and a correlation is presented between the tRNA molecules and the corresponding amino acids as tabulated by the genetic tableau. Proc Natl Acad Sci U S A, 1979 Mar, 76(3), 1126 - 30 Specialized transducing phage lambda carrying the genes for coupling factor of oxidative phosphorylation of Escherichia coli: increased synthesis of coupling factor on induction of prophage lambda asn; Kanazawa H et al.; Studies were made of the synthesis of the coupling factor complex (F1--F0) of oxidative phosphorylation after prophage induction of a set of Escherichia coli strains lysogenic for defective transducing phage lambda asn, lambda uncA, or lambda bglC . The transducing phages had been isolated from a strain of E . coli carrying prophage lambda cI857 S7 within the bglB gene located near the unc gene cluster {Miki, T., Hiraga, S., Nagata, T . & Yura, T . (1978) Proc . Natl . Acad . Sci . USA 75, 5099--5103} . When lysogenic cells carrying lambda asn and lambda cI857 S7 were induced at high temperature, synthesis of the F1-ATPase portion of the complex increased to severalfold that of the noninduced cells . In contrast, no increase was observed upon thermoinduction of cells carrying lambda uncA or lambda bglC . The number of membrane sites that could bind purified F1-ATPase also increased significantly upon induction by lambda asn but not by lambda uncA or lambda bglC . In addition, F1-depleted membranes prepared from lambda asn-induced bacteria required more dicyclohexylcarbodiimide to seal the proton pathway than did those from noninduced bacteria . These results strongly suggest that lambda asn carries a set of bacterial genes coding for all the F1 polypeptides (the alpha, beta, gamma, delta, and probably the epsilon subunits) and at least some of the genes involved in formation of F0 polypeptides . Although lambda uncA carries the structural gene (uncA) for the alpha subunit of F1-ATPase, it apparently does not carry the whole set of F1--F0 genes. J Bacteriol, 1979 Mar, 137(3), 1219 - 26 Arrangement of the genes coding for ribosomal ribonucleic acids in Neurospora crassa; Free SJ et al.; We have cloned and characterized Neurospora crassa ribosomal deoxyribonucleic acid (rDNA) . The rDNA is found as a tandemly repeated 6.0-megadalton sequence . We have mapped a portion of the rDNA repeat unit with respect to its sites for 13 restriction endonucleases and defined those regions coding for the 5 . 8S, 17S, and 26S ribosomal ribonucleic acids (rRNA's) . We have also isolated several clones containing 5S rRNA sequences . The 5S rRNA coding sequences are not found within the rDNA repeat unit . We found that the sequences surrounding the 5S rRNA coding regions are highly heterogeneous. Infect Immun, 1979 Mar, 23(3), 700 - 5 Behavior of Escherichia coli K antigens K88ab, K88ac, and K88ad in immunoelectrophoresis, double diffusion, and hemagglutination; Guinee PA et al.; Porcine enteropathogenic Escherichia coli strains were found to possess a variant of the K88 antigen provisionally termed K88ad . We propose to include this antigen into the international E . coli typing scheme . Ultrasonic extracts of field strains of E . coli possessing the K88ab, K88ac, or K88ad antigen and their E . coli K-12 K88+ transconjugants showed a specific K88 precipitation line in immunoelectrophoresis and double diffusion only when grown at 37 degrees C, but not when grown at 18 degrees C . By using agarose gels, K88ab, K88ac, and K88ad antigens showed anodic mobility in immunoelectrophoresis . When using Difco Noble agar gels, K88ad was not mobile or anodic, K88ab was cathodic; K88ac of 17 strains was cathodic and of 24 strains was anodic . The immunoelectrophoretic behavior of a K88 antigen (K88ab, K88ac, or K88ad) did not alter after transfer of the corresponding plasmid to E . coli K-12 . Anodic and cathodic K88ac antigens could not be distinguished serologically . The differences between the results obtained in Noble agar gels and agarose gels are due to electro-endosmotic flow . We describe a procedure which increases the detection level of K88+ transconjugants in a mating mixture . It is based on the specific mannose-resistant attachment of K88+ cells to guinea pig erythrocytes. Can J Microbiol, 1979 Mar, 25(3), 335 - 9 Response of various cell lines to Escherichia coli toxic products; Konowalchuk J et al.; Seven cell lines were compared with Y-1 and Vero cells for morphological response to E . coli toxic products . FL and WI-38, like Y-1 and Vero, responded to heat-labile enterotoxin . FL, HEp-2, HeLa, and BGM, like Vero but not Y-1, responded to heat-labile cytotoxin . FL was comparable to Vero in sensitivity to these two toxic products. Proc Natl Acad Sci U S A, 1979 Mar, 76(3), 1099 - 103 Escherichia coli adenylate cyclase complex: regulation by the proton electrochemical gradient; Peterkofsky A et al.; Sugars such as glucose are transported into Escherichia coli by a coupled phosphorylation mechanism (the phosphoenolpyruvate:sugar phosphotransferase system, PTS) . Transport of sugars through the PTS results in inhibition of adenylate cyclase {ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1} activity by a mechanism involving a change in the state of phosphorylation of PTS proteins . Other sugars (e.g., lactose) are transported without modification by a mechanism involving proton cotransport, which requires a proton motive force across the cell membrane . We show here that uptake of sugars through the lactose transport system results in inhibition of adenylate cyclase activity if the proton symport mechanism is also active . The protonophore carbonyl cyanide m-chlorophenylhydrazone also inhibits adenylate cyclase activity . These data suggest that the steady-state electrochemical proton gradient regulates the activity of adenylate cyclase . We propose that sugar-dependent inhibition of adenylate cyclase activity may occur by either of two mechanisms . Sugars transported by the PTS inhibited adenylate cyclase activity by dephosphorylation of a regulatory protein, while sugars transported by the proton motive force system inhibit adenylate cyclase activity as a result of collapse of the proton electrochemical gradient. Biol Bull Acad Sci USSR, 1979 Mar-Apr, 6(2), 172 - 8 Effect of beta-indoleacetic acid on RNA and protein synthesis in Escherichia coli; Drobysheva NA et al.; The effect of different concentrations of beta-indoleacetic acid (IAA) on RNA and protein synthesis and the stability of associations of E . coli ribosomes at 3 mM Mg2+ was investigated . IAA in a concentration of 100 microgram/ml had a slight stimulatory effect on the incorporation of {14C}leucine into proteins and stimulated the incorporation of {14C}uracil into the RNA of intact cells more noticeably . In the presence of 2500 microgram/ml of the auxin RNA synthesis was inhibited by 65% and protein synthesis by 40-50% as compared to the control . In a cell-free system containing poly(U) IAA in concentrations of 10-100 microgram/ml increased polyphenylalanine synthesis by an average of 30%, while at high concentrations IAA noticeably inhibited its synthesis . It was found that the proportion of stable ribosomes in lysates obtained from cells incubated with IAA in concentrations of 250 and 1000 microgram/ml decreased to 18 and 3% . It is suggested that the inhibition of protein synthesis in E . coli by IAA is due to inhibition of the initiation of translation. Biochim Biophys Acta, 1979 Feb 27, 561(2), 396 - 402 Utilization in vitro of deoxyuridine triphosphate in DNA synthesis by DNA polymerases alpha and beta from calf thymus; Yoshida S et al.; DNA polymerases alpha and beta (EC 2.7.7.7.) from calf thymus could utilize dUTP as a substrate for DNA synthesis as well as DNA polymerase I of Escherichia coli . Deoxyuridylate was incorporated into DNA by replacing deoxythymidylate and supported the further elongation of DNA chains on activated DNA or on the intiated homopolymers, poly(dA) . (dT)10 and poly(rA) . (dT)10 . The rate of the incorporation of deoxyuridylate into DNA varied from 50 to 160% of that of deoxythymidylate, depending on the nature of the template primers and the species of DNA polymerase used . The apparent Km values for dUTP were very similar to those for dTTP . Uracil DNA-glycosylase excised efficiently the uracil residues in products of DNA polymerase reactions with either activated calf thymus DNA or initiated homopolymers. Biochim Biophys Acta, 1979 Feb 27, 561(2), 435 - 44 A purified nucleoprotein fragment of the 30 S ribosomal subunit of Escherichia coli; Spitnik-Elson P et al.; A '13 S' nucleoprotein fragment was isolated from a nuclease digest of Escherichia coli 30-S ribosomal subunits and purified to gel electrophoretic homogeneity . It contained two polynucleotides, of about 1.1 . 10(5) and 2.5 . 10(4) daltons, which separated when the fragment was deproteinized . The major protein components were S4, S7 and S9/11, with S15, S16, S18, S19 and S20 present in reduced amount. Biochim Biophys Acta, 1979 Feb 27, 561(2), 312 - 23 Genomic integrity of T1 DNA after gamma-and ultraviolet irradiation; Martin-Bertram H et al.; T1 DNA, gamma-irradiated in the phage particle or irradiated with ultraviolet light was checked for structural integrity by kinetics of melting and reannealing . gamma-Irradiated DNA differed in all thermokinetic properties by a factor of 3-4 from DNA degraded by mechanical or enzymatical treatments . Ultraviolet irradiation caused much smaller effects than gamma-irradiation . Considering the frequency of pyrimidine dimers in relation to the gamma-ray induced lesions, strong evidence can be derived, that in addition to single base damages, local denatured regions are produced by gamma-irradiation . Such regions, formed possibly by direct absorption of radiation energy in DNA, i.e . by primary ionizations, are associated with base lesions and are passed over during reannealing. Biochim Biophys Acta, 1979 Feb 27, 561(2), 294 - 300 Escherichia coli Hfr-DNA degradation in endonuclease I-deficient minicells; Khachatourians GG; {3H}Thymidine (dThd)-labelled Hfr DNA was transferred by conjugation into Escherichia coli F- minicells harvested from an endonuclease I-deficient (endI-) strain and its iosgenic wild type (endI+) parent . The susceptibility of this DNA to attack by DNAase was examined . The kinetics of in vivo conversion of {3H}dThd-labelled DNA into acid soluble radioactivity was examined . This activity, attributed to exonuclease action was the same for both strains . Contribution of endonuclease I was measured by an analysis of changes in weight-average (Mw) and number-average (Mn) molecular weight distribution of DNA molecules recovered from minicells . Reduction in Mw was greater in the endI-strain . The ratio Mn/Mw changed drastically during the incubation period of endI- minicells, but remained unchanged in the endI+ strain . These experiments suggest that the presence of the endI- mutation in minicell-producing strain chi1268 leads to a greater loss in M2 of Hfr DNA conjugally transferred into the minicells. Biochim Biophys Acta, 1979 Feb 26, 576(2), 263 - 8 Presence of the epsilon-(gamma-glutamic)lysine crosslink in cellular proteins; Matacic SS et al.; The epsilon-(gamma-glutamic)lysine bond is a covalent interaction which has been found to crosslink polypeptide chains of a number of extracellular proteins . Among known covalent bonds crosslinking protein chains, it is unique in that it is formed directly by enzymatic catalysis, a property which may also endow Glu-Lys crosslink formation with important intracellular functions . We found glutamic-lysine bonds to be present in the procaryote, Escherichia coli, in primitive eucaryotes such as the slime mold, Physarum polycephalum, and the ciliate, Paramecium aurelia, and in muscle cells of a bird and a mammal . Our data show that, although Glu-Lys bonds occur in low concentrations in cellular proteins, they are nevertheless widely distributed . Evidence is also presented indicating that the low levels of the Glu-Lys bonds we measure in the proteins of various cells types are not artifacts of our analytical procedures. Mol Gen Genet, 1979 Feb 26, 170(2), 187 - 9 Methylgroups of ribosomal protein L11 are not related to the synthesis of ppGpp; Rohl R et al.; Ribosomes carrying either a normally methylated or an undermethylated L11, respectively, were tested with respect to the stringency reaction in the presence of crude stringent factor . Systems with either kind of ribosomes synthesize ppGpp with the same efficiency . The ppGpp synthesis in presence of ribosomes with undermethylated L11 depends also on stringent factor, mRNA and deacylated tRNA whereas aminoacyl-tRNA and peptidyl-analogous tRNA show no effect . Thus, the absence of methylation in L11 does not influence the stringency reaction. J Biol Chem, 1979 Feb 25, 254(4), 1408 - 13 Recognition sequence of the dam methylase of Escherichia coli K12 and mode of cleavage of Dpn I endonuclease; Geier GE et al.; The recognition sequence for the dam methylase of Escherichia coli K12 has been determined directly by use of in vivo methylated ColE1 DNA or DNA methylated in vitro with purified enzyme . The methylase recognizes the symmetric tetranucleotide d(pG-A-T-C) and introduces two methyl groups per site in duplex DNA with the product of methylation being 6-methylaminopurine . This work has also demonstrated that Dpn I restriction endonuclease cleaves on the 3' side of the modified adenine within the methylated sequence to yield DNA fragments possessing fully base-paired termini . All sequences in ColE1 DNA methylated by the dam enzyme are subject to double strand cleavage by Dpn I endonuclease . Therefore, this restriction enzyme can be employed for mapping the location of sequences possessing the dam modification. J Biol Chem, 1979 Feb 25, 254(4), 1016 - 21 Biosynthesis in Escherichia coli of sn-glycerol 3-phosphate, a precursor of phospholipid . Palmitoyl-CoA inhibition of the biosynthetic sn-glycerol-3-phosphate dehydrogenase; Edgar JR et al.; Homogeneous biosynthetic sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) of Escherichia coli was potently inhibited by palmitoyl-CoA and other long chain acyl-CoA thioesters . The concentration dependence of this inhibition was not cooperative . Enzyme activity was inhibited 50% at 1 microM palmitoyl-CoA; thus, this inhibition occurred at concentrations below the critical micellar concentration of palmitoyl-CoA . Palmitoyl-CoA was a reversible, noncompetitive inhibitor with respect to both NADPH and dihydroxyacetone phosphate . Palmitoyl-CoA did not affect the quaternary structure of the enzyme . This inhibition could be prevented or reversed by the addition of phospholipid vesicles prepared from E . coli phospholipids . Palmitoyl-CoA did not alter the kinetics of inhibition by sn-glycerol 3-phosphate, which is a proven physiological regulator of this enzyme . Decanoyl-CoA, dodecanoyl-CoA, myristoyl-CoA, palmitoyl-(1,N6-etheno)CoA, stearoyl-CoA, and oleoyl-CoA inhibited sn-glycerol-3-phosphate dehydrogenase at concentrations below their critical micellar concentrations . Palmitate inhibited sn-glycerol-3-phosphate dehydrogenase activity 50% at 200 microM . Palmitoyl-carnitine, deoxycholate, taurocholate, and dodecyl sulfate were more potent inhibitors than Triton X-100, Tween-20, or Tween-80 . Palmitoyl-acyl carrier protein at concentrations up to 50 microM had no effect on sn-glycerol-3-phosphate dehydrogenase activity . The possible physiological role of long chain fatty acyl-CoA thioesters in the regulation of sn-glycerol 3-phosphate and phospholipid biosynthesis in E . coli is discussed. J Biol Chem, 1979 Feb 25, 254(4), 1013 - 5 Evidence for a direct role of tRNA in an amino acid transport system; Yoo SH et al.; The transport of phenylalanine by the general aromatic transport system in spheroplasts of Escherichia coli 9723 has been found to be stimulated by exogenous tRNA . Neither periodate-treated tRNA nor phenylalanine-charged tRNA stimulated, and the latter inhibited, phenylalanine uptake . Among preparations of specific tRNAs, tRNAPhe and tRNATyr were effective in stimulating the uptake of phenylalanine and tyrosine, respectively, and tRNAGlu and tRNAVal gave no detectable stimulation of phenylalanine or tyrosine transport . The preparation of tRNATyr was 10 times as active as unfractionated tRNA and gave as much as 167% stimulation of tyrosine transport . Correspondingly, the preparation of tRNAPhe was at least 3.5 times as active as the unfractionated tRNA and 2.5 times as active as the preparation of tRNATyr in stimulation of phenylalanine transport . Preliminary results in fractionation of the active component of tRNA for stimulating phenylalanine uptake show that the major activity resides in minor isoacceptor(s) tRNAPhe rather than the major component tRNAPhe, and the slight activity of preparations of tRNATyr is probably due to a contamination of the active tRNAPhe . Other preliminary results indicate that this type of stimulation occurs with uptake of other amino acids and their tRNA. Biochim Biophys Acta, 1979 Feb 20, 551(1), 157 - 68 Unmasking of an essential thiol during function of the membrane-bound enzyme II of the phosphenolpyruvate beta-glucoside phosphotransferase system of Escherichia coli; Haguenauer-Tsapis R et al.; beta-Glucoside transport by phosphoenolpyruvate-hexose phosphotransferase system in Escherichia coli is inactivated in vivo by thiol reagents . This inactivation is strongly enhanced by the presence of transported substrates . In a system reconstituted from soluble and membrane-bound components, only the particulate component, the membrane-bound enzyme IIbgl appeared as the target of N-ethylmaleimide inaction . The same feature was found in the case of methyl-alpha-D-glucoside uptake via enzyme IIglc . It is shown that the sensitizing effect of substrates is specific and not generalized, methyl-alpha-D-glucoside only sensitizes enzyme IIglc and p-nitrophenyl-beta-D-glucoside only sensitizes enzyme IIbgl towards N-ethylmaleimide inactivation . The inactivation of enzyme IIbgl by thiol reagents is also promoted in vivo by fluoride inhibition of phosphoenolpyruvate synthesis . In toluene-treated bacteria, the presence of phosphoenolpyruvate protects against inactivation by thiol reagents of p-nitrophenyl-beta-D-glucoside phosphorylation . Both results suggest that the inactivator resistent form of enzyme IIbgl is an energized form of the enzyme. Biochemistry, 1979 Feb 20, 18(4), 637 - 47 Photocross-linking analysis of the contact surface of tRNA Met in complexes with Escherichia coli methionine:tRNA ligase; Rosa JJ et al.; Photoinduced covalent cross-linking has been used to identify a common surface of four methionine-accepting tRNAs which interact specifically with the Escherichia coli methionine:tRNA ligase (EC 6.1.1.10) . tRNA--ligase mixtures were irradiated, and the covalently linked complexes were isolated and digested with T1 RNase (Schimmel & Budzik, 1977) . The fragments lost from the elution profile of the T1 RNase digest were considered to have been cross-linked to the protein and therefore in intimate contact with the enzyme . Only specific cognate tRNA--ligase pairs produce covalently linked complexes . The four substrate tRNAs used in this study have substantially different sequences, but all showed a common cross-linking pattern, supporting the view that the sites cross-linked to the enzyme reflect the functionally common contact surface rather than particularly photoreactivity regions of tRNA . The cross-linked contact surface is comprised of three regions: (1) the narrow groove of the anticodon stem and its extension into the anticodon loop; (2) the 3' terminal residues; and (3) the 3' side of the "T arm" . Unlike previous studies with other tRNAs, the D arm is not involved and significant radiation damage is suffered by the tRNA which must be taken into account in the analysis . The results are consistent with and complement chemical modification studies {Schulman, L . H., & Pelka, H . (1977) Biochemistry 16, 4256}. Biochim Biophys Acta, 1979 Feb 19, 583(1), 73 - 80 The regulatory effects of growth rate and cyclic AMP levels on carbon catabolism and respiration in Escherichia coli K-12; Wright LF et al.; Cyclic AMP levels in glucose and succinate-limited and ammonia-limited glucose-containing continuous cultures of Escherichia coli were measured at different bacterial growth rates . Intracellular cyclic AMP concentrations were fairly constant (about 5 micrometer) at all dilution rates used when glucose was limiting . In ammonia-limited glucose cultures the cyclic AMP content was much lower (about 0.3 micrometer) . In succinate-limited cultures cyclic AMP levels fell from 2.7 to 0.8 micrometer as dilution rate increased from 0.05 to 0.4 h-1 . The effects of cyclic AMP on respiratory and carbon catabolic enzyme levels were studied . There was no indication of a direct cyclic AMP involvement in the regulation of these cellular functions . It seems more likely that the variations in enzyme levels observed resulted from variation of the specific growth rate of cultures. Mol Gen Genet, 1979 Feb 16, 170(1), 49 - 56 In vitro system for the replication of the mini R1 factor Rsc11; Bezanson GS et al.; The in vitro synthesis of the mini R1-factor, Rsc11, was achieved using a soluble Escherichia coli cell-extract system . Triton X-100 lysates of the K12 strain 1101 (Rsc11) fractionated by Sephadex G25 chromatography supported the incorporation of labeled deoxyribonucleotides into covalently-closed circular (30S) and open-circular (25S) plasmid DNA as well as other molecules of various sizes . DNA synthesis required the presence of all four ribonucleotides and was rifampicin sensitive . Pulse-chase experiments indicated that this reaction is discontinuous . A dependence on ATP and sensitivity to nalidixic acid suggested this system capable of the replicative synthesis of Rsc11 DNA . Density-shift analysis confirmed this . In addition to hybrid, fully-heavy plasmid supercoils were synthesized indicating that more than one round of replication was completed . Approximately one-third of the molecules available to the system participate in this reaction. Mol Gen Genet, 1979 Feb 16, 170(1), 103 - 7 ColE1 plasmid mobility: essential and conditional functions; Warren GJ et al.; Sequences essential for the conjugal transfer of ColE1 can be divided into a cis-acting site and a region encoding trans-acting products . Each of these was successively cloned into a non-transmissible plasmid vector . The resulting chimera was transmissible by the conjugative plasmids F'lac,pro (incFI) and R64drd11 (incIalpha) . The sequences encoding colicin E1, immunity, and incompatibility were absent from this chimera: therefore they are not essential for the conjugal transmission of the ColE1 plasmid . In contrast to ColE1, however, the same chimera was deficient in conjugal transfer initiated by R751 (incP) and R388 (incW) . This suggests that ColE1 sequences other than those cloned in the chimeric plasmid are necessary for its mobilization by R751 and R388 . Three such regions were revealed by screening a series of ColE1 insertion mutants for transfer by R751 and R388 . Two of these regions encode no other known function while the third is encoded by a region which overlaps the gene for colicin E1 itself. Science, 1979 Feb 16, 203(4381), 614 - 25 Total synthesis of a gene; Khorana HG; The method developed for the total synthesis of a given DNA containing biologically specific sequences consists of the following . The DNA in the double-stranded form is carefully divided into short single-stranded segments with suitable overlaps in the complementary strands . All the segments are chemically synthesized starting with protected nucleosides and mononucleotides . The 5'-OH ends of the appropriate oligonucleotides are then phosphorylated with the use of {y-32P}ATP and polynucleotide kinase . A few to several neighboring oligonucleotides are then allowed to form bihelical complexes in aqueous solution, and the latter are joined end to end by polynucleotide ligase to form covalently linked duplexes . Subsequent heat-to-tail joining of the short duplexes leads to the total DNA . The methods are described for the construction of a biologically functional suppressor transfer RNA gene . The total work involved (i) the synthesis of a 126-nucleotide-long bihelical DNA corresponding to a known precursor to the tyrosine suppressor transfer RNA, (ii) the sequencing of the promoter region and the distal region adjoining the C-C-A end, which contained a signal for the processing of the RNA transcript, (iii) total synthesis of the 207 base-pair-long DNA, which included the control elements, as well as the Eco R1 restriction endonuclease specific sequences at the two ends, and (iv) full characterization by transcription in vitro and amber suppressor activity in vivo of the synthetic gene. Eur J Biochem, 1979 Feb 15, 94(1), 65 - 75 The 'DNA-membrane complex' of Escherichia coli B/r . Its composition and properties and the fate of nascent and genome DNA during DNA synthesis; Robertson W et al.; The composition and properties of 'DNA-membrane complex' of Escherichia coli B/r have been investigated . The 'complexes' contain most of the DNA and membrane of the cells, and about 50% and 25% of the RNA and protein respectively . The properties of DNA synthesized by the 'complexes' are described and the process is concluded to be largely mediated through polymerase I . Nascent DNA synthesized by the 'DNA-membrane complexes' was of two main classes, one of molecular weight around 600,000--800,000 and the other of higher molecular weight . Polynucleotide ligase activity was not detectable . The onset of synthesis coincided with the dissociation of at least 70% of the genome DNA and all of the nascent DNA from the 'complexes' and was concomitant with the action of a nuclease on parental DNA . This nuclease activity was not ATP-dependent. Eur J Biochem, 1979 Feb 15, 94(1), 315 - 20 Ribosomal translocation assayed by the matrix-bound poly(uridylic acid) column technique; Belitsina NV et al.; The system of translation of cellulose-bound poly(uridylic acid) by Escherichia coli ribosomes has been used for preparation of pre-translocation state ribosomes in columns . Translocation has been induced by passing the elongation factor G (EF-G) with GTP or its non-cleavable analog (guanosine 5'-{beta, gamma-methylene}triphosphate) through the column . A method for quantitative comparison of translocation rates, and thus of effectiveness of translocation-inducing factors, has been proposed . The method is based on an analysis of the profile of deacylated tRNA elution resulting from translocation in the column . The determination of the rate and amount of translocation has been done under different ionic conditions . It has been found that the Mg2+ concentration is a decisive factor of translocation in vitro: at high Mg2+ (30 mM) EF-G cannot induce translocation, and lowering the Mg2+ concentration (to 10 mM) is required for EF-G to become effective . Sufficiently low Mg2+ (3 mM) itself has proved to induce fast and complete translocation, without EF-G. Eur J Biochem, 1979 Feb 15, 94(1), 207 - 14 The kinetics of Schiff-base formation during reconstitution of D-serine apodehydratase from Escherichia coli with pyridoxal 5'-phosphate; Reed TA et al.; Schiff base formation during reconstitution of D-serine dehydratase (Escherichia coli) from its apoenzyme and pyridoxal 5'-phosphate (pyridoxal-P) has been studied by rapid kinetic techniques using absorbance changes at 436 nm . Three distinct reaction phases have been observed . The first is a very rapid change during which pyridoxal-P is initially bound to the apoenzyme . This step has an equilibrium constant of 1500 M-1 and a forward reaction rate of the order of 2.6 x 10(6) M-1 s-1 . The second phase shows a first-order rate constant with a value dependent on pyridoxal-P and corresponds to a first-order step with a forward rate constant of 3.04 s-1 interacting with the initial equilibrium . The final phase is a slow first-order reaction, the rate constant of which is approximately 0.01 s-1 and is independent of pyridoxal-P concentration . The active pyridoxal species has been shown to be the free pyridoxal-P as opposed to hemiacetal or hemimercaptal forms. Experientia, 1979 Feb 15, 35(2), 183 - 4 lambda-Prophage induction by furocoumarin photosensitization; Baccichetti F et al.; Furocoumarin photosensitization induces lambda-prophage from lysogenic Escherichia coli cells; this effect is clearly due to the photoreaction that furocoumarins give with DNA, and it appears connected with the formation of monoadducts rather than of diadducts (cross-links). Nature, 1979 Feb 15, 277(5697), 538 - 41 Escherichia coli mutants accumulating the precursor of a secreted protein in the cytoplasm; Bassford P et al.; The maltose binding protein of Escherichia coli is secreted into the external periplasmic compartment of the cell . This article describes a selection procedure for the isolation of mutants which fail to export this protein . These mutants probably result from alterations in the amino terminal 'signal sequence', causing the maltose binding protein produced to accumulate in the cytoplasm in its precursor form. Mol Biol Rep, 1979 Feb 15, 4(4), 253 - 6 Evaluation of several enrichment procedures for the isolation of recombinant plasmid DNA; Rosner A et al.; A number of methods for the selective enrichment of recombinant plasmids were examined; these include alkaline phosphatase treatment of the restricted pBR322 vector, as well as a combination of this and S1 nuclease treatment of the ligated mixture of pBR322 and pCR1 plasmids or S . griseus DNA followed by D-cycloserine treatment to enrich for cells carrying recombinant molecules . The relative efficiencies of these methods were compared. J Biol Chem, 1979 Feb 10, 254(3), 955 - 63 Specific gene transcription in yeast nuclei and chromatin by added homologous RNA polymerases I and II; Tekamp PA et al.; When treated at pH less than 4.5, yeast nuclei or chromatin lose endogenous RNA synthetic activity . This activity is regained by addition of exogenous RNA polymerases . The specificity of transcription in this system by homologous RNA polymerases I and III has been investigated by gel electrophoresis, hybridization analysis, and RNase T1 mapping . Exogenous RNA polymerase I selectively transcribes rRNA genes . The transcription of these genes by polymerase I is 30- and 8-fold more selective than RNA polymerase III and Escherichia coli polymerase holoenzyme, respectively . Exogenous RNA polymerase III synthesized RNAs similar in size to authentic 5 S RNA, 4.5 S pre-tRNA, and 4 S tRNA . Eleven per cent of this RNA is 5 S RNA as determined by hybridization . Neither polymerase I nor E . coli polymerase synthesizes detectable quantities of RNA in this size range . AT1 ribonuclease digestion of 5 S RNA synthesized by exogenous RNA polymerase III acting on acid-treated chromatin gives a fragment pattern corresponding to that of 5 S RNA . Thus, RNA polymerase III transcribes the entire 5 S gene in this system. J Biol Chem, 1979 Feb 10, 254(3), 688 - 92 A new nucleotide involved in the stringent response in Escherichia coli . Guanosine 5'-diphosphate-3'-monophosphate; Pao CC et al.; A novel nucleotide has been detected in Escherichia coli subjected to the stringent response . However, this nucleotide does not accumulate in relA+ cells subjected to heat shock, in which guanosine 5'-diphosphate-3'-diphosphate does accumulate but stable RNA synthesis is not restricted . The intracellular level of this new nucleotide thus correlates well with control of stable RNA synthesis . Chemical and enzymatic analysis shows that the new nucleotide is guanosine 5'-diphosphate-3'-monophosphate . It is suggested that this nucleotide may play a role in stringent control of stable RNA synthesis. J Biol Chem, 1979 Feb 10, 254(3), 627 - 33 Increased synthesis of phosphatidylserine decarboxylase in a strain of Escherichia coli bearing a hybrid plasmid . Altered association of enzyme with the membrane; Tyhach RJ et al.; A strain of Escherichia coli bearing a hybrid plasmid containing the psd gene, starved for isoleucine by the addition of valine, produces amounts of phosphatidyl-serine decarboxylase, a membrane-bound enzyme, about 40-fold higher than wild type . At least 98% of the enzyme from cells with high levels of decarboxylase is isolated in the inner, cytoplasmic membrane fraction if the cells are broken by osmotic lysis of spheroplasts following treatment with lysozyme/EDTA . In contrast, if cells containing these large amounts of enzyme are disrupted by sonication, 40 to 45% of the activity is recovered in the 100,000 times g supernatant fraction, whereas with wild type cells, only 5 to 10% is recovered in this fraction . About half of the decarboxylase in membranes saturated with the enzyme is thus only loosely bound, and readily removed by sonication, but not by osmotic lysis . This apparent saturation of the membrane with decarboxylase seems specific, since two other membrane-bound enzymes, phosphatidyl-glycerophosphate synthetase, and CDP-diglyceride synthetase, are not displaced into the supernatant fraction upon sonication . Fractionation on columns of agarose and by centrifugation through gradients of sucrose revealed that the decarboxylase in the supernatant is associated with lipid, in a complex with an apparent molecular weight of at least 5 times 10(6). J Biol Chem, 1979 Feb 10, 254(3), 571 - 4 Oxidative deamination of epsilon-aminolysine residues and formation of Schiff base cross-linkages in cell envelopes of Escherichia coli; Mirelman D et al.; Oxidative deamination of the epsilon-amino group of lysyl residues to form allysine is the initial reaction in the cross-linking of collagen and elastin in vertebrates . The allysyl residues, generated by lysyl oxidase in this reaction, condense with either other allysyl residues or epsilon-amino groups of lysyl or hydroxylysyl to form aldol or Schiff base cross-links . This paper presents evidence that similar allysyl residues and Schiff base cross-links are synthesized in cell envelopes of Escherichia coli . Acid hydrolysis followed by amino acid analysis of envelopes either reduced with NaB{3H}4 or labeled with {14C}lysine and reduced with NaBH4 yielded allysine and two labeled fragments with elution profiles and molecular weights (250 and 330) consistent with Schiff base products derived at least in part from allysine . When {6-3H}lysine-labeled cell envelopes were incubated at 37 degrees C, gradual release of tritiated water occurred . This suggests that an enzymatic reaction catalyzes the deamination of lysine in E . coli membranes and that the higher molecular weight proteins detected in stationary phase or in log phase cell envelopes after NaBH4 reduction occur as a result of formation of Schiff base cross-links. J Biol Chem, 1979 Feb 10, 254(3), 852 - 62 Evidence for multiple electronic forms of two-electron-reduced lipoamide dehydrogenase from Escherichia coli; Wilkinson KD et al.; Results are presented which demonstrate that the 2-electron-reduced lipoamide dehydrogenase (EC 1.6.4.3) from Escherichia coli is a mixture of species . In catalysis, this enzyme cycles between the oxidized and the 2-electron-reduced forms . Three spectrally distinct species are indicated in the pH range 5.8 to 8.0 from measurements of the fluorescence and visible spectra during dithionite titration . These have the following properties . 1) A fluorescent form where the FAD is oxidized and the active center disulfide is reduced . This species is unable to charge transfer and predominates at low pH . 2) A form in which there is a facile charge transfer between thiolate and FAD (epsilon530 - 3300 M-1 cm-1) . This species, which predominates at high pH, is very similar to the 2-electron-reduced pig heart enzyme at high pH . 3) A form where the flavin is reduced and the disulfide is oxidized . The spectra of these three species have been determined . Anaerobic reduction of the enzyme with stoichiometric dihydrolipoamide leads to the formation of the charge transfer species in less than 1 s . Subsequently, in a process requiring about 12 s, the charge transfer complex relaxes to a mixture of species observed in dithionite titrations . The pH dependence of the oxidation-reduction potential, the fluorescence, the charge transfer absorbance (530 nm), and the 455 nm absorbance indicates the presence of a base which is able to stabilize the thiolate anion generated upon reduction of the active center disulfide . The pH dependence of the oxidation-reduction potential indicates that the reduction of the enzyme by dihydrolipoamide involves 2 protons as well as 2 electrons . These potentials are somewhat more positive than those determined for the pig heart enzyme and thus explain the ready further reduction of the E . coli enzyme to the 4-electron-reduced enzyme . The pH-independent formation constant (Kf) for the disproportionation of 2-electron-reduced enzyme (2EH2 in equilibrium E + EH4) is about 55 as calculated from dithionite titrations . Therefore at equilibrium there is about 80% 2-electron-reduced enzyme, 1-% oxidized enzyme, and 10% 4-electron-reduced enzyme . The spectrum of fully formed 2-electron-reduced enzyme has been calculated at several pH values from these data . The results confirm the previous conclusion that lipoamide dehydrogenase from E . coli is qualitatively similar to the pig heart enzyme, differing only in certain quantitative features such as the distribution between the various forms at the 2-electron-reduced level. Biochemistry, 1979 Feb 6, 18(3), 528 - 37 T7 RNA polymerase: promoter structure and polymerase binding; Oakley JL et al.; The sequences of two promoters recognized by the phage-specificied T7 RNA polymerase are presented . The two are identical in sequence but for one base pair from the initiation point (as determined by the 5' sequence of the transcripts), denoted +1, to position -15 . The common : formula: (see text), sequence also includes a region of hyphenated twofold symmetry indicated by the boxes, with the twofold axis as the center of the six base-pair box . The heavy line indicates the extent of homology . The first promoter (A) is demonstrated to lie within gene 1, the gene for the polymerase itself, and 40 bases into the message transcribed from this promoter is found the R Nase III site separating genes 1 and 1.1 . Binding of T7 RNA polymerase to these promoters is associated with a hyperchromic blue shift of the base chromophores consistent with partial melting of the base pairs at the promoter . Binding of T7 RNA polymerase to these promoters disappears at low pH and low temperature and is accompanied by a consequent loss of polymerase activity . The pH dependence of the binding step is adequately described by a single pK of 7.0 . Polymerase catalytic activity, but not promoter binding, requires a single free sulfhydryl group of the enzyme with a pKa of approximately 7.8. Biochem J, 1979 Feb 1, 177(2), 385 - 92 Anomalous reaction of 4-chloro-7-nitrobenzofurazan with thiol compounds; Nitta K et al.; The kinetics of reaction of 4-chloro-7-nitrobenzofurazan with thiol groups at pH values above 5 cannot be accounted for solely on the basis of formation of a single product, the 4-thio derivative . Spectroscopic observations indicate that, in addition to the 4-thio derivative, at least two other products are formed . One of these, referred to as P1, is most likely a reversible complex of thiol compound and 4-chloro-7-nitrobenzofurazan of the Meisenheimer type . The other product, P2, which forms primarily when thiol compound is in a large excess, does not appear to result from direct reaction of thiol group and 4-chloro-7-nitrobenzofurazan, but may be a reaction of product P1 and thiol compound . The coloured product, P2, will react further with proteins, such as bovine serum albumin and Escherichia coli RNA polymerase . This reaction irreversibly destroys the catalytic activity of RNA polymerase . The implications of these observations for utilization of 4-chloro-7-nitrobenzofurazan as a protein-modifying agent are discussed. Br J Urol, 1979 Feb, 51(1), 19 - 23 Surgical treatment of the massively dilated primary megaureter in children; Rabinowitz R et al.; From 1964 to 1975, of 83 children with primary megaureter 33 had unilateral and 8 bilateral massively dilated ureters that required surgical treatment, and of these 73% had febrile infection, 5% were septic and 10% were azotaemic . Surgical treatment consisted of ureteric reimplantation with excision of the atonic distal segment, preceded by diversion when indicated . Tailored reimplantation was successful in 98% of ureters and non-tailored reimplantation was uniformly unsuccessful . All failures were successfully corrected by tailored reimplantation . Thus the requirements for successful surgical management of massive ureteric dilatation in children secondary to an atonic ureteric segment are excision of the atonic segment, transvesical or extravesical tailoring of the ureter and reimplantation. Can J Biochem, 1979 Feb, 57(2), 107 - 11 Levels of glutathione in Escherichia coli; Loewen PC; Log phase cells of Escherichia coli growing in minimal medium contain a basal level of glutathione (5 pmol/mL per Klett unit) which can increase more than sixfold when the cells reach stationary phase . Since the addition of cysteine alone to log phase cells illicits the same response, the increase in the intracellular pool of glutathione appears to be influenced by the amount of cysteine available for glutathione synthesis . Glucose depletion at low cell densities resulted in a decrease in the glutathione pool while the addition of amino acids other than cysteine did not affect the glutathione pool . Depletion of ammonia or proline as the nitrogen source also resulted in a decrease in the glutathione pool to one-third of the original basal levels as did a shift to anaerobic growth . The large glutathione pool in stationary phase cells dropped from 31.5 to 4.5 pmol/mL per Klett unit within 30 min of transfer to fresh medium . There was no apparent correlation between changes in the glutathione and coenzyme A--glutathione disulfide (CoASSG) pools after a variety of metabolic disruptions. Gene, 1979 Feb, 5(2), 159 - 75 Mapping of restriction sites in the argF gene of Escherichia coli by partial endonuclease digestion of end-labeled DNA; Moore SK et al.; A detailed physical map depicting the cleavage sites generated by ten different restriction endonucleases was prepared for the argF region of the Escherichia coli K-12 genome carried on a 1650 base pair fragment capable of directing the in vitro synthesis of ornithine transcarbamylase (OTCase; ec 2.1.3.3) under the control of arginine holorepressor . The method employed was originally developed by Smith and Birnstiel (1976), and involved the electrophoretic sizing of partial endonuclease digestion products of DNA radiolabeled at one end . This novel technique proved to be rapid, simple, amenable to the simultaneous mapping of numerous cleavage sites, and provided the essential information for determining the map order of restriction fragments . A facile method which involved magnesium phosphate as the DNA-binding agent was presented for the isolation of DNA fragments . The discovery of a 117 base pair leader sequence in the argF gene is also discussed. Genetics, 1979 Feb, 91(2), 215 - 27 Localized mutagenesis for the isolation of temperature-sensitive mutants of Escherichia coli affected in protein synthesis; Champney WS; Two variations of the method of localized mutagenesis were used to introduce mutations into the 72 min region of the Escherichia coli chromosome . Twenty temperature-sensitive mutants, with linkage to markers in this region, have been examined . Each strain showed an inhibition of growth in liquid medium at 44 degrees, and 19 of the mutants lost viability upon prolonged incubation at this temperature . A reduction in the rate of in vivo RNA and protein synthesis was observed for each mutant at 44 degrees, relative to a control strain . Eleven of the mutants were altered in growth sensitivity or resistance to one or more of three ribosomal antibiotics . The incomplete assembly of ribosomal subunits was detected in nine strains grown at 44 degrees . The characteristics of these mutants suggest that many of them are altered in genes for translational or transcriptional components, consistent with the clustering of these genes at this chromosomal locus. Mutat Res, 1979 Feb, 59(2), 157 - 65 Characterization of DNA adenine methylation mutants of Escherichia coli K12; Bale A et al.; The phenotypic traits of 7 independently isolated dam mutants of Escherichia coli have been examined . The mutant strains differ from the wildtype in the following respects: (1) decreased DNA adenine methylase activity in vivo and in vitro; (2) a 14--85-fold increase in spontaneous mutability; (3) decreased survival after ultraviolet irradiation; (4) a 10--21-fold increase in spontaneous induction of lambda phage from lysogens; (5) a 3--17-fold increase in the level of recombination; and (6) inviability of double mutants containing dam- and recB- or recC- . Unmethylated fd phage chromosomes are able to replicate normally in dam- mutants . A mutant strain in which the dcm gene is deleted is viable, showing that the dcm gene product is dispensible for growth. J Gen Microbiol, 1979 Feb, 110(2), 285 - 9 Synthesis of vitamin B6 by a mutant of Escherichia coli K12 and the action of 4'-deoxypyridoxine; Scott TA et al.; Mutants of Escherichia coli K12 blocked in the oxidation of pyridoxine 5'-phosphate ('Oxidase' mutants) excreted pyridoxine at an initial rate of 19 pmol h-1 (10(8) bacteria)-1, i.e.0.6 nmol h-1 (mg dry wt)-1, when starved for pyridoxal . Glycolaldehyde, L-phosphoserine, DL-serine and, to a lesser extent, L-leucine stimulated the rate of pyridoxine excretion, but there was no significant stimulation by 2'-hydroxypyridoxine . 4'-Deoxypyridoxine inhibited or stimulated growth of the "Oxidase' mutant, depending on the relative concentrations of added pyridoxal and 4'-deoxypyridoxine . It was concluded that stimulation of growth by 4'-deoxypyridoxine was due to its conversion to pyridoxal. J Gen Microbiol, 1979 Feb, 110(2), 275 - 83 The isolation and characterization of three types of vitamin B6 auxotrophs of Escherichia coli K12; Hockney RC et al.; Approximately 500 vitamin B6 auxotrophs were isolated from 18 independent cultures of Escherichia coli strain CR63 . None grew in minimal medium supplemented with 2'-hydroxypyridoxine . Eighteen auxotrophs which had arisen independently were further characterized . All of them were defective in vitamin B6 synthesis rather than in an aminotransferase involved in vitamin B6 utilization . Two different phenotypes were recognized: 'Oxidase' mutants which grew only when supplied with pyridoxal or pyridoxal 5'-phosphate and 'Pre Pn' mutants which would also grow with pyridoxine or pyridoxine phosphate . "Oxidase' mutants were confined to a single linkage group, but data from interrupted mating experiments established that 'Pre Pn' mutants fall into two linkage groups which are possibly identical to pdxA and pdxB . All mutations in the in the pdxA region were allelic rather than located in two closely linked genes. Immunology, 1979 Feb, 36(2), 355 - 65 Time-dependence and selectivity of immunosuppressive agents; Berenbaum MC; The effects of a variety of agents on the mouse antibody response to sheep red cells (SRC) and Escherichia coli lipopolysaccharide (LPS) were investigated . Agents formed two broad groups according to the time-dependence of their effects . Class I agents inhibited responses if given at any time over a broad period from before administration of antigen to 1-2 days afterwards . They included ionizing radiation, cortisone acetate, cholera toxin and several alkylating agents . Class II agents inhibited responses substantially only if given 1-2 days after the antigen . These agents were all either antimetabolites or vinca alkaloids . Class II agents had only moderate selectivity . When given at the time of maximal effectiveness they usually suppressed the response to SRC more than that to LPS . Some class I agents had striking selectivity . Radiation, cortisone acetate, and cholera toxin markedly suppressed the SRC response but had much less effect on the LPS response . In contrast, mitoclomine and its analogues, which are lipid-soluble alkylating agents, suppressed the response to LPS far more than that to SRC . Possible reasons for these differences in time-dependence and selectivity are discussed. Biochem J, 1979 Feb 1, 177(2), 757 - 9 Oxidation--reduction potentials of molybdenum and iron--sulphur centres in nitrate reductase from Escherichia coli; Vincent SP; The potentials of the couples Mo(IV)--(Mo(V) and Mo(V)--Mo(VI) in nitrate reductase from Escherichia coli K12 were measured as + 180 mV and + 220 mV respectively at pH 7.14 . The potentials associated with two other e.p.r . signals, believed to be due to iron--sulphur centres, were measured as + 50 mV and + 80 mV. Appl Environ Microbiol, 1979 Feb, 37(2), 266 - 73 Deoxyribonucleic acid strand breaks during drying of Escherichia coli on a hydorohobic filter membrane; Asada S et al.; Cells of Escherichia coli mounted on a hydrophobic filter membrane were dried under various vapor pressures . A mutant defective in deoxyribonucleic acid repair (uvrA recA) was more sensitive to drying at a water activity of 0.53 or below than the parent strain but not at a water activity of 0.75 and above . Sucrose gradient studies showed that single- and double-strand breaks of deoxyribonucleic acid occurred at a water activity of 0.53 or below, but no breaks could be observed at a water activity of 0.75 or above . These results were observed in all cells rehydrated with 0.03 M tris (hydroxymethyl) aminomethane-hydrocholoride buffer solution at 0 or 37 degrees C, in the presence or absence of oxygen, with saturated water vapor or with a hypertonic solution followed by a gradual dilution . Freezable water was detected in the cells only at a water activity above 0.75 by differential scanning calorimetry . Removal of unfreezable water of cells in the drying, therfore, might induce deoxyribonucleic acid strand breaks. Acta Pathol Microbiol Scand {C}, 1979 Feb, 87C(1), 67 - 71 Effect of mycoplasmas on phagocytosis and immunocompetence in rats; Thomsen AC et al.; The purpose of this study was to evaluate some possible mechanisms by which mycoplasmas may facilitate a subsequent bacterial infection . The effector of M . arthritidis, M . hominis and U . urealyticum on lymphocyte reactivity to mitogens, subsequent antibody response, and the ability of neutrophils to carry out phagocytosis was investigated . In vitro, large doses of M . arthritidis and M . hominis depressed the reactivity of lymphocytes and the phagocytic ability of neutrophils . In vivo, inoculation of myoplasmas had no effect on reactivity of lymphocytes from rats, and antibody response to subsequently injected E . coli was normal . However, peritoneal neutrophils from rats injected intraperitoneally with large doses of M . arthritidis and M . Hominis were invalidated in their ability to cause phagocytosis of E . Coli . U . urealyticum had no observable effect on lymphocytes and neutrophils in vitro and in vivo. Mol Gen Genet, 1979 Feb 1, 169(3), 345 - 7 Analysis of lincomycin resistance mutations in Escherichia coli; Hummel H et al.; High level lincomycin resistant strains of Escherichia coli were isolated and screened for altered ribosomal proteins and functions . Amongst 58 strains investigated by electrophoresis one had an altered ribosomal protein S7, another one a mutated L14 and two showed altered L15 proteins . A correlation between these alterations and lincomycin resistant growth could not be demonstrated by genetic analysis for any of the mutants . In vitro, however, extracts from the two L15 mutants were less sensitive to inhibition by the drug . A gene locus (linR) responsible for the lincomycin resistance phenotype was mapped at min 30 of the Escherichia coli chromosome near tyrR; it seems to be identical to the previously described linB locus (Apirion, 1967); however, in contrast to these reports it does not seem to alter any ribosomal function. Mol Gen Genet, 1979 Feb 1, 169(3), 325 - 36 Investigations of the F conjugation gene traI:traI mutants and lambdatraI transducing phages; Willetts N et al.; A series of traI point and deletion mutants of Flac, and a traM mutant, were characterised . Complementation tests with an amber Flac traI mutant confirmed their genotypes, and in addition all the traI mutants, but not the traM mutant, were complemented by pRS31 (PSC101 traDI) and EDlambda109 (lambdatraI) . Judging from the efficiencies of plating of F-specific phages, none of the mutations affected pilus formation . The traI products of F and of the F-like plasmid R1 were interchangeable with each other but not with that of R100, while the traM product of F could not be replaced by those of R1 or of R100 . Neither traI nor traM were needed for conjugal transfer of ColE1 . Three lambda transducing phages carrying traI were isolated by in vivo or in vitro techniques, and characterised by genetic complementation tests, by analysis of the fragments produced by restriction endonucleases, and by measurement of heteroduplex molecules . The genetic structures together with the sizes and F coordinates, of the transfer regions carried by the phages were thereby determined . Comparison of the proteins synthesised in UV-irradiated cells by one of the lambdatraI phages with those made by a derivative carrying an amber traI mutation, allowed the traI product to be identified as a protein of molecular weight 174,000 . In addition, the molecular weights of the traD (84,000), traS (18,000), and traT (25,000) products made by the lambdatraSTD1 phage EDlambda107 were measured . The possible roles of the traI and traM products in conjugation are discussed. Mol Gen Genet, 1979 Feb 1, 169(3), 315 - 23 Mutants of Escherichia coli defective in the degradation of guanosine 5'-triphosphate, 3'-diphosphate (pppGpp); Somerville CR et al.; A new class of mutants of E . coli exhibiting altered metabolism of ppGpp and pppGpp has been isolated, and mapped at a locus designated gpp, near min 83 on the genetic map . These mutants accumulate elevated levels of pppGpp during amino acid starvation or carbon source downshift, and exhibit a reduced rate of pppGpp degradation in vivo . The in vitro evidence suggests that the gpp mutants are defective in a 5'-nucleotidase, which specifically hydrolyzes pppGpp to ppGpp . Certain combinations of gpp and spoT mutations are inviable . A gpp spoT double mutant, constructed by employing a leaky spoT mutation, was found to have a slower rate of pppGpp degradation than the gpp mutant alone . This result indicates that spoT also participates in pppGpp degradation . The inviability of certain gpp spoT combinations is attributed to the inability of the double mutants to degrade pppGpp . This is supported by the observation that selection for increased growth rate on the double mutant results in the recovery of relA mutations . Various effects of the gpp mutation upon the pppGpp and ppGpp pools provide additional support for a scheme in which pppGpp is the major precursor of ppGpp. Mol Gen Genet, 1979 Feb 1, 169(3), 299 - 314 Mutations affecting the formation of acetohydroxy acid synthase II in Escherichia coli K-12; Smith JM et al.; Genetic mapping experiments have established that two recently isolated valine-resistant mutants of the K-12 strain of Escherichia coli have lesions lying between ilvE and rbs . These lesions allowed expression of the ilvG gene, specifying the valine-insensitive acetohydroxy acid synthase (synthase II) and an increased expression of the ilvEDA operon . In this respect, they resembled an earlier described ilvO lesion that was reported to lie between ilvA and ilvC . All three lesions were cis-dominant in cis-trans tests . Reexamination of the earlier studied ilvO lesion revealed that it, too, lies between ilvE and rbs . Valine-sensitive derivatives with lesions presumed to be in ilvG were selected from each of the valine-resistant strains . In two of the valine-resistant strains, the ilvG mutations were on the rbs side of ilvO, indicating a gene order rbs-ilvG-ilvO-ilvE-ilvD-ilvA-ilvC . In one of the recently isolated valine-resistant stocks, however, the apparent ilvG mutation was found to be between ilvE and the aline resistance marker . This finding suggests that either ilvO and ilvG mutations are interspersed or there is another locus, ilvR, that behaves phenotypically like ilvO and which lies between ilvG and rbs. Mol Gen Genet, 1979 Feb 1, 169(3), 289 - 97 Deletion mapping of the ilvGOEDAC genes of Escherichia coli K-12; Baez M et al.; A set of lambdadilv phage has been examined that carry overlapping segments of isoleucine-valine structural and regulatory genes derived from the ilv cluster at 83 min on the Escherichia coli K-12 chromosome . The ilv genes present in these phage, and their order, have been determined by transduction of auxotrophs, escape synthesis, and deletion mapping . The order of ilv genes in the phage, and hence the order in the host chromosome, was found to be ilvG-ilvO-ilvEDA-ilvC . Lysogens containing lambdadilv phage were constructed for dominance analysis of regulatory mutations in the ilvO and ilvA genes . The ilvO671 allele is cis-dominant to ilvO+, while the ilvA538 allele is trans-recessive to ilvA+ . Thus, the ilvO gene, that is identified by cis-dominant regulatory mutations that result in increased ilvG and ilvEDA expression, is situated between and may be contiguous with ilvG and ilvEDA. Mol Gen Genet, 1979 Feb 1, 169(3), 271 - 8 Analysis of rpsD mutations in Escherichia coli . III . Effects of rpsD mutations on expression of some ribosomal protein genes; Olsson MO et al.; Relative rates of production and steady state levels of ribosomal proteins were determined in a temperature sensitive rpsD (S4) mutant of Escherichia coli . Some proteins (S4, S12, S13) were overproduced in the mutant at permissive temperature but steady state levels of all examined ribosomal proteins were normal . In a rpsD+/rpsD+ homodiploid strain the relative rates of production of ribosomal proteins were not affected by the increased gene dose . In a rpsD+/rpsD heterodiploid strain only wild type, but not mutant S4, was found . In such a strain S4, S7, S12 and probably S13 is overproduced . It is implied that S4 is involved in the regulation of expression of proximal genes of the two transcriptional units including the genes coding for S4 itself and S12, respectively . A degradation system for ribosomal proteins, which is rapid enough to be of regulatory significance, is demonstrated. Mol Gen Genet, 1979 Feb 1, 169(3), 259 - 69 Analysis of rpsD mutations in Escherichia coli . II . Physiology of some representative mutants; Olsson MO; The effects of ribosomal ambiguity mutations (ram A-) on the assembly of ribosomal 30S subunits in Escherichia coli were studied in some representative mutant strains . It was found that the inability of these strains to produce active 30S subunits at nonpermissive temperatures is correlated with a halt in the accumulation of protein S4 . It is demonstrated that 30S-precursor particles lacking this protein accumulate and break down at nonpermissive temperatures and that most of the 30S proteins as well as the 17S RNA constituting these particles are similarly unstable . These findings are discussed and related to the finding that merodiploid strains containing genes for both mutant and wild type protein S4 do not accumulate the mutant form of the protein . Experiments indicating that ribosomal precursor particles are associated with polysomes are presented . The implications of these findings are discussed and it is suggested that the assembly of ribosomes is tightly coupled to the synthesis of ribosomal proteins. Mol Gen Genet, 1979 Feb 1, 169(3), 245 - 50 Genetics of ribosomal protein methylation in Escherichia coli . III . Map position of two genes, prmA and prmB, governing methylation of proteins L11 and L3; Colson C et al.; Two genes governing ribosomal protein methylation have been located on the map of Escherichia coli by conjugation and transduction crosses between wild-type and prm (protein methylation) mutants . The Prm phenotype of recombinants was determined by an in vitro assay of methylgroups incorporation into protein . Gene prmA, governing methylation of protein L11 is situated at minute 71 on the map and is cotransduced with aroE (30%) and with rpsL (5%) . Gene prmB, governing methylation of protein L3 is at minute 50, very close to aroC (98.5% co-transduction) . A cold-sensitive phenotype was found associated with mutation prmB and was used to score a large number of recombinants in a three factor cross . The results of this cross suggest the order aroC -prmB - purF . The striking symmetrical clustering of aro, prm and rim (ribosome maturation) genes is discussed. J Virol, 1979 Feb, 29(2), 808 - 10 Chromosomal location of the attachment site for the PA-2 prophage in Escherichia coli K-12; Pugsley AP et al.; The chromosomal attachment site for the PA-2 prophage is located between dsd and aroC at the approximately 50 min on the Escherichia coli K-12 genetic map . The attachment site is designated attPA-2. J Med Microbiol, 1979 Feb, 12(1), 123 - 30 Escherichia coli K antigen in relation to serum-induced lysis and phagocytosis; Van Dijk WC et al.; The presence of capsular polysaccharides (K antigens) and their relation to phagocytosis and sensitivity to the lytic action of serum of 26 strains of E . coli isolated from stools of healthy volunteers and from blood cultures were studied . Four of 12 strains isolated from stool cultures and 12 (86%) of the 14 strains isolated from blood cultures possessed K antigen . Three of the 12 strains isolated from stool cultures and seven of the 14 isolated from blood cultures were resistant to uptake by polymorphonuclear leucocytes; these resistant strains contained large amounts of K antigen . By contrast 10 strains, three with low amounts of K antigen and seven without detectable amounts of K antigen, were readily phagocytosed . Thus it appears that K antigen renders E . coli resistant to phagocytosis . Only four (15%) of the 26 strains were sensitive to serum lysis and there was no correlation between the presence of K antigen and the resistance to serum lysis. J Clin Microbiol, 1979 Feb, 9(2), 197 - 9 Sensitivity, precision, and accuracy of the Y1 adrenal cell enterotoxin assay; Tenney JH et al.; The Y1 adrenal cell assay for heat-labile enterotoxin (LT) was found to be relatively insensitive for filtrates of toxigenic Escherichia coli H10407 . When observations were made blindly and subjected to rigorous controls, reliable detection of LT occured only at filtrate dilutions from 1:4 to 1:10 . Detection of LT was unreliable when E . coli H10407 was mixed with another enteric organism . Overall, 7.1% of observers' reports from a single assay were imprecise, and 2.6% would have resulted in errors in detection of LT; however, triplicate assays prevented false positive reports of LT detection . Routine testing for LT production in the clinical diagnostic laboratory awaits a simpler, more sensitive, and more direct method of testing stool specimens. Int J Pept Protein Res, 1979 Feb, 13(2), 146 - 51 Soluble di- and aminopeptidases in Escherichia K-12 . Dispensible enzymes; Hermsdorf CL et al.; As part of a study of the peptidase content of Escherichia coli K-12, two peptidase-deficient amino acid auxotrophs isolated and characterized by Miller as pepD- (strain CM17) and pepD- pepN- pepA- pepB- pepQ- (strain CM89) were examined for the presence of several peptidases previously obtained from strain K-12 in this laboratory . The soluble fraction of each mutant was found to lack the broad-specificity strain K-12 dipeptidase DP and the strain CM89 fraction also lacked activity characteristic of the strain K-12 aminopeptidases AP, L, and OP; like strain CM17, strain CM89 contained the tripeptide-specific aminopeptidase TP . Strain CM89 (but not CM17) appeared to contain little if any activity attributable to the ribosome-bound aminopeptidase I of strain K-12 . Whereas loss of DP, AP, OP, and aminopeptidase I activity may be attributed to the pepD-, pepB-, pepN-, and pepA- mutations, respectively, the reason for the loss of L activity remains uncertain . Grown responses of strain CM89 in liquid media containing di- or tripeptides were in accord with absence of enzymes catalyzing rapid hydrolysis of dipeptides . In synthetic liquid media supplemented with the required amino acids per se or with peptone, cultures of both CM strains grew more slowly than strain K-12 and produced smaller cell-yields than those produced by strain K-12. Proc Natl Acad Sci U S A, 1979 Feb, 76(2), 760 - 4 A general method for maximizing the expression of a cloned gene; Roberts TM et al.; We present a method, utilizing a combination of restriction endonuclease cleavage and digestion with Escherichia coli exonuclease III and Aspergillus orizae nuclease S1, that allows us to position a restriction fragment bearing the promoter of the lacZ gene of E . coli at virtually any distance in front of any cloned gene . In particular, we have used this method to examine the effect on protein production of gene-promoter separation for the cro gene of phage lambda and to produce plasmids that, upon transformation into appropriate E . coli hosts, direct the synthesis of up to 190,000 cro protein monomers per cell. Proc Natl Acad Sci U S A, 1979 Feb, 76(2), 736 - 40 Origin and direction of DNA replication of plasmid RSF1030; Conrad SE et al.; An in vitro replication system has been used to study the origin and direction of replication of the covalently closed, circular DNA of plasmid RSF1030, a nonconjugative R factor . We have enriched for replicative intermediates in these studies either by isolating them on the basis of their unique structure or by limiting the extent of synthesis in the in vitro system . Circular molecules that have replicated to various extents migrate to characteristic positions in agarose gels, thus providing a rapid and efficient method for isolating partially replicated forms . Alternatively, replicative intermediates can be isolated directly from reaction mixtures that contain dideoxyTTP (ddTTP), a compound that limits the average extent of synthesis in vitro . Electron microscopic analysis of such intermediates linearized with either Hpa I or BamHI indicates that RSF1030 replicates in vitro from a unique origin located 70% from one end of Hpa I-cleaved molecules and 47% from the BamHI site . The unidirectional mode of replication has been confirmed by the order in which the six HincII fragments of RSF1030 DNA are labeled in vitro when synthesis is limited to various extents with ddTTP . Finally, a physical map of RSF1030 has been constructed using the restriction endonucleases BamHI, Hpa I, and HincII, and the origin and direction of replication have been defined relative to the map. Proc Natl Acad Sci U S A, 1979 Feb, 76(2), 580 - 4 Nucleotide sequence of the origin of replication of the Escherichia coli K-12 chromosome; Meijer M et al.; The origin of replication, oriC, of the Escherichia coli chromosome was mapped within a DNA segment of 422 base pairs . The nucleotide sequence of this segment was determined . The source of DNA for the sequence analysis was a minichromosome constructed in vivo, consisting exclusively of chromosomal DNA and a minichromosome constructed by cloning in vitro . The nucleotide sequence of the replication origin is characterized by a high degree of repetitiveness due to both inverted and direct repeats . Sequence homologies were found between portions of the replication origins of E . coli and phages lambda and G4 . This suggests similarities in some steps in the initiation of replication of the different replicons. Proc Natl Acad Sci U S A, 1979 Feb, 76(2), 575 - 9 Nucleotide sequence of Escherichia coli K-12 replication origin; Sugimoto K et al.; From subfragments of an EcoRI fragment (9 kilobase pairs) that contained the replication origin of the Escherichia coli chromosome and had been cloned as a recombinant with a nonreplicating DNA fragment coding for ampicillin resistance, small derivative plasmids were constructed . The smallest of these, pTSO151, contained a segment of 463 base pairs as the chromosomal component . Another plasmid, pSY134, constructed from BamHI digests of the EcoRI fragment and mini-F(pMF21), contained a region of 422 base pairs identical with a corresponding region in pTSO151 . We conclude that the replication origin of E . coli chromosome is located within this 422-base-pair segment . The nucleotide sequence of this segment is presented. Nucleic Acids Res, 1979 Feb, 6(2), 525 - 44 The DNA sequence at the T7 C promoter; McConnell DJ; Restriction fragments of T7 DNA which selectively bind E . coli RNA polymerase have been identified . These include fragments located close to the beginning of gene 1 where according to Minkley and Pribnow (1973) there is a promoter called C . The smallest fragment from this region which binds RNA polymerase has been sequenced . It contains a promoter-like sequence, at an appropriate distance from the sequence TACA which Minkley and Pribnow suggested should lie at the initiation site of C . RNA synthesised in vitro from these fragments has been sequenced . The RNA sequence corresponds to the sequence to the right of the C promoter . The C promoter differs significantly from the A1 A2 and A3 promoters in sequence . Its structure and position suggest it plays a role in T7 infection. Nucleic Acids Res, 1979 Feb, 6(2), 507 - 23 The interaction of RNA polymerase II from wheat with supercoiled and linear plasmid templates; Lilley DM et al.; Interactions between a plant RNA polymerase II and ColE1 based plasmid DNA templates have been studied . Gel electrophoresis indicates that the enzyme binds to both supercoiled and linear species . Using the totally double stranded pMB9/SmaI fragment it is shown that transcription of completely base paired DNA is ten-fold lower than that of denatured or supercoiled plasmid, and reflects the presence of fewer initiation sites . A small proportion of the transcript remains tightly bound to supercoiled templates . 3' oligodeoxycytidine extensions on pMB9/SmaI serve to promote transcription of the linear double stranded form . Using restriction kinetics it is shown that there is a small enhancement of polymerase binding at the pMB9 tetracycline promoter, but that the selectivity of binding at this locus is lower than for the natural bacterial polymerase. J Biochem (Tokyo), 1979 Feb, 85(2), 423 - 32 Phosphoenolpyruvate carboxylase of Escherichia coli . The role of lysyl residues in the catalytic and regulatory functions; Naide A et al.; Phosphoenolpyruvate (PEP) carboxylase {EC 4.1.1.31} of E . coli was inactivated by 2,4,6-trinitrobenzene sulfonate (TNBS), a reagent known to attack amino groups in polypeptides . When the modified enzyme was hydrolyzed with acid, epsilon-trinitrophenyl lysine (TNP-lysine) was identified as a product . Close similarity of the absorption spectrum of the modified enzyme to that of TNP-alpha-acetyl lysine and other observations indicated that most of the amino acid residues modified were lysyl residues . Spectrophotometric determination suggested that five lysyl residues out of 37 residues per subunit were modified concomitant with the complete inactivation of the enzyme . DL-Phospholactate (P-lactate), a potent competitive inhibitor of the enzyme, protected the enzyme from TNBS inactivation . The concentration of P-lactate required for half-maximal protection was 3 mM in the presence of Mg2+ and acetyl-CoA (CoASAc), which is one of the allosteric activators of the enzyme . About 1.3 lysyl residues per subunit were protected from modification by 10 mM P-lactate, indicating that one or two lysyl residues are essential for the catalytic activity and are located at or near the active site . The Km values of the partially inactivated enzyme for PEP and Mg2+ were essentially unchanged, though Vmax was decreased . The partially inactivated enzyme showed no sensitivity to the allosteric activators, i.e., fructose 1,6-bisphosphate (Fru-1,6-P2) and GTP, or to the allosteric inhibitor, i.e., L-aspartate (or L-malate), but retained sensitivities to other activators, i.e., CoASAc and long-chain fatty acids . P-lactate, in the presence of Mg2+ and CoASAc, protected the enzyme from inactivation, but did not protect it from desensitization to Fru-1,6-P2, GTP, and L-aspartate . However, when the modification was carried out in the presence of L-malate, the enzyme was protected from desensitization to L-aspartate (or L-malate), but was not protected from desensitization to Fru-1,6-P2 and GTP . These results indicate that the lysyl residues involved in the catalytic and regulatory functions are different from each other, and that lysyl residues involved in the regulation by L-aspartate (or L-malate) are also different from those involved in the regulation by Fru-1,6-P2 and GTP. J Bacteriol, 1979 Feb, 137(2), 885 - 90 Biochemical characterization of nonintegrated plasmid-folded chromosome complexes: sex factor F and the Escherichia coli nucleoid; Miller JR et al.; The existence of nonintegrated plasmid-chromosome complexes has been deduced in previous work from the cosedimentation of covalently closed, circular plasmids with host folded chromosomes . In the present work, it is shown that about 70 to 90% of the covalently closed, circular F deoxyribonucleic acid could be released in vitro from chromosome complexes by ribonuclease treatment but not by protease, Sarkosyl, or ethidium bromide . Consistent with the in vitro studies, Escherichia coli cells treated for 5 min with rifampin, an inhibitor of ribonucleic acid initiation, released upon lysis 90% of their plasmid deoxyribonucleic acid as freely sedimenting molecules. J Bacteriol, 1979 Feb, 137(2), 846 - 53 Acetohydroxy acid synthase I of Escherichia coli: purification and properties; Grimminger H et al.; Several properties of the three acetohydroxy acid synthases of Escherichia coli have been compared in crude extracts . The three enzymes can be readily distinguished from each other . Acetohydroxy acid synthase I, the product of the ilvB gene, has been purified to near homogeneity . The purification was made possible by the fact that the enzyme was maintained in buffers of a high ionic strength or in buffers containing glycerol . Density gradient centrifugation studies indicated that the enzyme exists as a dimer of subunits of similar (60,000) molecular weight in buffers containing glycerol with or without two of the cofactors . Mg2+ and thiamine diphosphate . When flavine adenine dinucleotide was added along with Mg2+ and thiamine diphosphate, an increase in the rate of sedimentation occurred that was thought to be due to a rapid tetramer-dimer interconversion . The addition of pyruvate, the substrate, along with the three cofactors, resulted in a further increase in sedimentation rate, due presumably to an increase in the tetramer-to-dimer ratio . The addition of valine to the complete system resulted in maintenance of the enzyme in the dimeric state concomitant with inhibition of enzyme activity. J Bacteriol, 1979 Feb, 137(2), 830 - 8 Effect of host lex, recA, recF, and uvrD genotypes on the ultraviolet light-protecting and related properties of plasmid R46 in Escherichia coli; Waleh NS et al.; The ability of plasmid R46 to reduce the lethal but enhance the mutagenic effect of ultraviolet (UV) irradiation was tested in sets of Escherichia coli K-12 derivatives, wild type or with different mutations affecting DNA repair capacity, but otherwise isogenic . UV protection and enhancement of UV mutagenic effect were obtained in uvrA6, uvrB5, uvrD3, and recF143 hosts, but not in a recA56 strain . The plasmid gave some UV protection in two lexA1 and two lexA101 strains and in one lexA102 host, but produced no such effect in another lexA102 host . The plasmid restored UV mutagenic effect in a lexB30 strain, the yield of induced mutants per survivor of irradiation (10 J/m2) being about the same for the lexB30(R46) and lex+(R46) strains; by contrast the plasmid, though it reduced the UV sensitivity of the lexB30 strain, did not make it as UV-resistant as the lex+ R-strain. J Bacteriol, 1979 Feb, 137(2), 818 - 23 Lipoprotein synthesis in Escherichia coli spheroplasts: accumulation of lipoprotein in cytoplasmic membrane; Kanazawa H et al.; Synthesis of cell envelope proteins was studied in ethylenediaminetetraacetic acid-lysozyme spheroplasts of Escherichia coli ML30 . The rate of incorporation of {3H}arginine into proteins in spheroplasts was about 30% of that of intact cells . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins synthesized in spheroplasts revealed the preferential synthesis of five polypeptides, one of which has been identified as the free form of murein lipoprotein . Lipoprotein synthesized in spheroplasts was found to be of same molecular size as that of mature lipoprotein . No prolipoprotein was observed even with a short pulse-labeling with {3H}arginine . On the other hand, significant accumulation of newly synthesized lipoprotein in the cytoplasmic membrane fraction of spheroplasts was observed . These results suggest that the processing of prolipoprotein occurs in the cytoplasmic membrane fraction of the cell envelope. J Bacteriol, 1979 Feb, 137(2), 802 - 10 Promoter-like mutants with increased expression of the Escherichia coli uridine phosphorylase structural gene; Mironov AS et al.; From an Escherichia coli K-12 strain lacking adenylate cyclase (cya) and cyclic AMP receptor protein (crp), two mutants were isolated that synthesize uridine phosphorylase constitutively . The mutations differ from one another and also from a wild type in the maximum rate of uridine phosphorylase synthesis . They have constitutive expression of the uridine phosphorylase gene (udp) in the presence of repressor protein coded by the cytR regulatory gene and decrease the sensitivity of the udp gene simultaneously with catabolite repression . Both mutations cause a high level of udp expression whether they are in a cya crp or in a cya+ crp+ background . Another mutation (udpP1) isolated previously alters the response of udp gene to the ctyR repressor and produces a higher constitutive level of uridine phosphorylase in a cytR+ than in a cytR background when bacteria are grown in glucose . The synthesis of uridine phosphorylase in this mutant is dependent on an intact cyclic AMP-cyclic AMP receptor protein complex . All mutations studied are cis-acting and extremely closely linked to the udp structural gene, and appear to affect the uridine phosphorylase promoter-operator region . The data obtained are in accordance with a suggestion that the cytR repressor protein normally asserts its function by preventing the positive action of cyclic AMP-cyclic AMP receptor protein complex. J Bacteriol, 1979 Feb, 137(2), 790 - 4 Membrane-bound deoxyribonucleic acid from Escherichia coli: effects of replication, protein synthesis, and ribonucleic acid synthesis; Yaffe E et al.; The experiments presented in this paper suggest that the shift observed in sedimentation of deoxyribonucleic acid from cells of Escherichia coli subjected to amino acid starvation is related to inhibition of ribonucleic acid synthesis rather than to its release from the membrane at the termination of replication. J Bacteriol, 1979 Feb, 137(2), 740 - 5 Deoxyribonucleic acid-membrane interactions near the origin of replication and initiation of deoxyribonucleic acid synthesis in Escherichia coli; Craine BL et al.; A previously reported salt-sensitive binding of deoxyribonucleic acid (DNA) to the cell envelope in Escherichia coli, involving approximately one site per chromosome near the origin of DNA replication, is rapidly disrupted in vivo by rifampin or chloramphenicol treatment and by amino acid starvation . DNA replication still initiates with this origin-specific binding disrupted, even when the disruption extends over the period of obligatory protein and ribonucleic acid synthesis that must precede initiation after release of cells from amino acid starvation . Thus the origin-associated membrane-DNA interaction is not necessary either for the initiation event itself or for the maturation of a putative initiation apparatus in E . coli. J Bacteriol, 1979 Feb, 137(2), 1063 - 5 Chromosomal regulation of sexual expression in Escherichia coli; Lerner TJ et al.; We report a genetic analysis of a recessive chromosomal mutation of Escherichia coli K-12 that is responsibel for masking the sexual expression of an F factor that it carries . We call this new bacterial gene, which is closely linked to thr, fex, for F expression. J Bacteriol, 1979 Feb, 137(2), 1059 - 62 The relA locus specifies a positive effector in branched-chain amino acid transport regulation; Quay SC et al.; The regulation of branched-chain amino acid transport and periplasmic binding proteins was studied in Escherichia coli strains which were isogenic except for the relA locus, the gene for the "stringent factor," which is responsible for guanosine tetraphosphate synthesis . The strain containing the relA mutation could not be derepressed for the synthesis of leucine transport or binding proteins when shifted from a medium containing all 20 amino acids in excess to one in which leucine was limiting . The relA+ strain showed normal derepression under these conditions. Infect Immun, 1979 Feb, 23(2), 325 - 9 Relationship between enterotoxin production and serotype in enterotoxigenic Escherichia coli; Merson MH et al.; We examined the relationship between serotype and enterotoxin production in 109 enterotoxigenic Escherichia coli strains isolated from 109 patients with severe cholera-like diarrhea in Dacca, Bangladesh . Of 69 strains producing both heat-labile and heat-stable toxins, 59 (86%) belonged to the one of four O serogroups, and 56 (81%) of these strains belonged to one of six O:K:H serotypes . In contrast, 34 strains producing only heat-stable toxin were distributed among 15 O serogroups, and six strains producing only heat-labile toxin were distributed among six O serogroups . Twelve strains producing heat-labile and heat-stable toxins and five strains producing heat-stable toxin were found which had the same serotype (O78:K-:H12) and biotype . It appears that at least in one geographic setting E . coli strains producing both heat-labile and heat-stable toxins are more restricted in their O groups and O:K:H serotypes than E . coli that produce only heat-stable toxin and that certain serobiotypes may commonly include strains which produce both toxin types. Eur J Biochem, 1979 Feb 1, 93(3), 495 - 503 Protein interactions in the outer membrane of Escherichia coli; Palva ET; Specific protein interactions in Escherichia coli outer membrane were analyzed using chemical cross-linking with truly cleavable reagents and symmetrical two-dimensional sodium dodecyl sulphate/polyacrylamide gel electrophoresis . The major outer membrane proteins were shown to form cross-linked complexes . These include multimers of lambda receptor, protein I, II, III and the free form of lipoprotein . Lipoprotein was also found to be cross-linked to proteins II and III . The identity of many of these complexes was verified using appropriate mutants missing the proteins in question . No new protein interactions were detected in the mutants even when three of the major proteins were missing . Proteins II, III and the free form of lipoprotein could also be cross-linked to the peptidoglycan layer of the cell wall. J Immunol, 1979 Feb, 122(2), 619 - 22 Characterization of a congenitally LPS-resistant, athymic mouse strain; Vogel SN et al.; C57BL/10ScN (nu/nu) mice have B cells and macrophages unresponsive to a phenol-water extracted preparation of Escherichia coli K 235 LPS . This unresponsiveness was demonstrated in vitro by the inability of spleen cells to incorporate 3H-thymidine after a 48 hr incubation with LPS (Ph) and by the inability of LPS (Ph) to inhibit macrophage phagocytosis of 51 Cr-labeled, opsonized sheep erythrocytes . Furthermore, macrophage cultures stimulated with LPS (Ph) produced low levels of LAF and PGE2 when compared with macrophages from the LPS-sensitive C3H/HeN and C3H/HeN (nu/nu) strains . Therefore, the C57BL/10ScN (nu/nu) strain is similar in its LPS unresponsiveness to the well-characterized C3H/HeJ and C57BL/10ScCR strains . The combination of endotoxin unresponsiveness and the athymic nature of this mouse strain may provide a powerful new tool for studying the cellular events mediating endotoxicity. J Hyg (Lond), 1979 Feb, 82(1), 115 - 21 Escherichia coli in gastroenteritis of children in London and Jamaica; Ellis-Pegler RB et al.; The jejunal and stool flora of children with gastroenteritis in London and in Jamaica was examined . Although bacterial colonization of the small bowel was commonly detected, it was unusual to find the same serotype of E . coli in both jejunum and stool, and none of the jejunal strains of E . coli produced either heatlabile or heat-stable enterotoxin . Some strains of E . coli causing infant gastroenteritis are neither toxigenic nor invasive, and other mechanisms must be sought to account for their pathogenicity. J Bacteriol, 1979 Feb, 137(2), 839 - 45 High and selective resistance to mecillinam in adenylate cyclase-deficient or cyclic adenosine 3',5'-monophosphate receptor protein-deficient mutants of Escherichia coli; Aono R et al.; Adenylate cyclase-deficient (cya) mutants of Escherichia coli K-12 were selectively and highly resistant to mecillinam (FL1060) among several beta-lactam antibiotics in the absence of cyclic adenosine 3',5'-monophosphate (cAMP) . They became sensitive to the drug in the presence of cAMP . Also, cAMP receptor protein-negative (crp) mutants, with the exception of strain 5333, were highly resistant to mecillinam in the presence and in the absence of cAMP . Mecillinam exerted two distinct and sequential effects in both cya+ strains and cya strains supplemented with cAMP: (i) rounding of cells and (ii) cessation of cell division . The first effect was accompanied by a decrease in growth rate, whereas the second effect was accompanied by enlargement and lysis of the rounded cells . The second effect of mecillinam was dependent on inoculum size and cAMP . When the cell density was above about 10(6) cells per ml, the rounded cells stopped dividing but did not lyse . In the absence of cAMP, cya strains neither stopped dividing nor lysed; they were resistant to the second, lethal effect of mecillinam. J Bacteriol, 1979 Feb, 137(2), 1043 - 7 Lipid and lipopolysaccharide composition of Escherichia coli surface-altered mutants selected for resistance to levallorphan, tetracaine, and polymyxin; Dame JB et al.; Certain mutants of Escherichia coli with an altered permeability barrier have an essentially normal lipopolysaccharide, fatty acid, and phospholipid content, with a slight increase in the membrane protein:lipid ratio . The phospholipid metabolism of the lev and tec strains shows an abnormal response to growth in the selective agents levallorphan and tetracaine, respectively. J Bacteriol, 1979 Feb, 137(2), 1037 - 9 Transport of alpha-p-nitrophenylgalactoside by the lactose carrier of Escherichia coli; Putzrath RM et al.; alpha-p-Nitrophenylgalactoside was found to be accumulated by the lactose transport-system of Escherichia coli . This fact may help to resolve the differences in the reported number of sugar binding sites of the lactose transport protein in nonenergized and energized membrane vesicles. Tohoku J Exp Med, 1979 Feb, 127(2), 161 - 7 Prevention of tissue factor generation in mononuclear cells by agents known to increase intracellular cyclic AMP; Tono-oka T et al.; Agents known to increase intracellular levels of cyclic 3', 5'-adenosine monophosphate (cyclic AMP) were examined for their effect on tissue factor generation by mononuclear cells cultured with E . coli endotoxin . Aminophylline, an inhibitor of phosphodiesterase, and epinephrine, a beta-adrenergic agent, showed an inhibitory effect, and these effects were reversible . Moreover, dibutyryl cyclic AMP also exhibited the effect . Dibutyryl cyclic GMP, however, did not enhance the tissue factor generation by mononuclear cells . On the basis of these observations, it was concluded that the phenomenon of tissue factor generation by mononuclear cells is a biological event, and that intracellular cyclic AMP has a possible role in modulating this phenomenon. J Bacteriol, 1979 Feb, 137(2), 705 - 10 Proton translocation in cytochrome-deficient mutants of Escherichia coli; Brookman JJ et al.; Cytochrome-deficient cells of a strain of Escherichia coli lacking 5-amino-levulinate synthetase have been used to study proton translocation associated with the reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase region of the electron transport chain . Menadione was used as electron acceptor, and mannitol was used as the substrate for the generation of intracellular NADH . The effects of iron deficiency on NADH- and D-lactate-menadione reductase activities were studied in iron-deficient cells of a mutant strain unable to synthesize the iron chelator enterochelin; both activities were reduced . The NADH- menadione reductase activity in cytochrome-deficient cells was associated with proton translocation and could be coupled to the uptake of proline . However proton translocation associated with the NADH-menadione reductase activity was prevented by a mutation in an unc gene . It was concluded that there is no proton translocation associated with the NADH-dehydrogenase region of the electron transport chain in E . coli and that the proton translocation obtained with mannitol as substrate is due to the activity of membrane-bound adenosine triphosphatase. Can J Biochem, 1979 Feb, 57(2), 188 - 96 Purification of UDP-N-acetylenolpyruvoylglucosamine reductase from Escherichia coli by affinity chromatography, its subunit structure and the absence of flavin as the prosthetic group; Anwar RA et al.; The enzyme UDP-N-acetylenolpyruvoylglucosamine reductase (EC 1.1.1.158) was purified to homogeneity from Escherichia coli by affinity chromatography on a NADP-agarose column . The evidence suggests that the enzyme (molecular weight 35,000) is composed of two nonidentical subunits of molecular weight 21,500 and 13,500, respectively . The absorption spectrum of the purified enzyme shows no absorption band around 450 nm and thus does not support the previous suggestions that the enzyme is a flavoprotein . However, the A280: A260 ratio gives a value of 0.86 which suggests the presence of tightly bound nucleotide . A quantitative transfer of tritium from 1,4-{4-3H}NADPH to UDP-N-acetylenolpyruvoylglucosamine to form UDP-N-E13H}acetylmuramic acid was also observed, which clearly shows that the enzyme is not a flavoprotein. Appl Environ Microbiol, 1979 Feb, 37(2), 279 - 82 Formation of N-nitrosamines from seconday animes and nitrite by resting cells of Escherichia coli B; Kunisaki N et al.; In the presence of resting cells of Escherichia coli B, the formation of N-nitrosamines from nitrite and secondary amines, such as dimethylamine and piperidine, was proportional to the incubation time and to the cell concentration . Optimum pH was 8.0 . Boiled cells were incapable of nitrosating secondary amines . Although these experiments were carried out by using intact cells of E . coli B, the reaction followed Michaelis-Menten kinetics, and the apparent Km values calculated from Lineweaver-Burk plots were 0.12 +/- 0.03 M for dimethylamine and 0.07 +/- 0.02 M for nitrite . The apparent Km for piperidine was 0.15 +/- 0.05 M . The nitrosation was inhibited by high substrate concentrations . These results suggested that the formation of n-nitrosamines by resting cells of E . coli B apparently depends on their enzyme activities. Chromosoma, 1979 Jan 31, 70(3), 293 - 304 Transcription and hybridization of 125I-cRNA from flow sorted chromosomes; Sawin VL et al.; Metaphase chromosomes from the Chinese hamster cell line M3-1 were separated by means of a flow sorter . Two chromosome fractions were used for this study: A, which consisted of 95% pure chromosome no . 1, and B, which was 90% pure chromosome no . 2 . The DNA of 10(6) chromosomes of each type was purified, and a 125I-cRNA transcript was synthesized in a reaction containing E . coli RNA polymerase and carrier-free 125I-CTP (1.7 Ci/mumole) . The cRNA product synthesized with template DNA from 10(5) sorted chromosomes contained more than 10(6) dpm . The electrophoretic mobility profiles of the cRNAs on 7.5% SDS acrylamide gels demonstrated that more than 50% of the ribo-polymers were equal to or longer than marker E . coli met-tRNAf . In hybridization reactions 21% and 17% of the transcripts from Chinese hamster whole cell and sorted chromosome DNA hybridized to Chinese hamster DNA and did not hybridize significantly over background in reactions containing calf DNA at Crt values of 1.3 and 1.9 x 10(2) mole sec/l . Labelled cRNAs transcribed from the DNA of sorted chromosomes hybridized with the DNA of each sorted chromosome fractions at a Crt of 0.6 mole sec/l . This study demonstrated that the DNA can be (1) recovered from small numbers of highly purified flow sorted chromosomes, (2) used as template by E . coli RNA polymerase and (3) used to prepare a cRNA in reactions containing polymerase and carrier-free 125I-CTP to yield a product which can be employed for hybridization analysis. Mol Gen Genet, 1979 Jan 31, 169(2), 213 - 8 DNA sequence of the transposable element IS1; Johnsrud L; The nucleotide sequence of an IS1 element recently transposed into the lacI gene is reported . This sequence is nearly identical to one previously reported for another IS1 element (Ohtsubo and Ohtsubo, 1978) . The implications of this similarity are discussed . The sizes of potential polypeptides encoded in the IS1 DNA have been determined and possible roles for these peptides in the illegitimate recombination events mediated by the element are considered. Mol Gen Genet, 1979 Jan 31, 169(2), 205 - 11 Regulation of the biosynthesis of aminoacyl-tRNA synthetases and of tRNA in Escherichia coli . IV . Mutants with increased levels of leucyl- or seryl-tRNA synthetase; Theall G et al.; Spontaneous revertants of a temperature-sensitive Escherichia coli strain harboring a thermolabile leucyl-tRNA synthetase and seryl-tRNA synthetase were selected for growth at 40 degrees C . Among these, strains were found with increased levels of both thermolabile synthetases . Two distinct genetic loci were found responsible for enzyme overproduction . leuR, located near xyl, causes elevated levels of leucyl-tRNA synthetase; while serR, located near leu, causes elevated levels of seryl-tRNA synthetase. Mol Gen Genet, 1979 Jan 31, 169(2), 173 - 81 Template specificity of Qbeta and SP phage RNA replicases as studied by replication of small variant RNAs; Fukami Y et al.; Template specificity of two RNA-dependent RNA polymerases (Qbeta and SP RNA replicases) was examined using "variant RNAs" as template . Three variant RNAs, one (8S) generated by Qbeta replicase and two (6S and 5.2S) generated by SP replicase, were isolated from the reaction mixtures incubated in the absence of exogenous template RNA . All these RNAs were found to be active as template for both Qbeta and SP replicases, though homologous RNA exhibited activities about three times higher than heterologous RNA with either enzyme, in agreement with the results obtained in phage RNA-dependent reactions . In these reactions, faithful replication of variant RNA was observed, and the amount of RNA synthesized was in a many-fold excess over the template RNA added . We also found that the heterologous RNA-dependent reactions were suppressed by increasing the concentration of salts or decreasing the concentration of substrates . Under such conditions, replication of heterologous variant RNA was almost completely suppressed, while the amount of homologous variant RNA synthesized was only reduced to 50% of that synthesized under the standard conditions . Thus the template specificity of the two RNA replicases seems to be expressed more strictly in these replication systems. Mol Gen Genet, 1979 Jan 31, 169(2), 137 - 46 Genetics and biochemistry of the peptidoglycan-associated proteins b and c of Escherichia coli K12; Verhoef C et al.; To collect information on synthesis and regulation of the peptidoglycan-associated pore-forming outer membrane proteins b and c, mutants resistant to phages Me1 and TuIa were analyzed . Genetic analysis showed three linkage groups, corresponding with the genes tolF (phenotype b-c+), meoA (phenotype b+c-) and ompB (phenotypes b-c-, b-c+, b++c- and b++c+/-) . It has recently been described that also a b+c- phenotype can occur in the latter linkage group {Chai, T., Foulds, J., J . Bacteriol . 130, 781-786 (1977)} . Among ompB (b-c+)/meoA (b+c-) double mutants strains were found with the b+c- phenotype, showing that ompB is not the structural gene for protein b . Studies on purified proteins b and c showed profound differences between the two proteins with respect to the electrophoretic mobility of fragments obtained by treatment with cyanogen bromide, trypsin and chymotrypsin . The amino acid in position three of the amino-termini of proteins b and c, isolated from isogenic strains, were identified as isoleucine and valine respectively . Both the genetic and biochemical results are consistent with a model recently published {Ichihara, S., Mizushima, S., J . Biochem . (Japan) 83, 1095-1100 (1978)} which predicts that tolF and meoA are the structural genes for the proteins b and c respectively and that ompB is a regulatory gene whose product regulates the levels of both proteins. Biochim Biophys Acta, 1979 Jan 26, 561(1), 223 - 31 The enzymatic hydrolysis of the phosphate ester bond in some thionucleotides; Lee CC et al.; We prepared the 5'- and 3'-O-phosphorothioate esters of the antitumor agent O2 : 2'-anhydro-1-beta-D-arabinosylcytosine . We also included in this study esters of 2'-thio-2'-deoxycytidine, namely, 2'-S-dCyd-2' : 3'-P, 2'-S-dCyd-2'-P, and 2'-S-dCyd-3'-P, along with natural nucleotides . These compounds were subjected to the action of Escherichia coli alkaline phosphatase, potato acid phosphatase, and bovine pancreatic ribonuclease A . The data were analyzed by Lineweaver-Burk plots to obtain Km and KI values . Only 2'-S-dCyd-2'-P was a substrate for alkaline phosphatase; the anhydro-araCyt phosphorothioates were good competitive inhibitors, while 2'-S-dCyd-3'-P did not associate with the enzyme . Acid phosphatase hydrolyzed all four monoesters investigated, including the S-phosphorothioate . The cyclic phosphorothioate, 2'-S-dCyd-2' : 3'-P was neither hydrolyzed by, nor associated with, ribonuclease A . ORD spectroscopy was also used in an attempt to relate the structural features of analogs to the peculiarity of their hydrolysis. J Biol Chem, 1979 Jan 25, 254(2), 518 - 24 The monomeric glutamyl-tRNA synthetase of Escherichia coli . Purification and relation between its structural and catalytic properties; Kern D et al.; The glutamyl-tRNA synthetase has been purified to homogeneity from Escherichia coli with a yield of about 50% . It is a monomer with a molecular weight of 56,000 and has the same kinetic properties as those of the alpha chain of the dimeric alphabeta-glutamyl-tRNA synthetase described previously (Lapointe, J., and Soll, D . (1972) J . Biol . Chem . 247, 4966-4974) . It is the smallest amino-acyl-tRNA synthetase purified from E . coli and contains no important sequence repetition . It is also the only monomeric aminoacyl-tRNA synthetase reported so far to contain no major sequence duplication . Considering its structural and mechanistic similarities with the glutaminyl- and the arginyl-tRNA synthetases of E . coli, we propose the existence of a relation between the true monomeric character of the glutamyl-tRNA synthetase (as opposed to monomers with sequence duplications) and its requirement for tRNA in the activation of glutamate . A single sulfhydryl group of the native enzyme reacts with 5,5'-dithiobis(2-nitrobenzoic acid) causing no loss of enzymatic activity, whereas four such groups per enzyme react in the presence of 4 M guanidine HCl. J Biol Chem, 1979 Jan 25, 254(2), 327 - 32 The molecular basis of leucine auxotrophy of quinone-treated Escherichia coli . Active site-directed modification of leucyl-tRNA synthetase by 6-amino-7-chloro-5,8-dioxoquinoline; Wiebauer K et al.; Leucyl-tRNA synthetase from Escherichia coli is rapidly inactivated by 6-amino-7-chloro-5,8-dioxoquinoline (quinone), a model substance for cytostatic quinones . Loss of activity follows pseudo-first order kinetics . The quinone masks essential--SH groups that are reactive with N-ethylmaleimide . Specific protection of the enzyme by leucine provides evidence for active site-directed modification . Half-maximal protection is found at a concentration of 150 micron which is identical with the dissociation constant of the enzyme.substrate complex . The competitive inhibitor leucinol also protects the enzyme from inactivation by the quinone . MgATP enhances the protective effect of leucinol about 250-fold, thus substantiating recently published findings on synergistic coupling of ligands to aminoacyl-tRNA synthetases . The results support the assumption that the bacteriostatic quinone directly interferes with leucyl-tRNA synthetase in growing cells . Active-site-directed inhibition of the enzyme could adequately explain the phenotypically observed auxotrophy for leucine of quinone-treated E . coli. J Biol Chem, 1979 Jan 25, 254(2), 253 - 4 Effect of hydroxyurea on T4 ribonucleotide reductase; Berglund O et al.; Phage T4-induced ribonucleotide reductase, purified to homogeneity, catalyzes the reduction of the four ribonucleotides CDP, UDP, ADP, and GDP to the corresponding deoxyribonucleotides . The enzyme is an order of magnitude more sensitive to hydroxyurea than the corresponding Escherichia coli enzyme . Fifty per cent inhibition occurs at 10 micrometer hydroxyurea . Inhibition is complete at a high concentration of the drug, and there is no differential effect on the four substrates . Treatment of T4 ribonucleotide reductase or its isolated subunits with hydroxyurea does not lead to their irreversible inactivation. J Biol Chem, 1979 Jan 25, 254(2), 249 - 52 Purification of the mannitol-specific enzyme II of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system; Jacobson GR et al.; The inducible, mannitol-specific Enzyme II of the phosphoenolpyruvate:sugar phosphotransferase system has been purified approximately 230-fold from Escherichia coli membranes . The enzyme, initially solubilized with deoxycholate, was first subjected to hydrophobic chromatography on hexyl agarose and then purified by several ion exchange steps in the presence of the nonionic detergent, Lubrol PX . The purified protein appears homogeneous by several criteria and probably consists of a single kind of polypeptide chain with a molecular weight of 60,000 (+/- 5%) . In addition to catalyzing phosphoenolpyruvate-dependent phosphorylation of mannitol in the presence of the soluble enzymes of the phosphotransferase system, the purified Enzyme II also catalyzes mannitol 1-phosphate:mannitol transphosphorylation in the absence of these components . A number of other physical and catalytic properties of the enzyme are described . The availability of a stable, homogeneous Enzyme II should be invaluable for studying the mechanism of sugar translocation and phosphorylation catalyzed by the bacterial phosphotransferase system. Biochim Biophys Acta, 1979 Jan 25, 576(1), 128 - 33 Methionyl-tRNA synthetase of Escherichia coli . A zinc metalloprotein; Posorske LH et al.; The native dimeric form of methionyl-tRNA synthetase of Escherichia coli contains two zinc atoms per dimer, one per subunit . The bound zinc is retained upon trypsin modification which yields a monomer with one zinc atom . The enzymatic activity of both the dimeric forms is reversibly inhibited by 1,10-phenanthroline but not by its non-chelating analogues . In addition, the native enzyme binds two Mn2+ per dimer with a binding constant of approx . 70 micron but no binding is observed with the trypsin-modified monomer. J Chromatogr, 1979 Jan 21, 168(2), 489 - 94 Determination of 6-methyladenine in DNA by high-performance liquid chromatography; Yuki H et al.; A method for the determination of 6-methyladenine (6MA) by high-performance liquid chromatography (HPLC) has been developed . DNA bases were separated by using the strong cation-exchange resin Zipax SCX . Purine bases were obtained by hydrolysis and dialysis of DNA and analysed by HPLC . 6MA in DNA from Escherichia coli was determined by the proposed method . It is suggested that the method could be applicable to analyses of 6MA from other biological sources. Mol Gen Genet, 1979 Jan 16, 169(1), 67 - 78 Mechanisms of recombination by the RecBC and the RecF pathways following conjugation in Escherichia coli K12; Mahajan SK et al.; The recombinational processes directed by the RecBC and the RecF pathways following conjugation in E . coli have been compared . The viable recombinant products of the RecF pathway show a higher incidence of mismatch correction, higher percentage of heterogeneous clones produced by single ex-conjugants and a much slower rate of integration and segregation compared to the RecBC pathway . There are reasons to suspect that the product of recB and recC genes may be necessary for conversion of the single stranded donor DNA in the zygote to double stranded DNA . Theoretical considerations suggest that an exchange involving only one strand of DNA may be a much slower process, with more stringent homology requirement for the entire exchanged segment, than a double strand exchange of a comparable length; the latter should be much faster, with stringent homology requirements for only the terminal regions of the exchanged segments . It is suggested that the RecF pathway mainly mediates replacement of relatively long stretches of single strands of recipient DNA by the corresponding strands of donor DNA while the RecBC pathway mediates exchange of mostly double stranded DNA between the donor and the recipient; in addition, the RecBC pathway may also catalyze the integration of very small segments of single strands of the donor DNA . A model based on the above basic hypothesis is described . It is further suggested that the enzymes exonucleaseV and exonucleaseI Control the relative yields of the recombinants produced by the two pathways by regulating the supply of the donor substrates required by these pathways; the former diverts the potential substrate of the RecF pathway (single stranded DNA) to the duplex substrates of the RecBC pathway while the latter destroys the substrates of the RecF pathway, especially in absence of exonucleaseV. Mol Gen Genet, 1979 Jan 16, 169(1), 49 - 57 The control region of the F sex factor DNA transfer cistrons: physical mapping by deletion analysis; Thompson R et al.; A technique has been developed which allows the isolation of random deletions extending from unique restriction enzyme sites in plasmid DNA molecules . The method involves transformation of E . coli cells with linear plasmid DNAs generated by restriction enzyme cleavage . We have used this technique to map DNA transfer genes in the tra control region of F sex factor DNA . Deletions within EcoRI fragment f6 of F DNA have been isolated and used to assign physical locations to tra genes by a combination of genetic complementation tests, restriction enzyme analysis, DNA heteroduplexing and the analysis of the proteins synthesised in minicells and in vitro . Deletion analysis has also allowed the identification of the traK gene product. Biochem J, 1979 Jan 15, 178(1), 97 - 101 Glucose transport of Escherichia coli growing in glucose-limited continuous culture; Hunter IS et al.; Dilute cultures of wild-type Escherichia coli K12 and of derivatives impaired in one or other Enzyme-II component of the glucose phosphotransferase system were grown in continuous culture under glucose limitation . Cells harvested from the chemostat took up {U-14C}glucose from 0.1 mM solutions at rates directly related to the rates at which those cells had grown; the activity of the phosphotransferase system in those cells, rendered permeable with optimal accounts of toluene, parallels the ability of the cells to take up glucose . The capacity of these systems was rate-limiting for growth under the negligibly low glucose concentration in the chemostat, but was adequate to account for the stimulation of respiration observed when the cells were presented suddenly with excess glucose. Biochem J, 1979 Jan 15, 178(1), 133 - 7 Energy coupling to the transport of inorganic phosphate in Escherichia coli K12; Rosenberg H et al.; The nature of the energy source for phosphate transport was studied in strains of Escherichia coli in which either one of the two major systems (PIT, PST) for phosphate transport was present . In the PIT system, phosphate transport is coupled to the proton-motive force . The energy source for the PST system appears to be phosphate-bond energy, as has been found in other systems involving binding proteins . High concentration gradients of phosphate (between 100 and 500) are established by both systems. Eur J Biochem, 1979 Jan 15, 93(2), 345 - 53 Defect in the split proteins of 30-S ribosomal subunits and under-methylation of 16-S ribosomal RNA in a polyamine-requiring mutant of Escherichia coli grown in the absence of polyamines; Igarashi K et al.; Polyphenylalanine synthesis was carried out with Escherichia coli Q13 50-S ribosomal subunits and reconstituted 30-S particles containing different combinations of 23-S core particles and 30-S subunit split proteins obtained from a polyamine-requiring mutant of E . coli during its growth in the presence or absence of putrescine . It was concluded that the defect in the amount of some kinds of 30-S subunit split proteins was responsible for the decrease of polypeptide synthesis in a polyamine-requiring mutant of E . coli grown in the absence of polyamines . The methylation of 16-S RNA during growth in the absence of putrescine was decreased, while the degree of methylation of 23-S RNA did not change significantly . The decrease in methylation of 16-S RNA in the absence of putrescine was due mainly to a decrease of methylation of adenine . The relationship between the decrease of polypeptide synthetic activity of 30-S ribosomal subunits obtained from a polyamine-requiring mutant of E . coli grown in the absence of polyamines and the decrease of methylation of 16-S RNA is discussed. Eur J Biochem, 1979 Jan 15, 93(2), 339 - 43 Cobalt(III) labeling of methionyl-tRNA synthetase from Escherichia coli; Kalogerakos T et al.; Native and trypsin-modified methionyl-tRNA synthetases from Escherichia coli were found to be inactivated by incubation in the presence of Co(III) complexes of ATP, stabilized either by imidazole or phenanthroline, or by oxidation in situ to Co(III) of the substrate ATP-Co(II) . It has been shown that the inactivation proceeds by specific labeling of the catalytic ATP-Mg(II) site of the synthetases . The enzymes are completely inactivated by the incorporation of one cobalt atom and one ATP molecule per active site . The inactivated enzymes may be stored for a long period without significant reactivation or removal of the cobalt label . In the presence of dithiothreitol or 2-mercaptoethanol, the labeled enzymes recover full activity with concomittant release of the bound label molecules. Tijdschr Diergeneeskd, 1979 Jan 15, 104(2), 88 - 9 {Infections with strains of Escherichia coli indistinguishable from non-pathogenic porcine coliform organisms in animals other than swine (author's transl)}; van Ulsen FW; A report on sixty cases in which so-called porcine coliform organisms were isolated post-mortem from various species of animal . Clinically, these were cases of abortion (cattle, dogs) and enterotoxaemia (sheep, dogs, cats, rabbits, goats, guinea pigs, deer and birds). Eur J Biochem, 1979 Jan 15, 93(2), 245 - 56 Mechanism of carbamoyl-phosphate synthetase . Binding of ATP by the rat-liver mitochondrial enzyme; Rubio V et al.; This paper demonstrates, by pulse-chase techniques, the binding to rat liver mitochondrial carbamoyl phosphate synthetase of the ATP molecule (ATPB) which transfers its gamma-phosphoryl group to carbamoyl phosphate . This bound APTB can react with NH3, HCO-3 and ATP (see below) to produce carbamoyl phosphate before it exchanges with free ATP . Mg2+ and N-acetylglutamate, but not NH3 or HCO-3, are required for this binding; the amount bound depends on the concentration of ATP (Kapp = 10--30 microns ATP) and the amount of enzyme . At saturation at least one ATPB molecule binds per enzyme dimer . Binding of ATPB follows a slow exponential time course (t1/2 8--16 s, 22 degrees C), independent of ATP concentration and little affected by NH3, NCO-3 or by incubation of the enzyme with unlabelled ATP prior to the pulse of {gamma-32P}ATP . Formation of carbamoyl phosphate from traces of NH3 and HCO-3 when the enzyme is incubated with ATP follows the kinetics expected if it were generated from the bound ATPB, indicating that the latter is a precursor of carbamoyl phosphate ('Cbm-P precursor') in the normal enzyme reaction . This indicates that the site for ATPB is usually inaccessible to ATP in solution but becomes accessible when the enzyme undergoes a periodical conformational change . Bound ATP becomes Cbm-P precursor when the enzyme reverts to the inaccessible conformation . Pulse-chase experiments in the absence of NH3 and HCO-3 (less than 0.2 mM) also demonstrate binding of ATPA (the molecule which yields Pi in the normal enzyme reaction), as shown by a 'burst' in 32Pi production . Therefore, (in accordance with our previous findings) both ATPA and ATPB can bind simultaneously to the enzyme and react with NH3 and HCO-3 in the chase solution before they can exchange with free ATP . However, at low ATP concentration (18 micron) in the pulse incubation, only ATPB binds since ATP is required in the chase (see above) . Despite the presence of two ATP binding sites, the bifunctional inhibitor adenosine(5')pentaphospho(5')adenosine(Ap5A) fails to inhibit the enzyme significantly . A more detailed modification of the scheme previously published {Rubio, V . & Grisolia, S . (1977) Biochemistry, 16, 321--329} is proposed; it is suggested that ATPB gains access to the active centre when the products leave the enzyme and the active centre is in an accessible configuration . The transformation from accessible to inaccessible configuration appears to be part of the normal enzyme reaction and may represent to conformational change postulated by others from steady-state kinetics . The properties of the intermediates also indicate that hydrolysis of ATPA must be largely responsible for the HCO-3-dependent ATPase activity of the enzyme . The lack of inhibition of the enzyme by Ap5A indicates substantial differences between the Escherichia coli and the rat liver synthetase. C R Seances Acad Sci D, 1979 Jan 15, 288(2), 275 - 7 {A vector system designed to allow excretion of a protein encoded by a cloned gene}; Marchal C et al.; We describe a method which should allow construction of bacterial strains able to export a given protein through the cytoplasmic membrane . The principle is to fuse the structural gene of the protein to the proximal part of gene lamB, the structural gene of the lambda receptor, an outer membrane protein of E . coli K-12. Biochem J, 1979 Jan 15, 178(1), 103 - 7 Artificially induced active transport of amino acid driven by the efflux of a sugar via a heterologous transport system in de-energized Escherichia coli; Bentaboulet M et al.; Consistent with the model of an H+ cotransport, amino acid uptake can be driven by a proton gradient generated by an efflux of sugar when the normal energy sources are suppressed . Heterologous countertransport is completely inhibited by uncouplers unlike homologous countertransport . Positive coupling was obtained with methyl thiogalactoside/proline, methyl thiogalactoside/phenylalanine, gluconate/proline; however, the poor coupling efficiency suggests a more complex sequence of reactions. Mol Gen Genet, 1979 Jan 11, 168(3), 293 - 8 Repair of ultraviolet-light damaged ColE1 factor carrying Escherichia coli genes for guanine synthesis; Kibe A et al.; Hybrid ColE1 plasmids called ColE1-coslambda-qua A or ColE1-coslambda-gal can be efficiently tranduced into various E . coli K-12 cells through packaging into lambda phage particles . Using these plasmids, repair of ultraviolet-light (UV) damaged ColE1 DNAs was studied in various UV sensitive E . coli K-12 mutants . (1) The host mutations uvrA and uvrB markedly reduced host-cell reactivation of UV-irradiated ColE1-coslambda-guaA . (2) Pre-existing hybrid ColE1 plasmids had no effect on the frequency of lambda phage-mediated transduction of another differentially marked hybrid ColE1 DNAs . (3) ColE1-coslambda-guaA and ColE1-coslambda-gal DNAs could temporarily but not stably co-exist in E . coli K-12 recA cells . (4) The presence of ColE1-coslambda-gal in uvrB cells promoted the repair of super-infected UV-irradiated ColE1-coslambda-guaA about 7-fold . (5) The same ColE1-coslambda-gal plasmid in a uvrB recA double mutant did not have this promoting effect . These results indicate that the effect of resident hybrid ColE1 plasmids is manifested by the host recA+ gene function(s) and suggest that ColE1 plasmid itself provides norecA+-like functions. Mol Gen Genet, 1979 Jan 10, 168(2), 197 - 209 Control of the initiation of DNA replication in Escherichia coli . II . Function of the dnaA product; Zahn G et al.; General growth parameters and the kinetics of DNA replication have been determined in merogenotes carrying different combinations of the dnaA+ and the dnaA5 allele . The strain which is homozygous diploid for dnaA5 is different from all other combinations in cell volume, DNA per mass ratio, number of replication points per chromosome, and polymerization rate of DNA . From this we deduce that the dnaA product is a positively acting regulatory protein in initiation . In an appendix we show that in combinations between the dnaA5 and dnaA204 alleles the phenotype of dnaA5 is dominant. Mol Gen Genet, 1979 Jan 10, 168(2), 185 - 95 Control of the initiation of DNA replication in Escherichia coli . I . Negative control of initiation; Tippe-Schindler R et al.; A transient stimulation of the initiation of DNA replication during inhibition of protein synthesis has been demonstrated in dnaA5 and dnaA46 mutants . This suggests the existence of a negatively acting control protein (not the dnaA product) which decays rapidly or is removed during a block in protein synthesis . The stimulation of initiation is dependent on a previous accumulation of initiation specific proteins, which suggests that in addition to the negative control other control mechanisms are effective. Mol Gen Genet, 1979 Jan 10, 168(2), 173 - 84 Repetition of tetracycline resistance determinant genes on R plasmid pRSD1 in Escherichia coli; Mattes R et al.; The 30 megadalton (Mdal)-conjugative, fi- plasmid pRSD1 determines inducible tetracycline resistance (Tc) in Escherichia coli . As shown by restriction analysis, a 3.5 Mdal-EcoRI fragment of pRSD1 spliced into the small plasmid pRSD2124 comprises the entire Tc determinant (tet) region . A restriction map of pRSD1 is presented which includes the location of the tet region and of an "underwound" loop not related to Tc (Burkardt et al., 1978) . Selective amplification of tet genes is demonstrated by three lines of evidence . (i) The resistance level of cell harbouring pRSD1 increases approximately tenfold by induction with 10 microgram/ml of tetracycline . Further growth in the presence of 100 microgram/ml of the drug ("tetracycline stress") selects for cells with even higher resistance levels (about 300 microgram/ml) in rec+ cells . In a recA strain, a smaller proportion of cells attains these high resistance levels suggesting the involvement of host recombination . (ii) Electron micrographs of pRSD1-DNA isolated from tetracycline-stressed cells reveal a heterogeneous population of circular DNA molecules ranging between 1.7 and 21.6 micron . The distribution of contour lengths shows a discrete pattern ascribed to the presence of autonomous single- and multiple-copy Tc determinants and to intact plasmids containing zero to six tet regions in tandem repeats . (iii) This interpretation is supported by heteroduplex and restriction analyses which demonstrate the presence of multiple copies of the 3.5 Mdal-element encompassing the tet region in pRSD1 molecules selected by tetracycline stress . It has been concluded that gene amplification leading to tandem repetition of the tet region ensues in pRSD1 . Such plasmids confer increased tetracycline resistance and can, thefore, be selected by high doses of the drug. Biochemistry, 1979 Jan 9, 18(1), 208 - 13 Rapid purification of biologically active individual histone messenger RNAs by hybridization to cloned DNA linked to cellulose; Childs G et al.; We describe a rapid and simple method for the purification of biologically active messenger RNAs . The method allows the isolation in a few hours of specific mRNAs from either whole cell or polysomal RNA even if the RNA represents less than 1% of the starting molecules . We used, as a model, cloned sea urchin (Strongylocentrotus purpuratus) histone gene fragments linked to cellulose by the method of B . E . Noyes & G . R . Stark (1975) Cell 5, 301--310) as hybridization probes to isolate specific histone mRNAs from whole cell and polysomal RNA extracts . RNAs isolated in this manner maintain their biological activity, serving as templates for histone proteins in a wheat-germ, cell-free protein translation system . In addition, radiolabeled histone-specific RNA purified from cleavage stage sea urchin embryos, pulse labeled for short periods of time and analyzed on denaturing polyacrylamide gels, was the same size as mature histone mRNA's. Biochemistry, 1979 Jan 9, 18(1), 193 - 8 Triphasic concentration effects of gentamicin on activity and misreading in protein synthesis; Tai PC et al.; Gentamicin is shown to exert a triphasic concentration effect on peptide synthesis in vitro with natural messengers . Low concentrations (up to 2 micron) caused slowing and a decrease in total synthesis, but little misreading (assayed with extracts lacking Glu-tRNA); the inhibition was greater with an initiating system (with phage RNA as messenger) than with pure chain elongation on purified endogenous polysomes of Escherichia coli . Moderate concentrations (up to 100 micron) slowed synthesis less, markedly increased its duration in the noninitiating system, and strongly stimulated misreading; at optimal concentrations total synthesis was even greater than normal . Moreover, with phage RNA these concentrations increased the synthesis of large polypeptides . We conclude that binding of gentamicin to its first site causes inhibition but little misreading; binding to additional site(s) partly reverses the inhibition by first-site binding and markedly stimulates misreading, and the misreading appears to favor "readthrough" of termination codons . In the third phase (greater than 100 micron) synthesis is slowed again but the pattern of misreading does not appear to be altered; this effect need not involve a specific further action on the ribosome. Biochemistry, 1979 Jan 9, 18(1), 1 - 11 Lactose carrier protein of Escherichia coli . Transport and binding of 2'-(N-dansyl)aminoethyl beta-D-thiogalactopyranoside and p-nitrophenyl alpha-d-galactopyranoside; Overath P et al.; The elevated level of lactose carrier protein present in cytoplasmic membranes derived from Escherichia coli strain T31RT, which carries the Y gene of the lac operon on a plasmid vector (Teather, R . M., et al . (1978) Mol . Gen . Genet . 159, 239--248), has allowed the detection of a complex between the carrier and the fluorescent substrate 2'-(N-dansyl)-aminoethyl beta-D-thiogalactopyranoside (Dns2-S-Gal) . Binding is accompanied by a 50-nm blue shift in the emission maximum of the dansyl residue . The complex (dissociation constant, KD = 30 micron) rapidly dissociates upon addition of competing substrates such as beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside or upon reaction with the thiol reagent p-chloromercuribenzenesulfonate . Binding of both Dns2-S-Gal and p-nitrophenyl alpha-D-galactopyranoside (alpha-NPG) occurs spontaneously in the absence of an electrochemical potential gradient across the membrane . Comparison of equilibrium binding experiments using Dns2-S-Gal or alpha-NPG and differential labeling of the carrier with radioactive amino acids shows that the carrier binds 1 mol of substrate per mol of polypeptide (molecular weight 30 000) . In addition to specific binding to the lactose carrier, Dns2-S-gal binds unspecifically to lipid vesicles or membranes, as described by a partition coefficient, K = 60, resulting in a 25-nm blue shift in the emission maximum of the dansyl group . Both Dns2-S-Gal and alpha-NPG are not only bound by the lactose carrier but also transported across the membrane by this transport protein in cells and membrane vesicles . The fluorescence changes observed with dansylated galactosides in membrane vesicles in the presence of an electrochemical gradient (Schuldiner et al . (1975) J . Biol . Chem . 250, 1361--1370)) are interpreted as an increase in unspecific binding after translocation. Biochemistry, 1979 Jan 9, 18(1), 202 - 7 Cation-induced regulatory mechanism of GTPase activity dependent on polypeptide initiation factor 2; Beaudry P et al.; Initiation factor IF-2 ribosome dependent GTP hydrolysis (uncoupled GTPase) presents a bell-shaped pH profile which is shifted by changes in ionic strength . At low ionic strength (I = 25 mM) the maximal hydrolytic activity occurs at pH 7.5; when the ionic strength is increased the pH optimum of the reaction is shifted toward more acidic values . Such behavior can be satisfactorily explained as the effect of an electrostatic potential developed by a neighboring polyanion, presumably RNA, on the catalytic site . The addition of fMet-tRNAfMet or AcPhe-tRNAPhe and messenger RNA (coupled GTPase) changes the ionic strength--pH characteristics of the reaction . Thus there is an effect, direct or indirect, of components located at the ribosomal P site . Investigation of the effect of neighboring polyanions on the catalytic activity of the factor-dependent ribosomal GTPases can be seen to provide information about their functional significance that is complementary to that gained from direct structural studies. Biochemistry, 1979 Jan 9, 18(1), 96 - 101 Binding of bleomycin to DNA: intercalation of the bithiazole rings; Povirk LF et al.; At pH 5.5, binding of bleomycin relaxed supercoiled ColE1 DNA without breaking it . Binding of tripeptide S, a fragment of the drug containing the bithiazole rings, also relaxed and then recoiled supercoiled DNA, at pH 5.5 and at pH 8.0, where bleomycin is normally active . The unwinding angle was 12 degrees . Both compounds lengthened linear DNA by 3.1 A per molecule bound, and linear dichroism (303--315 nm) of bleomycin bound to linear DNA oriented in an electric field indicated the presence of a chromophore making an angle of 59--61 degrees with the helix axis . These results strongly suggest that bleomycin binding to DNA involves intercalation of the bithiazole rings . In 0.1 M Na Cl at pH 8, supercoiled ColE1 DNA was broken at a rate 50% greater than relaxed closed circular ColE1 DNA . Since supercoiling increases the affinity of DNA for intercalators, this result suggests that intercalative binding is involved in bleomycin-induced breakage of DNA. C R Seances Acad Sci D, 1979 Jan 8, 288(1), 151 - 4 {Construction of derivatives of lambda phage carrying the lamB gene inserted downstream from the promotors of the lactose operon}; Marchal C et al.; The expression of gene lamB, the structural gene for the lambda receptor in E . coli K-12, has been put under the control of the promoter of the lactose operon . This has been done by in vitro recombination using vectors which are derivatives of phage lambda. Mol Gen Genet, 1979 Jan 5, 168(1), 81 - 6 The use of sym-triazine trichloride in RNA-protein cross-linking studies with Escherichia coli ribosomal subunits; Oste C et al.; The reagent sym-triazine trichloride is used as a bifunctional reagent to generate RNA-protein cross-links within intact ribosomal subunits from E . coli . The reaction takes place in a stepwise manner, involving substitution of one chlorine atom at 12 degrees and pH 8, and substitution of the second at 40 degrees and pH 6 . The cross-linked proteins are analysed by two-dimensional electrophoresis, and the existence of a stable cross-linkage is demonstrated by isolating protein-oligonucleotide complexes from 32P-labelled subunits . The proteins cross-linked are S3 and S4 in the 30S subunit, and L2 in the large subunit, together with smaller amounts of other proteins . The reagent should prove useful in topographical studies of the E . coli ribosome as it is a rigid molecule and generates very short cross-links. Mol Gen Genet, 1979 Jan 5, 168(1), 69 - 80 Degradation of Escherichia coli DNA: evidence for limitation in vivo by protein X, the recA gene product; Satta G et al.; DNA is more extensively degraded after it is damaged in recA mutants of E . coli than in wild type cells . All data presented here are consistent with the recA gene product, protein X, being an inhibitor of nalidixic acid induced degradation of the bulk DNA (but not of newly replicated DNA) . Production of protein X also is correlated with appearance of various "S.O.S." repair functions . Evidence was obtained by comparing the rates of protein X synthesis and solubilization of uniformly-labeled DNA in intact cells, incubated in the presence of nalidixic acid . A set of mutants at the lexA locus produced protein X at different rates and degraded their DNA at rates which were inversely correlated to their rates of protein X production . A low concentration of rifampicin quite specifically inhibited protein X production by wild type E . coli, and allowed more rapid DNA degradation . After the DNA was damaged by the incubation of cells in the presence of nalidixic acid, cells preloaded with protein X degraded their DNA more slowly . We propose that protein X could protect DNA against degradation by binding to single-stranded regions, thereby inhibiting nuclease action. Mol Gen Genet, 1979 Jan 5, 168(1), 37 - 47 Introduction of an active enzyme into permeable cells of Escherichia coli: acquisition of ultraviolet light resistance by uvr mutants on introduction of T4 endonuclease V; Shimizu K et al.; Plasmolysed cells of Escherichia coli N212 (uvr A recA) acquired ultraviolet resistance when the cells were exposed to high concentrations of T4 endonuclease V . With increasing concentrations of T4 enzyme, survivals of plasmolysed cells after ultraviolet irradiation increased while colony-forming ability of unirradiated plasmolysed cells was not significantly affected by the enzyme treatment . Under appropriate conditions more than 200 fold increase in survivals was observed . When plasmolysed cells were treated with a pre-heated enzyme preparation or enzyme fractions derived from T4v1 (endonuclease V-deficient mutant)-infected cells, only little or no reactivation took place . Permeabilization of cells prior to the enzyme treatment was essential for the effective reactivation . Treatment of intact cells with the T4 enzyme did not cause any reactivation . Cells treated with 20 mM EGTA or 50 mM CaCl2 in cold were reactivated to certain extents by the enzyme, but the extents of the reactivation were far less compared to those of plasmolysed cells . Plasmolysed cells of strains carrying a mutation in one of uvrA, uvrB and uvrC genes were reactivated by introduction of T4 endonuclease V, as was the uvrA recA double mutant . UvrD mutants were also reactivated, but rather slightly . However, wild type strain as well as strains having a mutation in recA or polA gene were not reactivated . From these results it was suggested that T4 endonuclease V, taken up into permeable cells, can function in vivo to replace defective functions, which are controlled by the uvr genes . The conditions established in the present study may be used for introduction of other proteins into viable bacterial cells. Mol Gen Genet, 1979 Jan 5, 168(1), 27 - 36 Plasmid replication functions . III . Origin and direction of replication of a "mini" plasmid derived from R6-5; Synenki RM et al.; Replicating DNA molecules of the mini R6-5 plasmid, pKTO71, were purified by equilibrium centrifugation in two successive ethidium bromide-caesium chloride gradients, converted to linear forms by cleavage with either HindIII or BglII restriction endonuclease, and examined in the electron microscope . Determination of the replication fork positions in 65 replicating molecules demonstrated that replication is initiated at a unique location on the plasmid and that it proceeds uni-directionally from this site . The direction of replication is such that the origin-proximal BglII cleavage site is replicated late or, in the case of the parent R6-5 plasmid, is such that the R-determinant region of the molecule is replicated early . The origin of replication, located by these experiments at R6-5 coordinate 98.6 kb, is clearly distinct from that of the R6-5 incompatibility determinant which has been shown to be located on an adjacent PstI-generated DNA fragment whose termini have R6-5 coordinates 96.8 and 97.9 kb . This result indicates that the incompatibility function is not an origin DNA sequence. Biochim Biophys Acta, 1979 Jan 4, 582(1), 145 - 53 Biosynthesis of enterochelin in Escherichia coli K-12: separation of the polypeptides coded for by the entD, E, F and G genes; Woodrow GC et al.; Four enzymic components, coded for by the entD, entE, entF and entG genes, involved in the biosynthesis of enterochelin from 2,3-dihydroxybenzoate have been separated from cell extracts of mutant strains of Escherichia coli K-12 . The starting material for fractionation of the E, F and G components was a cell extract of an entD mutant strain, which yielded the E, F and G enzymic components uncontaminated by a functional D component . The D component was isolated from cell extracts of an entE mutant strain . The conversion of 2,3-dihydroxybenzoate and L-serine into enterochelin is dependent on the presence of all four enzymic components . The E and F components were shown to catalyze ATP-pyrophosphate exchange reactions dependent on 2,3-dihydroxybenzoate and L-serine, respectively, whereas fractionated extracts of the entE and entF mutant strains lacked these reactions . These data provide firm evidence that the E and F components are involved in the initial activation of the substrates . The D and G components are necessary for subsequent and, as yet, undefinedd reactions. Eur J Biochem, 1979 Jan 2, 93(1), 173 - 80 Messenger RNA of the large subunit of ribulose-1,5-bisphosphate carboxylase from Chlamydomonas reinhardi . Isolation and properties; Sano H et al.; Polysomes specifically synthesizing the large subunit of ribulose-1,5-bisphosphate carboxylase were isolated from Chlamydomonas reinhardi cells by the indirect immunoprecipitation method . Electrophoretic analysis showed that the immunoprecipitated polysomes were of chloroplast origin . The mRNA coding for the large subunit which was purified from immunoprecipitated polysomes migrated at the 19-S position on sucrose density gradients, and its molecular weight was estimated to be 7.3 x 10(5) by acid-urea/agarose gel electrophoresis . The mRNA was translated in vivo with a cell-free protein-synthesizing system derived from Escherichia coli to give full-length large-subunit polypeptides. Eur J Biochem, 1979 Jan 2, 93(1), 147 - 56 An investigation of the conformational properties of ribosomes using N-ethylmaleimide as a probe; Ghosh N et al.; The reactivity of ribosomal proteins towards N-ethylmaleimide has been examined in a variety of ribosome and ribosomal subunit preparations from Escherichia coli . The data show that samples which would be regarded as equivalent operationally can differ significantly in conformation, as judged by reactivity, depending on the method of preparation . The washing of ribosomes with high concentrations of salt has a particularly dramatic effect on protein reactivity . The implications of these results for our understanding of ribosome conformation and for the further study of conformation by chemical reactivity are discussed. Arzneimittelforschung, 1979, 29(1), 59 - 63 Interaction of indolmycin in the metabolism of tryptophan in rat liver; Werner RG et al.; When compared with other tryptophan analogues, indolmycin is a potent inhibitor of tryptophan pyrrolase and tryptophan decarboxylase, both enzymes involved in tryptophan catabolism . Otherwise the decarboxylation of 5-hydroxytryptophan is only slightly affected by indolmycin . The indolmycin derivative, 2-methylamino-5-(1-naphthylmethyl)-oxazolidin-4-one (YX-CX 22 XX) demonstrates only a slightly weaker inhibitory effect in the tryptophan-tRNA-ligase system in E . coli but does not show any significant action on the tryptophan metabolism in the eukaryotic system. Tex Rep Biol Med, 1979, 39, 157 - 72 Cardiac performance in hemorrhagic shock; Downing SE; Blood loss of sufficient magnitude to over-ride compensatory mechanisms and result in a lowering of arterial pressure will ultimately lead to irreversible circulatory collapse . Identification of the organ or tissues which may trigger a terminal cascade remains controversial . The weight of evidence supports the view that cardiac performance deteriorates with prolonged oligemic hypotension, although this may not be the initiating or sole reason for irreversible failure of the circulation . Controversy regarding the heart as an important target organ is no doubt in part due to the multiplicity of preparations and protocols, and variety of methods used to characterize cardiac function . We have used ventricular function curves to calibrate LV performance in terms of pump function, while arterial pressure remains at a pre-determined level . With this approach, a progressive decline in stroke volume for a given LV end diastolic pressure is consistently observed in hemorrhagic shock (AP, 30 mmHg) . If arterial pressure is briefly re-elevated at 30 minute intervals, permanent deterioration is prevented . However, if the hypotension is sustained for 2 hours, LV performance remains depressed following pressure re-elevation . Among the mechanisms responsible for deterioration of performance, coronary perfusion pressure (CPP) exerts a pivotal role . Thus, no LV depression occurs after 2 hours of shock provided CPP is maintained at normotensive levels . But if myocardial O2 availability falls below 10 ml/min/100 gm of heart, both O2 uptake and extraction decline and this is uniformly accompanied by cardiac failure . This likely reflects mitochondrial damage and impaired aerobic metabolism . These changes are potentiated by the appearance of metabolic acidosis and failure of sympathetic neurohumoral activity . Both factors directly reduce myocardial contractility, but assume much greater importance during shock . While E . coli endotoxin has been shown to reduce cardiac performance, the relative importance of bacterial products which may enter the circulation during hemorrhagic shock in uncertain . Reduced O2 availability, metabolic acidosis and adrenergic failure appear the major determinants of diminished cardiac performance and thereby may contribute to irreversible collapse of circulatory function. Nucleic Acids Symp Ser, 1979, (6), s119 - 22 Recognition of various arginine transfer ribonucleic acids with arginyl-tRNA synthetase purified from human placenta; Katon N et al.; Arginyl-tRNA synthetase has been purified approximately 550 fold from crude extract of human placenta by the following purification steps: Ammonium sulfate fractionation, chromatographies of DEAE-cellulose and CM-Sephadex and Sephadex G-100 gel filtration . Final preparation of this enzyme has specific activity of 123 nmole of arginyl-tRNA formed per mg of protein and was free from other aminoacyl-tRNA synthetase activities . Recognition of various arginine tRNAs with this enzyme was studied using kinetic analysis of arginylation of arginine tRNA and also arginine tRNA dependent ATP-PPi exchange reaction . Affinity of this enzyme with arginine tRNA was determine from Vmas/Km values and it was in the order of rabbit, Chum salmon, B . subtilis, E . coli and yeast in both systems. Arch Virol, 1979, 61(4), 321 - 5 Recovery of immune responsiveness to rabies vaccine after treatment of mice with cyclophosphamide; Turner GS; Mice treated with cyclophosphamide (Cy) recovered the ability to mount delayed type hypersensitivity reactions within 3 days . By contrast, antibody production to a T cell independent antigen (E . coli lipopolysaccharide) and to human diploid cell strain rabies vaccine did not occur unless the immunization of Cy-treated mice was delayed for 7-10 days . No significant resistance to rabies infection was recorded at 10 days but was demonstrable if 14 days had elapsed between Cy treatment and vaccination. Circ Shock, 1979, 6(3), 277 - 83 Hypoxanthine in lethal canine endotoxin shock; Aasen AO et al.; Endotoxin shock was induced in Labrador retriever dogs by intravenous infusion of a lethal dose of Escherichia coli endotoxin (2 mg/kg body weight) over a three-hour period in order to study plasma hypoxanthine concentrations . The animals succumbed within 14 hours after start of the infusion . Terminally when aortic blood pressure dropped below 30 mm Hg and bradycardia had developed, the animals were resuscitated by external cardiac massage, artificial ventilation, and volume therapy . During shock no significant alteration of plasma hypoxanthine concentrations occurred . During the 12-minute period of resuscitation, however, hypoxanthine concentrations of both arterial and venous plasma increased rapidly compared to the initial values . The changes of the hypoxanthine concentration revealed an exponential pattern . The likely explanation for this phenomenon is is that during shock bypoxanthine was accumlated in the tissues due to tissue hypoxia and that the metabolite was washed out into the circulation during resuscitation. Circ Shock, 1979, 6(3), 261 - 9 Recovery from endotoxin shock after extracorporeal perfusion without anticoagulation; Beller-Todd B et al.; The purpose of this study was to determine the effect of an extracorporeal nonanticoagulated perfusion system on survival from endotoxin shock in anesthetized closed-chest dogs . Dogs weighing approximately 18 kg were perfused four hours or used as nonperfused controls . In the perfused animals, blood was diverted from the distal aorta via plastic tubing at 1,000 ml/min into a reservoir and returned by means of a roller-type pump to the femoral veins . Whole blood clotting times increased from a control value of seven minutes to greater than 24 hours within 45 minutes of perfusion in the absence of exogenous anticoagulation . After blood became incoagulable, animals were infused with 3 mg/kg Escherichia coli endotoxin during a 30-minute period . Systemic pressures declined during the initial period but returned to base-line values; glucose remained at normal levels and all six dogs thus treated remained healthy survivors after seven days . On the other hand, animals infused with endotoxin without extracorporeal perfusion demonstrated hypotension, hypoglycemia, and diarrhea, and five of six dogs died within 36 hours. Circ Shock, 1979, 6(3), 201 - 11 Myocardial function in feline endotoxin shock: a correlation between myocardial contractility, electrophysiology, and ultrastructure; McCaig DJ et al.; The nature of the myocardial depression observed in patients with septic shock, and in animals late in shock induced by endotoxin, is still under examination . These studies, in cats and kittens administered an LD80 dose of E coli endotoxin, were designed to examine the relationship between changes in myocardial contractility, in cellular electrophysiology and in ultrastructural morphology . There was no difference between tension developed in vitro by cardiac muscle removed from cats five hours after endotoxin administration and from cats not administered endotoxin . There was also no difference in their responses to calcium chloride or to anoxia . The action potential characteristics of ventricular muscle isolated from endotoxin treated cats were also not different from control, and ultrastructural damage was minimal and not extensive . Endotoxin (100 microgram/ml) had no effect on cardiac muscle in vitro, even after a one-hour contact time . It is concluded that the integrity of the myocardium is maintained even late in shock and that endotoxin has no direct effects on the heart. Avian Dis, 1979 Jan-Mar, 23(1), 174 - 8 Toxicity of endotoxin to chicks; Adler HE et al.; Large doses of purified lipopolysaccharide (LPS) purified from Escherichia coli induced clinical signs but no mortality in chicks . Five chicks survived a mean dose of 517 mg/kg . One individual that received LPS at 577 mg/kg recovered from clinical manifestations within two days . Attempts failed to produce a generalized Shwartzman-like reaction with two intravenous inoculations of LPS at about 24-hour intervals . Prior injection of uric acid did not protect chicks from LPS by intravenous exposure. Intervirology, 1979, 11(5), 314 - 6 Phage Mu mutants with increased transduction abilities; Teifel J et al.; Mutants of the Escherichia coli phage Mu with increased transduction frequencies are describe . These mutants can be subdivided as (i) mutants in which only the marker used for detection is affected and (ii) mutants in which all other markers tested are also effected, but not to the same extent . The possible mechanisms of the altered transducing properties are discussed. Exp Pathol (Jena), 1979, 17(1), 3 - 11 Renal cortical tubules in experimental malakoplakia . Phagocytic alteration of tubular epithelium; Ormos J et al.; Intrarenal injection of a crude E . coli extract (endotoxin-antigen-complex) induced malakoplakia in rats . Beside the granulation tissue the proximal tubular epithelium showed a strong phagolysosomal response--especially in two-three weeks--, thus becoming very similar to the Hansemann cells of the malakoplakia granulation tissue . This malakoplakia alteration of the epithelium if very severe sometimes led to necrosis or it was segregated inside the epithelial cells . Later on an atrophy of the tubules developed similar to the atrophy of different etiology, but the remaining cells often contained a striking number of residual bodies . It is suggested that the tubular granulated cells of megalocytic interstitial nephritis regarded as identical with renal cortical malakoplakia also have a tubular epithelial origin. Scand J Immunol, 1979, 9(1), 53 - 60 Evidence that soluble products released by PHA-stimulated human lymphoid cells activate immunosuppressive monocytes; Larsson EL et al.; Soluble mediators, lymphokines, released by stimulated lymphoid cells can modify immunological responses in several ways . In this investigation we have examined whether the supernatants of phytohaemagglutinin (PHA)-activated human lymphocytes (active SUPs) contain factors that can suppress proliferative responses of lymphocytes in vitro . The results have shown that crude preparations of peripheral lymphoid cells incubated for 24 h in active SUPs can suppress the responses of cocultured autologous lymphocytes to PPD tuberculin in vitro . Their suppressive activity was not abolished by mitomycin treatment . Some reduction of phytomitogen responses was also noted . Maximal suppressive activity was obtained within 24 h of incubation in active SUPs and it could not be induced in cell preparations depleted of monocytes-macrophages . Similar results were obtained by treating lymphoid cells with lipopolysaccharide from Escherichia coli, which is a known activator of monocytes . These results thus show that lymphokines released by stimulated lymphoid cells can activate monocytes-macrophages in such a way that they become immunosuppressive. Adv Shock Res, 1979, 2, 277 - 87 Endotoxin-induced intravascular coagulation (DIC) and its therapy; Prager RL et al.; Anticoagulants in the form of heparin, dipyridimole, steroids, prostaglandin E1, Macrodex, and antithrombin III were administered in separate experiments prior to endotoxin infusion in the dog . The pattern of disseminated intravascular coagulation (DIC) developed consistently when endotoxin alone was administered . Heparin dosages from 1 to 10 mg/kg did not influence the appearance of thrombocytopenia but effectively eliminated the decrease in fibrinogen levels ordinarily found . Antithrombin III (AT III), obtained from the National Red Cross, administered in a dose designed to provide a doubling of the circulating AT III, reduced the fibrinogen utilization to a similar degree as heparin without affecting the platelet loss . Dipyridimole, as administered, was ineffective in this model, and did not alter the development of thrombocytopenia or the hypofibrinogenemia . Steroids, Macrodex, and prostaglandin E1 had minimal effect on the coagulopathy . Our finding would suggest that the endotoxin effect on dog platelets id direct, and not mediated by thrombin, and that the role of heparin in the clinical management of DIC should be considered only in instances in which renal complications exist. Adv Shock Res, 1979, 2, 249 - 56 Cerebrospinal fluid composition during endotoxin shock in the dog; Raymond RM et al.; Cerebrospinal fluid (CSF) electrolytes, lactate, pyruvate, total proteins, and osmolality, together with arterial plasma electrolytes, total proteins, osmolarity, and systemic arterial blood pressure were monitored during 2 mg/kg and 5 mg/kg E coli endotoxin shock in spontaneously breathing dogs . During four hours of shock, Na+, K+ osmolarity, and total proteins did not change in either plasma or CSF . CSF lactate and lactate/pyruvate ratio were significantly elevated, whereas pyruvate was decreased throughout the four hours of shock . Systemic arterial blood pressure was below control during the entire shock period . Saline control experiments yielded no significant changes in all parameters monitored . These data show that during four hours of shock the brain has shifted form aerobic to anaerobic metabolism the degree of which is not sufficient to cause any detectable changes in membrane permeability or alterations in Na+/K+ active processes. Adv Shock Res, 1979, 2, 245 - 8 Effect of endotoxin on hepatic pyruvate kinase activity in normal and diabetic dogs; Sharma C et al.; The influence of endotoxic administration (0.5 mg/kg) on hepatic pyruvate kinase activity in normal (nondiabetic) and diabetic dogs was investigated . Pyruvate kinase activity was not affected four hours after endotoxin injection in nondiabetic dogs . However, it was stimulated by 288% in dogs that were made diabetic prior to the administration of endotoxin . Since pyruvate kinase activity in diabetic dogs without endotoxin treatment was not affected, these findings suggest that the stimulatory effect of endotoxin on pyruvate kinase activity in diabetic dogs may be associated with insulin deficiency and/or high ambient blood glucose level. Adv Shock Res, 1979, 2, 163 - 75 Myocardial effects of endotoxin shock: characterization of an isolated heart muscle model; Parker JL et al.; Atrial myocardium of guinea pigs was used to study effects of Escherichia coli endotoxin shock on inotropic characteristics of heart muscle free from noncardiac influences of the in vivo shock state . In vitro exposure of atrial muscle to large concentrations of endotoxin (10-1,000 microgram/ml, final concentration) for a prolonged period (90 minutes) had no effect on myocardial contractility . However, atrial muscle isolated from endotoxin-shocked guinea pigs exhibited clear evidence of mechanical depression, as reflected by markedly low values for both isometric contractile tension and maximal rate of tension development (dT/dt) . Also, since systolic and diastolic time intervals of myocardial contractions were not discernibly affected by shock, the contractile deficit represented a true inotropic dys-function and was not due simply to a temporal change in the active state of the muscle . The shock-induced inotropic disorder was permanent enough to persist in vitro for several hours of observation . However, if the Ca++ concentration of the bathing medium was increased from 2.5 mM to maximally effective concentrations ( greater than 4.5 mM), contractile strength of heart muscle from the shocked group was equal to corresponding responses of control muscles . Present findings verify myocardial contractile dysfunction associated with in vivo endotoxin administration and provide characterization of a test system that should prove useful for further study of functional changes occurring to the heart in shock. Adv Shock Res, 1979, 2, 137 - 51 The pony as a model for septic shock; Sembrat RF et al.; This study was conducted to determine the feasibility of using alert, conscious ponies as a model for septic shock in man . Ten ponies were given 0.7-5 X 10(9) organisms/kg of body weight of live E coli intravenously over one hour . All ponies died and exhibited signs of low cardiac output septic shock . significant decreases were found in cardiac index to 3.15 +/- 0.1 liters/min/m2 (P less than 0.05), white blood cell count to 1,930 +/- 100 cells/m3 (P less than 0.05), preterminal blood glucose to 75 +/- 5 mg/dl (P less than 0.05), PaO2 to 75.7 +/- 5.7 mm Hg (P less than 0.05), and pH to 7.15 +/- 0.5 (P less than 0.05) . Increases were noted in systemic resistance to 3,869 +/- 322 dynes/dic/cm-5 (P less than 0.05), pulmonary resistance to 770.8 +/- 11.12 dynes/sec/cm-5 (P less than 0.05), pulmonary arterial pressure to 41 +/- 7 mm Hg (P less than 0.05), pulmonary wedge pressure to 19.5 +/- 2.5 mm Hg (P less than 0.05), intrapulmonary shunt to 16.43 +/- l.73% (P less than 0.05), early blood glucose to 204 +/- 9.0 mg/dl (P less than 0.05), and excess lactate concentration to 53.06 +/- 5.3 mg/dl (P less than 0.05) . From these data it appears that the septic pony shows changes similar to low output septic shock documented in man. Acta Univ Carol Med Monogr, 1979, 94, 1 - 114 Acute endotoxin shock in the dog and an attempt at its therapy; Vyhnanek F; The present study aimed at monitoring of hemodynamic changes proceeding in a standard model of acute endotoxin shock in the dog, and mainly at an attempt at the therapy of its early phases . Acute endotoxin shock represents a grave state with rapidly advancing changes in blood circulation, therefore we studied the effects of selected drugs, intended to regulate the circulation conditions in the course of shock . In a standard model in the dog, in which endotoxin shock had been induced by intravenous injection of Escherichia coli endotoxin dosed 1.75 mg.kg-1, the effects of dopamine and hydrocortisone were investigated . The course of the hemodynamic changes can be divided into three phases, as follows . In the first phase, during endotoxin infusion, the blood pressure sank steeply, and the myocardial contractility, left ventricular pressure, and renal arterial blood flow decreased . The central venous pressure rose . After the conclusion of endotoxin infusion the second phase of shock began . It was characterized by a transitory partial correction of hemodynamics . In the third phase all hemodynamic indicators gradually worsened . An especially grave phenomenon was the reduction of renal arterial blood flow . The fatal effects of the administered endotoxin dose were preventable by infusion of dopamine, or hydrocortisone, or their combination . Dopamine plus hydrocortisone raised the survival quota of animals in endotoxin shock, enhanced the renal arterial blood flow, maintained the diuresis, enhanced myocardial contractility, and elevated left ventricular blood pressure. Pol Arch Weter, 1979, 22(1), 87 - 99 {Activity of various enzymes in the blood, liver, intestines and kidneys in spontaneous colibacteriosis of piglets}; Malinowska A; The purpose of the studies was to determine the activity of enzymes in the serum and pig organs with colibacteriosis in the form of oedema and stomach-intestines disease playing a diagnostic role in the determination of the organ damage degree . Studies were carried out on 29 pigs . The activity of LAP, GPT, GOT, AM, FDPA, Lp, AP and AcP was determined in the serum liver, empty intestines and kidneys of the diseased and control pigs . It resulted from the experiments that in the serum of all the diseased animals the activity of FDPA and GOT was considerably increased and that of AP decreased . So, activity determination does not allow to differentiate both forms of colibacteriosis . AM activity increases only in the serum of oedema pigs . In colibacteriosis of pigs the determination of FDPA activity is the most sensitive enzymatic test . In both diseases the activity of this enzyme increases considerable in the serum and all the studied organs . The activity of the studied enzymes shows that in both forms of collibacteriosis of pigs liver, intestines and kidneys are damaged. Ciba Found Symp, 1979, (75), 227 - 51 Studies of the ATP dependence of protein degradation in cells and cell extracts; Goldberg AL et al.; Experiments with metabolic inhibitors in vivo indicate that intracellular protein degradation requires the continuous production of ATP . We have established soluble cell-free preparations from rabbit reticulocytes, rat liver, and Escherichia coli that degrade abnormal protein in an ATP-dependent fashion . These enzymes appear to be responsible for the selective breakdown of abnormal protein that may result from mutations, biosynthetic errors or intracellular denaturation . Experiments with inhibitors indicate that this process and the degradation of many short-lived normal proteins does not occur in the lysosome . The cell-free extracts prepared from these crude extracts hydrolyse {14C} globin by a process stimulated 2--3-fold by ATP and to a lesser extent by GTP, CTP or UTP . These activities degrade globin to large peptides which are then cleaved by soluble peptidases . The ATP-stimulated protease that partially purified from rat liver cytoplasm is also stimulated by pyrophosphate . This protease has an apparent molecular weight of 480,000 . In contrast, the E . coli enzyme has an apparent molecular weight of 115,000 and is completely dependent on ATP, after partial purification by ion exchange and gel chromatography . This enzyme can be distinguished from six other proteolytic enzymes from E . coli active at pH 7.8 . E . coli contains, in addition, four proteases that are not stimulated by ATP and degrade globin to acid-soluble material . We have also demonstrated in E . coli and reticulocytes other proteases that appear specific for small protein substrates and may play a role in the later steps in protein breakdown . The ATP-stimulated endoproteases appear to catalyse the rate-limiting steps in intracellular protein breakdown . However, the actual role of ATP in the degradative process is not known. Infection, 1979, 7 Suppl 6, 551 - 3 {In vitro activity of oral cephalosporins (author's transl)}; Wiedemann B et al.; The in vitro activity of cephradine, cephalexin, and cefaclor was studied in comparison with that of cephalothin . Cefaclor inhibits peptidoglycan synthesis somewhat more than cephalexin and cephradine . There is no marked difference in the stability of the three substances in the presence of beta-lactamases, and the outer membrane of Escherichia coli is no barrier to permeation of any of the three antibiotics. Acta Biol Med Ger, 1979, 38(9), 1259 - 69 Lethal cellular changes induced by near ultraviolet radiation; Tyrrell RM; There is clear evidence that significant quantities of lesions are induced in DNA by near-UV radiation and that these lesions, although susceptible to repair, may lead to cell death because of the simultaneous disruption of DNA repair systems by the same wavelengths . No particular DNA lesion can be linked to cell death in wild type strains . However, there are good grounds for speculating that a type of near-UV lesion exists which is rapidly "fixed" as a lethal event in cells as a result of the oxygen-dependent disruption of repair . There is a strong indication that the relative ability of various near-UV wavelengths to sensitize cells to heat, chemicals or other radiations is directly related to their efficiency in disrupting DNA repair systems in general . Some important specific questions remain . For example, it is important to ask why breaks formed at 365 nm and 405 nm, although apparently requiring a pol dependent pathway for their repair, do not produce the predicted lethal biological action in the strains tested . In general terms it is hoped to provide more comprehensive physico-chemical data in support of, or contradicting, the proposed model. Ciba Found Symp, 1979, (72), 191 - 204 Studies on the nature and regulation of the cellular thio:disulphide potential; Ziegler DM et al.; Microsomal fractions separated from homogenates of liver, kidney and corpora lutea contain a monooxygenase (dimethylaniline monooxygenase {N-oxide forming}, EC 1.14.13.8) that catalyses NADPH- and oxygen-dependent oxidation of cysteamine to cystamine . The monooxygenase purified to homogeneity from hog liver also catalyses oxygenations of diverse xenobiotics, but it does not catalyse oxidation of any other physiological sulphur- or nitrogen-containing compounds . All the available evidence indicates that cysteamine is the physiological substrate for the monooxygenase, and the oxidation of this thiol to the disulphide may be a significant source of disulphide maintaining the cellular thiol:disulphide potential . The concentration of protein-low molecular weight mixed disulphide is a function of this potential . Changes in concentration of this protein-mixed disulphide reflect changes in thiol:disulphide balance . At constant substrate concentrations the potential would depend primarily on activity of the cytosol glutathione reductase (NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2) relative to that of the membrane-bound monooxygenase . In hepatic tissue from adult mice and hamsters there is a correlation between the concentration of protein-mixed disulphide and the activity of the monooxygenase relative to the reductase . Hepatic glutathione reductase is relatively constant in mice, but the monooxygenase is much higher in the female than in the male . After gonadectomy monooxygenase activity decreases in the female and increases in the male . Activities are restored to control levels by treating males with testosterone and females with progesterone . Testosterone decreases and progesterone increases activity . These two hormones apparently regulate the level of this enzyme in hepatic tissue. Acta Physiol Acad Sci Hung, 1979, 54(4), 389 - 99 Age-related differences in thermoregulatory responses to endotoxin in rabbits; Szekely M et al.; E . coli endotoxin evokes fever in rabbits immediately after birth . In 0--3 day-old rabbits the fever is monophasic and brown fat thermogenesis is mainly responsible for the reaction . In 6--10 day-old animals the fever is usually biphasic and increased heat conservation also contributes to the response . An inverse relationship exists between the endotoxin dose and the latent period before the onset of fever, while the height of the fever is independent of the endotoxin dose . The response is similar as that of adult rabbits, except that after all endotoxin doses the latent period is longer and the magnitude of the response slightly smaller in the newborn. Zentralbl Bakteriol Naturwiss, 1979, 134(7), 624 - 6 {On the antiviral effect of a triazole compound (author's transl)}; Menzel G et al.; 4-{Hydroxy-3-piperid-N-yl-prop-1-yl}-5-methyl-2-phenyl-triazole (HMPT) reduced the plaque formation caused by M 12, f 2, and Q beta and retarded the liberation of phages . The concentration of tobacco mosaic virus in primarily infected tobacco leaves was decreased . HMPT inhibited the development of local lesions . Free phages and TMV particles werenot influenced by HMPT. Adv Exp Med Biol, 1979, 121B, 455 - 69 Studies on the endotoxin induced tumor resistance; Nowotny A et al.; In summary, this report has discussed the immunologic mechanisms involved in the enhancement of nonspecific resistance to tumor by endotoxin . The optimal conditions for tumor protection involved pretreatment with approximately 25 mug LPS administered at the siteof subsequent tumor challenge . In an attempt to relate endotoxin structural components to the ability to enhance TUR, a variety of whole LPS's, endotoxic glycolipids and PS preparations were compared . While all of the intact LPS's and several of the glycolipids were effective in enhancing TUR, some endotoxic glycolipids were totally inactive although they were equally toxic . Some lipid-free PS preparations were also active although less than whole LPS . Evidence was presented to suggest that mechanism for enhancement of TUR involves B cells and macrophages but not T cells . The mechanism also involves the production of soluble factors which are released into the serum of mice in response to LPS or PS . These factors can transfer and mediate the antitumor effects of LPS . The preinfection of mice with BCG enhanced the activity of LPS and PS in the production of antitumor activity. Acta Microbiol Acad Sci Hung, 1979, 26(4), 351 - 61 Elimination of F'lac plasmid by different psychotropic drugs and some related compounds; Farkas S et al.; Desipramine, trimipramine, protriptyline, noxiptyline, promazine, trimeprazine, triflupromazine and chlorprothixene methoiodide eliminated the F'lac plasmid of Escherichia coli, while thiazinanum, toluidine blue, lidocaine and procaine were ineffective in this respect . The plasmid eliminating action of the drug ceased in the presence of 0.05 M magnesium sulphate . Methylene blue did not inhibit plasmid elimination by the psychotropic drugs, and in presence of the dye even lidocaine and procaine became effective . Based on plasmid elimination in the presence of methylene blue and on the selective effects of the lon- mutant, the plasmid eliminating mechanism of psychotropic drugs seems to differ from that of acridine orange. Acta Physiol Acad Sci Hung, 1979, 54(1), 33 - 41 Comparison of the effector mechanisms during endotoxin fever in the adult rabbit; Szelenyi Z et al.; In unanaesthetized adult rabbits an intravenous dose of E . coli endotoxin evoked a febrile rise in colonic temperature at ambient temperatures of 9 to 31 degrees C . The rise in colonic temperature and oxygen consumption did not depend on the ambient temperature, while, among the heat loss effectors, in warmer environments only the depression of respiratory heat loss and in cooler environments only ear skin vasoconstriction contributed to the febrile rise in colonic temperature . In moderately warm environments the endotoxin first induced a maximum inhibition of respiratory frequency and this was followed by vasoconstriction . Later, a transient rise in oxygen consumption occurred . During defervescence the timing of the effectors was reversed . The results showed that a febrile response is not necessarily characterized by simultaneous changes in the thermoregulatory effector mechanisms. Acta Physiol Acad Sci Hung, 1979, 53(4), 469 - 77 Endotoxin fever in capsaicin treated rats; Szekely M et al.; The fever elicited by 10 micrograms/kg intravenous E . coli endotoxin was significantly higher in capsaicin desensitized rats than in controls, but there was an upper limit to the rise in body temperature in both groups . Capsaicin desensitization permitted the participation of various heat producing mechanisms even if the initial body temperature was high . The fever course was characteristically biphasic, similar as in control rats, thus desensitization did not alter the factors mediating the various phases of the endotoxin response. Microbios, 1979, 25(101-102), 167 - 76 Variations in the cell cycle of slow-growing Escherichia coli 15T-; Urban JE; DNA synthesis was measured via 3H-thymidine pulses, and patterns of incorporation were compared to cell number increases during synchronous division of Escherichia coli 15T- (555-7) in M9.01% aspartate . The timing of chromosome initiation, duration of chromosome synthesis, and the D period were found to vary over the three cycles of division analysed . In addition, the D period measured by comparing 3H-thymidine incorporation and concomitant division, differed from D period measurements made by timing division cessation in DNA synthesis inhibitors. Acta Microbiol Acad Sci Hung, 1979, 26(3), 255 - 62 An altered heart-labile enterotoxin (LT') produced by Escherichia coli serogroup O55 strain; Ketyi I et al.; An Escherichia coli serogroup O55 strain produced heat-labile enterotoxin only, which exerted unusual effects on cell cultures; it caused elongation of CHO and HeLa cells but no changes in Y-1 cells . Injection of this substance, designated LT', into mouse foot pad and rabbit skin caused a well-expressed necrotic effect beside the LT-like activity . LT' showed no cytotoxic effect and failed to produce mouse lung oedema . The strain was not haemolytic . According to Sephadex G-100 fractionation, the LT' had a high molecular weight . The LT' and the necrotic activities could not be separated by fractionation . Neutralization experiments suggested an antigenic relationship between LT and LT' . The antigenic deficiency of LT' was closely related to the common antigenic component of LT and choleragen . The necrotic effect caused by crude LT' was neutralized only by the homologous serum. Acta Physiol Acad Sci Hung, 1979, 53(3), 279 - 83 Nutritional state and endotoxin fever of newborn rabbits; Szekely M; At thermoneutral environments 6-10 day-old well-fed rabbits responded to 20 microgram/kg I.V . E . coli endotoxin with biphasic fever: temperature peaks at 60 and 120-150, and a transient fall between 60 and 90 min after endotoxin injection . In rabbits starved for 24 hours, and in runt rabbits body temperature did not rise, but a decline started 60 min after endotoxin administration, corresponding to the transient fall observed in well-fed animals and continuing until about the 100-120th min; thereafter body temperature tended to stabilize at the low level. Acta Physiol Acad Sci Hung, 1979, 53(3), 265 - 77 Endotoxin fever in the rat; Szekely M et al.; In rats intravenous injections of E . coli endotoxin at thermoneutral or slightly warmer environmental temperatures resulted in biphasic febrile response: two rises of temperature being separated by a transient fall . At an ambient temperature of 20 degrees C the change in body temperature still had a biphasic pattern, however, the fall was the dominant change . Each part of the response was the result of a coordinated reaction which involved heat production mechanisms (including interscapular and periaortic brown fat thermogenesis) and heat loss effectors (tail vasomotor changes) separately or in combination . Beside ambient temperature, the initial body temperature at the start of endotoxin action exerted an important role in determining which of the effector functions would be involved in the response. Vet Med Nauki, 1979, 16(8), 72 - 7 {Immunobiological activity of various blood serum fractions from cattle and swine . The dynamics of Ig in specific serum against colibacillosis in pigs}; Iotov M et al.; The level of the IgM and IgG agglutinating antibody specific hyperimmune serum against coli-bacteriosis and its fractions were studied . The serum was obtained at various stages of the preparation, hyperimmunization and exploitation of serum producing swine by using the 2-mercapto-ethanol test . It was established that serum of the total globulin, alpha-beta-globulin and gamma-globulin fractions contains IgM and IgG agglutinating antibodies against E . coli . These immunoglobulin classes are synthetized at all stages of serum producing swine preparation, hyperimmunization and exploitation . The quantitative IgM:IgG ratios at the individual stages vary . At the initial stages and until the 10th cycle of exsanguination (phlebotomy) IgM antibodies predominate . After that, at the following stages IgG antibodies begin gradually to predominate . Up to the 20th cycle of exsanguination both globulins are synthetized, but the 2-mercapto-ethanol non-sensitive IgG antibodies are in greater quantity. Pharmazie, 1979, 34(9), 557 - 9 Problems involved in the specification and interpretation of quantitative structure-activity relationships . Part 1: A modified type of structure-activity equations; Mager H et al.; It has been demonstrated that ordinary regression coefficients of a quantitative structure-activity equation depend strongly on the scaling both of the biological parameter and the explanatory variables (regressors) . Even in the case of only two regressors this fact may lead to challengable interpretations regarding the relative performances of the explanatory variables, say physicochemical parameters . The use of standardized variables and, consequently, of standardized regression coefficients is proposed in order to get a better insight into the mechanistic features of the mode of action of the congeneric series under investigation . The procedure has been illustrated with examples adapted from the literature. Acta Microbiol Acad Sci Hung, 1979, 26(2), 135 - 7 Endotoxin tolerance in rats treated with antilymphocyte serum; Bertok L et al.; Rats were treated with rabbit anti-rat lymphocyte serum (ALS) . The subsequent selective immunosuppressive effect on the immune response of thymus dependent antigen was shown by immunization with sheep red blood cells (SRBC) . Endotoxin tolerance could be evoked in ALS treated animals . This suggests that the establishment of endotoxin tolerance is independent of thymus function and makes it possible to enhance the nonspecific resistance of immunosuppressed patients with a transplanted organ. Mol Gen Genet, 1979, 177(1), 85 - 9 Integration of IS3 into IS2 generates a short sequence duplication; Sommer H et al.; The Gal+ allele IS2-43 is known to segregate Gal- clones . Among 11 Gal- segregants, one was shown to be due to the integration of IS3 into IS2-43 . Precise excision of the integrated IS3 element occurred at a rate of 5 x 10(-9)/cell/generation . DNA sequence analysis revealed that the termini of the IS3 element have the relation of imperfect inverted repeats and it is now flanked by a 3bp or 4bp duplication, a size which has not been seen before with other elements. Mol Gen Genet, 1979, 177(1), 155 - 61 Expression of incompatibility by derivatives of the broad host-range inc P-1 plasmid RK2; Meyer RJ; A segment of DNA encoding incompatibility on the inc P-1 plasmid pRK248 was identified by the analysis of deletions generated in vitro, and then cloned into several unrelated and mutually compatible plasmids . These derivatives were tested for expression of P-1 incompatibility . It was demonstrated by transformation experiments that P-1 plasmids were efficiently eliminated from an E . coli host following introduction of any one of the derivatives . However, all the derivatives were compatible with each other . The cloned segment of pRK248 DNA is itself capable of autonomous replication, without being cloned into any plasmid, if plasmid-specified gene products are provided in trans . This satellite plasmid is eliminated from the cell by the inc P-1 plasmid pRK286 . The results argue against a partitioning mechanism as the basis for P-1 incompatibility but are consistent with incompatibility being the consequence of negative regulation of copy number . For the inc P-1 system, susceptibility of the plasmid to elimination, but not its ability to eliminate, requires that the P-1 replication system is active. Contrib Microbiol Immunol, 1979, 6, 146 - 58 Deletions, rearrangements and tandem duplications of recombinant plasmids containing yeast ribosomal DNA; Cohen A et al.; Recombinant DNA plasmids, formed by the insertion of yeast ribosomal DNA into Escherichia coli plasmids pSC101 or pMB9, underwent deletions, rearrangements and tandem duplications . Independently derived deletion products of plasmids, constructed using pMB9 as a vector, are indistinguishable from each other . These deletion products from multimers composed of tandem repeats . In at least one case a plasmid, constructed by inserting an SmaI fragment of yeast ribosomal DNA into pSC101, underwent deletion and rearrangement to form a product in which a segment, consisting of part of the pSC101 sequence and part of the yeast ribosomal DNA sequence, was duplicated to form a tandem repeat . Deletion and rearrangement take place in Rec+, recA- and recB- recC- cells . The rate of deletion in Rec+ cells is higher than in recA- cells . The rate of deletion in minicell-producing, X-ray resistant strains is much higher than in other Rec+ strains.
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