Escherichia coli

Proc Natl Acad Sci U S A, 1977 Jan, 74(1), 44 - 8
Selective and accurate transcription of the Xenopus laevis 5S RNA genes in isolated chromatin by purified RNA polymerase III; Parker CS et al.; Chromatin isolated from immature oocytes was found to contain an endogenous RNA polymerase activity (RNA nucleotidyltransferase; nucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) that synthesizes predominately 5S RNA . However, the levels of total RNA synthesis and 5S RNA synthesis in chromatin were each stimulated 10- to 50-fold by an exogenous RNA polymerase III purified from X . laevis oocytes . The 5S genes in chromatin were transcribed by the exogenous enzyme in a highly selective (3000-fold above random) and predominately asymmetric fashion . A significant fraction of 5S RNA sequences were also found in a discrete transcript, approximately 5S in size . Total RNA synthesis was significantly stimulated when chromatin was transcribed by oocyte RNA polymerase I, murine RNA polymerase II, and low levels of Escherichia coli RNA polymerase . However, these enzymes did not significantly stimulate 5S RNA synthesis above the endogenous levels . Both homologous oocyte RNA polymerase I and III and E . coli RNA polymerase transcribed the 5S genes in deproteinized DNA to approximately the same extent (severalfold above random) and both the sense and anti-sense strands of the gene were transcribed . It appears, therefore, that both chromatin-associated components and a purified RNA polymerase III are necessary and sufficient for the selective and accurate transcription of the 5S RNA genes in vitro.

Microbios, 1977, 18(71), 7 - 25
Oxygen toxicity: comparative sensitivities of membrane transport, bioenergetics and synthesis in Escherichia coli; Brown OR et al.; The oxygen sensitivities of basic cell functions were compared to evaluate their significance as potential causes of the reversible growth inhibition produced in Escherichia coli by exposure to hyperbaric oxygen . Growth and net incorporation of radioactive glucose into cell structure, and specifically in to protein, were completely inhibited in approximately 1/20 of a generation by a gas phase containing 4.2 atmospheres of oxygen . The inhibition occured before there was significant decrement in cellular glucose transport, respiration, or intracellular concentration of adenosine triphosphate . The data indicate that fundamental steps leading to protein biosynthesis from glucose should be examined in the search for specific primary sites of oxygen toxicity.

Folia Microbiol (Praha), 1977, 22(3), 168 - 72
The activity of Escherichia coli DNA-dependent RNA polymerase on DNA templates of different origin . The effect of cAMP; Jiresova M et al.; DNA isolated from different T phages served as a better template for the synthetic activity of unmodified Escherichia coli RNA polymerase in the in vitro system than did the host DNA . cAMP significantly stimulated the activity of such a preparation of RNA polymerase . The stimulation was more pronounced with the host DNA template than with phage DNA . However, the synthetic activity of Escherichia coli RNA polymerase was greater in the presence of cAMP than without it when phage DNA served as the template.

Acta Neurol Scand Suppl, 1977, 63, 145 - 56
In vitro lymphocyte transformation of MS patients to paramyxovirus antigens; Cunningham-Rundles S et al.; The immune response of 21 MS patients and 21 healthy volunteers was tested in vitro lymphocyte transformation assay . The stimulating antigens were measles, mumps, parainfluenza, E . coli, S . aureus and C . albicans . MS patients were found to respond similarly to the normal controls to all antigens tested with the exception of mumps antigen . The maximum responses of the MS patients to this antigen were significantly lower than those observed for the normals . However, since 95 per cent of the MS lower maximum responses were still within the positive range, the immunological significance of this phenomenon will require further clarification.

Proc Natl Acad Sci U S A, 1977 Jan, 74(1), 49 - 53
Nucleotide sequence of the 5' end of araBAD operon messenger RNA in Escherichia coli B/r; Lee N et al.; The transcription reaction in vitro provides a means of analyzing the nucleotide sequence of the mRNA of the araBAD operon . By controlling the time of synthesis, we obtained araBAD mRNA of varying lengths beginning from the 5' end . These 5' fragments were freed of lambda RNA transcripts by successive hybridizations to the sense strands of a pair of lambda ara transducing phages that carry ara genes in opposite orientations . The purified 5' fragments were ordered by their times of appearance during synchronized RNA elongation and by nearest neighbor analyses . The results, when combined with the knowledge of the NH2-terminal sequence of the product of the first cistron (L-ribulokinase gene araB), establish the nucleotide sequence of the first 69 bases at the 5' end of the araBAD operon mRNA . The AUG starter codon for L-ribulokinase is located at positions 29-31 . The sequence is: 5' A-C-C-C-G-U-U-U-U-U-U-U-U-G-G-A-U-G-G-A-G-U-G-A-A-A-C-G-A-U-G-G-C-G-A-U-U-G-C-A-A-U-U-G-G-C-C-U-C-G-A-U-U-U-U-G-C-A-G-U-G-A-U-U-C-U-G-(U)- . . .3'.

J Gen Virol, 1977 Jan, 34(1), 73 - 85
Protection of mice against encephalomyocarditis virus infection by preparations of transfer RNA; Stebbing N et al.; Preparations of bacterial transfer RNA (tRNA), give dose-dependent protection of mice against encephalomyocarditis (EMC) virus infection at up to I mg tRNA per mouse with maximum response when the tRNA is administered around 6 h before infection . Protection occurs with intraperitoneally and intravenously administered tRNA against infections by both these routes . In some experiments significant protection occurs by single treatments of tRNA up to 24 h after infection with virus doses of I X LD100 . Some tRNA preparations of eukaryotic origin do not give significant protection . Protection is not a feature of all species of bacterial tRNA; partially purified valine, tyrosine and phenylalanine tRNAs from Escherichia coli are not protective . tRNA treatment does not induce circulating interferon nor does it 'hypo-reactivate' the protective effect of poly (I).poly (C) treatment of mice . Humoral and cell mediated immune responses do not seem to be involved in tRNA mediated protection since first, cytosine arabinoside treatment does not affect protection by tRNA; second, serum from mice treated with tRNA and an EMC vaccine does not protect other mice against infection, and third, mice that survive normally lethal infections as a result of tRNA treatment are generally just as susceptible to re-infection as previously untreated, uninfected mice . Silica treatment abolishes protection of mice by tRNA implying that macrophages are necessary . However, tRNA does not seem to act by clearance of virus particles since vaccination of mice by inactivated EMC virus is not affected by tRNA treatment . These results are considered in relation to the presence of a tRNA-like structure in EMC virus RNA and protection of mice by other single stranded polynucleotides.

J Gen Virol, 1977 Jan, 34(1), 177 - 82
Aminoacylation of encephalomyocarditis virus RNA; Lindley IG et al.; RNA extracted from purified encephalomyocarditis (EMC) virus (EMC-RNA) can be aminoacylated with synthetase praparations from Escherichia coli, beef and rabbit liver . The extent of aminoacylation is between 0-024 and 0-080 moles per mole EMC-RNA and occurs only with serine . Either removal of possible low mol . wt . contaminants with 3 M-sodium acetate nor periodate oxidation of the virus RNA affects its aminoacylation capacity.

J Bacteriol, 1977 Jan, 129(1), 564 - 6
Role of the rel gene product in the control of cyclic adenosine 3',5'-monophosphate accumulation; Braedt G et al.; The presence of a relA mutant allele affects the kinetics of cyclic adenosine 3',5'-monophosphate accumulation during downshift from glucose to succinate . The nucleotide accumulates at the normal rate early in the downshift transition but continues to accumulate for a longer time in the relA mutant, leading to a two- to threefold excess by the end of the diauxic lag . Evidence is presented that this effect occurs independently of the accumulation of ppGpp.

Folia Microbiol (Praha), 1977, 22(3), 198 - 205
Membrane mutation affecting energy-linked functions in Escherichia coli K 12; Brana H et al.; A small-colony forming variant of Escherichia coli with a mutation in the ncf gene was analysed . The alternation of the protein composition in the cytoplasmic membrane and the interaction with K and E group colicins indicated a membrane mutation . The effect of this mutation on some membrane-bound processes, the activity of Mg2+-activated ATPase, the growth on different carbon sources and the active transport of amino acids, is described . This mutation does not exert any effect on the electron transport system.

Proc Natl Acad Sci U S A, 1977 Jan, 74(1), 193 - 7
A mechanism of duplex DNA replication revealed by enzymatic studies of phage phi X174: catalytic strand separation in advance of replication; Scott JF et al.; The enzyme system for duplicating the duplex, circular DNA of phage phi X174 (replicative form) in stage II of the replicative life cycle was shown to proceed in two steps: synthesis of the viral (+) strand }stage II(+)}, followed by synthesis of the complementary (-) strand }stage II(-)} {Eisenberg et al . (1976) Proc . Natl . Acad . Sci . USA 73, 3151-3155} . Novel features of the mechanism of the stage II(+) reaction have now been observed . The product, synthesized in extensive net quantities, is a covalently closed, circular, single-stranded DNA . The supercoiled replicative form I template and three of the four required proteins--the phage-induced cistron A protein (cis A), the host rep protein (rep), and the DNA polymerase III holoenzyme (holoenzyme)--act catalytically; the Escherichia coli DNA unwinding (or binding) protein binds the product stoichiometrically . In a reaction uncoupled from replication, cis A, rep, DNA binding protein, ATP, and Mg2+ separate the supercoiled replicative form I into its component single strands coated with DNA binding protein . In the presence of Mg2+, cis A, nicks the replicative form I; rep, ATP, and Mg2+ achieve strand separation with a concurrent cleavage of ATP and binding of DNA binding protein to the single strands . rep exhibits a single-stranded DNA-dependent ATPase activity . These observations suggest that the rep enzymatically melts the duplex at the replicating fork, using energy provided by ATP; this mechanism may apply to the replication of the E . coli chromosome as well.

J Bacteriol, 1977 Jan, 129(1), 544 - 6
Pleiotropic phenotype of an Escherichia coli mutant lacking leucyl-, phenylalanyl-transfer ribonucleic acid-protein transferase; Deutch CE et al.; A mutant of Escherichia coli that lacks leucyl-, phenylalanyl-transfer ribonucleic acid-protein transferase had diminished activities of L-phenylalanyl-transfer ribonucleic acid synthetase and tryptophanase, grew faster than its parent with aspartic acid as the sole nitrogen source, accumulated higher levels of enterochelin in the medium during iron limitation, and exhibited an abnormal morphology.

Mutat Res, 1977-78, 47(3-4), 141 - 60
A review of the genetic toxicology of chlorinated dibenzo-p-dioxins; Wassom JS et al.; Information from both published and unpublished sources considered relevant to the understanding of the genetic toxicology of chlorinated dibenzo-p-dioxins is summarized in this review . Interest in writing this paper was stimulated by the fact that this class of compounds, particularly 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has gained notoriety as an extreme environmental and industrial hazard . The potential for human exposure occurs in the work place when dioxins are formed during the synthesis of a number of commercially important compounds such as 2,4,5-trichlorophenoxyacetic acid, hexachlorophene, and pentachlorophenol . Environmental contamination may result from manufacturing processes and from dioxin contaminants in marketed products . Research on dioxins as potential mutagens was initiated because of their structural similarity to acridines, a class of known intercalating agents . To date, only 4 dioxin compounds have been evaluated for mutagenicity: the di-, tetra-, and octa-chlorinated derivatives and the unsubstituted dibenzo-p-dioxin . Since only a few of the many possible structural forms of dioxins have been tested, no definite conclusions can be made about their potential mutagenicity . Furthermore, the positive mutagenicity and cytological effects reported thus far with the few dioxin isomers examined seems to depend on the position of chlorine substitution . The most active form of the molecule is the 2,3,7,8-derivative (TCDD) . Data available for assessing the mutagenic potential of TCDD are conflicting and scarce . Differences in testing results reported in these studies could be attributed to solubility problems with the test chemical, treatment protocols, purity of test samples, or toxicity . Because there are conflicting data, additional experiments are needed before the mutagenic potential of TCDD and other dioxins can be determined . Studies exploring the promoting effect of dioxins on the mutagenicity of other compounds are also recommended because experiments have shown TCDD to be an extremely active liver enzyme inducing agent that enhances the mutagenicity of certain polycyclic hydrocarbons such as 3-methylcholanthrene in vitro . The importance of discerning the hazards to human health from dioxin compounds became apparent after an accidental release of TCDD from a chemical plant contaminated the Seveso, Italy area in July 1976 . This accident revealed that insufficient data were available to properly evaluate the long-term health risks posed by dioxin compounds . Several research projects were therefore initiated after the Seveso incident; it is hoped that many of the questions concerning the mutagenicity of TCDD and possibly of other dioxin congeners will be answered as a result of this work.

Acta Univ Carol Med Monogr, 1977, (77 Pt 1), 113 - 7
Intestinal gradient of enterokinase activity in different species of animals; Malis F et al.; The proximodistal gradient of enterokinase activity was studied in mucosal homogenates of the duodenum, jejunum, ileum, caecum (or megacaecum) and sigmoid flexure of various mammals . The study was carried out in groups of 5 monkeys, guinea pigs, dogs and rats and also in gnotobiological rats--conventional (n = 5), germ-free (n = 10) and monocontaminated (n = 5) with Escherichia coli 0 86 . Enterokinase activity was likewise determined in duodenal mucosal homogenates prepared from material resected at operation from adult humans (n = 5) and from a group of monkeys (n = 5), with and without the addition of Triton X-100 . Enzymatic activity was determined by a modification of Nordstrom and Dahlqvist's method, using pH-stat titration . In all the animals, enterokinase values were unequivocally the highest in the duodenal mucosa; in the other intestinal segments it displayed a marked aboral decrease, so that we found about 30% of duodenal activity in the jejunum, trace amounts in the ileum and zero values in the caecum and the sigmoid flexure . In the individual animals, the enterokinase activity values fell in the sequence monkey greater than guinea-pig greater than dog greater than rat . Enterokinase activity in the human duodenum was practically the same as in the rat duodenal mucosa . No reciprocal differences were found in gnotobiological rats . Either whole homogenate or the supernatant of triton-treated homogenate can be used for the demonstration of enterokinase in laboratory practice . The only part which can be employed for diagnostic purposes in duodenal mucosa.

Acta Microbiol Pol, 1977, 26(2), 129 - 35
Dependence of efficiency of DNA transfer and recombinat formation on cell cycle of Hfr in Escherichia coli K-12; Mycielski R et al.; The efficiency of DNA transfer during the Hfr cell cycle, studied with the use of 3H-thymidine, is the highest in the first half of the cycle . The efficiency of recombinant formation in the Hfr cell cycle demonstrates a similar periodicity only when the ratio of 1 Hfr cell to 8 or more F- cells in mating mixture is maintained . The absence of such changes in the number of recombinants during the cell cycle of a donor with relative excess of Hfr cells seems to be caused by limitation of the number of recombinants by the competent recipent cell fraction.

Acta Microbiol Pol, 1977, 26(2), 119 - 28
Kinetics of pair formation during cell cycle of Hfr and F in Escherichia coli K-12; Mycielski R et al.; Pair formation ability in mating mixture of synchronized Hfr or F- culture and asynchronous culture of appropriate partner was studied . The obtained results allow to conclude that the pair formation ability of F- cells is stable over the whole cell cycle whereas in Hfr cells is it subject to cyclic changes with maximum in the first half of the cycle.

Acta Microbiol Pol, 1977, 26(1), 19 - 26
On the combined action of drugs on the lambda prophage induction in Escherichia coli K12 cells; Gajcy H et al.; The capability of methotrexate, jododeoxyuridine and 5-fluorouracil to induce lambda prophage was compared when given alone or in combination . All these drugs were found to cause inducing conditions in Escherichia coli K12(lambda) cells . Combined action of jododeoxyuridine and methotrexate resulted in a pronounced increase in the number of free phages compared with that resulting the treatment either with methotrexate of jododeoxyuridine alone . Treatment with 5-fluorouracil caused inactivation of plaque forming ability in cells induced with methotrexate.

Microbios, 1977, 19(77-78), 205 - 12
Transport capacity, alkaline phosphatase activity and protein content of glutaraldehyde-treated cell forms of Escherichia coli; Gorman SP et al.; Studies on the comparative transport capacity of various cell forms of Escherichia coli suggest that glutaraldehyde acts only in the outer regions of the cell envelope and to such an extent that transport of alpha-aminoisobutyric acid is reduced by 50% . Alkaline phosphatase activity in whole cells was severely impaired in the presence of alkaline glutaraldehyde and in NaCl-washed cells both acid and alkaline glutaraldehyde (0.01%) caused approximately 80-90% reduction in enzyme activity in 10 min . Protein content was reduced by only 10-15% with this concentration of glutaraldehyde, and cell volume decreased by the same extent . These results are discussed in terms of the mode of action of the disinfectant.

J Neurosci Res, 1977, 3(1), 63 - 72
Endotoxin alters spontaneous transmitter release at the frog neuromuscular junction; Person RJ; The direct neurotoxic effects of E . coli endotoxin (ETX) on spontaneous transmitter release were tested at the frog sartorius muscle neuromuscular junction . Spontaneous transmitter release was monitored by intracellularly recording miniature end-plate potentials (MEPPs) . Junctions were continuously exposed to standard concentrations of 10 microgram/ml of 3 ETX samples, 2 of which produced a significant elevation of MEPP frequency followed by a decline of frequency to very low rates . The third ETX sample, known to have a decreased canine lethality, was without effect on MEPP frequency . No significant changes in MEPP amplitude were evident . The rate of change in MEPP frequency, but not the peak frequency, was reduced by lowering ETX concentrations . Alterations in MEPP frequency induced by ETX were prevented by removing Ca++ and antagonized by high {K+}out . The results suggest that ETX alters ion conductance channels, particularly those for Ca++, in the presynaptic terminal membrane.

Circ Shock, 1977, 4(4), 369 - 77
Endotoxemia and large intestinal blood flow in subhuman primates; Swan KG et al.; The hemodynamic effects of Escherichia coli endotoxin (LD80) were measured in the large intestine of anesthetized Rhesus monkeys to determine whether this organ contributes to the pathogenesis of experimental shock . Inferior mesenteric arterial blood flow (IMF) was measured with an electromagnetic flowmeter . Pressures within the aorta (AP) and portal vein (PP) were recorded . Distribution of colon blood flow was measured with radioactive microspheres: Ce, Sr, and Cr were injected into the left heart . Reference blood samples were obtained from a femoral artery . Mean control IMF was 22.9 +/- 2.2 (SE) ml/min . Aortic pressure was 113 +/- 11 mm Hg, and PP was 6 +/- 1 mm Hg . Arterial blood pH was 7.43 +/- 0.02; pO2 and pCO2 were 93.4 and 37.1 mm Hg, respectively . All parameters were measured at hourly intervals for 4 hr . Neither IMF nor its distribution within the colon changed during the entire observation period . Aortic pressure fell to a low of 60 +/- 6 mm Hg (p less than 0.02) at 3 hr; PP, pO2 and pCO2 were unchanged by endotoxin . Arterial blood pH fell to 7.315 +/- 0.020 at 4 hr (p less than 0.01) . These observations indicate that the colon is not a "target organ" of endotoxic shock in subhuman primates, despite considerable hypotension and metabolic disturbances subsequent to near lethal endotoxemia.

C R Seances Soc Biol Fil, 1977, 171(3), 516 - 25
{Guanyl cyclase activity in the EF-T elongation factor of Escherichia coli}; Macchia V et al.; Highly purified EF-Ts from E . coli does contain guanylate cyclase activity, which is absent from other purified transfer factors, such as EF-Tu and EF-G . Guanylate cyclase activity has been characterized by its sensitivity to inhibitors and substrate specificity . Although the physicochemical properties of guanylate cyclase are closely related to those of EF-Ts, it does not appear to be a contaminant of this transfer factor, but a specific enzyme . The possible role of guanylate cyclase in protein synthesis is discussed.

Nucleic Acids Res, 1977, 4(5), 1513 - 37
Shear degradation of DNA; Adam RE et al.; A concentric-cylinder flow-birefringence instrument is used to generate sufficient shear fields to break T2 DNA (M = 1.2 X 10(8)) and E . coli DNA (M = 2.5 X 10(9)) in dilute solution . Breakage is monitored in situ by measuring the change in birefringence relaxation after the flow has been stopped . The breakage of T2 DNA follows first-order kinetics . Rate constants are obtained as functions of shear rate and viscosity (varied by adding glycerol) . The data are fitted by a modified Arrhenius equation, assuming that stess increases the rate by lowering the activation energy . The rate increases with temperature, pH, and water concentration, and appears to be a base-catalyzed hydrolysis of the phosphate-ester linkage . La3+ ions catalyze the reaction . E . coli DNA was reduced to half molecules at a shear stress of 0.4 dynes/cm2, which is about 2500 times less than that required for T2 . The difference in rates is accounted for in part by the difference in size of the two, but may also reflect the presence of many single-strand nicks in the coli DNA.

J Pak Med Assoc, 1977 Jan, 27(1), 262 - 4
L-asparaginase--antileukemia agent; Bukhari SZ et al.; This paper describes the most recent development in the field of biosynthesis of L-Asparaginase by means of various culture, purification by Column Chromatography, informations of the Catabolite repression of L-Asparaginase and its metabolism in mycobacteria . The separation, purification studies, idea about the effect of different PH on activity, quaternary structure and the effect of specific antibodies on catalytic activity of L-Asparaginase is discussed . This material shows the role of L-Asparaginase in acute lymphocytic leukemia.

Z Allg Mikrobiol, 1977, 17(3), 221 - 5
{Bistability in the activity of glutamine synthetase in ammonium-limiting chemostat cultures of Escherichia coli ML 30}; Muller PJ et al.; The approximative estimation of the function micron({NH+4}) in cultures of E . coli ML 30 had shown that bistability of the ammonium concentration in ammonium limited continuous cultures could be possible (BERGTER et al . 1977) . This phenomenon suggested a bistability in the regulation of ammonia assimilation . Therefore, the activity of one key enzyme of the two ammonia assimilation systems was measured . The distribution of the activity of glutamine synthetase in ammonia limited continuous cultures after different transition states confirmed this suggestion.

Acta Biochim Pol, 1977, 24(1), 53 - 8
Purification and properties of L-asparaginase EC-2 from Escherichia coli 055:B5; Pajdak E et al.; 1 . L-Asparaginase has been isolated from aerobically grown Escherichia coli 055:B5 and purified about 140-fold in a three-step procedure involving acidification to pH 4.5, ammonium sulphate fractionation and column chromatography on DEAE-Sephadex A-50 . The activity of the preparation is 140 U/mg protein . 2 . The enzyme acts within a broad pH range (pH 5-9) and is affected neither by PCMB, N-ethylmaleimide nor metal ions . 3 . Molecular weight of the isolated asparaginase is 130 000.

Nucleic Acids Res, 1977 Jan, 4(1), 85 - 98
Equimolar addition of oligoribonucleotides with T4 RNA ligase; Uhlenbeck OC et al.; T4 induced RNA ligase will join equimolar concentrations of two oligoribonucleotides, (Ap)3C and p(Up) 5, to form a single product, (Ap)3Cp(Up) 5, in high yield . The presence of the 3' phosphate on p(Up)5 prevents the oligomer from adding to itself . The pH optimum of the reaction is about 7.5, but less of the undesirable adenylated intermediate, App(Up) 5, forms at pH 8.2 . The reaction rate is a linear function of oligomer concentration from 3 micronM to 0.6 mM . The data suggest that T4 RNA ligase will be a useful enzyme for the synthesis of oligomers of defined sequence.

Bioinorg Chem, 1977, 7(1), 1 - 4
Selective reactions of nucleobases under biological conditions; Beck MT et al.; Pentacyanonitrosylferrate/II/ complex reacts under biological conditions (pH= 7.5, T= 25-40 degrees C, dilute solution) selectively with nucleobases . The reaction with adenine and guanine probably leads to nitrosation . A new compound formed in the reaction with adenine is prepared; both this compound and the pentacyanonitrosylferrate/II/ inhibits the multiplication of Escherichia coli.

J Bacteriol, 1977 Jan, 129(1), 108 - 14
Partial purification and some properties of delta1-pyrroline-5-carboxylate reductase from Escherichia coli; Rossi JJ et al.; delta1-Pyrroline-5-carboxylate (PCA) reductase {L-proline:NAD(P)+5-oxidoreductase, EC 1.5.1.2} has been purified over 200-fold from Escherichia coli K-12 . It has a molecular weight of approximately 320,000 . PCA reductase mediates the pyridine nucleotide-linked reduction of PCA to proline but not the reverse reaction (even at high substrate concentrations) . The partially purified preparation is free of competing pyridine nucleotide oxidase, PCA dehydrogenase, and proline oxidase activities . The Michaelis constant (Km) values for the substrate, PCA, with reduced nicotinamide adenine dinucleotide phosphate (NADPH) or NADH as cofactor are 0.15 and 0.14 mM, respectively . The Km values determined for NADPH and NADH are 0.03 and 0.23 mM, respectively . Although either NADPH or NADH can function as cofactor, the activity observed with NADPH is severalfold greater . PCA reductase is not repressed by growth in the presence of proline, but it is inhibited by the reaction end products, proline and NADP.

Mol Gen Genet, 1976 Dec 31, 142(4), 263 - 75
Reversion of the gal3 mutation of Escherichia coli: partial deletion of the insertion sequence; Ahmed A et al.; The gal3 mutation of E . coli is an insertion of a DNA sequence, 1,100 base pairs in length, into the operator-promoter region of the galactose operon . This mutation reverts spontaneously to gal+ by excision of the insertion to produce stable, inducible revertants, or by tandem duplications of the gal operon to produce unstable, constitutive revertants . The nature of a third class of revertants, which are stable and constitutive, is the subject of the present study . The stable, constitutive class of revertants included approximately 30% of all gal+ revertants obtained from a gal3 (lambda) strain . Although the constitutive reversions could be transduced by lambda, the efficiency was found to be extremely poor and the rare transductants which did appear seemed to originate from abnormal transducing particles . It was concluded that these reversions were not normally packaged by lambda . In order to facilitate the packaging of these reversions, the chlD-pgl region was deleted from the parent gal3 (lambda) strain . Unexpectedly, the gal3 mutation in the majority of these deletions reverted to produce stable, constitutive reversions exclusively . The explanation proposed was that the chlL-pgl deletions had also removed part of the gal operator-promoter . These revertants were not considered to be true representatives of the stable, constitutive class . The specificity of deletion end-points at the insertion was found only in the gal3 (lambda) strain, and not in gal+, gal+(lambda), or gal3 strains . Moreover, the frequency of spontaneous chlD-pgl deletions increased 10- to 15-fold in presence of the gal3 insertions . A lambdagal phage bearing a true stable, constitutive reversion (galc200) was isolated from the revertant strain by subsequent deletion of the chlD-pgl segment (delta31) . Electron micrographs of lambdagal+ and lambdac200 delta31(chlD pgl) DNA heteroduplexes were interpreted to indicate that the stable, constitutive reversion had arisen by a deletion of 3/4 of the gal3 insertion sequence . The main conclusions are: (i) the stable, constitutive reversions of gal3 can arise by partial deletions of the insertion sequence, apparently by elimination of the nucleotide sequence which causes polarity; (ii) the chlD-pgl deletions may exhibit preferential termination at the right extremity of the gal3 insertion in presence of prophage lambda; and (iii) the gal3 insertion appears to inhibit the production of lambdagal particles by providing a nucleotide sequence which is recognized and degraded by a specific endonuclease . It is suggested that inhibition of transducing particle formation by gal3 and the preferred termination of deletions at gal3 might represent related phenomena.

Thromb Haemost, 1976 Dec 31, 36(3), 495 - 502
Severe antithrombin III deficiency in an infant associated with multiple arterial and venous thromboses; Mendelsohn G et al.; Inherited antithrombin III (AT-III, heparin cofactor) deficiency is a rare condition, presenting with thrombotic disease in adult life . This paper reports an 8 months old South African Black male infant with multiple large vessel venous and arterial thromboses, and E . coli septicaemia . This was associated with an extremely low plasma AT-III level . Micronodular cirrhosis and intracytoplasmic hyaline globules in the liver cells were present . These globules were eosinophilic, and PAS-positive after diastase . They measured approximately 5 mu to 30 muin diameter, occurred singly in the liver cells and were located mainly in the periportal areas . The histological findings in the liver are similar to those observed in alpha 1-antitrypsin (AAT) deficiency in which the intracytoplasmic globules represent accumulation of altered AAT . Immunochemical studies carried out on formalin fixed tissue failed to detect cross reaction material with anti-alpha 1 antitrypsin or anti-AT III antiserum . This is the first case report of AT-III deficiency presenting in infancy . It is also the first case associated with distinctive liver pathology . The available data presented are insufficient to distinguish between an inborn defect and acquired caused of the severely depressed AT-III plasma level and the distinctive liver pathology.

J Mol Evol, 1976 Dec 30, 8(4), 317 - 28
Selective disadvantage of non-functional protein synthesis in Escherichia coli; Andrews KJ et al.; Comparison of growth rates of isogenic strains that synthesize varying levels of beta-galactosidase during continuous culture on non-inducing medium indicates that synthesis of low levels of non-functional protein has a small but possibly significant effect upon growth rate.

Biochemistry, 1976 Dec 28, 15(26), 5769 - 75
Location of accessible bases in Escherichia coli formylmethionine transfer RNA as determined by chemical modification; Schulman LH et al.; Chemical modification of Escherichia coli tRNAfMet with 1 M chloroacetaldehyde, pH 5.5-6.0 at 25 degrees C, has been found to result in alteration of six cytidine and five adenosine residues in the molecule . The modified cytidine residues are the same as those previously found to be reactive with sodium bisulfite at pH 6.0 . The accessible adenosine residues are A36 in the anticodon, A58 in the T psi C loop, and A73, A74, and A77 in the 3; terminal sequence . No modification of adenosine residues in the dihydrouridine or variable loops or of adenosine residues on the 3' side of the anticodon loop could be detected . Treatment of fMet-tRNAfMet with chloracetaldehyde gave the same pattern of midofication as was observed with deacylated tRNAfMet . Chemical modification of E . coli tRNAfMet with 2 sodium bisulfite, pH 7.0 at 25 degrees C, resulted in selective modification of exposed uridine residues in the tRNA . Only three sites were found to be reactive: U18 in the dihydrouridine loop, U37 in the anticodon, and U48 in the variable loop . The overall pattern of chemical modification of tRNAfMet is very similar to that found by others for yeast tRNAPhe, supporting the idea that many of the tertiary interactions in the two tRNAs are the same . The adenosine residue at position 58 in the center of the T psi C loop of the initiator tRNA shows unusual reactivity, however, being modified by chloroacetaldehyde at the same rate as the 3' terminal adenosine residue . This result is in sharp contrast to the uniform resistance of nucleotides in the T psi C loop of yeast tRNAPhe to chemical modification.

Biochemistry, 1976 Dec 28, 15(26), 5776 - 83
Physicochomecial studies on interactions between DNA and RNA polymerase . Isolation and mapping of a T7 DNA fragment containing the early promoters for Escherichia coli RNA polymerase; Hsieh T et al.; The cleavage sites in the early promoter region of coliphage T7 have been mapped for four restriction enzymes . They are, from the left end in base pairs, 1100 and 740 for Hinf; 680, 320, 530, 240, 77, and 67 for Hind II; 620 and 530 for Hpa II; 790 for Alu I . The nucleotide sequence between the Hind II site at 680 base pairs from the left end and the Hinf site at 740 base pairs from the left end has been determined, from which the start point of the promoter A3 is located at 720 base pairs from the left end . The start points of the other two major promoters A1 and A2 are deduced to be at 460 and 580 base pairs from the left end, respectively, from the chain lengths of the in vitro transcripts off the 1100 base-pairs long Hinf fragment . Similar to the sequences of a pL and pR promotors of phage lambda and a sequence in Simian Virus 40 used by Escherichia coli RNA polymerase as a promotor, the sequence of the A3 promotor of T7 also has a Hind II restriction site approximately 30 base pairs upstream to the start point of RNA synthesis . No such Hind II sites exist, however, for the A1 and A2 promoters . Experiments on the protection of some of the restriction sites on the 1100 base-pairs-long Hinf fragment by RNA polymerase binding support the electron microscopic observations of others that, in addition to the three sites A1, A2 and A3, there is at least a fourth site at which E . coli RNA polymerase can bind strongly . In addition to the Hind II site at 680 base pairs from the left end and the Hinf site at 740 base pairs from the left end, which are presumably protected by the binding of a single RNA polymerase at the A3 site, the Hind II site at 240 base pairs from the left end is also protected at a level of 5 polymerase molecules/DNA fragment . The possible existence of several minor promotor sites in the early promotor region, in addition to the three major promotor sites, is discussed.

Biochemistry, 1976 Dec 28, 15(26), 5743 - 53
Kinetics of ribosome dissociation and subunit association studied in a light-scattering stopped-flow apparatus; Gorisch H et al.; The association-dissociation kinetics of ribosomes from Escherichia coli have been studied under various conditions in a light-scattering stopped-flow apparatus . The dissociation reaction at 2 mg/ml at 25 degrees C, induced by lowering the MgCl2 concentration from 18 to 3 mM, can best be described by three independent first-order processes with rate constants of 15 s-1, 0.9 s-u, and 3 X 10(-2) s-1, the slowest process comprising about 60% of the overall reaction . The fraction of ribosomes dissociating with the fastest rate (15 s-1) is concentration dependent and becomes negligible at 0.1 mg/ml9 Ribosomes treated with puromycin also show three dissociation rates with essentially the same rate constants as the nontreated samples . The dissociation induced by a high KCl concentration (0.85 MKCl, 18 mM MgCl) also shows three first-order phases with the same rate constants as for the dissociation induced by lowering the MgCl2 concentration . The formation of 70S ribosomes from 30S and 50S subunits, induced by increasing the MgCl2 concentration from 2 to 21 mM, follows second-order biphasic kinetics . A detailed analysis of the kinetic results shows that the two principal ribosomal forms must have one type of subunit in common . When the association data are analyzed assuming that the kinetic heterogeneity arises from two forms of only one subunit, the rate constants are found to be 6.4 X 10(6) and 1.05 X 10(6) M-1 s-1 . Sequential flow experiments show that the rapid and slow association species are to be identified, respectively, with phases II (0.9 s-1) and III 0.03 s-1) of dissociation . Relaxation measurements show that these correspond to type B ("loose") and A ("tight") ribosomes, respectively . Tight and loose ribosomes were isolated by sucrose density centrifugation, and dissociation and association kinetic studies confirmed the above assignments . Furthermore, the rate constants for these ribosoems agreed within experimental error with rate constants derived from analysis of the multiphasic kinetic data . The association rate constants are for ribosomes dissociated by dilution with the appropriate buffer immediately before recording the kinetics of association . Ribosomes dissociated by dialysis overnight against 2 mM MgCl2 show an association rate constant for the slower association reaction (type-A ribosome) that is about four times smaller, whereas the rate constant for the faster process is roughly the same . The activation energies of the dissociation reactions, whether induced by lowering the MgCl2 concentration or increasing the KCl concentration, and the association raaction induced by increasing the MgCl2 concentration are less than 3.5 kcal/mol . The rate constants of the dissociation at 3 mM MgCl2 and of the association reaction at 21 mM MgCl2 do not vary between pH 7.2 and 8.4 . When 30S and 50S subunits are flowed against buffer containing 20 mM spermidine, the association process is monophasic, with an association constant k - 6 X 10(6) M-1 s-1...

J Biol Chem, 1976 Dec 25, 251(24), 779 - 84
Cross-linking of initiation factor IF2 to proteins L7/L12 in 70 S ribosomes of Escherichia coli; Heimark RL et al.; Initiation factor IF2 bound to the 70 S initiation complex with 5'-guanylyl methylenediphosphonate was treated with the cross-linking reagent, dimethyl-3,3'-dithiobispropionimidate . Covalent cross-linking of the factor to ribosomes was demonstrated by stabilization of initiation factor IF2-70S complexes during centrifugation at high salt concentrations . Specific cross-linking of the factor to the 50 S proteins L7/L12 was shown by: (a) co-precipitation of the 50 S proteins L7/L12 by antibodies made against initiation factor IF2 and (b) the appearance of radioactive bands containing {32P}phosphoryl initiation factor IF2 in regions of elevated molecular weight following polyacrylamide/dodecyl sulfate gel electrophoresis . One major band had an apparent molecular weight of 132,000, consistent with its being a dimer containing one copy of L7/L12 (13,000) and initiation factor IF2 (120,000) . This cross-linked species was shown to contain {32P}phosphoryl initiation factor IF2 and 35S-labeled L7/L12 in separate experiments . It is concluded that L7/L12, which were shown previously to be required for the binding of the factor, are located at or near the initiation factor IF2 binding site on the 70 S initiation complex.

Mol Gen Genet, 1976 Dec 22, 149(3), 329 - 33
Transfer gene expression during fertility inhibition of the Escherichia coli K12 sex factor F by the I-like plasmid R62; Gasson M et al.; Further understanding of how the FinQ fertility inhibition system of the I-like plasmid R62 inhibits transfer of the sex factor F has been gained by the use of a genetic assay for individual transfer gene products . The technique involved construction of a series of Flac plasmids carrying suppressible mutations in individual transfer genes together with a FinQ inhibitor-insensitive traQ mutation . The transfer of the Flac double mutants from a strain carrying wild-type Fhis and R62 then indicated the effect of R62-encoded transfer inhibition on the expression of individual F transfer genes . During such inhibition the products of genes traJ, traA, traE, traB and traC were present in quantities sufficient to permit efficient F transfer, whereas the levels of the traF, traH, traG and traD gene products were so reduced as to limit F transfer . These findings and a failure to obtain recombination between traC and traQ mutations suggest that the R62 fertility inhibition system terminates transcription of the transfer operon between traC and traF.

Mol Gen Genet, 1976 Dec 22, 149(3), 323 - 8
Transfection of Escherichia coli by Mu DNA; Kahmann R et al.; Infectivity of Mu DNA was demonstrated in Ca+ +-treated Escherichia coli cells that lacked the nucleases Exo V and Endo I . The efficiency of transfection is about 10(-7) per phage equivalent . Infectivity is destroyed by denaturation of Mu DNA, and cannot be restored by renaturation.

Mol Gen Genet, 1976 Dec 22, 149(3), 297 - 302
Isolation and characterization of temperature-sensitive mutants of Escherichia coli with altered ribosomal proteins; Isono K et al.; The ribosomal proteins of temperature-sensitive mutants of Escherichia coli isolated independently after mutagenesis with nitrosoguanidine were analyzed by two-dimensional gel electrophoresis . Out of 400 mutants analyzed, 60 mutants (15%) showed alterations in a total of 22 different ribosomal proteins . The proteins altered in these mutants are S2, S4, S6, S7, S8, S10, S15, S16, S18, L1, L3, L6, L10, L11, L14, L15, L17, L18, L19, L22, L23 and L24 . A large number of them (25 mutants) have mutations in protein S4 of the small subunit, while four mutants showed alterations in protein L6 of the large subunit . The importance of these mutants for structural and functional analyses of ribosomes is discussed.

Mol Gen Genet, 1976 Dec 22, 149(3), 291 - 6
The effects of the relA gene on the synthesis of aminoacyl-tRNA synthetases and other transcription and translation proteins in Escherichia coli A; Blumenthal RM et al.; The effects of a partial restriction of valyl-tRNA aminoacylation on the synthesis of aminoacyl-tRNA synthetases, ribosomal proteins, and other translation and transcription proteins were examined in otherwise isogenic stringent (relA+) and relaxed (relA1) derivatives of E . coli B . The synthesis of individual ribosomal proteins, elongation factor G, and to a lesser extent elongation factors Tu and Ts, and the valyl- and arginyl-tRNA synthetases was found to be subject to the influence of the stringent control system . The synthesis of the alpha and beta subunits of RNA polymerase and several of the aminoacyl-tRNA synthetases, in contrast, is either not subject to the influence of the stringent control system, or is subject to additional regulatory constraints.

Mol Gen Genet, 1976 Dec 22, 149(3), 279 - 89
Biosynthetic regulation of individual proteins in relA+ and relA strains of Escherichia coli during amino acid starvation; Reeh S et al.; An isogenic pair of Escherichia coli mutants (relA+ tufB valSts and relA1 tufB valSts) has been cultured at several temperatures to establish various degrees of limitation for valyl-tRNA synthetase . The biosynthetic rate of 16 identifiable proteins, most of which are components of the transcription and translation apparatus, was measured by pulse-labelling with {35S}-methionine, followed by protein separation using two-dimensional gel electrophoresis (O'Farrell, 1975) . No single pattern of response to amino acid starvation of biosynthetic rate was observed . EF-Ts, L12 and S6 were found to be under strong stringent and relaxed regulation; EF-G, EF-Tu-A and S1 are under strong stringent, but weak relaxed regulation; EF-Tu-B, alpha, VRS, IRS and ARS are under week stringent and weak relaxed regulation; beta is under weak stringent regulation and does not respond at all to relaxed conditions; the biosynthetic rate of a protein called stringent starvation protein is strongly stimulated, relative to other proteins, in the starved stringent strain.

Mol Gen Genet, 1976 Dec 22, 149(3), 273 - 7
Hyper-recombination in dam mutants of Escherichia coli K-12; Marinus MG et al.; F-prime heterogenotes of dam-3 bacteria segregate F-prime homogenotes at a frequency 30-200 times higher than the isogenic dam+ strain . A hyperrecombination mutant which shows increased recombination between chromosomal duplications was characterized as a dam mutant . The dam-3 allele causes a reduction in linkage of proximal unselected markers in transconjugants and increases the recombination frequency between a pair of closely linked markers . It is concluded that dam mutations confer a hyperrecombination phenotype to the cell.

Mol Gen Genet, 1976 Dec 22, 149(3), 255 - 65
A transcriptional barrier in the regulatory region of the tryptophan operon of Escherichia coli: its role in the regulation of repressor-independent RNA synthesis; Pouwels PH et al.; A study was made of the influence of the growth rate of Escherichia coli bacteria on the transcription of the tryptophan (trp) operon, in various trp repressor negative strains . Selective measurement of the levels of RNA transcribed from the regulatory region (reg) of this operon and from the structural genes, respectively, has revealed that the increase of the rate of trpRNA synthesis with bacterial growth rate (Rose and Yanofsky, 1972) is due to a decrease of the frequency of termination of transcription at the transcriptional barrier in the regulatory region of the operon . In a mutant strain of E . coli with a deletion covering the promotor distal part of the regulatory region of the trp operon where the barrier is located, the rate of trpRNA synthesis is not affected by the growth rate . In suA- strains, in which Rho factor activity is reduced the bacterial growth rate does not affect the rate of synthesis of trpRNA . This result suggests that in wild type bacteria Rho factor contributes to the control of the transcription of the trp operon . In bacteria with a mutation rendering Tryptophanyl-tRNA synthetase (TRSase) inactive (trpS- strains) the rate of trpRNA synthesis is affected by the growth rate in the same way as in the isogenic wild type bacteria . This result indicates that TRSase plays no obligatory role in the control of trpRNA synthesis through a mechanism of termination and anti-termination of transcription, at least not in the studied strains, which carried a relA mutation.

Mol Gen Genet, 1976 Dec 22, 149(3), 243 - 9
A virus-specified mechanism for the prevention of multiple infection--T7- and T3-mutual and superinfection exclusion; Hirsch-Kauffmann M et al.; Co- and superinfection of cells with T3/T7 result in exclusion (mutual or superinfection exclusion) . The exclusion mechanism is also directed against homologous (or identical) virus . Exclusion is established after the adsorption but before the genome becomes available for gene expression or replication, that is only one virus per cell develops . The exclusion is triggered by a constituant of the viral particle . An early T7 gene (M gene) (Schweiger et al., 1975) is essential for the formation of exclusion competent virions.

Biochim Biophys Acta, 1976 Dec 22, 453(2), 418 - 25
UDP-glucose dehydrogenase from Escherichia coli . Purification and subunit structure; Schiller JG et al.; UDPglucose dehydrogenase from Escherichia coli has been purified 330-fold with an overall yield of 27% . A single homogeneous subunit was demonstrated by ultracentrifugation in 6 M guanidium chloride and by dodecyl sulfate-polyacrylamide gel electrophoresis . Since the molecular weight of the intact dehydrogenase is in the order of 86 000 and the subunit weight determined by the dodecyl sulfate-polyacrylamide gel electrophoresis is 47 000, the enzyme consists of two polypeptide chains . The sole amino terminal acid shown by the dansylation technique was arginine . Forty-four tryptic peptides were obtained by peptide mapping, in agreement with the number of arginine and lysine residues/mole protein {43} determined by amino acid analysis . The data are consistent with the presence of two identical or very similar polypeptide chains in E . coli UDPglucose dehydrogenase.

Biochim Biophys Acta, 1976 Dec 22, 453(2), 383 - 90
A study of the enzymic dephosphorylation of beta-casein and a derived phosphopeptide; West DW et al.; beta-Casein, and the phosphate containing peptide derived from it by tryptic digestion, have been dephosphorylated by the action of two phosphatases . Escherichia coli alkaline phosphatase (EC 3.1.3.1) has been shown to remove the phosphates from these substrates in two distinct stages . Substrate molecules retaining three of the original phosphoseryl residues accumulate during the reaction and are resistant to further dephosphorylation at low enzyme concentrations . In contrast bovine spleen phosphoprotein phosphatase (EC 3.1.3.16) achieves complete dephosphorylation of these substrates sequentially without any of the intervening species showing resistance to the action of the enzyme . The phosphopeptide has been partially dephosphorylated by the action of the two phosphatases and the resultant peptides containing three phosphoseryl residues compared in their reactivity toward the E . coli alkaline phosphatase . The results obtained are discussed in relation to the mode of action of the two enzymes.

C R Acad Sci Hebd Seances Acad Sci D, 1976 Dec 20, 283(16), 1815 - 8
{Antiglycogen activity of rabbit antisera raised against a strain of Escherichia coli 013}; Zweibaum A et al.; Rabbit antisera raised against a strain of E . coli 013 (Su 4321/41 013 K11 H11) contain high titres of antiglycogen antibodies . This activity was evidenced through positive immunofluorescent reactions observed on liver and muscle sections . Such reactions disappeared when the sections were previously treated with alpha-amylase . They were inhibited with Oyster glycogen, phenolalcohol, perchloric acid, and trichloroacetic liver extracts . These same substances had no inhibiting activity when treated with alpha-amylase . The immunofluorescent reactions were negative on livers from Mice treated with endotoxins and insulin which are known to deplete the stores of liver glycogen . The antisera gave interfacial precipitates with glycogen and liver extracts . The same substances, as well as the LPS from E . coli 013, had a specific inhibiting activity in a radioimmunoassay test using glycogen linked to bovine serumalbumine labeled with 125I.

Chromosoma, 1976 Dec 16, 59(2), 89 - 101
Electron microscopy of membrane-free folded chromosomes from Escherichia coli; Kavenoff R et al.; Membrane-free folded chromosomes were purified from log-phase cultures of Escherichia coli and prepared for electron microscopy by aqueous (Kleinschmidt and Zahn) spreading . The appearance of the chromosomes depended on the salt concentrations in spreading . At certain salt concentration, the chromosomes resembled rosettes, with supercoiled loops of DNA radiating from a central core containing RNA . The rosettes support previous models deduced from physical studies of folded chromosomes . Apparently, cores contain must of the visible RNA, and the organization of the core is linked to the organization of the DNA loops.

Biochem J, 1976 Dec 15, 160(3), 813 - 6
Effects of dicyclohexylcarbodi-imide on proton translocation coupled to fumarate reduction in anaerobically grown cells of Escherichia coli K-12; Gutowski SJ et al.; The addition of dicyclohexylcarbodi-imide to anaerobic cells of Escherichia coli K12 decreases both the observed extent of proton translocation coupled to fumarate reduction by endogenous substrates and the t 1/2 of proton re-entry after such translocation, but does not affect fumarate uptake . Dicyclohexylcarbodi-imide also inhibits fumarate reductase activity in cell extracts.

Biochem J, 1976 Dec 15, 160(3), 721 - 6
Peptidyltransferase activity of ribosomes and a ribosome precursor from a mutant of Escherichia coli; Sims PF et al.; Escherichia coli strain 15-28 is a mutant with a defect in ribosome synthesis that caused the accumulation of ribonucleoprotein ('47S') particles during exponential growth . These particles are precursors to 50S ribosomes that lack three ribosomal proteins . Peptidyltransferase activity and binding at the peptidyl site of the peptidyltransferase centre are greatly decreased in 47S particles . Both these activities are lower in the 50S and 70S ribosomes of strain 15-28 than in its parent . Unusual assembly of the larger ribosomal subunit in strain 15-28 may produce completed ribosomes with diminished biological activity.

Biochem J, 1976 Dec 15, 160(3), 505 - 19
A study of the influence of magnesium ions on the conformation of ribosomal ribonucleic acid and on the stability of the larger subribosomal particle of rabbit reticulocytes; Cox RA et al.; Mg2+ was shown to affect the conformation of rRNA over the range of 0.03-1.2M-KCl . The species studies were Escherichia coli S-rRNA and L-rRNA (the RNA moieties of the smaller and larger subribosomal particles respectively) and rabbits S-rRNA and L-rRNA . 2 . The addition of Mg2+ to rRNA in reconstitution buffer (0.35M-KCl0.01M-Tris/HCl, pH7.2) at 20 degrees C let to an increase in bihelical secondary structure through the formation of additional (mainly A-U) base-pairs (e.g . an additional approx . 58 A-U base-pairs per molecule of E . coli S-rRNA as judged by u.v . difference spectrophotometry...

Experientia, 1976 Dec 15, 32(12), 1572 - 3
Arabinose nucleoside triphosphates are no inhibitors for DNA-dependent RNA polymerases; Muller WE; 1-Beta-D-arabinofuranosylcytosine-5' -triphosphate and 9-beta-D-arabinofuranosyladenosine-5' -triphosphate were found to have no inhibitory potency for both mammalian DNA-dependent RNA polymerase II and E . coli DNA-dependent RNA polymerase.

Biochim Biophys Acta, 1976 Dec 14, 455(3), 889 - 99
Release of outer membrane fragments from normally growing Escherichia coli; Hoekstra D et al.; A complex containing lipopolysaccharides, phospholipids and proteine separated from the medium by gelfiltration on Sephadex G-200 or by centrifugation . Electron microscopy revealed that this material is released as vesicles and membrane fragements . To determine the origin of these fragments, they were compared to outer and cytoplasmic membranes with respect to keto-deoxyoctulosonic acid, phospholipid, and protein content, phospholipid composition, fatty acid composition, protein distribution on sodium dodecyl sulfate-polyacrylamide gels, buoyant density, and content of several membrane marker enzymes . The results of this comparison indicate that the membrane fragments found in the culture supernatant of normally growing Escherichia coli consist of practically unmodified outer membrane . Possible mechanisms as to the cause of the release of outer membrane fragments, and its relationship to cell-division, are discussed.

Biochemistry, 1976 Dec 14, 15(25), 5474 - 80
Demonstration of ribosome-dependent photoinduced chain breakage of the 16S ribosomal ribonucleic acid component of the Escherichia coli 30S ribosomal subunit; Gorelic L; The effects of 253.7-nm radiation on the structural integrities of free and ribosome-bound 16S ribosomal ribonucleic acid (rRNA) have been elucidated . Exposure of aqueous solutions of Escherichia coli 30S ribosomal subunits to 253.7-nm radiation results in RNA-chain scission and the formation of single-stranded breaks in double-stranded regions of the ribosome-bound 16S rRNA . The minimum doses of incident 253.7-nm radiation required for the first detection of the two types of RNA chain breaks are 2 X 10(20) quanta for single-strand breaks in double-stranded regions of the ribosome-bound 16S rRNA,and at least 5 X 10(20) quanta for RNA-chain scission . In contrast to the photosensitivity of ribosome-bound 16S rRNA toward chain breakage, free 16S rRNA seems to be resistant toward photoinduced chain breakage at doses of incident 253.7-nm radiation up to at least 10(21) quanta.

Biochim Biophys Acta, 1976 Dec 13, 454(3), 429 - 35
Hybridization of bean chloroplast transfer RNAs to chloroplast DNA; Steinmetz A et al.; Bean (Phaseolus vulgaris) chloroplast tRNAsLeu and tRNAsPhe hybridize to chloroplast DNA, whereas the corresponding cytoplasmic tRNA species do not, suggesting that chloroplast transfer RNAs are coded for by chloroplast DNA . The hybridization of the three chloroplast tRNAsLeu or of the two tRNAsPhe isoacceptors is not additive, and the isoacceptors compete with each other in the hybridization to chloroplast DNA, suggesting that these isoacceptors are coded for by the same gene(s) and differ only in the extent of post-transcriptional modification . Although hererologous aminoacylation reactions and comparisons of base composition suggest a resemblance between chloroplast and procaryotic tRNAs, only a slight cross hybridization reaction was observed between chloroplast and Escherichia coli leucyl- or phenylalanyl-tRNAs and DNAs.

Biochim Biophys Acta, 1976 Dec 13, 454(3), 558 - 66
Main binding sites of the carcinogen, 4-nitroquinoline 1-oxide in nucleic acids; Tada M et al.; 4-Hydroxyaminoquinoline 1-oxide, the reduced metabolite of 4-nitroquinoline 1-oxide, was reacted with homopolyribonucleotides through the catalysis of an activating enzyme . It bound specifically to poly(G), poly(A) and poly(X) but negligibly to poly(C), poly (U) and poly(I) . Chromatographic analysis of the acid hydrolysates of carcinogen-bound polynucleotides revealed that the reaction of the carcinogen with polynucleotides yielded two guanine, one adenine and two xanthine adducts . The same kinds of guanine and adenine adducts were found in DNA or RNA isolated from Escherichia coli and mammalian cells that had been exposed to the carcinogen . Analysis of nucleic acids isolated from 4-hydroxyaminoquinoline 1-oxide-treated cells revealed that 4-hydroxy-aminoquinoline reacts in vivo preferentially with guanines, to a less, but significant, extent with adenines and not significantly with pyrimidines.

Biochim Biophys Acta, 1976 Dec 13, 454(3), 567 - 77
The sucrose gradient and native DNA S20,W, an examination of measurement problems; Clark RW et al.; Sedimentation coefficients of T7, T2H AND T4 DNA were determined with isokinetic sucrose gradients in both 0.1 M and 1 M NaCl . The s values were completely equivalent to those measured by analytical ultracentrifuge and no reduction of s20,w was observed due to the presence of sucrose (anomalous sedimentation) . s20,w values are calculated on the basis of both partial specific volume (v) and apparent specific volume (0) . Using the latter value s20,w molecular weight relations are derived for 0.1 M and 1 M NaCl solvents . The glucosylation of T2H and T4 DNA appears to influence s20,w in a manner disproportionate to the molecular weight added by glucose.

Biochim Biophys Acta, 1976 Dec 13, 454(3), 504 - 13
Effect of stringent and relaxed control on transcription of the tryptophan operon from the ptrp promoter and the PL promoter in trp phage; Kuwano M et al.; Tryptophan (trp) mRNA synthesis from the authentic trp promoter (Ptrp) is apparently arrested upon translation blockage, while trp mRNA synthesis as a result of read-through from the Pl promoter of the N gene in trp phage is not so affected . When translation is blocked at a nonpermissive temperature in the temperature-sensitive mutants of Escherichia coli rel carrying altered ribosomal elongation factors G (strain CP78G) and Ts (strain HAK88), CP78G and HAK88 show relaxed and stringent phenotypes respectively in control of RNA synthesis . Under conditions causing translation blockage in both mutants, Pl-promoted synthesis of trp mRNA is not depressed while Ptrp-promoted synthesis of trp mRNA is blocked . The insensitivity of Pl-promoted transcription of the translocated trp operon to stringent control is also confirmed by using a strain 10b6s rel carrying a temperature-sensitive valyl-tRNA synthetase . In contrast to the above observation, transcription of the trp operon from either the Pl and Ptrp promoters in a 10b6r rel infected with trp is inhibited greatly at the nonpermissive temperature . This effect occurs even if the level of trp transcription observed at 30 degrees is already low, thus suggesting that translational machinery is intrinsically abnormal in the rel strain.

Biochim Biophys Acta, 1976 Dec 13, 454(3), 469 - 79
Characterization of the RNA transcribed in vitro from native mammalian DNA by Escherichia coli RNA polymerase; Alonso A et al.; High-molecular-weight native mouse DNA was transcribed with Escherichia coli RNA polymerase under low salt conditions, and the nature of the DNA sequences transcribed determined by molecular hybridization . The results indicated that E . coli RNA polymerase does not transcribe the sequences in native mouse DNA randomly under these conditions . First, hybridization with a large excess of mouse DNA showed that no more than 5% of the RNA synthesized had been transcribed from repeated sequences in the DNA . Second, hybridization with tracer amounts of labelled non-repeated mouse DNA indicated that the bulk of the RNA had been transcribed from less than 1% of the non-repeated sequences and only about 10% had been transcribed from a further 25% of these sequences; the remaining non-repeated sequences in the DNA, amounting to 50% of the genome, were not represented in the RNA synthesized in vitro to any detectable extent . Third, the proportion (40%) of complementary DNA transcribed from mouse-liver nuclear polyadenylated RNA which hybridized with the RNA synthesized in vitro was significantly greater than would have been expected if transcription had been random . The data have also been interpreted as indicating the presence of two types of initiation site for E . coli RNA polymerase in the non-repeated sequences in mouse DNA . The frequencies of their occurrence have been calculated to be one per 150 000 base-pairs and one per 500 base-pairs, respectively.

Eur J Biochem, 1976 Dec 11, 71(2), 509 - 18
Symmetry and asymmetry of the pyruvate dehydrogenase complexes from Azotobacter vinelandii and Escherichia coli as reflected by fluorescence and spin-label studies; Grande HJ et al.; Fluorescence-lifetime measurements of FAD bound to lipoamide dehydrogenase from Azotobacter vinelandii and Escherichia coli were performed . It is shown from these results that the two FAD groups in the isolated dimeric enzyme, as well as in the enzyme in the intact complex of E . coli, are in non-equivalent surroundings . This contrasts with the near equivalence of the FAD groups of both the enzyme and complex isolated from A . vinelandii . Reduction of the complex with Mg2+, thiamine pyrophosphate and pyruvate or with NADH enables the attachment of a maleimide analogue specifically to the lipoyl moieties of the transacetylase(s) . Spin label {N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)maleimide} introduced in such a way proves the existence of at least two different micro-environments around the lipoyl moieties in complex isolated from A . vinelandii . Electron paramagnetic resonance spectra of the specifically spin-labelled complexes from E . coli and A . vinelandii, when dissolved in tricine {N-tris(hydroxymethyl)-methylglycine} buffer, show interactions of at least two electron spins with each other, which indicate that the lipoyl moieties are rather close together . Fluorescent label {N-(1-anilinonaphthyl-4)maleimide} is specifically attached to the lipoyl moiety of the high-Mr transacetylase of the freshly isolated complex from A . vinelandii . From the large differences in the apparent lifetimes tau p and tau m, as detected by phase fluorimetry, it is shown that this fluorscent label is distributed in different micro-environments . The differences observed in energy transfer between fluorescent label, attached to the lipoyl moiety of the high-Mr transacetylase, indicate different conformations of the complex from A . vinelandii . Upon introduction of the label after reduction with NADH a much larger energy transfer, thus a shorter distance, is observed between the label and FAD than when reduction is performed with Mg2+, thiamine pyrophosphate and pyruvate . A similar conformation dependence upon reduction is found for the pyruvate dehydrogenase complex from E . coli . It is thus proposed that the transacetylase of E . coli and the high-Mr transacetylase of A . vinelandii are both non-symmetrically distributed within the complex.

Eur J Biochem, 1976 Dec 11, 71(2), 483 - 91
Characterization and site of action of a soluble protein that stimulates peptide-bond synthesis; Glick BR et al.; A recently identified soluble protein, named EF-P, stimulates peptide bond synthesis from ribosomal-bound N-formylmethionyl-tRNA and the aminoacyl-tRNA analog, puromycin . Using this model of peptide bond formation we have purified this activity approximately 100-fold from ribosome-free extracts of Escherichia coli . In order to study the mechanism by which the EF-P factor stimulates peptide bond formation, we examined and compared the requirements and site of action of the spontaneous and the EF-P-mediated synthesis of peptide bonds . We find that "enzymic" peptide bond synthesis (+EF-P) is characterized by relatively broad temperature and NH4Cl optima, a sharp Mg2+ optimum at 12 mM, and an apparent pKa of approximately 8.5 . The characteristics of enzymic peptide bond synthesis closely resemble those reported for native peptidyl-puromycin formation rather than other models of peptide synthesis . Factor EF-P requires both 30-S and 50-S subunits for activity . The 30-S particle is inactive by itself and may function in the reaction merely to bind the fMet-tRNA substrate . Both the peptidyl transferase and the EF-P binding site may be part of the 50-S subunit . Unlike all other propagation factors, EF-P does not require the 50-S ribosomal proteins L7 and L12 and may therefore occupy a different ribosomal site.

Eur J Biochem, 1976 Dec 11, 71(2), 459 - 67
Enzyme-linked sandwich immunoassay of macromolecular antigens using the rabbit antibody-coupled glass rod as a solid phase; Hamaguchi Y et al.; A highly sensitive sandwich immunoassay of macromolecular antigens using the rabbit antibody Fab' - beta-D-galactosidase complex and the rabbit antibody immunoglobulin-G-coupled glass rod as a solid phase is described . The Fab' fragments of rabbit antibody IgG are conjugated with beta-D-galactosidase from Escherichia coli using N,N'-o-phenylenedimaleimide . Rabbit antibody IgG is coupled to the aminoalkylsilyl glass rods (3 mm in diameter and 5 mm in length) using glutaraldehyde . A wide range of the concentrations of rabbit IgG fraction (20-2000 mug/ml) is effective for coupling, and the amount of rabbit immunoglobulin G coupled can be controlled . The smallest amounts of ornithine delta-aminotransferase from rat liver, human immunoglobulin G and 2,4-dinitrophenyl human immunoglobulin G that can be determined are 0.03, 0.3 and 0.04 fmol, respectively . The sensitivity of the assay for these antigens is affected mainly by the non-specific binding of the complexes to the solid phase and the ability of antigen molecules, adsorbed on the solid phase, to bind specifically the complexes . The assay with the rabbit antibody immunoglobulin-G-coupled glass rods is simpler and more sensitive than that with the rabbit antibody immunoglobulin-G-coupled Sepharose 4B.

Eur J Biochem, 1976 Dec 11, 71(2), 437 - 42
Thermodynamic studies on the specificity of L-isoleucine-tRNA ligase of Escherichia coli MRE 600 . Calorimetric investigations on binding of amino acids and isoleucinol to the enzyme; Hinz HJ et al.; The association enthalpies, delta Ha, involved in the reactions between L-isoleucine:tRNA ligase (AMP-forming) from Escherichia coli MRE 600 (EC 6.1.1.5) and various amino acids have been determined calorimetrically in 50 mM potassium phosphate buffer, at pH 7.5, in the presence of 1 mM dithioerythritol . The delta Ha values for binding of L-isoleucine, L-leucine, L-valine, L-norvaline and L-2-amino-3S, 4-dimethyl pentanoic acid agree within the limits of experimental error in magnitude (3.7 +/- 0.9 kcal mol-1 or 15.5 +/- 3.8 kJ mol-1 at 25 degrees C) and variation with temperature (delta cp = -430 +/- 20 cal mol-1 K-1 or 1799 +/- 84 J mol-1 K-1) . In view of the large differences in the equilibrium constants for the corresponding binding equilibria, the identical association enthalpies suggest that the enthalpic contribution to the Gibbs free energy of binding, delta Ga, cannot be responsible for the specificity of the interaction of the enzyme with the amino acids . It has rather to be inferred that the entropic term, delta Sa, is decisive in discriminating the correct amino acid . Analogous calorimetric binding studies on the reaction between L-isoleucinol and the enzyme suggest that the absence of the carboxyl group renders the association enthalpy more positive (by 4-5 kcal mol-1 or 16.7-20.9 kJ mol-1) with respect to that of the amino acids . The variation with temperature of the delta Ha values, however, practically parallels that of the amino acids.

Eur J Biochem, 1976 Dec 11, 71(2), 327 - 36
Chorismate mutase/prephenate dehydratase from Escherichia coli K12 . 2 . Evidence for identical subunits catalysing the two activities; Gething MJ et al.; On the basis of amino acid composition, tryptic fingerprints and the determination of amino acid sequences around the four cysteine residues, it can be concluded that chorismate mutase/prephenate dehydratase from Escherichia coli K12 consists of identical, or closely similar subunits . It follows from this that the mutase and dehydratase activities of the enzyme are probably catalysed on the one subunit.

Eur J Biochem, 1976 Dec 11, 71(2), 317 - 25
Chorismate mutase/prephenate dehydratase from Escherichia coli K12 . 1 . The effect of NaCl and its use in a new purification involving affinity chromatography on sepharosyl-phenylalanine; Gething MJ et al.; A new simplified procedure for the purification of chorismate mutase/prephenate dehydratase, based on affinity chromatography on Sepharosyl-phenylalanine, has been developed . The method utilizes the effect of NaCl on the binding properties of the enzyme . NaCl inhibits both the mutase and dehydratase activities of the enzyme . In each case this inhibition is cooperative indicating homotropic interactions between NaCl binding sites on the enzyme . In addition NaCl induces homotropic cooperative effects between chorismate binding sites and between prephenate binding sites . NaCl also increases the sensitivity of the enzyme to inhibition by phenylalanine.

Eur J Biochem, 1976 Dec 11, 71(2), 577 - 83
Terminal riboadenylate transferase from Escherichia coli . Characterization and application; Sano H et al.; Catalytic properties of terminal riboadenylate transferase from Escherichia coli and the products of the enzymic reaction were investigated . The kinetic analysis revealed that the reaction obeys the sequential ordered bi-bi mechanism . The application of conditions elaborated in this study resulted in the synthesis of products of defined size and efficient primer utilization . The tRNA(rA)n obtained was a good template for the synthesis of complementary DNA with reverse transcriptase.

Eur J Biochem, 1976 Dec 11, 71(2), 443 - 9
Interaction of DNA with DNA-binding proteins . The characterization of protein HD from Escherichia coli and its nucleic acid complexes; Berthold V et al.; DNA-binding protein HD with a monomer molecular weight of 9000 was isolated from Escherichia coli cells . The protein occurs as a tetramer under native conditions and binds to single and double-stranded DNA and also to RNA . DNA complexed with protein HD is a poor template for DNA synthesis by E . coli polymerase I, II or III holoenzyme . Exonuclease III is hindered in degrading HD-protein-covered double-stranded DNA, whereas exonuclease I can digest complexed single-stranded DNA . Transcription is liqhtly stimulated in the presence of protein HD.

J Biol Chem, 1976 Dec 10, 251(23), 7709 - 16
Nucleotide sequence and in vitro processing of a precursor molecule to Escherichia coli 4.5 S RNA; Bothwell AL et al.; A precursor molecule to the stable 4.5 S RNA species of Escherichia coli has been found to accumulate at 42 degrees in a strain thermosensitive for the function of ribonuclease P . The precursor molecule is 130 nucleotides long . Twenty-two extra nucleotides, starting with pppGp, precede the mature sequence at its 5' terminus . At least 1 extra uridine residue can be found at the 3' terminus . The precursor to 4.5 S RNA is cleaved in vitro by RNase P to generate a 5' end identical to that of the mature 4.5 S RNA.

J Biol Chem, 1976 Dec 10, 251(23), 7577 - 80
Disappearance of specific proteins from the membranes of colicin-treated cells; Knepper JE et al.; Two colicins that affect energy metabolism in Escherichia coli (colicins K and E1) are shown to cause loss of specific membrane proteins from treated cells . Disappearance of these proteins after treatment with colicin K occurs at low multiplicities and is independent of ATPase (EC 3.6.1.4) and phospholipase A (EC 3.1.1.4) activities . The uncouplers carbonyl cyanide m-chlorophenylhydrazone and dinitrophenol do not alter the pattern of membrane proteins.

Mycopathologia, 1976 Dec 10, 60(1), 57 - 62
Methylation and aging in Rhizoctonia solani; Gottlieb D et al.; Our earlier studies had shown that as fungi age, many of their vital functions decrease; in Rhizoctonia solani, protein synthesis is one of the functions so affected . We now find that the ability to methylate tRNA, a vital component of the protein synthesizing system, also decreases with age . This methylation of Escherichia coli tRNA by R . solani methylase preparations increased with the concentration of enzyme and with time of incubation; in both cases the rate of increase was considerably higher for preparations from young cells than for those from old cells . The methylation reaction also increased with the concentration of substrate tRNA, with temperature, at least to 45 degrees C, and with pH to 9.0 . Methylase preparations from R . solani methylated both exogenous E . coli tRNA and yeast tRNA, but were only weakly active on isolated R . solani tRNA . However, acid-precipitated methylases from R . solani were very effective in methylating the homologous exogenous tRNA . Regardless of the source of the tRNA used as substrate, the methylases from older cells were always less active than those from young cells from the same mycelium . No methylase inhibitor was detected in the fungus.

J Biol Chem, 1976 Dec 10, 251(23), 7669 - 74
A new endoribonuclease from Escherichia coli . Ribonuclease N; Misra TK et al.; A new ribonuclease called RNase N was isolated from Escherichia coli . It is a nonspecific endoribonuclease that can cleave rRNA, poly(U), and poly(C) to small oligonucleotides and 5'-mononucleotides . It requires monovalent cations and is inhibited by divalent cations . It is suggested that this enzyme plays a role in the decay of rRNA,under various starvation conditions and perhaps in the decay of mRNA.

J Biol Chem, 1976 Dec 10, 251(23), 7530 - 8
Catalytic mechanisms of glutamine synthetase enzymes . Studies with analogs of possible intermediates and transition states; Wedler FC et al.; Glutamine synthetase enzymes isolated from pea seeds and from Escherichia coli are observed to behave differently in experiments designed to probe reaction mechanism . Although both enzymes were found to bind and release substrates in random order mechanisms (Wedler, F.C . (1974) J . Biol . Chem, 247, 5080-5087), isotopic exchanges with partial reaction systems indicative of a gamma-glutamylphosphate intermediate are catalyzed only by the pea seed enzyme . The E . coli system fails to catalyze any exchanges at appreciable rates unless all substrates are present . This negative result implies either an absolute conformational requirement for bound substrates or that the putative complex (E-Glu-P-MgADP) is exceedingly tight . To test the latter, a nonreactive structural analog of gamma-glutamyl-phosphate, namely 3-(phosphonoacetylamido)-L-alanine (PA2LA), has been synthesized . With the E . coli enzyme PA2LA was found to bind no more tightly than L-glutamate and is strictly competitive versus L-glutamate (Ki = 3 mM) . Thus, failure to catalyze partial exchange reactions indicative of gamma-Glu-P is probably not attributable to tight complex formation . The binding of PA2LA with the pea seed enzyme apparently involves a two-step process: a rapid, reversible step in which PA2LA binds 10-fold more tightly than L-glutamate, followed by a slow (but reversible) process involving very tight PA2LA binding, apparently with enzyme isomerization promoted by nucleotide . The specificity of the two enzymes toward L-methionine-SR-sulfoximine, Met(O)(NH), was also different...

Mol Gen Genet, 1976 Dec 8, 149(2), 225 - 8
Identity of a gene responsible for suppression of aminoacyl-tRNA synthetase mutations with rpsT, the structural gene for ribosomal protein S20; Buckel P; Sepcialized transducing lines of phage lambda carrying segments between thr and car from the E . coli chromosome have been isolated . With help of these phages it has been shown that the gene sups20 (Bock et al., 1974) corresponds to rpsT, the structural gene for ribosomal protein S20.

Mol Gen Genet, 1976 Dec 8, 149(2), 201 - 10
Revertants from RNase III negative strains of Escherichia coli; Apirion D et al.; E . coli strains carrying the rnc-105 allele do not show any level of RNase III in extracts, grow slower than rnc+ strains at temperatures up to 45 degrees C and fail to grow at 45 degrees C . Revertants which can grow at 45 degrees C were isolated . The vast majority of them still do not grow as fast as rnc+ strains and did not regain RNase III activity . The mutation(s) which caused them are suppressor mutations (physiological suppressors) which do not map in the immediate vicinity of the rnc gene . A few of the revertants regain normal growth, and contain normal levels of RNase III . They do not harbor the rnc-105 allele and therefore are considered to be true revertants . By using purines other than adenine it was possible to isolate rnc + pur- revertants from an rnc- pur- strain with relative ease . They behaved exactly like the true rnd+ revertants isolated from rns- strains at 45 degrees C . A merodiploid strain which contains the rnc+ gene on an episome behaves exactly like an rnc+ strain with respect to growth and RNA metabolis, eventhough its specific RNase III activity is about 60% of that of an rnc+ strain; thus the level of RNase III is not limiting in the cell . The rnc- strains show a characteristic pattern of transitory molecules, related to rRNA, 30S, 25S, "p23" and 18S, which are not observed in rnc+ strains . This pattern is unchanged in rnc- strains and in the revertants which are still lacking RNase III, regardless of the temperature in which RNA synthesis was examined (30 degrees to 45 degrees C) . On the other hand, in the rnc+ strains as well as in the true revertants and the rnc+/rnc- merodiploid, the normal pattern of p16 and p23 is observed at all temperatures . These findings suggest that all the effects observed in RNase III- strains are due to pleiotropic effects of the rnc-105 allele, and that the enzyme RNase III is not essential for the viability of the E . coli cell.

Mol Gen Genet, 1976 Dec 8, 149(2), 151 - 8
REPLICAtion of small plasmids in extracts of Escherichia coli: requirement for both DNA polymerases I and II; Staudenbauer WL; The role of the three E . coli DNA polymerases (pol I, II, and III) in the replication of Col E1 DNA and other small plasmids with similar replicative properties was investigated in a soluble in vitro system prepared by freeze-thaw lysis of chloramphenicol-treated cells (Staudenbauer, 1976) . Extracts from isogenic mutants of the polA, polB and polC gene loci deficient in pol I, II, and III respectively were examined for their replicative capacity . It was found that polA and polC extracts are deficient in the synthesis of supercoiled plasmid DNA, whereas the polB mutation has not effect . Deficient extracts could be complemented by addition of purified pol I and pol III holoenzyme . Analysis of the in vitro synthesized DNA by alkaline gradient centrifugation indicates that pol I is involved in an early step of the replication cycle whereas pol III is required at a later stage . These conclusions are confirmed by inhibition studies employing arabionsylcytosine triphosphate (aCTP) which is shown to interfere with pol III as well as pol II . The strong inhibitory effect of aCTP on plasmid replication is not influenced by the polB mutation and mimicks the effects of thermal inactivation of polC extracts . It is suggested that aCTP blocks plasmid ENA replication in vitro by interfering with pol III function

Mol Gen Genet, 1976 Dec 8, 149(2), 135 - 43
Gal mRNA initiated within IS2; Rak B; A strain of E . coli carrying IS2 in the control region of the gal operon has been found to revert to a constitutive phenotype . These revertants can be divided into two classes, which differ in their rate of enzyme synthesis and gal transcription . One revertant synthesizes the gal messenger at a low level (low level revertants), the other at a higher level that exceeds that of the induced wild type two- to three-fold (high level revertants) . In both cases it has been shown that the gal messenger is covalently bound to IS2-RNA, which is transcribed from IS2 in the original orientation . The evidence suggests, that the section of the IS2-element, from which this RNA is transcribed in the case of the high level revertants is smaller than 50 nucleotides long: i.e . the resolution of the hybridization method used for the detection of sequence homologies exceeds that of the electron microscopical heteroduplex technique.

Biochim Biophys Acta, 1976 Dec 8, 452(2), 625 - 8
The incorporation of tritium from tritium-enriched water into UDP-N-acetylglucosamine and UDP-N-acetylmannosamine catalyzed by UDP-N-adetylglucosamine 2-epimerase from Escherichia coli; Salo WL; Uridine diphosphate N-acetylglucosamine 2-epimerase from Escherichia coli 014 K7 H- catalyzes the reversible epimerization of uridine diphosphate N-acetylglucosamine to uridine diphosphate N-acetylmannosamine . During epimerization, tritium from tritium-enriched water is incorporated into both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylmannosamine . The position of incorporation is C-2 of the N-acetylhexosamine moieties.

Biochim Biophys Acta, 1976 Dec 8, 452(2), 566 - 79
Phosphoenolpyruvate carboxylase of Escherichia coli . Studies on multiple conformational states elicited by allosteric effectors with a fluorescent probe, 1-anilinonaphthalene-8-sulfonate; Yoshinaga T; Conformational change of phosphoenolpyruvate carboxylase (orthophosphate: oxaloacetate carboxy-lyase (phosphorylating), EC 4.1.1.31) induced by allosteric effectors was investigated using a hydrophobic probe, 1-anilinonaphthalene-8-sulfonate (ANS) . Kinetic experiments suggested that ANS binds with the enzyme at the sites which are not involved in the catalytic and regulatory functions, though it partially inhibits the enzyme activity with half-saturation concentration (S0.5) of 38.5 muM . Binding experiments showed that a maximum of 2 mol of ANS are able to bind with 1 mol of the enzyme subunit presumably with an equal dissociation constant to each other (34.5 muM) . Flourescence emission of ANS was markedly increased by binding with the enzyme . L-Aspartate, the allosteric inhibitor, and CoASAc and fructose 1,6-bisphosphate (Fru-1,6-P2) the allosteric activators, produced various degrees of change in fluorescence, when added singly or in combinations . The changes were shown to be attributable to the allosteric interactions between the enzyme and effectors from some criteria such as structural specificity, half-saturation concentrations, and heterotropic-homotropic interactions of the ligands . It was concluded from these analyses that the enzyme can be in at least four conformational states which are distinct from each other . Especially noteworthy is the finding that the enzyme, upon simultaneous binding of CoASAc and Fru-1,6-P2, takes a new conformation which is enterely different from those induced by sole binding of each effector . In addition, the heterotropic interaction between the activator and the inhibitor was observed through conformational change by the ANS method, as observed in the kinetic studies.

Mol Gen Genet, 1976 Dec 8, 149(2), 229 - 37
Biochemical, genetic, and regulatory studies of alanine catabolism in Escherichia coli K12; Franklin FC et al.; E . coli K12 was found to utilise both D-and L-stereoisomers of alanine as sole sources of carbon, nitrogen and energy for growth . This capability was absolutely dependent upon the possession of an active membrane-bound D-alanine dehydrogenase, and was lost by mutants in which the enzyme was defective . The Michaelis constant for the enzyme with D-alanine as substrate was 30 mM, and the pH optimum about 8.9 . D-alanine was the most active substrate, L-alanine was inactive and several other D-amino acids were 10--50% as active as D-alanine . Oxidation of D-alanine was linked to oxygen via a cytochrome-containing respiratory chain . Synthesis of the dehydrogenase was induced 16 to 23-fold by incubation with D- or L-alanine, but only D-alanine was intrinsically active as an inducer . L-alanine was active either as a substrate or inducer only in t he presence of an uninhibited alanine racemase which converted it to the D-isomer . The map-location of their structural genes between ara and leu, together with other similarities, indicate that D-alanine dehydrogenase and the "alaninase" of Wijsman (1972a) are the same enzyme . Both D- and L-alanine were intrinsically active as inducers of alanine racemase synthesis . The synthesis of both D-alanine dehydrogenase and alanine racemase was found to be regulated by catabolite repression.

Biochim Biophys Acta, 1976 Dec 6, 449(3), 401 - 11
An evaluation of N-phenyl-1-naphthylamine as a probe of membrane energy state in Escherichia coli; Cramer WA et al.; Colicin El and the uncoupler of oxidative phosphorylation, trifluoromethoxy-carbonylcyanidephenylhydrazone (FCCP), cause an increase in the fluorescence intensity of N-phenyl-1-naphthylamine bound to whole cells of Escherichia coli . It has been shown elsewhere that this fluorescence increase correlates well with de-energization . Addition of glucose causes a large cyanide-sensitive decrease of intensity, tentatively associated with energization, with the emission spectrum almost returning to the original trace with a peak at 417 nm . These data suggest that there may be a measurable competition between de-energization and energization of the cell membrane, and that the probe fluorescence intensity may be a general indicator of membrane energy level . The conclusions reached about cellular energy level from measurements of the probe fluorescence intensity correlate partly (a, b below, not c) with the energy level assayed physiologically through rates of active transport; (a) FCCP is found to be a poor inhibitor of proline transport if cells are first incubated with glucose, showing eutger cinpetition between the processes of energization and de-energization or an increase in the envelope permeability barrier to FCCP caused by glucose addition . (b) Cyanide blocks the fluorescence decrease caused by glucose and inhibits proline and serine transport, consistent with the decrease in probe fluorescence intensity indicating an increase in membrane energization . However, (c) it appears that the amplitude of the fluorescence intensity decrease caused by glucose addition in the presence of FCCP and colicin E1 greatly exaggerates the extent of real membrane energization . Glucose added after uncoupler can cause only a small increase, and after colicin, a negligible increase in the proline transport rate, indicating that the magnitude of the fluorescence intensity decrease after glucose addition is not a true measure of membrane energization, but rather seems to amplify this energization greatly . Glucose addition does not cause a decrease in fluorescence intensity in cells treated with EDTA to remove lipopolysaccharide and an apparent barrier to the probe . The rotational relaxation time of the probe in intact cells appears to correlate somewhat better with the cellular energy level than does intensity.

Biochim Biophys Acta, 1976 Dec 6, 449(3), 376 - 85
Mutations affecting the reduced nicotinamide adenine dinucleotide dehydrogenase complex of Escherichia coli; Young IG et al.; A strain carrying a point mutation affecting the NADH dehydrogenase complex of Escherichia coli has been isolated and its properties examined . The gene carrying the mutation (designated ndh) was located on the E . coli chromosome at about minute 23 and was shown to be cotransducible with the pyrC gene . Strain carrying the ndh- allele were found to be unable to grow on mannitol and to grow very poorly on glucose unless the medium was supplemented with succinate, acetate or casamino acids . The following properties of strains carrying the ndh- allele were established which suggest that the mutation affects the NADH dehydrogenase complex but apparently not the primary dehydrogenase . Membrane preparations possess normal to elevated levels of D-lactate oxidase and succinate oxidase activities but NADH oxidase is absent . NADH is unable to reduce ubiquinone in the aerobic steady state and reduces cytochrome b very slowly when the membranes become anaerobic . NADH dehydrogenase, measured as NADH-dichlorophenolindophenol reductase is reduced but not absent . NADH oxidase is stimulated by menadione although not by Q-3 or MK-1 and in the presence of menadione, cytochrome b is reduced normally by NADH . Further mutants affected in NADH oxidase were isolated using a screening procedure based on the growth characteristics of the original ndh- strain . The mutantions carried by these strains were all cotransducible with the pyrC gene and the biochemical properties of the additional mutants were similar to those of the original mutant . The properties of the group of ndh- mutants established so far suggest that they are affected in the transfer of reducing equivalents from the NADH dehydrogenase complex to ubiquinone.

Biochim Biophys Acta, 1976 Dec 2, 455(2), 412 - 32
Correlated x-ray diffraction and freeze-fracture studies on membrane model systems . Perturbations induced by freeze-fracture preparative procedures; Costello MJ et al.; Lipid-water and protein-lipid-water phases have been examined by X-ray methods before and after freezing . Frozen samples have been subsequently fractured and replicated, thus permitting an evaluation of the nature of structural perturbations in samples examined by freeze-fracture electron microscopy . Important results are summarized: (1) Freezing low water content (approx . less than 25%) phases causes perturbations in the packing of hydrocarbon chains . The results suggest that freezing liquied paraffin chains produces a condensed "glass-like" packing . (2) Additional perturbations occur in high water content samples . After freezing, much smaller lamellar repeat distances, intense ice reflections, and extensive perturbation of fracture faces are consistant with the expulsion of water from between lamellae . Presence of glycerol generally relieves these perturbations but in some cases introduces additional lattice disorder . (3) Surprisingly, cooling by a stream of cold N2 gas (-140 degrees C) produces qualitatively the same results as rapid cooling in liquid Freon-22 (-160 degrees C) . (4) Complex perturbations occur in phases containing integral membrane proteins . Interesting results have been obtained with cytochrome b5-lecithin lamellar associations which display both smooth and rough fracture faces without clearly defined particles.

Arch Biol Med Exp (Santiago), 1976 Dec, 10(1-3), 78 - 84
Microinjection of tRNA into amphibian oocytes; Allende JE et al.; The microinjection technique affords us the possibility to introduce purified components into living cells and to answer the question of what effects the change introduced has on cellular metabolism . This technique can therefore be used to test the hypothesis that transfer RNA plays a regulatory role in cellular protein synthesis . Prior to these experiments it is important, however, to test whether transfer RNA microinjected into amphibian oocytes is stable and functional inside this cells . These two questions are answered affirmatively in this report . The stability of tRNA was tested by following the content of TCA precipitable counts inside the oocytes at different times after microinjection of radioactive yeast and E . coli tRNA and by polyacrilamide gel electrophoresis of the material recovered from the cell . The results clearly indicate that tRNAs are resistant to the action of occyte ribonucleases that degrade other RNAs such as 5S RNA . The functionality of the injected tRNA was tested by assaying the intracellular aminoacylation of microinjected yeast tRNA . The aminoacylation of bulk yeast (3H) tRNA introduced into Xenopus laevis oocytes was tested by the capacity of the material recovered 5 hours after injection into the cell to form a ternary complex with wheat protein synthesis elongation factor 1 and GTP . The complex only forms with aminoacyl-tRNA and not with unacylated tRNA . This method showed that at least 80% of the tRNA introduced into the cell was aminoacylated in vivo . A direct assay for internal aminoacylation made use of microinjection of pure tRNAPhe and subsequent determination by phenol extraction of (14C)Phe-tRNA content of oocytes that had been incubated for 2 hours in a medium containing (14C)phenylalanine . The results obtained showed that the oocytes could internally aminoacylate 200-500 times more tRNAPhe that the cell normally contains . Appropiate controls demonstrated that the aminoacylation was aminoacid and tRNA specific and that periodate oxidized tRNAPhe could not be in vivo aminoacylated but tRNAPhe deprived of its Y base could accept the aminoacid . A brief study demonstrated that bulk yeast tRNA and tRNAPhe without its Y base did not inhibit endogenous protein synthesis but a similar amount of tRNAPhe caused 50% inhibition and periodate-oxidized tRNAPhe a 95% inhibition.

Nucleic Acids Res, 1976 Dec, 3(12), 3359 - 67
End group of naturally terminated and UV lesion terminated T7 in vitro RNA; Ali R et al.; The 3' terminal nucleosides of RNA transcribed in vitro by E . coli RNA polymerase from T7 DNA and UV irradiated TN DNA were determined . The 3' terminal nucleoside of naturally terminated (t1 termination site) RNA cytidine . In the case of RNA terminated at UV lesions, it is cytidine in 0 per cent of the molecules and adenosine in the remaining 30 per cent . Cytidine trialcohols are labile in high concentrations of KOH and at high temperature and appear to convert to uridine.

J Biochem (Tokyo), 1976 Dec, 80(6), 1411 - 22
Effects of heating in dodecyl sulfate solution on the conformation and electrophoretic mobility of isolated major outer membrane proteins from Escherichia coli K-12; Nakamura K et al.; Major outer membrane proteins of Escherichia coli K-12 with apparent molecular weights ranging from 30,000 to 40,000 were resolved into four distinct bands by electrophoresis on an improved urea-sodium dodecyl sulfate (SDS)-polyacrylamide gel containing a high concentration of N,N'-methylenebisacrylamide . The electrophoretic mobilities of three of these proteins, O-8, O-9, and O-10, changed when they were heated in SDS solution . Proteins O-8, O-9, and O-10, were purified to near homogeneity without heating in SDS solution . Electrophoretic profiles of the purified proteins changed depending on the solubilization temperature in SDS solution . Infrared and CD spectra of these proteins revealed that they were extremely rich in beta-structured polypeptide, which is stable in SDS solution at room temperature . On the other hand, CD spectra typical of alpha-helix structure were obtained when the proteins were heated in SDS solution, indicating that a gross conformational change occurred in the protein molecules upon heating in SDS solution . The conformational change was confirmed by the abnormal profiles of Ferguson plots in gel electrophoretic analysis . It was concluded that conformational changes in the protein molecules are responsible for the heat modifiability of these proteins in SDS gel electrophoresis.

J Cell Physiol, 1976 Dec, 89(4), 551 - 9
Nucleoside transport systems in Escherichia coli K12: specificity and regulation; Munch-Petersen A et al.; Two nucleoside transport systems have been verified and separated by mating and recombination experiments . The recipient strain was a mutant which is negative for transport of all nucleosides . The two systems differ in specificity and in regulation . One system transports pyrimidine and adenine nucleosides . It is regulated by the cytR gene . The other system transports all nucleosides and is regulated by the cytR as well as by the deoR genes . Enzyme assays performed on whole cells of strains, able or unable to transport nucleosides, indicate that the nucleoside catabolizing enzymes are located inside the permeability barrier of the cell.

Curr Top Radiat Res Q, 1976 Dec, 11(3), 201 - 50
Irradiation of cells by single and double pulses of high intensity radiation: oxygen sensitization and diffusion kinetics; Epp ER et al.; The biological effects of ionizing radiation in living cells are the ultimate result of a long chain of events with the initial step being the local absorption of radiation . Whereas such physical abosrption is probably over within 10(-16) s after dose delivery, the biological consequences of radiation do not manifest themselves until very much later times . Between these two extremes of time, events occur relatively early at the molecular level which are undoubtedly critically related to the still unknown basic mechanisms of cellular radiation damage . These events may include not only the interaction of highly reactive species produced within the cell but also the diffusion of molecules such as oxygen or other chemical radiosensitizers.

West Afr J Pharmacol Drug Res, 1976 Dec, 3(2), 153 - 60
Generic inequivalence between two brands of chloramphenicol capsule; Bugaje UM et al.; Generic inequivalence between two brands of chloramphenicol capsules has been investigated . Both brands complied with official standards for drug content and capsule disintegration time, and exhibited similar anti-bacterial activity when tested in vitro . A slow dissolution rate was observed with one brand which could be related to a formulation defect which might explain reported therapeutic inefficacy.

Arch Biol Med Exp (Santiago), 1976 Dec, 10(1-3), 85 - 91
The stringent control mechanism . Binding of uncharged tRNA and stringent factor to Escherichia coli ribosomes; Richter D; The mechanism of the interaction of uncharged tRNA and stringent factor with the ribosomes during the in vitro synthesis of guanosine polypnophates (pppGpp and ppGpp) was studied . 70S ribosomes lacking proteins L7 and L12 were able to bind radioactive stringent factor but not EF-Tu or EF-G . In addition, ribosomes carrying EF-Tu, GMPPCP and Phe-tRNA bound as much stringent factor as 70S ribosomes alone . This evidence suggests that the stringent and elongation factors recognize nonidentical site (s) on the ribosomes . When a 70S - poly U - tRNA complex was formed in the absence of stringent factor and was subsequently incubated with Nac-Phe-tRNA or Phe-tRNA only the binding of Nac-Phe-tRNA to the complex was inhibited . However, when the 70S-poly U-tRNA complex was formed in the presence of stringent factor and then was incubated with Nac-Phe-tRNA or Phe-tRNA only the binding of Phe-tRNA was inhibited . These results indicate that the stringent factor directs uncharged codon-specific tRNA into the acceptor site of the ribosome.

Zentralbl Bakteriol {Orig B}, 1976 Dec, 163(5-6), 509 - 23
{Kinetics of the release of testbacteria from the artificially contaminated hand (author's transl)}; Koller W et al.; It is demonstrated that the release of testbacteria from the finger tips of artificially contaminated hands is influenced to a very small degree only by application of the finger tip method when compared to the effect of ablutions . Therefore, without apprehension of influencing the post-values this method may be employed in order to assess pre-values as required by the test-model of Rotter and Mittermayer (5) for the evaluation of procedures for the hygienic disinfection of hands . In addition, it can be shown directly that it is irrelevant for the number of bacteria released after a germ-reducing procedure, whether the finger tip-method has been used or not, preceedingly . In contrast, all technical variations that aim at the restitution of the supposed loss of testbacteria (like renewed contamination after the assessment of prevalues) change the postvalues measurably and must be avoided, therefore . Drying of bacterial suspensions on the hands, as after artificial contamination, reduces the release of viable testbacteria depending on the time . This effect is stronger on the palms than on the finger tips . The influence of handbrushes on the release of testbacteria with simultaneous useage of detergents compares well with the effect of an alcohol disinfection . However, this method is not suitable to eliminate pathogens safely.

J Biochem (Tokyo), 1976 Dec, 80(6), 1247 - 58
Nature of Escherichia coli mutants deficient in detergent-resistant and/or detergent-sensitive phospholipase A; Doi O et al.; 1 . Escherichia coli K-12 mutants deficient in detergent-resistant (DR) and detergent-sensitive (DS) phospholipases A and deficient in DS phospholipase A were isolated . 2 . The growth, compositions of phospholipids and fatty acids and turnover of phospholipids of three mutants (DR-, DS-, DR-DS-) were compared with those of their parent (DR- was isolated in a previous study) . 3 . Autodegradations of membrane phospholipids of 18,000 X g supernatants and precipitates of the homogenates of these three mutants were also compared with those of the parent, and the effects of various detergents and organic solvents on these activities were examined . 4 . We could not identify any significant physiological role for DR or DS phospholipase A.

Can J Biochem, 1976 Dec, 54(12), 1061 - 8
Further studies on aspartate transcarbamoylase: molecular weight of the c3r6 complex and analysis of succinate inhibition in the native enzyme; Chan WW; The complex which is formed when excess regulatory subunits (r2) of aspartate transcarbamoylase (EC2.1.3.2) are added to a dilute solution of the catalytic subunit (c3) has been studied by gel-filtration on Sephadex G-200 . The elution volume indicates a Stokes' radius of between 5.42 and 5.92 nm, depending on the method of calculation . Using the sedimentation coefficient of 7.7S previously determined, the molecular weight is estimated to be close to 200 000, in support of the c3r6 structure proposed earlier for the complex . The calculated frictional coefficient indicates abnormal hydrodynamic properties which are probably due to unusual structure characteristics . The pattern of succinate inhibition of native aspartate transcarbamoylase has also been analyzed . At low concentrations, succinate activates the enzyme, presumably by converting it from the taut state to the relaxed (r) state . Further increase in the succinate concentration leads to competitive inhibition of the R state . Using a novel procedure for analysis of the data, the Michaelis constant for aspartate of the R state has been estimated to be about 7mM . This value is close to the Km of c3r6 for aspartate, measured under identical conditions . The result therefore provides further evidence suggesting that the c3r6 complex resembles the R state of the native enzyme.

Biophys Chem, 1976 Dec, 6(1), 23 - 9
Dependence of the sedimentation of high molecular weight DNA on centrifuge speed; Hutchinson F et al.; A theory by Zimm {B.H.Zimm, Biophys . Chem . 1-(1974)279} predicts that for a given centrifuge speed, there is a broad maximum in a plot of the sedimentation coefficient of DNA against molecular wight . Experimental measurements of these maxima for various centrifuge speeds were made for double-helical DNA in neutral sucrose gradients and singlestrand DNA in alkaline gradients . The measurements are in quantitative agreement with the theory, providing good evidence for its validity . The existence of the maximum shows that there is a limit to the sedimentation rate under specified conditions for KNA in the linear form . By implication, DNA observed to sediment faster than this limit is not in the linear form to which most sedimentation theory is applicable.

Mutat Res, 1976 Dec, 41(2-3), 185 - 200
Misrepair of overlapping daughter strand gaps as a possible mechanism for UV induced mutagenesis in UVR strains of Escherichia coli: a general model for induced mutagenesis by misrepair (SOS repair) of closely spaced DNA lesions; Sedgwick SG; It has been previously reported that an inducible form of post-replication repair appeared to be required for UV induced mutagenesis in a uvrA strain of Escherichia coli . It is shown here that the numbers of daughter strand gaps requiring inducible repair were similar to the numbers calculated to be overlapping one another in opposite daughter chromosomes . An estimation of survival with no repair of these gaps resembled the survival predicted with no mutagenesis . It is the thus proposed that inducible post-replication repair causes mutagenesis by the repair of overlapping daughter strand gaps . A general model for induced mutagenesis is presented . It is proposed that (a) some DNA lesions introduced by any DNA damaging agent may be close enough to interfere with constitutive repair replication of each other, (b) these lesions induce a repair system (SOS repair) which involves the recA+ lexA+ and polC+ genes (c) repair and concomitant mutagenesis occurs during repair replication by the insertion of mismatched bases opposite the noncoding DNA lesions.

J Gen Microbiol, 1976 Dec, 97(2), 257 - 65
Growth on D-lyxose of a mutant strain of Escherichia coli K12 using a novel isomerase and enzymes related to D-xylase metabolism; Stevens FJ et al.; Escherichia coli K12 cannot grow on D-lyxose, but a mutant was isolated which can utilize D-lyxose as sole source of carbon and energy for growth . D-Lyxose is transported into the bacteria by the D-xylose permease . The mutant constitutively synthesizes a new isomerase which is not inducible in the parent strain under any of the conditions tested . This enzyme, whose native substrate appears to be D-mannose, fortuitously converts D-lyxose into D-xylulose . Its structural gene is located at around 85 min on the E . coli genetic map, away from other known isomerase genes . D-Xylulose is subsequently catabolized by the enzymes of the normal D-xylose metabolic pathway . D-Mannose isomerase was partially purified and some of its properties were examined.

J Cell Physiol, 1976 Dec, 89(4), 545 - 9
Carbohydrate uptake by Escherichia coli; Kornberg HL; In contrast to active transport, the uptake of carbohydrates via the phosphoenolpyruvate-dependent phosphotransferase system (PTS) leads to the appearance in the cell of the sugar initially as a 1- or 6-phosphate ester . The components of the PTS that transfer phosphate to the sugar are not absolutely specific for any one sugar . Both their synthesis and their activity are controlled; in the latter, "fine" control, glucose-6-phosphate appears to play an important role . Studies of growth on, and uptake of, galactose by E.coli mutants devoid of components of the PTS and also devoid of active transport systems for galactose, suggest that proteins effecting facilitated diffusion of hexoses may be part of, or be closely associated with, the sugar-specific components of the PTS.

J Cell Physiol, 1976 Dec, 89(4), 529 - 41
Cell envelope proteins involved in the transport of maltose and sn-glycerol-3-phosphate in Escherichia coli; Boos W; Two types of proteins are discussed in their role of facilitating the transport of maltose and sn-glycerol-3-phosphate in E . coli . The first protein is the receptor for phage lambda, known to be an outer membrane protein . By facilitating the diffusion of maltose and the higher maltodextrins through the outer membrane the effect of the lambda receptor is to decrease the Km of the transport system without influencing the Vmax of substrate flux . The second protein is a periplasmic protein that is induced by growth on glycerol and is essential for transport of sn-glycerol-3-phosphate in whole cells but not in membrane vesicles . This protein has solely been identified by the use of a two-dimensional polyacrylamide gel electrophoresis of periplasmic proteins in wild-type and mutants defective in sn-glycerol-3-phosphate transport.

Eur J Biochem, 1976 Dec, 71(1), 201 - 10
Isolation and properties of an inhibitor of Escherichia coli RNA polymerase; Crimaldi A et al.; An inhibitor of the Escherichia coli RNA polymerase has been isolated from E . coli and has been partially characterized . The inhibitor, a polypeptide of molecular weight 70000, acts to shut off RNA synthesis at about the time that the first round of RNA synthesis is over, preventing any further RNA synthesis . The inhibitor apparently does not recognize specific termination sequences in the DNA template, since it works equally well with double-stranded DNA, single-stranded DNA and poly{d(A-T)} as templates for RNA synthesis, and because the RNA molecules synthesized from T7 DNA appear to be terminated at the same site either in the presence or absence of the inhibitor . Several experiments indirectly indicate that the inhibitor may reversibly bind to the RNA polymerase at the termination step, in a ratio of approximately one inhibitor molecule per polymerase molecule.

Eur J Biochem, 1976 Dec, 71(1), 171 - 6
Free 3'-OH group of the terminal adenosine of the tRNA molecule is essential for the synthesis in vitro of guanosine tetraphosphate and pentaphosphate in a ribosomal system from Escherichia coli; Sprinzl M et al.; tRNAPhe from yeast modified at the 3'-terminal adenosine residue or missing part of its CpCpA end was investigated for its ability to participate in the synthesis of guanosine tetraphosphate and pentaphosphate using Escherichia coli ribosomes . Only tRNA containing complete CpCpA end and an intact ribose residue in the terminal adenosine was active in this process . The absence of the 2'-hydroxyl group of the terminal adenosine does not influence the reaction, but the 3'-hydroxyl group is essential and cannot be replaced by an amino group or a hydrogen atom . tRNA in which the 3'-terminal adenosine is replaced by formycin is active in the induction of guanosine tetraphosphate and pentaphosphate synthesis, but the reaction proceeds relatively slowly.

Eur J Biochem, 1976 Dec, 71(1), 109 - 16
Sequence studies on D-serine dehydratase of Escherichia coli . Primary structure of the tryptic phosphopyridoxyl peptide and of the N-terminus; Schiltz E et al.; An improved procedure for large-scale production of crystalline D-serine dehydratase (EC 4.2.1.14) from Escherichia coli is described . The N-terminal sequence of the enzyme (Mr 45500) was determined in a solid-phase sequencer as Met-Glu-Asn-Ala-Lys-Met-Asn-Ser-Leu-Ile-Ala-Gln-Tyr-Pro-Leu-Val-Lys-Asp-Leu-Val-Ala-LEU-Lys . Four of the first five N-terminal residues are homologeous with tryptophanase, another pyridoxal-phosphate (P-Pxy) enzyme that catalyzes alpha,beta-elimination reactions . After borohydride reduction and tryptic digestion of the enzyme, a peptide was isolated showing the sequence Lys-Asp-Ser-His-Leu-Pro-Ile-Ser-Gly-Ser-Ile-Lys(P-Pxy)-Ala-Arg . No clear homology of this portion of the enzyme with tryptophanase or another pyridoxal-phosphate enzyme was observed.

Biochem J, 1976 Dec 1, 159(3), 677 - 82
Evidence for two lipoic acid residues per lipoate acetyltransferase chain in the pyruvate dehydrogenase multienzyme complex of Escherichia coli; Danson MJ et al.; The reaction of two maleimides, N-ethylmaleimide and bis-(N-maleimidomethyl) ether, with the pyruvate dehydrogenase multienzyme complex of Escherichia coli in the presence of the substrate, pyruvate, was examined . In both cases, the reaction was demonstrated to be almost exclusively with the lipoate acetyltransferase component, and evidence is presented to show that the most likely sites of reaction are the lipoic acid residues covalently bound to this component . With both reagents the stoicheiometry of the reaction was measured: 2 mol of reagent reacted with each polypeptide chain of lipoate acetyltransferase, implying that each chain bears two functionally active lipolic acid residues . This observation can be reconciled with previous determinations of the lipoic acid content of the complex by allowing for the variability of the subunit polypeptide-chain ratio that can be demonstrated for this multimeric enzyme.

Biochem J, 1976 Dec 1, 159(3), 503 - 11
The aminoacylation of transfer ribonucleic acid . Inhibitory effects of some amino acid analogues with altered side chains; Old JM et al.; Several amino acid analogues that are able to replace amino acid residues in binding positions of the biologically active C-terminal tetrapeptide amide sequence, Trp-Met-Asp-PheNH2, of the gastrins were examined for their ability to inhibit the aminoacylation of tRNA in an Escherichia coli and rat liver system . Although in both systems the amino acid side chains are involved in the recognition process, the structural requirements of the side chain in the two systems are not comparable . Analogues of methionine and phenylalanine behaved similarly in the E . coli and rat liver systems, whereas analogues of tryptophan behaved differently . From the results it is possible to suggest structural features of the amino acid side chains which are required for recognition by the aminoacyl-tRNA synthetases.

Z Immunitatsforsch Immunobiol, 1976 Dec, 152(4), 343 - 8
Effect of heparin on the priming with a protein antigen in mice; Jokay I et al.; Heparin has been found to stimulate or suppress the priming activity of a protein antigen (cock muscle phosphorylase-b) in mice depending on the various parameters (the dose of antigen, timing of administration, etc.) . The adjuvant effect of endotoxin on priming was potentiated by a single dose of heparin injected 10-12 hours after immunization . Optimal doses of heparin incorporated into Freund's incomplete adjuvant also enhanced the humoral immune response first of all the development of 7 S immunologic memory . The possible mechanisms are discussed.

Proc Natl Acad Sci U S A, 1976 Dec, 73(12), 4474 - 8
Novobiocin and coumermycin inhibit DNA supercoiling catalyzed by DNA gyrase; Gellert M et al.; Novobiocin and coumermycin are known to inhibit the replication of DNA iing of DNA catalyzed by E . coli DNA gyrase, a recently discovered enzyme that introduces negative superhelical turns into covalently circular DNA . The activity of DNA gyrase purified from a coumermycin-resistant mutant strain is resistant to both drugs . The inhibition by novobiocin of colicin E1 plasmid DNA replication in a cell-free system is partially relieved by adding resistant DNA gyrase . Both in the case of coliclls . DNA molecules which are converted to the covalently circular form in thepresence of coumermycin remain relaxed, instead of achieving their normal supercoiled conformation . We conclude that DNA gyrase controls the supercoiling of DNA in E . coli.

Proc Natl Acad Sci U S A, 1976 Dec, 73(12), 4374 - 8
Activation of long chain fatty acids with acyl carrier protein: demonstration of a new enzyme, acyl-acyl carrier protein synthetase, in Escherichia coli; Ray TK et al.; A soluble enzyme activity which catalyzes the synthesis of acyl-acyl carrier protein from acyl carrier proteins, a long chain fatty acid, and ATP has been demonstrated in E . coli . The reaction requires high concentrations of both Ca++ and Mg++ for activity, and cleaves ATP to AMP and PPi . The fatty acyl product has been identified as acyl-acyl carrier protein by its solubility, thioester linkage, molecular weight, charge, and biological activity . Several criteria indicate the enzyme is distinct from acyl-CoA synthetase . The fatty acid specificity of the enzyme suggests a role of acyl-acyl carrier protein synthetase in the incorporation of fatty acids into phospholipid.

Proc Natl Acad Sci U S A, 1976 Dec, 73(12), 4324 - 8
Endonuclease II, apurinic acid endonuclease, and exonuclease III; Kirtikar DM et al.; An endonuclease of Escherichia coli active on a DNA treated with methylmethane sulfonate has been separated from an endonuclease active on depurinated sites . The former enzyme is disignated here as endonuclease II, while the latter enzyme is designated as apurinic acid endonuclease . Endonuclease II is also active on DNA treated with methylnitrosourea, 7-bromomethyl-12-methylbenz{a}anthracene, and gamma-irradiation . A third fraction which contains activities for both depurinated and alkylated sites needs further study . Endonuclease II, molecular weight 33,000, has been purified 12,500-fold and does not have exonuclease III activity . Apurinic acid endonuclease, molecular weight 31,500, has been purified 11,000-fold and does not have exonuclease III activity . Exonuclease III, molecular weight 26,000, has been purified 2300-fold and does not have endonucleolytic activity at depurinated reduced sites or at alkylated sites in DNA . Therefore, these are three separate proteins . Exonuclease III can produce, presumably by its exonucleolytic activity, double-strand breaks in heavily alkylated DNA under conditions where it does not make single-strand endonucleolytic breaks at either depurinated-reduced or alkylated sites.

Nucleic Acids Res, 1976 Dec, 3(12), 3369 - 76
The nucleotide sequence of asparagine tRNA from Escherichia coli; Ohashi K et al.; The nucleotide seuquence of Escherichia coli asparagine tRNA was determined to be pU-C-C-U-C-U-G-s4U-A-G-U-U-C-A-G-D-C-G-G-D-A-G-A-A-C-G-G-C-G-G-A-C-U-Q-U-U-t6A-A-phi-C-C-G-U-A-U-m G-U-C-A-C-U-G-G-T-phi-C-G-A-G-U-C-C-A-G-U-C-A-G-A-G-G-A-G-C-C-AOH . Its D-stem and D-loop have almost the same sequence as Escherichia coli aspartate tRNA.

Nucleic Acids Res, 1976 Dec, 3(12), 3255 - 69
Kinetic studies on the cleavage of adenovirus DNA by restriction endonuclease Eco RI; Forsblom S et al.; The kinetics of cleavage of DNA from Adenovirus Type 1 (Ad1), Type 5 (Ad5) and Type 6 (Ad6) by restriction endonuclease EcoRI was investigated by quantitative evaluation of the fluorescence from ethidium stained DNA fragments separated on agarose gels . The apparent rate constants of cleavage at different cleavage sites have been determined and large differences in the cleavage rates of the individual sites within one type of DNA were found . From the kinetics of cleavage information on the sequence of the DNA fragments can be obtained . The order of the fragment A, B, C, D of Ad6 DNA obtained after complete cleavage by restriction endonuclease Eco RI was found to be A-D-C-B; the order of the corresponding fragments A, B, C of Ad1 and Ad5 DNA was found to be A-C-B.

J Hyg (Lond), 1976 Dec, 77(3), 393 - 400
Escherichia coli O 27 in adult diarrhoea; Hobbs BC et al.; Escherichia coli O 27 H 7 was found in 16 stool samples submitted during a Caribbean cruise (Cruise Z) by 29 patients reporting with diarrhoea . A retrospective search revealed E . coli O 27 H 7 in 11 of 20 and 2 of 14 stool cultures from patients on two previous cruises (Y and X respectively) and in a culture from fresh cream (Cruise Y) . The repeated occurrence of E . coli O 27 H 7 in the absence of any other apparent cause suggested that this serotype may have been responsible for the diarrhoea . The results of pathogenicity tests suggested that this strain elaborated heat-stable (ST) enterotoxin . The possibility that food may have been the vector is discussed.

Biochim Biophys Acta, 1976 Dec 1, 454(2), 375 - 81
The absence of DNA photoreactivation enzyme in yeast mitochondria; Daily OP et al.; Mitochondria isolated from haploid yeast cells by spheroplast lysis were purified by flotation on renografin gradients . Electron micrographs and respiratory control ratios revealed that the purified mitochondria were still intact and functional . Assays for photoreactivation enzyme using as substrate {3H}-thymine-labeled Escherichia coli DNA were performed on crude and purified mitochondrial preparations . While the crude preparation contained high amounts of photoreactivation enzyme, it appeared to be associated with contaminating nuclei . The purified mitochondria lacked any photoreactivation enzyme activity . We suggest that yeast mitochondria do not normally contain photoreactivation enzyme.

Ann Surg, 1976 Dec, 184(6), 672 - 8
Consumptive opsoninopathy: possible pathogenesis in lethal and opportunistic infections; Alexander JW et al.; Serum levels of properdin, Factor B and C3 and the ability of these sera to opsonize E . coli 075 were measured in 17 patients with surgical infections ranging in severity from mild to fatal . There was good direct correlation between severity of infection, serum levels of properdin and C3, and the ability of the serum to support opsonization . The levels of Factor B were not significantly reduced when measured by radial immunodiffusion, but immunoelectrophoresis showed conversion . Restoration of full opsonic activity was accomplished only by the addition of a combination of C3, Factor B, and properdin in excess . The findings provide evidence that severe bacterial infection causes a consumption of opsonic proteins which may result in a reduced ability of the patient's serum to opsonize bacteria and thereby further increase susceptibility to infection.

Am J Vet Res, 1976 Dec, 37(12), 1445 - 8
Intensification by glucocorticoids of induced acute leukocytic responses in the rabbit uterus; Hawk W et al.; Sexually mature does were given cortisone acetate, hydrocortisone acetate, or 9 alpha-fluoroprednisolone by intramuscular injection for 3 days, then the uterine lumens were inoculated with Escherichia coli . The glucocorticoids doubled the number of leukocytes that migrated into the uterine lumen during the acute inflammatory response . The corticoids had similar effects in estrous, ovariectomized, and pseudopregnant rabbits . The administration of hydrocortisone acetate nearly doubled the number of polymorphonuclear (PMN) leukocytes in the circulation within 24 hours . The high numbers of circulating PMN leukocytes were maintained over the 3 days of treatment . The increased leukocytic response to induced uterine infection was similar in magnitude to the increase in numbers of circulating PMN leukocytes; the corticoid-induced neutrophilia was probably responsible for the intensified leukocytic response to uterine infection.

AJR Am J Roentgenol, 1976 Dec, 127(6), 1015 - 9
Angiographic and ultrasonic findings in infected simple cysts of the kidney; Cho KJ et al.; This paper describes angiographic and ultrasonic findings in three patients with proven infected renal cyst . The clinical picture was that of inflammatory renal disease . B-mode ultrasonography showed the characteristic features of renal cyst; however, this technique cannot differentiate between infected and simple cysts . Selective renal angiography demonstrated the following, enabling differentiation from simple renal cyst; hypervascular rim, irregular inflammatory vessels, indistinct interface between cyst and adjacent parenchyma, and prominent capsular branches . Since percutaneous puncture of an infected renal cyst or abscess carries risk of infectious complications, angiography appears justified only for those patients with fever and flank pain who have cystic lesions in the kidney confirmed by echography.

J Nucl Med, 1976 Dec, 17(12), 1088 - 92
Sensitivity of the Limulus test and inhibitory factors in the radiopharmaceuticals; Murata H et al.; The basic sensitivity of the Limulus test and the inhibitory factors were examined in 21 radiopharmaceuticals commonly used in Japan . The sensitivity of the Limulus test using pre-gel was found to be 1 mug/ml for Escherichia coli endotoxin . This sensitivity is about ten times that of the rabbit test adopted by USP and JP . The Limulus test was applicable, with full sensitivity and without inhibitory reaction, for the evaluation of 99mTcO4-, 99mTc-albumin, 99mTc-MAA, 99,Tc-Sn-colloid, 131I-Hippuran, Na131I, Na251CrO49, 67Ga citrate, and 57Co-bleomycin as commercially supplied . On the other hand, with 111In-DTPA, 99mTc-phytate, 99mTc-pyrophosphate, 99mTc-DTPA, 131I-polyvinylpyrrolidone (PVP), 59FeCl3, Na232PO4, 198Au colloid, and selenomethionine (Se-75), the pH required adjustment to avoid inhibition of the gelation reaction . Benzyl alcohol showed an inhibitory effect on the gelation reaction at concentrations of more than 1% . Iodine-131-Bromsulphalein (BSP) and 131I-rose bengal showed intense inhibition of the gelation reaction.

J Bacteriol, 1976 Dec, 128(3), 853 - 4
Adenine methylation of Okazaki fragments in Escherichia coli; Marinus MG; In Escherichia coli polA lig-4 bacteria, the moles percent 6-methyladenine content of 10S deoxyribonucleic acid (Okazaki fragments) is 0.96 compared with 1.4 for bulk desoxyribonucleic acid.

J Bacteriol, 1976 Dec, 128(3), 815 - 26
Complexity of the ultraviolet mutation frequency response curve in Escherichia coli B/r: SOS induction, one-lesion and two-lesion mutagenesis; Doudney CO; Three distinct sections of the ultraviolet mutation frequency response (MFR) curve toward tryptophan prototrophy have been demonstrated in Excherichia coli B/r WP2 trp thy and its uvrA derivative in log-phase growth in minimal medium . The initial section, which appears fluence-squared, may reflect the necessity, if mutation is to result, for induction of two lesions, one located within the potentially mutated genetic locus and the other damaging deoxyribonucleic acid replication and resulting in inducation of the error-prone SOS repair function . A second linear section is ascribed to the continued induction, after exposure above that sufficient for complete SOS expression, of isolated lesions which lead to mutation in potentially mutated loci . The third section demonstrates an increased rate of mutagenesis and suggests the induction of two lesions in proximity which result in additional mutations . Split-exposure studies support the inducible nature of the SOS function and suggest that mutation frequency decline (MFD) is due to exicion resulting from or related to the prevention of SOS induction by inhibition of protein synthesis . Preirradiation tryptophan starvation of the uvr+ strain for 30 min decrease MFR in the first and second sections of the curve . Reduction of MFR in the third section requires more prestarvation time and is blocked by nalidixic acid . The decreased MFR of the first and second sections ascribed to promotion of postirradiation MFD based on excision and that of third section to completion of the chromosome during the prestarvation period.

J Bacteriol, 1976 Dec, 128(3), 810 - 4
Mapping of nrdA and nrdB in Escherichia coli K-12; Fuchs JA et al.; The structural genes coding for the B1 and B2 subunits of the enzyme ribonucleoside diphosphate reductase, nrdA (formerly designated dnaF) and nrdB, respectively, have been mapped in Escherichia coli . They are located at approximately 48 min . The gene order in this region of the E . coli chromosome was found to be purF glpT nrdB nrdA nalA cdd dcd his.

J Bacteriol, 1976 Dec, 128(3), 801 - 9
Deoxyribonucleic acid synthesis in a temperature-sensitive Escherichia coli dnaH mutant, strain HF4704S; Derstine PL et al.; The dnaH mutant strain HF4704S, isolated by Sakai et al . (1974), was examined for its effect on phiX174 deoxyribonucleic acid (DNA) synthesis . It was found to carry two mutations affecting DNA synthesis . One mutation had no affect on phiX174 DNA synthesis, but did affect the ability of the mutant cells to form colonies on agar medium at 41 degrees C, and caused host DNA synthesis to cease after 1 h at 41 degrees C . The mutant marker cotransduced with ilvD at a frequency of about 9% . It seems likely that this mutation is in the dnaA gene . The second mutation affected the ability of the mutant cells to form colonies on agar medium supplemented with only 2 mug of thymine per ml, and affected both host and phiX174 DNA synthesis in medium supplemented with only 2 mug of thymine per ml . Both effects could be overcone by adding excess exogenous thymine . We were not able to unambiguously determine the map position of this mutant locus . Our data show that the DNA synthesis phenotype of the mutant strain HE4704S is governed by both these mutations, neither of which directly affects the replication of phiX174 DNA.

J Bacteriol, 1976 Dec, 128(3), 776 - 84
Role and location of "protease I" from Escherichia coli; Kowit JD et al.; Pacaud and Uriel described an enzyme from Escherichia coli ("protease I") that hydrolyzes acetyl phenylalanine naphthyl ester (APNE) . We examined the possible involvement of this enzyme in intracellular protein degradation, its subcellular distribution, and its proteolytic activity . Although the APNE-hydrolyzing activity is localized primarily in the periplasm, proteolytic activity against casein was found in the periplasm, membrane, and cytoplasm with similar specific activities . The APNE-hydrolyzing enzyme did not appear to contribute to the proteolytic activity of the periplasm . A mutant deficient in APNE-hydrolyzing activity lacked all activity in the periplasm but showed a slight percentage of residual activity in the cytoplasm . Extracts of such cells were normal in their ability to hydrolyze casein . The mutant was indistinguishable from wild-type cells in its rate of protein degradation during growth or glucose starvation and in the ability to rapidly degrade puromycin-containing polypeptides . Nitrogen starvation, which increased protein breakdown severalfold, affected neither the total amount nor the distribution of APNE-hydrolyzing activity . The mutant showed no defect in its ability to cleave small phenylalanine-containing peptides released during protein degradation . The mutant and wild-type cells are equally able to hydrolyze exogenously supplied phenylalanyl peptides . These experiments suggest that the APNE-hydrolyzing enzyme is not required for protein degradation and that "protease I" is probably not a protease.

J Bacteriol, 1976 Dec, 128(3), 735 - 40
Protein synthesis and degradation in a leucine auxotroph of Escherichia coli; Aguanno JJ et al.; The synthesis and degradation of the soluble and sodium dodecyl sulfate-(SDS)-solubilized protein fractions of Escherichia coli were studied in both growing and nongrowing cultures . When separated according to molecular weight on SDS-polyacrylamide gels, the proteins of both fractions of growing cells undergo no measureable differential synthesis or degradation during logarithmic growth . However, when a leucine auxotroph is suspended in medium containing 5.3 muM leucine (a level that will not sustain growth), the SDS-solubilized protein of such a nongrowing culture shows a rapid synthesis of two protein components (32,000 and 12,000 daltons) found only in the out membrane.

J Bacteriol, 1976 Dec, 128(3), 701 - 7
lacY mutant of Escherichia coli with altered physiology of lactose induction; Flagg JL et al.; A mutant of Escherichia coli is described that grew on lactose only in the presence of isopropylthiogalactoside . This cell contained a defect in the lacY gene that resulted in the formation of a transport system with a poor affinity for lactose . The inability to grow on lactose alone was due to the failure of induction by this disaccharide . This failure of inducation was presumably due to a defect in lactose accumulation which resulted in significant reduction in the formation of allo-lactose, the true inducer of lac operon . These results are consistent with the view that the capacity to accumulate lactose plays an important physiological role in the induction of the enzymes necessary for its utilization.

Biochim Biophys Acta, 1976 Dec 1, 454(2), 298 - 308
Selenalysine and protein synthesis; De Marco C et al.; Selenalysine is a lysine analog having the gamma-methylene group substituted by a selenium atom . It has been demonstrated that selenalysine is activated and transferred to tRNAlys by either Escherichia coli or rat liver aminoacyl-tRNA synthetases, and inhibits lysine incorporation into polypeptides in protein-synthesizing systems from E . coli, rat liver or rabbit reticulocytes . All tests were performed in comparison with thialysine, a lysine analog having the gamma-methylene group substituted by a sulfur atom . In all the reactions studied, both thialysine and selenalysine act as competitive inhibitors of lysine . With respect to thialysine, selenalysine act as competitive inhibitors of lysine . With respect to thialysine, selenalysine shows a slightly lower activity as lysine inhibitor.

J Cell Physiol, 1976 Dec, 89(4), 561 - 6
Effect of uncoupler on "downhill" substrate efflux of Escherichia coli is dependent on (Mg2+, Ca2+) . Adenosine triphosphatase; Rotman B; Previous studies have shown that mutations in the unc gene of Escherichia coli K12 cause defects in energy transduction as well as a membrane-bound (Mg2+, Ca2+)-adenosine triphosphatase . We studied the effect of this mutation on the "downhill" efflux of methyl-beta-D-galactopyranoside, a suboli K12 did not show significant differences in substrate influx of efflux, a differential effect of an uncoupler, 2,4-dinitrophenol was demonstrated . In contrast to the unc+, dinitrophenol failed to inhibit significantly the rate coefficient of efflux in the unc- strain . Analysis of spontaneous unc+ revertants of the unc- mutant provided additional evidence that a functional unc gene is necessary for dinitrophenol inhibition of efflux . Other uncouplers tested in the unc+ strain showed different effects on efflux . While arsenate, azide and carbonyl cyanide p-trifluoromethoxyphenulhydrazone caused little or no effect, 2,4-dibromophenol and pentachlorophenol increased efflux by a considerable factor.

J Antibiot (Tokyo), 1976 Dec, 29(12), 1328 - 33
Effect of spectinomycin on peptide chain initiation; Reusser F; Spectinomycin inhibits the formation and causes dissociation of performed specific cell-free initiation complexes prepared with the trinucleotide A-U-G or R17 phage RNA, fmet-RNAF and either 30S ribosomal subunits or 70S ribosomes . Both the initiation factor and the Mg2+-induced processes are subject to inhibition by spectinomycin . The only exception is the Mg2+-induced 70S initiation system formed with A-U-G which is stimulated by spectinomycin.

Biochim Biophys Acta, 1976 Dec 1, 454(2), 285 - 97
Studies on the enzymatic synthesis of enterochelin in Escherichia coli K-12 . Four polypeptides involved in the conversion of 2,3-dihydroxybenzoate to enterochelin; Greenwood KT et al.; Four gene products involved in the enzymatic synthesis of enterochelin from 2,3-dihydroxybenzoate, L-serine and ATP (Luke, R.K.L . and Gibson, F . (1971) J . Bacteriol . 107,557-562; Woodrow, G.C., Young, I.G . and Gibson, F . (1975) J . Bacteriol . 124, 1-6) have been partially purified using a previously reported fractionation procedure (Bryce, G.F . and Brot, N . (1972) Biochemistry 11, 1708-1715) . The products of genes E, F and G have been separated from each other and correspond to the E1, E2 and E3 activities described by Bryce and Brot . These three gene products were not completely separated from the product of gene D . We refer to these gene products as components E, F, G and D of the enzymic apparatus for biosynthesis of enterochelin . Certain properties and functions of the four semi-purified components have been investigated . The E component is involved in the activation of 2,3-dihydroxybenzoate and the F component in the activation of L-serine . The D component physically associates with the F and G components during gel filtration and chromatography on DEAE Sephadex . It is proposed that the synthesis of enterochelin from L-serine and 2,3-dihydroxybenzoic acid is catalysed in vivo by a multienzyme complex, enterochelin synthetase.

Acta Pathol Microbiol Scand {C}, 1976 Dec, 84C(6), 495 - 500
Inhibition of the mitogenicity of the carrier molecule results in loss of immunogenicity of a hapten-LPS conjugate; Smith E et al.; Colistin methanesulfonate, a basic polypeptide similar to polymyxin E, has been shown to suppress the mitogenicity of lipopolysaccharide (LPS) from E . coli . It also inhibits the immunogenicity of a hapten-LPS conjugate . The inhibition was neither due to interference with the expression of hapten determinants, nor was it due to crossreactivity between the hapten and colistin methanesulfonate . As mitogenicity and immunogenicity was similarly affected, we conclude that activation of bursa-derived lymphocytes, in specific thymus-independent immune responses, does not take place in the absence of a mitogenic (non-Ig mediated) signal, thus supporting the hypothesis of the "one nonspecific signal" for B cell triggering.

Acta Pathol Microbiol Scand {B}, 1976 Dec, 84B(6), 321 - 5
Five new Escherichia coli K antigens, K95, K96, K97, K98 and K100; Orskov I et al.; Five Escherichia coli strains were established as antigenic test strains for five new polysaccharide K antigens: K95, K96, K97, K98 and K100 . K95 to K98 served already as test strains of O antigens O75, O77, O81 and O107 respectively . F147, which is test strain of K100, had O antigen O75.

J Biochem (Tokyo), 1976 Dec, 80(6), 1401 - 9
Interactions of outer membrane proteins O-8 and O-9 with peptidoglycan sacculus of Escherichia coli K-12; Hasegawa Y et al.; The outer membrane proteins O-8 and O-9 were specifically bound to the peptidoglycan sacculus in sodium dodecyl sulfate (SDS) solution . Other cellular proteins failed to interact with the peptidoglycan sacculus under the same conditions . When the outer membrane was preheated in SDS solution, the binding did not take place . Optimum binding was observed at pH 8 in the presence of 5 mM Mg2+ . A high concentration of sodium chloride strongly inhibited the binding . The effects of these factors on the bindings of O-8 and O-9 required neither the bound nor the free form of Braun's lipoprotein, nor was the binding of either protein necessary for the binding of the other . Proteins O-8 and O-9 were also found in the peptidoglycan sacculus when it was prepared from cells in SDS solution at 60 degrees . A dilution experiment showed that the complex was not an artifact . The mode of interaction between these proteins and peptidoglycan in the preparation was similar to that in the reassembled O-8-O-9-peptidoglycan complex, as judged from the sensitivity to sodium chloride and temperature . The physiological importance of the complex is discussed in relation to the assembly of the outer membrane on the cell surface.

Biophys Chem, 1976 Dec, 6(1), 1 - 8
HNMR of succinate binding to aspartate transcarbamylase . A comparison of results in D2O and H2O; Mosberg HI et al.; The interaction of succinate with asparatete transcarbamylase from Escherichia coli has been studied by magnetic resonance relaxation measurements of the dicarboxylic acid methylene protons in H2O solutions . The pH and temperature dependence of the relaxation in the presence of either native asparte transcarbamylase or its catalytic subunit in H2O solutions is qualitatively very similar to the corresponding situation utilizing D2O as the solvent . From previous result of measurements in D2O{C.B . Beard and P.G . Schmidt, Biochemistry 12(1973)2255} a mechanism was proposed involving 2 protonated groups affecting succinate binding and titratable over the pH range 7-10 . Quantitatively, fitting the data from H2O solutions to the mechanism yeilds values of the fitting parameters generally in good agreement with the D2O experiments . The main exceptions are the pKa values calculated for the two titratable groups . For these species the values obtained in the presence of the catalytic subunit are 6.7 and 7.8 in H2O solutions versus 7.3 and 8.6 in D2O solutions . In the presence of native enzyme the corresponding values are 6.8 and 8.3 in H2O versus 7.6 and 9.2 in D2O . These observed differences are consistent with differences in ionization constants of weak acids in D2O relative to H2O . The results imply that succinate interaction with the enzyme active site is similar in the two solvents.

Nucleic Acids Res, 1976 Dec, 3(12), 3423 - 38
Implications of electrostatic potentials on ribosomal proteins; Kliber JS et al.; Potentiometric studies of ribosomal particles 30S, 50S, and 70S, were designed to investigate possible implications of the electrostatic potentials developed by the 16S and 23S rRNA fractions . Release of protons and proton titrations of these ribosomal fractions were examined as a function of Mg2+ and K+ concentrations . The effects of these cations fit the polyelectrolyte theory remarkably well and are discussed accordingly.

J Trauma, 1976 Dec, 16(12), 993 - 9
Osteomyelitis following puncture wounds of the foot in children; Lang AG et al.; Review of the laboratory and clinical findings and treatment of eight patients with osteomyelitis of the foot after puncture wounds revealed that: 1) osteomyelitis after puncture wounds is a infrequent but potentially serious complication, with significant morbidity; 2) osteomyelitis is frequently preceded by inadequate primary care for simple puncture wounds, and when treatment is appropriate, osteomyelitis usually can be avoided; 3) P . aeruginosa is the most commonly recovered organism; 4) the clinical presentation is characterized by a lack of systemic toxicity, paucity of laboratory abnormalities, and evidence of a localized infection process and the patient may be asymptomatic for a few days to several months after the injury before presentation of the osteomyelitis; and 5) once the infection has become established, treatment must be aggressive, including surgical debridement.

Mol Cell Biochem, 1976 Nov 30, 13(2), 79 - 87
The host specificity system in Escherichia coli SK; Nikolskaya II et al.; E . coli SK has its own enzyme system providing DNA host specificity which differs from the known types of specificity in E . coli K12 and E . coli B . Modification and restriction are observed when the PBVI or PBV3 phages are transferred from E . coli SK to E . coli B or K12 (and back) . A methylase has been isolated from E . coli SK cells and partly purified . This methylase catalyzes in vitro transfer of the labelled methyl groups from S-adenosylmethionine (SAM) to DNA of both phage and tissue origin which gives rise to 5'-methylcytosine (5'MC) and 6'-methylaminopurine (6'MAP) . The methylase preparations isolated from the cells at the stationary growth have proved to be 1.5-1.7 times as active as the enzyme from the cells at the logarithmic growth stage . The extract of E . coli SK cells infected with the phage SD cannot methylate DNA in vitro . This fact is due to de novo synthesis of the enzyme which disintegrates SAM down to 5'-methyltioadenosine (5'MTA) and homoserine (HS) . This enzyme is not found in the cells infected with the SD phage in the presence of chloroamphenicole . The activity of the enzyme which disintegrates SAM is the highest between the 4th and the 5th minutes of infection . Thus it may be assumed that this enzyme, most probably, is an early virus specific protein and prevents in vivo methylation of the phage DNA.

Biochemistry, 1976 Nov 30, 15(24), 5246 - 53
Conformational changes of 30S ribosomes measured by intrinsic and extrinsic fluorescence; Spitnik-Elson P et al.; The intrinsic tryptophan fluorescence and the fluorescence of N-(3-pyrene)maleimide, a covalently bound sulfhydryl-specific extrinsic probe, have been used to study the conformation of the 30S ribosomal subunit of Escherichia coli . (a) The tryptophan fluorescenct spectrum of the free ribosomal proteins is shifted to shorter wavelengths than that of free tryptophan . When the proteins are incorporated into the organized structure of the ribosome, there is a small additional blue shift and the emission band becomes narrower . In 6 M urea, the spectrum of the proteins, whether free or in the ribosome, becomes identical with that of the amino acid, reflecting exposure of previously shielded tryptophan residues . (b) When magnesium-depleted ribosomes are unfolded at low ionic strength, the tryptophan fluorescence spectrum changes, although circular dichroism shows no change in alpha-helix content of the proteins . (c) Intrinsic and extrinsic fluorescence were both found to be sensitive to a limited and fully reversible transition that takes place when ribosomes are incubated under conditions that increase their activity in vitro . This suggests that both probes may be of use in monitoring conformational changes that occur under conditions consistent with activity . The kinetics of the concurrent changes in extrinsic fluorescence and aminoacyl-tRNA binding activity were compared . (d) Conditions are described for labeling ribosomes with N-(3-pyrene)maleimide without impairing their activity.

Biochemistry, 1976 Nov 30, 15(24), 5241 - 6
Double-stranded DNA in methanol-ethanol-buffer solvent system; Kay E; DNA in a solvent system consisting of roughly equal volumes of methanol and ethanol and 5% buffer has a conservative circular dichroism (CD) spectrum of very low intensity above 220 nm and an increase of epsilon258 comparable to that of denatured DNA (about 40%) . A direct comparison of this spectrum with the CD of single-stranded DNA reveals many differences, indicating DNA in this solvent system has a conformation different from that of denatured DNA . When the alcohols are removed, the B form conformation and normal epsilon258 are restored in native DNA, while single-stranded DNA remains denatured . A double-stranded structure of DNA in the methanol-ethanol-buffer solvent system is confirmed by the neutral cesium chloride density gradient centrifugation of DNA in which one chain is labeled with {14C}thymidine and the other {3H}5-bromodeoxyuridine . The doubly labeled DNA exposed to the alcohol solvent system has a centrifugal pattern identical with that of control DNA; the two radioactivities cosediment and form a superimposing band, distinctly different from that of single-stranded DNA; 3H-labeled (thymidine) chains sediment further than 14C-labeled chains (5-bromo-deoxyuridine) . Denatured DNA exhibits varying CD spectra depending on solvents . It is suggested that single-stranded DNA in different solvent systems assumes different modes of base stacking.

Biochemistry, 1976 Nov 30, 15(24), 5205 - 11
Membrane-associated phosphatidylglycerophosphate synthetase from Escherichia coli: purification by substrate affinity chromatography on cytidine 5'-diphospho-1,2-diacyl-sn-glycerol sepharose; Hirabayashi T et al.; The membrane-associated cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDP-diglyceride):sn-glycerol-3-phosphate phosphatidyltransferase (EC 2.7.8.5) from Escherichia coli has been solubilized wiTriton X-100 and purified 6000-fold to 85% of homogeneity . The major purification was attained using several modifications of the the CDP-diglyceride Sepharose affinity chromatography system described by Larson et al . (Larson, T.J., Hirabayashi, T., and Dowhan, W . (1976), Biochemistry 15, 974) . The native enzyme in Triton X-100 had an apparent molecular weight of over 200 000, as judged by Sepharose 6B gel filtration . The apparent size of the native enzyme appeared to be due to its association with Triton X-100, as judged by sucrose gradient centrifugation, polyacrylamide gel electrophoresis, and the lack of affinity for ion-exchange resins . The minimum subunit molecular weight of the enzyme, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, was 24 000 . This low molecular weight is consistent with the stability of enzyme to heat, urea, or sodium dodecyl sulfate denaturation . The purified enzyme had an absolute requirement for magnesium ion (KM = 50 mM) and Triton X-100 (0.5-6%) for activity when either CDP-diglyceride or dCDP-diglyceride was used as substrate . Kinetic analysis of the enzymatic reaction indicated an ordered sequential Bi-Bi reaction with the liponucleotide forming a dead-end complex at high concentration, which inhibited both the forward and reverse reactions . The enzyme would not hydrolyze the pyrophosphate bond of its lipid substrate or the phosphate esters of its lipid product but would catalyze a cytidine 5'-monophosphate dependent exchange reaction between glycero-3-phosphate and phosphatidylglycerophosphate.

Biochemistry, 1976 Nov 30, 15(24), 5212 - 8
Ribosomal-associated phosphatidylserine synthetase from Escherichia coli: purification by substrate-specific elution from phosphocellulose using cytidine 5'-diphospho-1,2-diacyl-sn-glycerol; Larson TJ et al.; Cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDPdiglyceride):L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthetase) is bound tightly to the ribosomes in crude extracts of Escherichia coli . After separation of the enzyme from the ribosomes by the method of Raetz and Kennedy (Raetz, C.R.H., and Kennedy, E.P . (1974), J . Biol . Chem . 249, 5038), we have purified the enzyme to 97% of homogenekty . The major portion of the overall 5500-fold purification was attained by substrate-specific elution from phosphocellulose using CDP-diglyceride in the presence of detergent . The purified enzyme migrated as a single band with an apparent minimum molecular weight of 54 000 when subjected to electrophoresis on polyacrylamide disc gels containing sodium dodecyl sulfate . The purified enzyme catalyzed exchange reactions between cytidine 5'- monophosphate (CMP) and CDP-diglyceride and between serine and phosphatidylserine . The enzyme also catalyzed the hydrolysis of CDP-diglyceride to form CMP and phosphatidic acid . dCDP-diglyceride was equivalent to CDP-diglyceride in all reactions catalyzed by the enzyme . In addition, the purified enzyme catalyzed the formation of phosphatidylglycerol or phosphatidylglycerophosphate at a very slow rate when serine was replaced as substrate by glycerol or sn-glycero-3-phosphate, respectively . These results suggest catalysis occurs via a ping-pong mechanism through the formation of a phosphatidyl-enzyme intermediate.

Biochemistry, 1976 Nov 30, 15(24), 5315 - 20
Fluorine-containing analogues of intermediates in the Shikimate pathway; Pilch PF et al.; The phosphoenolpyruvate analogue (Z)-phosphoenol-3-fluoropyruvate is a substrate for phenylalanine-inhibitable 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthase from Escherichia coli . In the presence of excess erythrose 4-phosphate, apparent KM values of 65 and 38 muM were observed for phosphoenol-3-fluoropyruvate and phosphoenolpyruvate, respectively . Because the apparent Vmax for phosphoenol-3-fluoropyruvate is only 1.17% of that for phosphoenolpyruvate, one can study the former as an inhibitor of 3-deoxy-arabino-heptulosonic acid-7-phosphate synthase . Kinetic experiments showed phosphoenol-3-fluoropyruvate to be competitive with respect to phosphoenolpyruvate . Two distinguishable Ki values of 8 and 48 muM were obtained . The product (3S)-3-deoxy--3-fluoro-arabino-heptulosonic acid 7-phosphate was purified, characterized, and shown to act as a substrate for 5-dehydroquinate synthase . 3-Deoxy-3-fluoro-arabino-heptulosonic acid 7-phosphate, in contrast to 3-deoxy-arabino-heptulosonic acid 7-phosphate reacts slowly or not at all with reagents specific for 2-keto-3-deoxy sugars and is relatively resistant to oxidative cleavage by sodium periodate . The expected product of periodate oxidation, 3-fluoro-3-formylpyruvate, cannot be detected . This observation was clarified by studies with model compounds.

C R Acad Sci Hebd Seances Acad Sci D, 1976 Nov 29, 283(14), 1597 - 600
{Multiplicity of beta lactamases: a problem of isoenzymes}; Labia R et al.; Analytical isoelectric focusing of beta lactamases is a very sensitive and powerful technique which generally shows a number of satellites near the main enzymatic spike . By this technique we studied two bacterial groups known to produce different beta lactamases, but only one by germ . Analytical isoelectric focusing showed that all bacteria from the same group produce the same satellite system which differs only in relative activity.

Nature, 1976 Nov 25, 264(5584), 328 - 33
Cell length, cell growth and cell division; Donachie WD et al.; When cells of E . coli reach a certain critical length, which is constant in all growth conditions and eqqal to twice the minimum cell length, they abruptly increase their rate of elongation and divide about 20 min later . Chromosome replication terminates at about this same cell length but is not the signal for the change in rate of cell elongation.

J Biol Chem, 1976 Nov 25, 251(22), 7011 - 20
Interconvertible forms of Escherichia coli dihydrofolate reductase with different affinities for analogs of dihydrofolate; Pattishall KH et al.; Dihydrofolate reductase from Escherichia coli exists as two species, which show large differences in their affinities for trimethoprim and for pyrimethamine . The two species are present in approximately equal proportions . Each possesses one binding site per mol with dissociation constants (KD) of 14 and 1400 nM, respectively, for the binding of trimethoprim in the binary complex, and of 5 and 47 nM for the pyrimethamine binary complex . In the formation of the ternary complex with NADPH, trimethoprim bound to dihydrofolate reductase as if all the enzyme existed as a single species with a KD for trimethoprim of 1.9 nM . Formation of the trimethoprim NADPH ternary complex thus involves strong cooperative effects, and interconversion of the two species . The binding of pyrimethamine in the ternary complex was indistinguishable from its binding in the binary complex, showing neither the cooperative effects, nor the interconversion of the two species observed with trimethoprim . The species with a low KD for trimethoprim and pyrimethamine could be isolated by selective proteolysis . It was quite stable, but could be converted to a mixture of the original species via formation of the ternary complex with trimethoprim, as predicted from the binding data . The results are interpreted in terms of a model in which the two species can be interconverted only via the formation of a common ternary complex, which can be formed after the binding of trimethoprim or dihydrofolate, but not pyrimethamine . The model is shown to be consistent with all data from the measurements of both binding and inhibition by both ligands.

Mol Gen Genet, 1976 Nov 24, 149(1), 87 - 99
The use of specialised transducing phages in the amplification of enzyme production; Moir A et al.; Two types of lambdatrp phages have been used as model systems to investigate ways of optimising the expression of bacterial genes from transducing phage genomes . Excellent yields of trp enzymes were achieved by infecting a trpR- host with Q- or Q-S- derivatives of lambdatrpAM1, which expresses its trp genese exclusively from the trp promoter . The five trp geneproducts constituted more than 50% of the total soluble protein of infected cells under these conditions, and an even higher proportion of the protein synthesized after infection . In a trpR+ host, phage DNA replication was easily able to override tryptophan-mediated repression by titration of the trp promoter were equally productive, while having the advantage of being much simpler to construct and propagate . lambdatrp phages lacking the trp promoter were used to investigate ways of optimising gene expression initiated at the phage promoter, PL . Though very powerful, the latter promoter is more difficult to harness then the trp promoter . Derepression of transcription from PL by the use of cro- mutations is accompanied by poor replication of transducing phage DNA . Attempts to circumvent this difficulty using virulent of cro,cII double mutants have not been successful . Nevertheless, cells infected with a lambdatrp phage expressing its trp genes exclusively from PL made up to 16 per cent of their protein as trp gene-products.

Mol Gen Genet, 1976 Nov 24, 149(1), 23 - 31
Cloning of calf thymus satellite I DNA in Escherichia coli; Gautier F et al.; The 1400 base pair repeat produced by digestion of calf satellite I DNA (phi = 1.714 g/cm3) with EcoRI, was cloned in E . coli . The hybrid plasmid (pGM 214) which contains the ColE1-Ap vector (pSF 2124) and the 1400 base pair fragment replicates stably in E . coli and can be amplified by chloramphenicol treatment . No clone was found in which more than one "repeat unit" of the satellite I DNA was present in the chimaera plasmid . Digestion of the original satellite I and the plasmid pGM 214 with R-SmaI shows that the satellite DNA replicated in E . coli is cleaved by the restriction endonuclease SmaI whereas the original satellite I DNA from calf thymus is not, suggesting that the satellite I contains a large amount of modified cytosine or guanosine, probably 5-methyl-cytosine . R-EcoRI* produces a number of fragments with the satellite I in the range of 300 base pairs to 1400 base pairs . A physical map of pGM 214 (and pSF 2124) with R-EcoRI, R-HincII, R-HindIII, R-SmaI, R-BamI and R-EclI was constructed . The 1400 base pair "repeat unit" in the pGM 214 is efficiently transcribed in vitro by purified RNA polymerase, starting from a pSF 2124 promoter . The restriction enzyme EclI produces a 350 base pair repeat with calf satellite II (phi = 1,722 g/cm3), whereas the satellite I is not cut by this enzyme.

Biochim Biophys Acta, 1976 Nov 19, 450(2), 137 - 41
{Fatty acid composition and phospholipid pattern in auxotrophs for unsaturated fatty acids (author's transl)}; Beaudoin AR; The relationship between fatty acid composition and phospholipid pattern has been studied in Escherichia coli auxotrophs for unsaturated fatty acids . 1 . The presence of a regulatory mechanism which enables the organism to maintain a given fluidity of the lipids has been corroborated using exogenous fatty acids which cause dramatic changes in fatty acid composition . 2 . The fatty composition of phosphatidic acid is different from that of the other classes of phospholipids . 3 . Changes in fatty acid composition are concomittant with the alteration of the phospholipid pattern . The ratio of phosphatidylglycerol to diphosphatidylglycerol is particularly sensitive to the physical characteristics of the exogenous unsaturated fatty acid . The relative increase in diphosphatidylglycerol is associated with membrane alterations.

Biochim Biophys Acta, 1976 Nov 19, 450(2), 269 - 72
Purification and positional specificity of sn-glycerol-3-phosphate acyltransferase from Escherichia coli membranes; Ishinaga M et al.; SN-Glycerol-3-phosphate acyltransferase was solubilized from membranes of Escherichia coli B and K-12 and purified on an affinity column of Sepharose 4B coupled with 6-phosphogluconic acid . Phosphatidylglycerol was required for activation and stabilization of the purified enzyme . The acyl residues were exclusively transferred to the position 1 of sn-glycerol 3-phosphate by the enzyme, regardless of whether the acyl-CoA was saturated or unsaturated.

Mol Gen Genet, 1976 Nov 17, 148(3), 337 - 40
Genetic analysis of mutants from Escherichia coli K12 unable to grow anaerobically without exogenous acceptor; Casse F et al.; The ana mutation leads E . coli to need an exogenous electron acceptor for anaerobic growth . The affected gene maps near 26 min between chl C and tdk on the chromosome of the organism.

Mol Gen Genet, 1976 Nov 17, 148(3), 271 - 9
Conditional lethal phosphatidylserine decarboxylase mutants of Escherichia coli . Mapping of the structural gene for phosphatidylserine decarboxylase; Hawrot E et al.; The final step in the biosynthesis of phosphatidylethanolamine, the major membrane lipid of Escherichia coli, is catalyzed by the membrane-bound enzyme, phosphatidylserine decarboxylase . A variation of a procedure for localized mutagenesis (Hong and Ames, 1971) was employed to generate conditional lethal mutants in phosphatidylserine decarboxylase . In our modification, an episome carrying the psd gene closely linked to purA+ was heavily mutagenized in vivo in a strain also lysogenic for phage P1CMclr100 . After induction of a phage lytic cycle, the purA+ marker was transduced to a purA- recipient . A majority of the Pur+ transductants thus contained a psd gene originating from the heavily mutagenized episomal strain . Three mutants were isolated in which temperature-sensitive growth is caused by thermosensitive phosphatidylserine decarboxylase activity that is defective in vivo at the non-permissive temperature . All 3 mutations were mapped at the same location as psd1, being cotransduced with melA, purA, and ampA . The gene order in this region, as determined by a phage Pl-meidated, three-factor cross is ampA-psd-purA . psd+ is dominant to the psd mutant alleles.

Biochemistry, 1976 Nov 16, 15(23), 5188 - 92
Physical characteristics of protein-deficient derived from the 50S ribosomal subunit; Giri L et al.; Sedimentation coefficients, diffusion coefficients, density increments, extinction coefficients, and molecular weights were determined for selected core particles from the 50S Escherichia coli ribosomal subunits . These core particles were found to be loosened structures containing less than half the protein present on the intact 50S subunits and totally lacking peptidyl transferase and polyphenylalanine synthesis activity unless reconstituted . The results of this study confirm that a loosening of the subunit takes place upon the removal of protein, but this loosening is not necessarily due to the loss of the protein.

Biochemistry, 1976 Nov 16, 15(23), 5126 - 31
Equilibrium between two forms of the lac carrier protein in energized and nonenergized membrane vesicles from Escherichia coli; Rudnick G et al.; p-Nitrophenyl alpha-D-galactopyranoside is a competitive inhibitor of lactose transport in membrane vesicles prepared from Escherichia coli ML 308-225 (Ki congruent to 6.6 muM) but is not accumulated by the vesicles . Binding of p-nitrophenyl alpha-D-{6-3H}galactopyranoside to membrane vesicles has been measured by flow dialysis . In the presence of D-lactate, ligand binds to the vesicles with a KD of about 6 muM, and a total of 2.3 nmol per mg of membrane protein is bound at saturation . In the absence of D-lactate, a small amount of binding can be detected (approximately 0.2 nmol per mg of membrane protein) with a similar affinity constant (KD congruent to 9 muM) . Binding inthe presence or absence of D-lactate is dependent upon a functional lac y gene product and upon the structural integrity of the vesicle membrane and is reversed by p-hydroxymercuribenzenesulfonate . Agents such as 2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, and valinomycin, alone or in combination, abolish D-lactate-dependent binding but do not affect binding in the absence of electron donors . The results confirm previous observations that the bulk of the lac carrier protein is unable to bind ligand unless the membrane is energized . and they also corroborate observations that a small amount of binding occurs in the absence of energy coupling . The findings are discussed in terms of a model in which the lac carrier protein exists in a state of dynamic equilibrium between two forms: (i) a low affinity, cryptic form which predominates in the absence of energy coupling; and (ii) a high affinity form, accessible from the external surface of the membrane, which predominates in the presence of an electrochemical gradient of protons (interior negative and alkaline).

Biochemistry, 1976 Nov 16, 15(23), 5083 - 7
Separation of transfer ribonucleic acids on polystyrene anion exchangers; Singhal RP et al.; The transfer RNA separation by chromatography on strong-base polystyrene exchange materials is examined and compared with the widely used reversed-phase chromatography . Results indicate important differences in some transfer RNA (tRNA) elution patterns by the anion-exchange chromatography, as compared with the reversed-phase chromatography . Transfer RNAs containing hydrophobic groups are adsorbed more strongly . The anion exchanger has twice the number of theoretical plates . Single peaks of tRNA2Glu and tRNA1Phe obtained from the reversed-phase column give multiple peaks only polystyrene anion-exchange chromatography . All six leucine tRNAs (Escherichia coli) and differences in tRNA populations synthesized during early and late stages of the dividing lymphocytes from normal human blood can be characterized by the anion-exchange chromatography . Different separation profiles are obtained by two separation systems for tyrosine tRNAs from mouse liver and mouse-plasma-cell tumor . The results indicate that, in contrast to the reversed-phase chromatography, strong-base-polystyrene anion-exchange chromatography is capable of separating tRNAs with minor structlral differences.

Biochemistry, 1976 Nov 16, 15(23), 5057 - 64
Studies on the noncooperative binding of the Escherichia coli DNA unwinding protein to single-stranded nucleic acids; Ruyechan WT et al.; The noncooperative binding of the Escherichia coli DNA unwinding protein to single-stranded DNA oligomers has been studied by means of equilibrium dialysis . Dialyses were performed under a number of solution and temperature conditions using oligomers of varying length and base compositions . The results of these studies, which include a Scatchard analysis of the binding, have allowed us to propose a model for the cooperative binding of the protein to single-stranded DNA . The results of experiments dealing with the interaction of the protein with single-stranded RNA are also presented.

Int J Cancer, 1976 Nov 15, 18(5), 672 - 8
Effect of localization of L-asparaginase as the concanavalin A conjugate on anti-tumor activity; Shier WT et al.; A method potentially capable of enhancing the effectiveness of therapeutic enzymes such as L-asparaginase was investigated . The method was suggested by the following properties that have been observed for lectins injected into tissues: (1) six lectins with differing specificities were retained near the site of injection in the feet of mice 10 to 100 times longer than several non-lectin proteins . Prolonged retention of 125I-labelled concanavalin A was also observed in other normal and malignant mouse tissues . (2) The retention of 125I-labelled concanavalin A was not affected by prior immunization against concanavalin A . (3) Electrophoresis of tissue extracts on sodium dodecyl sulfate-poly-acrylamide gels followed by radioautography indicated that the 125I-labelled concanavalin A retained in the tissue remained as intact in form as prior to injection . Since the therapeutic efficacy of many enzymes may be enhanced by localization at the intended site of action, in principle it should be possible to enhance the effectiveness of therapeutic enzymes by combining the tissue-localizing properties of a lectin with therapeutic effectiveness of the enzyme . A conjugate of E . coli L-asparaginase and concanavalin A has been prepared by covalent cross-linking with glutaraldehyde and has been shown to be retained in mouse tissue 90 times longer than the free enzyme . However, it is completely ineffective in the treatment of the L-asparaginase-sensitive lymphosarcoma 6C3HED in C3H/HeJ mice . The ineffectiveness of the conjugated enzyme may be associated with the interiorization of the conjugate by the cells of the tumor.

Eur J Biochem, 1976 Nov 15, 70(2), 483 - 92
RNA sequences associated with proteins L1, L9, and L5, L18, L25, in ribonucleoprotein fragments isolated from the 50-S subunit of Escherichia coli ribosomes; Branlant C et al.; 32P-labelled 50-S subunits from Escherichia coli ribosomes were hydrolysed under conditions known to give rise to two specific ribonucleoprotein fragments, containing proteins L1, L9, and L5, L18 and L25 respectively . RNA corresponding to these ribonucleoproteins was isolated and purified, and the various RNA fragments obtained were subjected to oligonucleotide analysis . The results showed that the RNA associated with proteins L1 and L9 was very similar to the RNA found with protein L1 after controlled digestion of 23-S-RNA - L1 complexes (described elsewhere); this RNA lies within a region 550-1000 nucleotides from the 3' terminus of 23-S RNA . The RNA associated with proteins L5, L18, and L25 consisted predominantly of two species of similar size . One was 5-S RNA, and the other a fragment of 23S RNA, lying within the region 450-1000 nucleotides from the 3' terminus.

Eur J Biochem, 1976 Nov 15, 70(2), 457 - 69
The binding site of protein L1 ON 23-S ribosomal RNA of Escherichia coli . 2 . Identification of the rna region contained in the L1 ribonucleoproteins and determination of the order of the RNA subfragments within this region; Branlant C et al.; Ribonucleoproteins were obtained by T1 ribonuclease digestion of reconstitued complexes of ribosomal protein L1 AND 23-S RNA from Escherichia coli . The RNA region of the main ribonucleoprotein 2 was totally digested with T1 ribonuclease . The oligonucleotide products were characterised and they showed that this region comprises 148 nucleotides located between the 550th and 1000th necleotides from the 3' end of the 23-S RNA . Of the other two ribonucleoproteins, the largest ribonucleoprotein 1 contained an extra RNA sequence, of at least 15 nucleotides, that was located at the 5' end of the RNA region . The smallest ribonucleoprotein 3 lacked an RNA section towards the 3' end of the region . The order of the RNA subfragments and the enzymic cutting positions in the whole RNA region are given for the ribonucleoproteins . It is shown that protein L1 most strongly protects a continuous section of 115 nucleotides at the 5' end of the main RNA region . Finally, evidence is presented for a methylated base, and for two sequence heterogeneities, in this region of the 23-S RNA.

Eur J Biochem, 1976 Nov 15, 70(2), 447 - 56
The binding site of protein L1 on 23-S ribosomal RNA of Escherichia coli . 1 . Isolation and characterization; Sloof P et al.; Ribonucleoproteins were prepared by ribonuclease digestion of a reconstitued complex of ribosomal protein L 1 and 23-S RNA from Escherichia coli . Three main ribonucleoproteins were identified . The largest was only obtained in an impure state at low ribonuclease concentration, whereas the two smaller ones, which were difficult to separate from one another electrophoretically, were stable over a range of enzyme concentrations . The two smaller ribonucleoproteins yielded a total of 13 RNA subfragments that were judged to be homogeneous electrophoretically . The latter were characterized for molecular weight and the subfragment composition of each of these ribonucleoproteins was established . Furthermore, the subfragments were shown to be maintained together in each ribonucleoprotein by RNA-RNA interactions . The primary and specific binding site of protein L1 was localized on one continuous RNA subfragment of about 110 nucleotides in length by two newly developed binding methods.

Eur J Biochem, 1976 Nov 15, 70(2), 601 - 10
Conformational studies on murein-lipoprotein from the outer membrane of Escherichia coli; Braun V et al.; Conformational studies on an isolated integral membrane protein are reported . Lipoprotein of Escherichia coli outer membrane was released from murein by treatment with either lysozyme or trypsin . The isolated lysozyme-released lipoprotein (lipoprotein I) contained 2 or 3 muropeptides covalently linked at the C-terminal end, while the trypsin-released lipoprotein (lipoprotein II) was free of muropeptides and lacked the C-terminal peptide Tyr-Arg-Lys . Circular dichroism spectra of the two preparations were essentially identical, and they show an alpha-helix content of about 80% . According to calculations based on the Chou-Fasman rules for proteins of known sequence, lipoprotein is 64% alpha-helix and 15% beta-structure . Infrared spectroscopy qualitatively supports these values . The conformation was stable in the pH range of 5 - 12 . Danaturation of lipoprotein by heat, 8 M urea, or sodium dodecylsulphate was a fully reversible, cooperative process . The thermal denaturation of lipoprotein occurs in two steps with transition points at 79.4 degrees C for lipoprotein I and at 85.1 degrees C for lipoprotein II . Lioprotein markedly changes conformation at dodecylsulphate concentrations where micelle formation sets in . The unusual behaviour of the lipoprotein convormation in sodium dodecylsulphate is discussed in relation to the lipoprotein conformation and aggregation within the membrane.

Eur J Biochem, 1976 Nov 15, 70(2), 339 - 47
Derivatives of guanosine triphosphate with ribose 2'-hydroxyl substituents . Interactions with the protein synthetic enzymes of Escherichia coli; Hamel E; This report describes the preparation of four methylated and phosphorylated derivatives of GTP, 2'-O-methylguanosine 5'-triphosphate (PPP-Me2' Guo), and guanosine 2'-monophosphate 5'-triphosphate (PPP-Guo-2'P), 3'-O-methylguanosine 5'-triphosphate (PPP-Me3'Guo), and guanosine 3'-monophosphate 5'-triphosphate (PPP-Guo-3'P) . These compounds were compared to GTP in their ability to support reactions catalyzed by Escherichia coli initiation factor 2(IF-2), elongation factor Tu (EF-Tu), and elongation factor G )EF-G) . As with previously studied GTP analogues, the nucleotide specificities of IF-2-dependent N-formylmethionylpuromycin formation and EF-Tu-dependent Ac-Phe2-tRNA formation were similar . There was little difference between the reactions supported by GTP, PPP-Me2' Guo, PPP-Me3' Guo, and PPP-Guo-3'P, but PPP-Guo-2'P was a poor substrate with both enzymes . A spectrum of activity was observed in EF-G-dependent formation of N-acetylphenylalanylphenylalanylpuromycin . While PPP-Me2' Guo was almost as effective as GTP in supporting translocation, PPP-Guo-2'P was a very poor substrate, having even less activity than guanosine 3'-diphosphate 5'-triphosphate . Intermediate activities were observed with PPP-Me3' Guo and PPP-Guo-3'P, the former nucleotide being more active than the latter.

Biochem J, 1976 Nov 15, 160(2), 185 - 94
Polypeptide-chain-elongation rate in Escherichia coli B/r as a function of growth rate; Young R et al.; By evaluating the kinetics of radioactive labelling of nascent and finished polypeptides, the peptide-chain elongation rate for Escherichia coli B/r at three different growth rates (mu) was determined to be 17 amino acids/s for the fast-growing cells (mu equals 1.3 and 2.0 doublings/h) and 12 amino acids/s for slow-growing cells (mu equals 0.67 doublings/h) . The results agree with the growth-rate-dependence of the rate of peptide-chain elongation found for the translation of newly induced beta-galactosidase messenger in this strain and under these conditions of growth {Dalbow & Young (1975) Biochem . J . 150, 13-20} . Together with the previously observed ribosome efficiency at these growth rates {Dennis & Bremer (1974) J . Mol . Biol . 84, 407-422} the results indicate that the fraction of ribosomes engaged in protein synthesis is about 0.8 at all three growth rates.

Eur J Biochem, 1976 Nov 15, 70(2), 509 - 16
Studies on 3-deoxy-d-arabinoheptulosonate-7-phosphate synthetase(phe) from escherichia coli k12 . 3 . Structural studies; Simpson RJ et al.; 1 . Investigations with structural analogues of phenylalanine indicated an absolute requirement for the aromatic ring and both the alpha-carboxyl and alpha-amino groups of phenylalanine for inhibition of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe) activity . Replacement of the alpha-H atom with a methyl group does not decrease the inhibition greatly . Varying degrees of inhibition were observed with o, m and p mono-substituted fluoro, chloro and hydroxy phenylalanines . D-Phenylalanine and several metabolites of the aromatic biosynthetic pathways do not inhibit enzymic activity . 2 . Circular dichroism studies indicated that the native enzyme possesses approximately 26% alpha-helix . Both circular dichroic and ultraviolet difference spectra indicated that the addition of phenylalanine to the synthetase induces a conformational change involving a small alteration of the secondary structure and large alterations in th interactions of some of the aromatic residues of the enzyme . In particular, a tryptophan residue moves from an extremly hydrophobic environment to one less hydrophobic . 3 . Kd for the binding of phenylalanine to the enzyme was determined spectrophotometrically to be 75 muM . 4 . Chemical modification studies suggested that a sulphydryl group and possibly a lysine residue may be implicated in the catalytic activity of the enzyme.

Eur J Biochem, 1976 Nov 15, 70(2), 501 - 7
Studies on 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe)from Escherichia coli K12 . 2 . Kinetic properties; Simpson RJ et al.; 1 . Co2+ is not a cofactor for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe) . 2 . The following analogues of phosphoenolpyruvate were tested as inhibitors of 3-deoxy-D-arabinoheptolosonate-7-phosphate synthetase(phe): pyruvate, lactate, glycerate, 2-phosphoglycerate, 2,3-bisphosphoglycerate, 3-methylphosphoenolpyruvate, 3-ethylphosphoenolpyruvate and 3,3-demethylphosphoenolpyruvate . The rusults obtained indicate that the binding of phosphoenolpyruvate to the enzyme requires a phosphoryl group on the C-2 position of the substrate and one free hydrogen atom at the C-3 position . 3 . The dead-end inhibition pattern observed with the substrate analogue 2-phosphoglycerate when either phosphoenolpyruvate or erythrose 4-phosphate was the variable substrate is inconsistent with a ping-pong mechanism and indicates that the reaction mechanism for this enzyme must be sequential . The following kinetic constants were determined:Km for phosphoenolpyruvate, 0.08 +/- 0.04 mM; Km for erythrose 4-phosphate, 0.9 +/- 0.3 mM; K is for competitive inhibition by 2-phosphoglycerate with respect to phosphoenolpyruvate, 1.0 +/- 0.1 mM . 4 . The enzyme was observed to have a bell-shaped pH PROFILE WITH A PH OPTIMUM OF 7.0 . The effects of pH ON V and V/(Km for phosphoenolpyruvate) indicated that an ionizing group of pKa 8.0-8.1 is involved in the catalytic activity of the enzyme . The pKa of this group is unaffected by the binding of phosphoenolpyruvate.

Eur J Biochem, 1976 Nov 15, 70(2), 493 - 500
Studies on 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe) from Escherichia coli K12 . 1.Purification and subunit structure; Simpson RJ et al.; 1 . 3-Deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe) from Escherichia coli K12 has been purified to near homogeneity . The purified enzyme has a specific activity of 67 units/mg which is about 1000 times that found in cell-free extracts of wild-tupe E . coli K12 . 2 . The minimum molecular weight of the enzyme was estimated by dodecylsuphate-gel electrophoresis to be 33000 . Re-estimation of the native molecular weight by gel filtration confirmed the previously determined value of 110000 . 3.Amino acid anaktsus abd tryptic fingerprints indicated that the subunits of the enzyme are very similar and possibly identical . 4.The purified enzyme does not contain Co2+.

Eur J Biochem, 1976 Nov 15, 70(2), 377 - 83
Enzyme reduction of disulfide bonds by thioredoxin . The reactivity of disulfide bonds in human choriogonadotropin and its subunits; Holmgren A et al.; The NADPH-dependent enzymic reduction of disulfide bonds in human choriogonadotropin and its two subunits, alpha and beta, was examined with thioredoxin and thioredoxin reductase from Escherichia coli . With 12 muM thioredoxin and 0.1 muM thioredoxin reductase at pH 7 all disulfide bonds in the alpha subunit could be reduced in 15 min . The reduction of disulfide bonds was recorded by a simple spectrophotometric assay at 340 nm, which allowed quantitation of the reduction rate and the number of disulfide bonds reduced . Partial reduction of the alpha subunit with thioredoxin followed by S-carboxymethylation with iodol{2-3H}acetic acid and analysis of tryptic peptides indicated that all S-S bonds in the alpha subunit were surface oriented and equally reactive . The usefulness of thioredoxin reduction of disulfide bonds as a chemical probe of protein structure was shown by the much slower reaction of disulfide bonds in the intact hormone as compared to its two biologically inactive subunits.

Biochim Biophys Acta, 1976 Nov 12, 454(1), 45 - 50
Reversibility of partial denaturation of DNA; Acuna MI et al.; The hypochromicity recovery of a partially denatured DNA sample when salt concentration is suddenly increased at an intermediate stage of the transition, is studied . The results of CsC1 gradient analysis, polyethylenglycol/dextran partition analysis, and the behaviour in a new thermal transition, support the view that the process is an intramolecular double chain renaturation as that induced by cooling . The degree of denaturation irreversibility is dependent on the size of DNA segments between two nicks and on the helicity value at which salt concentration-jump is performed.

Biochim Biophys Acta, 1976 Nov 12, 454(1), 97 - 113
General and specific effects of amino acid starvation on the formation of undermodified Escherichia coli phenylalanine tRNA; Fournier MJ et al.; The heterogeneity of undermodified phenylalanine tRNA produced in relaxed control E . coli during amino acid starvation was investigated . Examination of the RPC-5 elution profiles of tRNAPhe prepared from non-starved cells and cells starved of a variety of amino acids, including some known to be involved in the formation of modified bases revealed that: (1) only one species of fully modified tRNAPhe appears to occur in cells grown in enriched medium; (2) at least two chromatographically unique isoacceptor species are observed in addition to the normal tRNAPhe in starved cells; (3) the unique, undermodified species of tRNAPhe from leucine-starved cells, known to be deficient in dihydrouridine, pseudouridine, 2-thiomethyl-N6-(delta2-isopentenyl) adenosine and 3-(3-amino-3-carboxypropyl) uridine, co-elute with the unique species produced in cells starved of histidine or arginine or treated with puromycin or chloramphenicol; (4) additional unique species of tRNAPhe can be detected in methyl- and sulfur-deficient tRNA from methionine- and cysteine-starved cells; (5) analysis of phenoxyacetylated tRNA revealed that the chromatographically unique and normal species from starved cells contain subspecies deficient in 3-(3-amino-3-carboxypropyl) uridine; and (6) using phenoxyacetylation as a means of effecting the resolution of undermodified subspecies, a total of at least ten chromatographically unique subspecies of rRNAPhe were detected in an organism that appears to posses only one gene for tRNAPhe . Taken together, the results support the view that there are both general and specific effects of amino acid starvation on the post-transcriptional modification of tRNA.

Biochim Biophys Acta, 1976 Nov 12, 454(1), 9 - 20
The synthesis and enzymatic polymerization of 5-thio- and 5-methylmercurithio-pyrimidine nucleotides; Livingston DC et al.; The 5-thio and 5-methylmercurithio derivatives of UTP, dUTP and dCTP have been synthesized and tested as substrates for nucleic acid polymerases . The 5-thio-nucleotides were polymerized inefficiently by both RNA polymerase and DNA polymerase I of Escherichia coli . The 5-methylmercurithio derivatives of dUTP and dCTP were, however, utilized by DNA polymerase I, an enzyme insensitive to mercurial compounds, although they were potent inhibitors of all other polymerases tested . While polymers containing the 5-thio substituent possess structural abnormalities, most likely interstrand disulfide bridges, polymers containing 5-methylmercurithio groups appear normal . The latter polynucleotides are readily separated from non-sulfated polymers by chromatography on mercuriagarose.

Biochim Biophys Acta, 1976 Nov 12, 454(1), 185 - 92
Analysis of the kinetics of photoinduced crosslinkage of the protein and RNA components of the Escherichia coli 50 S ribosomal subunit; Gorelic L; The kinetics of photoinduced crosslinkage of the protein and ribosomal RNA components of the Escherichia coli 50 S ribosomal subunit were determined by two-dimensional gel electrophoresis . One group of ribosomal proteins was found to be reactive in the crosslinkage reaction at doses of 254 nm radiation of 2.9 X 10(3) ergs-mm-2 . A second group of 50-S ribosomal proteins was found to be unreactive in the crosslinkage reaction at doses of 254 nm radiation less than 8.6 X 10(6) ergs-mm-2, but were reactive at higher doses.

Biochim Biophys Acta, 1976 Nov 12, 454(1), 129 - 37
Rifampicin binding as a probe for subunit interactions in Escherchia coli RNA polymerase; Lowe PA et al.; The binding of the inhibitor rifampicin to RNA polymerase (alpha2betabeta') and its deficient subunit mixtures was investigated . The ability of beta to bind stoichiometric amounts of rifampicin was restored by formation of the alpha2beta subassembly . beta,beta' alpha, betabeta' and alpha2beta' were unable to bind rifampicin . RNA polymerase denatured with 6 M guanidine hydrochloride and dialysed against a renaturing buffer at 0degrees C ("renatured inactive enzyme") bound stoichiometric amounts of rifampicin but had lost the ability of bind dna . compared with native RNA polymerase "renatured inactive" enzyme possessed a markedly different tertiary structure as judged by limited proteolysis.

Proc Natl Acad Sci U S A, 1976 Nov, 73(11), 4135 - 9
Repair tracts in mismatched DNA heteroduplexes; Wagner R Jr et al.; Heteroduplexes with mismatches at four sites were constructed from separated strands of lambda DNA and used to transfect Escherichia coli under recombinationless conditions . The output phages from 967 single cells in one experiment and 1016 in another were analyzed to determine the pattern of mismatch repair . A wide range of repair frequencies was found among the mismatches studied . Repair involving two or more close sites in the same heteroduplex occurs much more often on thesamestrandthanonopposite strands.Analysis of the pattern of repair suggeststhat repair tracts initiate at mismatches, propagate preferentially in the 5'leads to 3' direction, and extend an average distance of ca 3000 nucleotides.

J Biol Chem, 1976 Nov 10, 251(21), 6823 - 30
Isotope labeling of free and aminoacyl transfer RNA synthetase-bound transfer RNA; Schoemaker HJ et al.; The structural organization of the complex of Escherichia coli Ile-tRNA synthetase and tRNA Ile has been studied by isotope labeling of purine units in the tRNA . Free or bound tRNA is incubated in tritiated water for 5 to 15 h at 37 degrees in order to incorporate tritium into the C-8 positions of purine units . (Previous work has shown that the labeling rate of a purine is very sensitive to its microenvironment.) Under conditions where exchange-out does not occur, the nucleic acid is digested with nucleases and purines are subsequently isolated from known locations in the structure . Four purines are substantially perturbed by bound Ile-tRNA synthetase; in each case, the rate of labeling is retarded in the presence of the synthetase . The four purines occur at or near the 3' terminus and at the interface of the dihydrouridine stem and loop . These bases occur in segments of the tRNA that previous photochemical cross-linking studies have identified as important for synthetase-tRNA interactions . It appears that the effects observed on these sites are caused by their direct interaction with or shielding by the bound synthetase . In addition, two other sites, one in the anticodon and one in the amino acid acceptor-T psi C helix, appear to be perturbed (retarded labeling rates) by the bound enzyme . The data also suggest there is no significant conformational change in the tRNA upon binding to the synthetase.

J Biol Chem, 1976 Nov 10, 251(21), 6735 - 8
Microcalorimetric study of the binding of thiodigalactoside to the lactose permease M protein of Escherichia coli; Belaich A et al.; The energetics of the binding of thiodigalatoside onto vesicles of Escherichia coli containing M protein is described . The Kd determined from equilibrium dialysis was 5-10(-5) M . The enthalpy change (deltaH) was measured by calorimetry . The derived deltaG and deltaH values allowed estimation of the entropic change associated with the binding reaction . The control experiments were made with membranes from cells that were not induced for the lac system . All the experiments were carried out in presence of 10(-2) M sodium azide to prevent any concentration of thiodigalactoside into the vesicles . It was concluded that such membrane vesicles which are in a de-energized state are able to bine thiodigalactoside specifically with a Kd corresponding to the Km of the entry of beta-galactoside measured with intact, active cells.

J Biol Chem, 1976 Nov 10, 251(21), 6717 - 23
Metabolic fate of 3,4-dihydroxybutyl-1-phosphonate in Escherichia coli; Tyhach RJ et al.; 3,4-Dihydroxy{3-(3)H}butyl-1-phosphonate, and analogue of glycerol 3-phosphate, is incorporated into a very polar lipid material by cultures of Escherichia coli strain 8 and in vitro by CDP-diglyceride:sn-glycerol-3-phosphate phosphatidyltransferase . These labeled lipids have been fractionated by column chromatography on DEAE-cellulose, revealing that only one labeled compound is formed in vitro, while four are synthesized in vivo . The main component of the material formed by intact cells has been shown to be identical with that produced enzymatically . This species has been identified as the phosphonic acid analogue of phosphatidylglycerophosphate {(1,2-diacyl)-sn-glyceryl-D-4'-phosphoryloxy-3'-hydroxybutyl-1'-phosphonate} . Hydrolysis of this novel lipid with phospholipase C resulted in the production of diglyceride and a water-soluble derivative of 3,4-dihydroxybutyl-1-phosphonate and inorganic phosphate in a molar ratio of 1.03/1 . Enzymatic analysis of the phosphonate liberated in this manner showed it to be the D enantiomer, thereby confirming the proposed structure of the lipid analogue . The analogue of phosphatidylglycerophosphate did not turn over and appeared to have no precursor-product relationship to the other labeled lipids derived from 3,4-dihydroxy{3-(3)H}butyl-1-phosphonate in vivo . Analysis of the other three labeled products revealed the tritium to be present on glycerol 3-phosphate and not intact phosphonate, indicating some metabolic degradation of the latter . Examination of cell components other than lipids revealed little incorporation of label, while a significant amount of tritium was found to be present in a distillable form, 3H2O . Experiments with mutants of E . coli lacking the known glycerol-3-phosphate dehydrogenases indicated that these enzymes are not responsible for the removal of tritium from from 3,4-dihydroxy{3-(3)H}butyl-1-phosphonate in vivo . Indirect evidence suggests that the inhibition of cell growth by this analogue is not due to its catabolic products.

J Biol Chem, 1976 Nov 10, 251(21), 6662 - 6
Effect of lipids on the reconstitution of D-lactate oxidase in Escherichia coli membrane vesicles; George-Nascimento C et al.; An unsaturated fatty acid auxotroph lacking D-lactate dehydrogenase activity has been isolated from Escherichia coli ML 308-225 dld-3 . While NADH oxidase activity in membrane vesicles prepared from the mutant cells grown in a variety of unsaturated fatty acids is comparable to that of previously isolated fatty acid auxotrophs, D-lactate oxidase activity is absent . However, D-lactate oxidase ativity can be restored when vesicles are incubated with a purified preparation of D-lactate dehydrogenase obtained from wild type cells . The effect of altering the fatty acid composition of the membrane on the reconstitution of D-lactate oxidase activity was examined . Binding of purified D-lactate dehydrogenase was not affected by either the lipid composition of the membrane vesicles or the temperature during reconstitution . However, the reconstitution of D-lactate oxidase activity was strongly influenced by the fatty acid composition of the membrane lipids . The temperature dependence of the reconstituted activity was analyzed . Temperature transitions were not observed with membrane vesicles supplemented with oleic or linolenic acid but palmitelaidic acid-enriched vesicles exhibited a transition temperature at 30 degrees . Attempts to reconstitute the elaidic-supplemented vesicles at temperatures below 30 degrees failed to yield active D-lactate oxidase and the vesicles aggregated . At 42 degrees, aggregation did not occur and D-lactate oxidase activity was obtained to a level of 10% of that found with membranes from the parent strain (ML 308-225) . These results suggest that although binding of D-lactate dehydrogenase is independent of the physical state of the membrane, reconstitution of the D-lactate oxidase activity in membrane vesicles is dependent upon the fatty acid composition of the phospholipid, hence the physical state of the membrane.

J Biol Chem, 1976 Nov 10, 251(21), 6646 - 52
Polyamine stimulation and cation requirements of rabbit liver tRNA nucleotidyltransferase; Evans JA et al.; We have examined the cation requirements of rabbit liver tRNA nucleotidyltransferase . The enzyme had an absolute requirement fo a divalent cation which could be satisfied by Mg2+, Mn2+ or Co2+ . In contrast to the Escherichia coli enzyme, we have found no evidence to implicate Zn2+ in the action of rabbit liver tRNA nucleotidyltransferase . We have also identified a second cation requirement which was satisfied by divalent or monovalent cation or polyamines . The polyamines, spermine and spermidine, were most effective leading to 3-fold higher rates of nucleotide incorporation than could be obtained at any concentration of the other cations . However, the polyamines were not an absolute requirement for enzyme activity . The polyamine stimulation of tRNA nucleotidyltransferase was not due to alteration of the pH or ionic strength of the reaction mixture or reactivation of denatured tRNA or enzyme . Polyamines also had no effect on the apparent Km values of the substrates . Spermine increased the specificity of the enzyme for tRNA substrates and also inhibited the reverse action . Our results suggest that polyamines may be the normal counterions for tRNA in vivo, and that they affect the rate-limiting step in tRNA nucleotidyltransferase catalysis.

J Biol Chem, 1976 Nov 10, 251(21), 6630 - 7
Purification of the carbodiimide-reactive protein component of the ATP energy-transducing system of Escherichia coli; Fillingame RH; The ATP-energy transducing system in membranes of Escherichia coli is inhibited by dicyclohexylcarbodiimide . The protein component of this complex with which carbodiimides covalently react to inhibit function was previously identified by labeling wild type and dicyclohexylcarbodiimide-resistant mutants with dicyclohexyl{14C}carbodiimide (Fillingame, R . H . (1975) J . Bacteriol . 124, 870-883) . This specific carbodiimide-reactive protein has now been purified . The protein was extracted from the membrane with chloroform:methanol and chromatographed on DEAE-cellulose and hydroxypropyl Spehadex G-50 in this sulvent mixture . The resultant 700-fold purification yielded a protein that was homogeneous on dodecyl sulfate-acrylamide gel electrophoresis and virtually free of phospholipid . It remained soluble in neutral chloroform:methanol throughout the purification procedure . The amino acid composition of the purified protein was extraordinary in that only 16% of the amino acids present could be considered polar . Histidine, serine, cysteine, and tryptophan were not found . Abnormally high contents of methionine, glycine, alanine, and leucine were present . One mole of lysine and threonine were found/mole of dicyclohexyl{14C}carbodiimide bound . The minimum molecular weight based on the amino acid composition was 8400 . The specific carbodiimide-reactive protein has also been purified without prior modification by dicyclohexylcarbodiimide . The unmodified protein eluted from DEAE-cellulose at a higher salt concentration than the dicyclohexylcarbodiimide-modified form, which suggested that the reaction with the carbodiimide neutralized the negative charge . Only one-third of the total carbodiimide-reactive protein in the membrane was modified by dicyclohexylcarbodiimide under conditions which maximally inhibited adenosine triphosphatase activity . These results rais the possibility that the carbodiimide-reactive protein may be present as an oligomer in the energy-transducing complex . The purification of the unmodified carbodiimide-reactive protein should permit assessment of tis biological function, particularly its role in the protein-translocation process that is catalyzed by this energy-transducing complex.

J Biol Chem, 1976 Nov 10, 251(21), 6544 - 9
Interactions of adenosine diphosphate-ribosylated elongation factor 2 with ribosomes; Bermek E; The binding of adenosine diphosphate-ribosylated elongation factor 2 (ADPRib-EF-2) to ribosomes was inhibited both in the presence and absence of GTP in proportion to the amounts of unmodified EF-2 added . Concomitant with this inhibition, an increase in the activity of ribosome-bound EF-2 in polyphenylalanine synthesis was observed . On the other hand, the addition of ADPRib-EF-2 reduced the rate of poly(Phe) synthesis observed in the presence of a saturating amount of EF-2 and increased the amount of EF-2 required for the half-maximal rate of poly(Phe) synthesis . Phe-tRNA, nonenzymatically bound to the ribosome in the presence of poly(U), inhibited the subsequent binding of ADPHRib-EF-2 . The same ribosomal population appeared to preferentially bind either aminoacyl-tRNA or ADPRib-EF-2 . The Scatchard plot of the binding of ADPRib-EF-2 to the ribosome in the presence of GTP revealed the presence of two ribosomal binding sites (or ribosomal populations) with apparent different affinities for the modified factor (K371 degrees d,1 = 6.6 nM and K37 degrees d,2 = 126 nM) . At saturating concentrations of ADPRib-EF-2, a maximum of about 1 molecule of the factor was bound per ribosome . The binding of ADPRib-EF-2 to the ribosome was stimulated by GTP . The binding of radioactive GTP to the ribosome was observed concomitantly with the binding of ADPRib-EF-2 . One mole of GTP was bound per mole of ADPRib-EF-2 . No significant difference could be found in the binding of GTP to ribosome required in the presence of either EF-2 or ADPRib-EF-2 . The binding of ADPRib-EF-2 to the ribosome required the presence of Mg2+ and reached a maximum at 5 mM . The binding was greatest at K+ concentrations below 20 mM . ADPRib-EF-2 was bound primarily to the large ribosomal subunit . A slight, but reproducible binding to the 40 S subunit was also observed . The addition of 40 S to 60 S subunits stimulated the binding of ADPRib-EF-2 . GTP displayed a stimulatory effect on the binding only in the presence of recombined subunits . Human ADPRib-EF-2 was bound to rat liver ribosomes as efficiently as to human tonsil ribosomes, while the binding to Escherichia coli ribosomes was insignificant.

J Biol Chem, 1976 Nov 10, 251(21), 6775 - 83
Escherichia coli acetate kinase mechanism studied by net initial rate, equilibrium, and independent isotopic exchange kinetics; Skarstedt MT et al.; The equilibrium constant of the reaction catalyzed by acetate kinase (E.C . 2.7.2.1.) was determined: K = (MgADP) (acetylphosphate)/(MgATP)(acetate) = 6.7 +/- 1.3 X 10 (-4) (pH 7.4, 25 degrees) . The respective free nucleotides uncomplexed to magnesium inhibit the net reaction in both directions: competitively with the respective magnesium nucleotide (MgATP or MgADP) and noncompetitively with the co-substrate (acetate or acetylphosphate) . Excess free magnesium also inhibits the net reaction in both directions . The inhibition is not competitive with the phosphoryl donor (MgATP or acetylphosphate) but is competitive with respect to the phosphoryl acceptor (acetate or MgADP) . A 50-fold increase in concentration of reactants at equilibrium in 0.1 M Tris/HCl, pH 7.4, at 25 degrees resulted in a rise to plateau levels of the acetate equilibrium acetylphosphate exchange rate (measured with {U-14C}acetate) and of the ATP equilibrium ADP exchange rate (measured with {U-14C}ADP and 10-fold higher than acetate equilibrium acetylphosphate), suggesting that there is no compulsory order of binding of magnesium nucleotide and co-substrate and that all chemical transformation steps cannot be much slower than the dissociation steps . The ATP equilibrium ADP exchange rate was independent of the presence or concentration of co-substrate, whereas the acetate equilibrium acetylphosphate exchange reaction occurred only in the presence of magnesium nucleotide, and the rate was directly related to the degree of saturation of enzyme with magnesium nucleotide . The independent ATP equilibrium ADP exchange, which presumably involves a phosphoenzyme intermediate, was progressively inhibited by increasingly elevated MgADP concentrations (when MgADP/MgATP greater than or equal 5), but increasing MgATP/MgADP was not inhibitory, suggesting that MgADP may bind to nonphosphorylated as well as phosphorylated enzyme, but that MgATP cannot bind to phosphorylated enzyme . While direct transfer of the phosphoryl group between nucleotide and co-substrate in a concerted mechanism has not been ruled out, an "activated ping pong" mechanism can also be proposed which is compatible with the isotopic exchange and initial net rate kinetic results . This mechanism includes a phosphoenzyme intermediate and requires enzyme-bount MgADP for phosphorylation of the enzyme by acetylphosphate.

J Biol Chem, 1976 Nov 10, 251(21), 6555 - 61
Photochemical studies and ultraviolet sensitization of Escherichia coli thymidylate kinase by various halogenated substrate analogs; Chen MS et al.; The effect of 5-iodo-2'-deoxyuridine monophosphate (IdUMP), various 5-halogenated-5'-azido-2', 5' -dideoxyuridine derivatives, 2'-deoxy-6-azauridine (AzdUrd), and its halogenated analogs on the ultraviolet sensitization of Escherichia coli thymidylate kinase has been investigated . Only those compounds iodinated in position 5 enhance the rate of ultraviolet inactivation of this enzyme . However, 5'-azido nucleosides with iodo, bromo, chloro, or fluoro substituents in position 5 neither protect nor sensitize thymidylate kinase to ultraviolet inactivation . Thymidine 5'-monophosphate partially protects the enzyme against ultraviolet inactivation either in the presence or absence of ultraviolet-sensitizing iodinated analogs . Magnesium ion does not enhance the ultraviolet inactivation of thymidylate kinase by 5-iodinated nucleoside analogs . The kinatic data support an active site-directed enhancement of the enzyme to ultraviolet inactivation by 5-iodo-2'-deoxyuridine monophosphate, since the concentration of IdUMP required to attain 50% maximal enhancement is 0.24 mM which is in good agreement with its Ki of 0.18 mM . When either {125I}IdUMP or {2-14C}IdUMP was irradiated with the enzyme, both radioactivities were associated with the enzyme, however only with the 14C analog was the amount bound at half-saturation essentially equal to the amount required to inactivate the enzyme by 50% . These data support the hypothesis that the active entity in the enhancement by IdUMP of thymidylate kinase inactivation during ultraviolet irradiation is the uridylate free radical which is formed photochemically from IdUMP . Photochemical studies of 6-azauracil (AzUra), 2'-deoxy-6-azauridine, and 5-iodo-2'-deoxy-6-azauridine (IAzdUrd) were performed . Photolysis of IAzdUrd in the presence of a hydrogen donor yields AzdUrd which upon further photolysis yields the photohydrate . The photohydrate of AzdUrd when incubated in the dark at pH 5.2 is 90% converted back to AzdUrd, whereas the photohydrate of AzUra is only partially (20%) converted to AzUra . The rate of deiodination of IAzdUrd is 2.1-fold greater than that of IdUMP . Although the Ki of IdUMP and IAzdUrd is similar, the increased photosensitivity of the aza analog accounts for the much greater enhancement of ultraviolet inactivation of thymidylate kinase . The ability of a compound to enhance the ultraviolet inactivation of deoxythymidylate kinase is correlated with the potential of the compound to produce a free radical rather than a photohydrate when the enzyme-substrate analog complex is irradiated.

Biochim Biophys Acta, 1976 Nov 8, 452(1), 13 - 29
Preparation of Escherichia coli pyruvate oxidase utilizing a thiamine pyrophosphate affinity column; O'Brien TA et al.; An improved procedure is reported for the purification of Escherichia coli pyruvate oxidase (pyruvate:ferricytochome b1 oxidoreductase, EC 1.2.2.2), a peripheral membrane flavo-enzyme, which is much more reproducible and requires considerably less time than the original purification scheme . The key element in this protocol is a new Sepharose-based affinity resin designed for the isolation of thiamine pyrophosphate-requiring enzymes . The synthesis, partial characterization, and use of two such affinity resins is described . Pyruvate oxidase is a pure, homogenous protein as it is eluted from the affinity resin . The enzyme is a tetramer with a subunit molecular weight of approx . 60 000 . The subunits appear to be identical . The isoelectric point of pyruvate oxidase is 5.6.

Biochim Biophys Acta, 1976 Nov 8, 452(1), 253 - 61
Studies on aspartase . III . Alteration of enzymatic properties upon trypsin-mediated activation; Mizuta K et al.; Highly purified aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) from Escherichia coli, already of full activity, is further activated 3.3-fold by limited treatment with trypsin . The activation requires a few minutes to attain maximum level, and hereafter the activity gradually decreases to complete inactivation . Prior or intermediate addition of soybean trypsin inhibitor results in an immediate cessation of any further change in the enzyme activity . Upon trypsin-mediated activation no appreciable change is detected in the molecular weight of the enzyme subunits as judged from sodium dodecyl sulfate polyacrylamide gel electrophoresis, nor in the pH vs . activity profile in the presence of added metal ions . However, S0.5 and hill coefficient for L-aspartate considerably increase upon activation . As the trypsin-mediated activation proceeds, a marked absorbance difference spectrum of the trypsin-treated aspartase vs . untreated aspartase appears with negative absorbance maxima at 278 and 285 nm . When the trypsin-activated enzyme is denatured in 4 M guanidine-HCl, followed by removal of the denaturant by dilution, the enzyme activity is readily restored to as much as 1.5 times that of the native enzyme, indicating that the trypsin-activated enzyme is rather a stable molecule.

Biochemistry, 1976 Nov 2, 15(22), 4866 - 75
beta-Galactosidase alpha complementation: properties of the complemented enzyme and mechanism of the complementation reaction; Langley KE et al.; Intracistronic alpha complementation involving Escherichia coli beta-galactosidase occurs between the cyanogen bromide peptide CB2, derived from residues 3-92 of beta-galactosidase (Langley, K.E., Fowler, A.V., and Zabin, I . (1975), J . Biol . Chem . 250, 2587), and the defective beta-galactosidase from the Z-deletion mutant strain M15 . The M15 protein, a dimer, lacks residues 11-41 of beta-galactosidase (Langley, K.E., Villarejo, M.R., Fowler, A.V., Zamenhof, P.J., and Zabin, I . (1975), Proc . Natl . Acad . Sci . U.S.A . 72, 1254) . The complemented enzyme formed from purified components has a molecular weight of 533 000+/-25 000, is therefore tetrameric, and has a probable stoichiometry of 1 CB2:1 M15 monomer . The complemented enzyme has the same Km for substrate as wild type enzyme, but is less stable to heat or urea treatment . The overall equilibrium constant for the complementation reaction is approximately 1-2 X 10(9) M-1 . Initial velocity studies indicate saturation kinetics when either component is fixed and limiting, with an apparent Kd of about 10(-6) M . A first-order rate constant of 0.05-0.1 min-1 was estimated . The kinetics favor a model of rapid complex formation, followed by slow conformational change, as the mechanism of activation . Ultraviolet difference spectroscopy indicated an increased absorbance in the 290-300 nm region as a result of the complementation reaction . The kinetics of the increase suggest that two processes, one rapid and the other slower, could be responsible . The temperature dependence of complementation (Ea approximately 24 000 cal) is also consistent with the rate-determining step being a conformational change.

Biochemistry, 1976 Nov 2, 15(22), 4792 - 6
Menaquinone biosynthesis: conversion of o-succinylbenzoic acid to 1,4-dihydroxy-2-naphthoic acid and menaquinones by Escherichia coli extracts; Bryant RW Jr et al.; Two intermediates in the biosynthetic pathway to bacterial menaquinones are o-succinylbenzoic acid and 1,4-dihydroxy-2-naphthoic acid . Cell-free extracts have been prepared from Escherichia coli which catalyze the conversion of labeled o-succinylbenzoic acid to the naphthoic acid and also to menaquinones . The naphthoate synthetase has been partially purified and found to require coenzyme A and ATP . The synthetase has an approximate molecular weight of 45 000 . The conversion of o-succinylbenzoic acid to menaquinones is stimulated by the presence of farnesyl pyrophosphate . The major menaquinone produced is then MK-3 with the farnesyl side chain.

Biochemistry, 1976 Nov 2, 15(22), 4786 - 91
Reactivity of ribosomal sulfhydryl groups in 30S ribosomal subunits of Escherichia coli and 30S-IF-3 complexes; Ewald R et al.; The reaction of 30S subunits with the SH group reagent N-ethylmaleimide (NEM) causes the loss of approximately 60% of their synethetic activity, but has little or no effect on the ribosomal binding of initiation factor IF-3 . The ribosomal binding of this factor, on the other hand, was found to significantly influence the rate and the extent to which several 30S ribosomal proteins react with radioactively labeled NEM when the reaction kinetics of individual ribosomal proteins toward NEM were compared in 30S and 30S-IF-3 complexes . Of the nine 30S ribosomal proteins which react with NEM, proteins S1, S11, S12, and S18 were found to have lower reactivities, while proteins S4 and S21 displayed higher reactivity in the presence of IF-3 . The reactivity of proteins S8, S13, and S17, on the other hand, appeared to be influenced little or not at all by the presence of the factor . These results are interpreted as supporting evidence for the premise that the binding of IF-3 results in a conformational change of the 30S subunit.

Proc Natl Acad Sci U S A, 1976 Nov, 73(11), 3989 - 93
Colicin E2 is DNA endonuclease; Schaller K et al.; Colicin E2 purified by conventional methods contains a tightly bound low-molecular-weight protein, as has been found with purified colicin E3 {Jakes,N.&Zinder,N.D.(1974) Proc . Natl . Acad . Sci . USA 71, 3380-3384} . Such E2 preparations do not cause DNA cleavage in vitro . After separation from the low-molecular-weight protein, colicin E2 retained the original in vivo killing activity, and in addition showed a high activity in vitro in cleaving various DNA molecules, such as a ColE1 hybrid plasmid and DNAs from Escherichia coli, lambda phage, chiX174 phage, and simian virus 40 . The low-molecular-weight protein ("E2-immunity protein") specifically prevented this in vitro DNA cleavage reaction, i.e., had an "immunity function." The results demonstrate that colicin E2 itself is a DNA endonuclease and explain the in vivo effects caused by E2 in sensitive cells as well as the mechanism of immunity in E2-colicinogenic cells.

Biokhimiia, 1976 Nov, 41(11), 1968 - 77
{DNA-methylase activities from animal mitochondria and nuclei: different specificity of DNA methylation}; Kudriashova IB et al.; DNA-methylase activities which methylate cytosine residues in homo- and heterologous DNA were detected in mitochondria and nuclei from rat liver and beef heart . Adenine modifying DNA-methylases in mitochondria and nuclei were not found . DNA from mitochondria and nuclei differ significantly in the methylation degree and in the pattern of the 5-methyl-cytosine distribution by pyrimidine isostichs as DNA in vivo and in vitro being methylated . Mitochondrial DNA methylase has the maximum activity at 30 degrees and pH 7.8 this enzyme(s) differ(s) from the nuclear one(s) in the pH dependence of its activity . After exhaustive in vitro methylation of various DNA by the nuclear enzyme DNA-methylase from mitochondria additionally introduces CH3 groups from S-adenosylmethionine into these DNA (about 3 times more CH3 groups than nuclear enzyme) . Nuclear DNA-methylase also methylates DNA which is previously fully-methylated by the mitochondrial enzyme, but to a lesser degree . In conditions of exhaustive DNA methylation mitochondrial enzyme introduces into E . coli B DNA about four times more methyl groups as compared to the nuclear one . After the methylation of E . coli B DNA by mitochondrial enzyme the label (3H-methyl) was detected predominantly in mono-, and in case of nuclear enzyme--in di- and tripyrimidine fragments . Mitochondrial DNA-methylase differs from the nuclear one in the nature of recognized DNA sequences; these enzymes seems to be represented by different proteins . The mitochondrial enzyme methylates shorter nucleotide sequences in DNA as compared to the nuclear DNA-methylase . All these data suggest there exist organoid specificity of genome methylation in animal cell and the modification-restriction systems in animal nucleus and mitochondria are different in character.

Eur J Biochem, 1976 Nov 1, 70(1), 233 - 40
Specific binding of a nonhistone chromosomal protein from lymphocyte to DNA; Bluthmann H; The isolation of a nonhistone chromosomal protein, NH30 000, from bovine lymphocytes by affinity chromatography on a DNA-agarose column is described . The procedure starts with nonhistone fraction NH4 obtained by hydroxyapatite chromatography as described previously {Bluthmann, H., Mrozek, S . & Gierer, A . (1975) Eur . J . Biochem . 58, 315-326} . Protein NH30 000 migrates as a single band with a molecular weight of 30 000 on sodium dodecylsulfate polyacrylamide gels . It contains, on a molar basis 25.3% acidic amino acids and 18.3% basic amino acid residues, 10.3% being lysine . End-group determination confirms that this protein is composed of one polypeptide chain with the NH2-terminal amino acid arginine . The binding of protein NH30 000 to native DNA of different molecular weights has been investigated by the nitrocellulose filter assay . The results suggest that in the presence of 0.2 M NaCl it binds about 30% of the DNA with binding sites every 1700 nucleotide pairs . Competition experiments show that protein NH30 000 exhibits a higher binding affinity for lymphocyte DNA in comparison to Escherichia coli DNA.

Acta Paediatr Scand, 1976 Nov, 65(6), 673 - 7
Immunologic studies in phenylketonuria; Passwell J et al.; Significantly reduced immunoglobulins were found in 22 patients with phenylketonuria . Tests of cellular immune function which included delayed skin hypersensitivity, T rosettes and PHA transformation were normal . Escherichia coli antibodies and the booster response to tetanus toxoid were also normal.

Klin Wochenschr, 1976 Nov 1, 54(21), 1055 - 6
{Lipid A antibody titers in Crohn's disease (author's transl)}; Schussler P et al.; Lipid A antibody titers and O antibody titers against E . coli were determined in 18 patients with Crohn's disease, 28 patients with ulcerative colitis, 24 patients with acute enteritis and in 68 healthy adults . The patients with Crohn's disease showed a statistically significant elevation of the lipid A antibody titers and of the O antibody titers against E . coli compared with each of the three other groups investigated . The results could be indicative of bacterial involvement in the pathogenesis of Crohn's disease . The determination of lipid A antibody titers may be useful for the differential diagnosis between Crohn's disease and ulcerative colitis.

Cell, 1976 Nov, 9(3), 439 - 48
DNA-dependent in vitro synthesis of fibosomal proteins, protein elongation factors, and RNA polymerase subunit alpha: inhibition by ppGpp; Lindahl L et al.; We have previously shown that the synthesis of ribosomal proteins (r proteins) in E . coli cells is under stringent control (Dennis and Nomura, 1974) . Since guanosine tetraphosphate (ppGpp) has been implicated in stringent control, we examined the effects of ppGpp on the in vitro synthesis of r proteins directed by DNA from transducing phage lambdafus3 and lambdarifd18 . lambdafus3 carries genes for protein elongation factors EF-Tu and EF-G, and RNA polymerase subunit alpha, in addition to genes for approximately 27 r proteins . lambdarifd18 carries genes for EF-Tu, RNA polymerase subunits beta and beta1, and a set of rRNAs, in addition to genes for approximately five r proteins . We have shown that low concentrations of ppGpp (0.2-0.3 mM) specifically inhibit DNA-dependent r protein synthesis in this system, and that this inhibition takes place directly, rather than as a consequence of the inhibition of rRNA synthesis by ppGpp . In addition, we have also shown that ppGpp inhibits the synthesis of EF-G, EF-Tu, and RNA polymerase subunit alpha, as well as rRNAs.

J Biochem (Tokyo), 1976 Nov, 80(5), 937 - 50
The primary structure of non-initiator methionine transfer ribonucleic acid from Bakers' yeast . I . Purification and complete digestion with ribonuclease T1 and pancreatic ribonuclease A; Koiwai O et al.; The methionine acceptor activity of a crude tRNA from bakers' yeast was resolved into two peaks (I and II) by column chromatography on DEAE-Sephadex A-25 with a 1 M phosphate system . Methionine tRNA from peak II was not formylated by E . coli methionyl-tRNA transformylase {EC 2.1.2.9.} after being charged with methionine, whereas that from peak I was formylatable under the same conditions . A substantial amount of unlabelled methionine tRNA, tRNAMetm, was highly purified from the peak II fraction by successive chromatographic procedures . The purified tRNAMetm was digested with pancreatic ribonuclease A {EC 3.1.4.22} and ribonuclease T1 {EC 3.1.4.8} . The digestion products were isolated into individual components and completely sequenced . The results of sequence analysis of the two RNase digests were in good agreement and indicated that the chain length of this tRNA is 76, including 13 modified nucleotides . These oligonucleotide fragments can be constructed into a unique total sequence, assuming a few conventional features of clover leaf structure for the tRNA was established by analyses of partial digestion products with RNase T1, as reported in the accompanying paper.

J Trauma, 1976 Nov, 16(11), 886 - 97
Comparison of hemodynamic and regional blood flow changes at equivalent stages of endotoxin and hemorrhagic shock; Rutherford RB et al.; Hemodynamic, respiratory, and regional blood flow measurements were carried out in two groups of monkeys at three roughly equivalent stages of endotoxin and hemorrhagic shock . Comparisons revealed characteristic differences at the two early stages, particularly in systemic vascular resistance and the pattern of distribution of cardiac output . However, at the final stage of shock, these patterns had merged and there were no characteristic differences between the two groups . The pathologic significance of these findings, in terms of the endotoxin theory of irreversible hemorrhagic shock and the realtive contributions of vasoactive humoral substances at various stages of the two forms of shock, is discussed.

Cell, 1976 Nov, 9(3), 393 - 407
Isolation, characterization, and structure of the folded interphase genome of Drosophila melanogaster; Benyajati C et al.; The intact interphase genome of Drosophila melanogaster has been isolated by sucrose gradient centrifugation after gentle lysis of tissue culture cells in 0.9 M NaCl-0.4% nonidet P40 . The non-viscous folded DNA sediments as a single broad 5000S peak in a complex with RNA (a fraction of the nuclear nascent RNA) and protein (all of the four intranuclesome histones: H2A, H2B, H3, and H4) . The folded DNA is supercoiled and can be relaxed to slower sedimenting forms either by intercalating ethidium or by nicking with DNAase I . Incomplete DNAase treatment gives partially relaxed complexes, indicating that each nick relaxes only a stretch of DNA (defined as a supercoiled DNA loop) without affecting the superhelical content of the rest of the genome . The concentration of superhelices in the Drosophila folded DNA is the same as in the E . coli and SV40 closed circular DNAs-that is, about one negative turn every 200 base pairs (bp) in 0.15 M NaCl at 26 degrees C . The estimated average size of the supercoiled DNA loops, about 85,000 bp, equals the size of the larger Drosophila chromomeres . Ethidium intercalation in 0.9 M NaCl both removes the negative superhelical turns and dissociates the four histones from the DNA . The four histones are dissociated in equimolar concentrations, and the relative proportion of histones displaced from the DNA is a function of ethidium concentration . The histones are completely dissociated from the folded DNA at the ethidium concentration . The histones are completely dissociated from the folded DNA at the ethidium concentration which removes all of the negative superhelices . Thus the data strongly suggest that the rotation of the Watson Crick helix which accompanies ethidium intercalation causes the loss of nucleosomes from the DNA . The results are interpreted in terms of a model for the folded Drosophila genome which has the DNA constrained (by both protein-DNA and RNA- DNA interactions) into independent supercoiled loops containing on the average 400 nucleosomes per loop . Each nucleosome is composed of a histone core with the DNA wound around it in a 360 degrees left-handed toroidal supercoil; each nucleosome toroidal supercoil plus its relaxed internucleosome DNA contains, on the average, 200 bp.

J Bacteriol, 1976 Nov, 128(2), 673 - 6
Ribonucleotide reductase activity in ether-treated cells of Agmenellum quadruplicatum; Gleason FK et al.; Ribonucleotide (cytidine 5'-diphosphate) reductase activity can be detected in situ in cells of the blue-green alga Agmenellum quadruplicatum, strain PR-6, after the cells are made permeable by treatment with ether . The Agmenellum reductase resembles the enzyme from Escherichia coli.

Mol Biol (Mosk), 1976 Nov-Dec, 10(6), 1221 - 30
{DNA compact form . 8 . X-ray diffraction study of DNA compact particles, formed in solutions containing polyethylene glycol}; Evdokimov IuM et al.; A comparative X-ray study of DNA compact particles, formed in PEG-containing solution from native DNA and from DNA molecules with altered secondary structure was carried out . Low-angle reflections, present in X-ray patterns of compact particles (in powder form) from native DNA, correspond to spacings of 84, 42 and 35 A, while wide-angle reflections correspond to spacings of 12.8; 8.4, 6.0, 4.5, 3.4 A . Low-angle reflections at 84 and 42 A are present also in X-ray patterns of compact particles, formed from DNA molecules with altered secondary structures . These two reflections are believed to be the results of an ordering of DNA molecules within the compact particles . The main features of this ordering appear in the first approximation to be independent on DNA secondary structure . The CD spectra of all types of compact particles, mentioned above, were also studied . It has been shown that the intense negative band (lambda approximately 270 nm) in a CD spectrum appeared only in the case of compact particles, formed from native DNA molecules . The nature of the revealed correlation between the 35 A reflection and the CD negative band is discussed . Data presented in the paper allow one to suppose that the 35 A reflection in X-ray patterns and the CD negative band result from specific interactions between double-stranded DNA fragments spatially brought together in compact particles . Such type of interaction is believed to be characteristic only of native DNA molecules.

Ann Sclavo, 1976 Nov-Dec, 18(6), 849 - 54
{The "Escherichia coli" responsable of gastroenteric symptoms (author's transl)}; Dessi S et al.; From 450 aerobic cultures of children stools specimens 212 Escherichia coli strains have been isolated: 122 serotypes were enteropathogens (of these 65 were pure cultures): it can not be concluded that only these strains are responsable of gastroenteric symptoms.

Res Vet Sci, 1976 Nov, 21(3), 303 - 8
The effect of colostrum or past colibacillosis on the adhesion of Escherichia coli to the small intestine of the pig; Nagy LK et al.; Nursing litters of vaccinated (K88 antigen) and non-vaccinated gilts as well as weaned piglets, which had recovered from natural colibacillosis, were exposed to Escherichia coli O149: K91(B), K88ac(L) orogastrically and 3-4 h after exposure a proportion of each group was killed for adhesion studies; others were kept for clinical observations . From killed piglets duodenal contents were collected and after washing of the duodenum by standardised techniques it was homogenised . Viable counts of E coli O149 in these specimens were carried out and compared . Colicounts in the duodenal contents and in the homogenate of washed duodenum of naturally infected piglets were also compared . There was less than tenfold difference in counts of K88-positive E coli between duodenal contents and washed duodenum of piglets which were (A) naturally infected, (B) colostrum deprived and experimentally infected, or (C) were from colostrum fed groups where those litter mates which were kept for clinical observation died of colibacillosis . There was 10(3)-10(5)-fold difference in E coli O149 counts between comparable specimens from piglets which (a) recently recovered from K88-positive E coli infection at the time of oral challenge or (b) were from colostrum fed groups where those litter mates which were kept for clinical observation survived oral challenge . It is concluded that adhesion of K88-positive E coli to the epithelium of the anterior small intestine of the pig is a feature of natural as well as experimental colibacillosis . Such adhesion may be prevented by (i) earlier natural infection of K88-positive E coli or (ii) by the ingestion of colostrum of K88 antigen vaccinated dams.

Mol Biol Rep, 1976 Nov, 3(2), 181 - 7
A model of promoter recognition along the two helical grooves of DNA; Ivanov VI; A speculative model based on published sequences to explain the specific binding of RNA polymerase of E . coli to promoters is suggested: (see article) . A fragment 24 base pairs long to the left of the initiation site must not contain the G's in the positions indicated by the circles and must contain T's (or G's) in the positions marked by the squares . In most known cases mutual disposition of the circle and square patterns is as shown above . In one case (the fd G3 promoter) the pattern of squares is shifted by 4 base pairs to the right relative to the pattern of universal non-G's (circles).

Mol Biol Rep, 1976 Nov, 3(2), 121 - 9
Ribosomal protein as substrate for a GTP-dependent protein kinase from yeast; Kudlicki W et al.; A protein kinase specific for casein and acidic ribosomal proteins was isolated and partly characterized . It was found that the enzyme utilizes GTP and ATP as phosphoryl donors . Its affinity for ATP was considerably higher than for GTP with the km values of 7.6 X 10(-6)M and 5.5 X 10(-5)M, respectively . Two-dimensional acrylamide gel electrophoresis revealed the phosphorylation of the same ribosomal proteins with either of the {gamma-32P} nucleotides used . It was also shown that one acidic protein (S1 or S2) of 40 S and two acidic proteins (L2 and L3) of 60 S ribosomal subunits were predominantly phosphorylated in vitro . The phosphorylated proteins: L2 and L3 seem to correspond to the proteins of L7 and L12 of E . coli ribosomes . The isolated kinase phosphorylated several basic ribosomal proteins though to a lower extent than the acidic ones.

Mikrobiologiia, 1976 Nov-Dec, 45(6), 1100 - 2
{Viability of Escherichia coli cultures}; Marchenkova TV et al.; In the logarithmic phase of batch cultures of E . coli B and MRE-600, a high percentage (upto 80%) of the cells did not grow . Viability of the cells was estimated by inoculation on plates, in agar drops, and on slides . As was established by mathematical planning experiments, the viability and growth rate of the culture depend on the micro-element composition of the medium.

Eur J Biochem, 1976 Nov 1, 70(1), 39 - 47
The mode of action of thiostrepton in the initiation of protein synthesis; Naaktgeboren N et al.; The inhibition by thiostrepton of the initiation of protein synthesis is exerted at a different level from the inhibition of reactions mediated by EF-Tu and EF-G in the elongation of protein synthesis . The presence of thiostrepton on the 50-S subunit completely prevents the binding of the EF-Tu - GTP - aa-tRNA complex and EF-G - GTP complex to the 70-S ribosome, resulting in cessation of protein synthesis at a concentration of 1 muM thiostrepton . On the other hand, during initiation thiostrepton impairs the coupling of the 50-S subunit with the 30-S initiation complex, indirectly causing inhibition of IF-2-dependent reactions . Impairment of the coupling is strongly influenced by the conditions of incubation . Since formation of formylmethionylpuromycin and the IF-2-dependent GTP hydrolysis are inhibited to the same extent and recycling of IF-2 can take place in the presence of thiostrepton, we conclude that the basic mechanism of inhibition of initiation differs from that of inhibition of elongation.

Eur J Biochem, 1976 Nov 1, 70(1), 33 - 8
Interaction of substrate analogues with Escherichia coli DNA-dependent RNA polymerase; Armstrong VW et al.; The inhibition of RNA polymerase with ATP and UTP analogues modified in the phosphate and ribose moieties has been investigated . 1 . Modification of the terminal phosphate with a loss of the negative charge {adenosine 5'-(3-O-methyl)triphosphate, Ki = 1.75 mM} substantially weakens the binding ability of these analogues to the enzyme whereas modification with retention of the charge is not so detrimental {adenosine tetraphosphate, Ki = 0.17 mM} . 2 . 2'-Modified analogues are only weak competitive inhibitors {2'-amino-2'-deoxyadenosine 5'-triphosphate, Ki = 2.3 mM} of their corresponding substrates {ATP, Km = 0.07 mM} whereas 3'-modified analogues are extremely potent in their inhibition {3'-amino-3'-deoxyadenosine 5'-triphosphate, Ki = 2.3 muM} . 3 . A difference was observed in the inhibition of the elongation step of RNA polymerase by ATP and UTP analogues . Thus ATP analogues showed a strong binding to the CT form of the poly{d(A-T)} ternary complex and only a weak binding to the CA form . UTP analogues, on the other hand, showed a similar binding to both forms of the complex.

Eur J Biochem, 1976 Nov 1, 70(1), 191 - 6
Heterogeneity of sites in isolated catalytic subunits of aspartate transcarbamoylase; Suter P et al.; Carbamoyl phosphate, a substrate of aspartate transcarbamoylase from Escherichia coli, binds with different modes of association in 3 sites in the unmodified catalytic subunits . Over a narrow pH range (6.6--8.0), positive, negative or no interactions are observed . Several substrate analogues also bind to 3 sites in the catalytic trimer . The association of pyridoxal phosphate, CTP and ATP, all competitive inhibitors of carbamoyl phosphate, exhibit negative interactions . Binding of succinate, an analogue of the second substrate, aspartate, is also characterized by heterogeneity . Dissociation constants to high and low-affinity sites differ by factors of 10-100 . These observations clearly indicate that, although not observed kinetically, the active sites in the catalytic subunits of aspartate transcarbamoylase are heterogenous.

Eur J Biochem, 1976 Nov 1, 70(1), 137 - 45
Arginyl-tRNA synthetase from Escherichia coli . Influence of arginine biosynthetic precursors on the charging of arginine-acceptor tRNA with {14C}arginine; Charlier J et al.; The behaviour of arginyl-tRNA synthetase (EC 6.1.1.19) in the presence of the arginine biosynthetic precursors, argininosuccinate, ornithine and citrulline, was studied in several Escherichia coli K12 strains and in E . coli W . The results of kinetic measurements with partially purified extracts indicate that the arginyl-tRNA synthetase of E . coli is not inhibited by the arginine precursors . The apparent affinity constant Km for arginine of the K12 enzyme is about 3.4 muM in the absence and in the presence of these precursors, whereas the W enzyme an apparently slightly lowered Km and a decreased {14C}arginyl-tRNA equilibrium level in the presence of argininosuccinate . This however was shown to be due to isotopic dilution of {14C}arginine by non-radioactive amino acid formed from argininosuccinate by argininosuccinate lyase (EC 4.3.2.1) contaminating the synthetase preparation . This finding emphasizes the necessity of using pure arginyl-tRNA synthetase in order to study the possible regulatory involvement of this enzyme in the control of the arginine regulon in vitro.

Nucleic Acids Res, 1976 Nov, 3(11), 3109 - 22
7-Methylguanine specific tRNA-methyltransferase from Escherichia coli; Aschhoff HJ et al.; A 7-methylguanine (m7G) specific tRNA methyltransferase from E . coli MRE 600 was purified about 1000 fold by affinity chromatography on Sepharose bound with normal E . coli tRNA . The purified enzyme catalyzes exclusively the formation of m7G in submethylated bulk tRNA of E . coli K12 met- rel- . The purified enzyme transfers the methyl group from S-adenosyl-methionine to initiator tRNA of B . subtilis and 0.8 moles m7G residues are formed per mole tRNA . It is suggested that the enzyme specifically recognizes the extra arm unpaired guanylate residue.

Nucleic Acids Res, 1976 Nov, 3(11), 3063 - 76
Repeating restriction fragments of human DNA; Manuelidis L; Human DNA digested with Hae III showed multiple repeats of a 170 base pair fragment . The most prominent band was the 340 base pair dimer, estimated to be 0.8% of the entire genome . Eco R1 and Hha I yielded fragments with similar electrophoretic mobility to the Hae III dimer . In each case this band was markedly enriched in DNA reassociating at a 0t of less than or equal to 1 . Hybridization of the Hae III dimer to gels eluted on to filters demonstrated that the multiple Hae III fragments and Eco R1 fragments contained compatible sequences . These sequences may comprise a distinct subclass of DNA.

Nucleic Acids Res, 1976 Nov, 3(11), 3015 - 24
Study on the structure-function relationship of polynucleotide phosphorylase: model of a proteolytic degraded polynucleotide phosphorylase; Guissani A et al.; It is already known that modification of E . coli polynucleotide phosphorylase by endogenous proteolysis induces drastic changes in both phosphorolysis and polymerisation reactions . The structural parameters of the proteolysed polynucleotide phosphorylase are described . The phosphorolysis of polynucleotide, which is quite progressive for the native enzyme, is shown to be only partially progressive for the degraded enzyme, owing to the loss of polymer attachment sites.

Nucleic Acids Res, 1976 Nov, 3(11), 3001 - 14
Studies on the ability of partially iodinated 16S RNA to participate in 30S ribosome assembly; Schendel PL et al.; Deproteinated 16S RNA was iodinated at pH 5.0 in an aqueous solution containing TlCl3 plus KI for 1-5 hours at 42 degrees C . Under these conditions 33 moles of iodine are incorporated per mole of RNA . As judged by sucrose gradient sedimentation, the iodinated RNA does not exhibit any large alteration in conformation as compared to unmodified 16S . The iodinated RNA was examined for its ability to reconstitute with total 30S proteins . Sedimentation velocity analysis reveals that the reconstituted subunit has a sedimentation constant of approximately 20S . In addition, protein analysis of particles reconstituted with 16S RNA iodinated for 5 hours indicates that proteins S2, S10, S13, S14, S15, S17, S18, S19, and S21 are no longer able to participate in the 30S assembly process and that proteins S6, S16 and S20 are present in reduced amounts . The ramifications of these results concerning protein-RNA and RNA-RNA interactions occurring in ribosome assembly are discussed.

J Med Chem, 1976 Nov, 19(11), 1295 - 9
Polyglutamyl and polylysyl derivatives of the lysine analogues of folic acid and homofolic acid; Plante LT et al.; A series of Nepsilon-poly-alpha-glutamyl and Nepsilon-polylysyl derivatives of Nalpha-pteroyllysine and Nalpha-homopteroyllysine, analogues of the naturally occurring gamma-polyglutamyl forms of folate, was prepared and tested as substrates for dihydrofolate reductase and as substrates and inhibitors of thymidylate synthetase . Nalpha-Dihydropteroyl-Nepsilon-(tri-alpha-glutamyl)lysine was 1.8 times as active as Nalpha-dihydropteroyl glutamate (dihydrofolate) as a substrate for L1210 murine leukemia dihydrofolate reductase . N-alpha-Dihydropteroyl-Nepsilon-(di-alpha-lysyl)lysine was 1.2 times as active as dihydrofolate in spite of its strong positive charge . The most active compound tested, Nepsilon-(tert-butyloxycarbonyl)lysine, was 3.5 times as active as dihydrofolate . None of the enzymatically prepared Nalpha-tetrahydropteroyllysine derivatives tested was as active as Nalpha-tetrahydropteroyl glutamate (tetrahydrofolate) as a substrate for E . coli thymidylate synthetase . However, there was a progressive increase in activity with the addition of each alpha-glutamyl residue, the Nepsilon-(penta-alpha-glutamyl)lysine being 88% as active as tetrahydrofolate . Nalpha-Tetrahydropteroyl-Nepsilon-(di-alpha-lysyl)lysine was the most active thymidylate synthetase substrate of the polylysine derivatives, being 67% as active as tetrahydrofolate . Addition or deletion of lysyl residues resulted in diminished activity . It is noteworthy that substrate activity is retained in spite of the positively charged poly(amino acid) side chain . None of the enzymatically prepared tetrahydrohomopteroyl derivatives tested was as active as Nalpha-tetrahydrohomopteroyl glutamate (tetrahydrohomofolate) as an inhibitor of E . coli thymidylate synthetase.

J Med Chem, 1976 Nov, 19(11), 1276 - 9
Phenylalanyl transfer ribonucleic acid synthetase from rat liver . Analysis of phenylalanine and adenosine 5'-triphosphate binding sites and comparison to the enzyme from Escherichia coli; Santi DV et al.; Inhibition of the ATP--PPi exchange reaction catalyzed by rat liver phenylalanyl-tRNA synthetase by structural analogues of L-phenylalanine and ATP has been examined and compared with data reported for the enzyme from E . coli . The phenylalanine binding sites are similar in the following characteristics . (1) The region that complexes the phenyl ring shows a strict requirement for the unsubstituted phenyl ring altough the rat liver enzyme is more tolerant in this respect . (2) The protonated amino group of phenylalanine is required for binding . (3) The region neighboring the binding site for the carboxylate of phenylalanine is diffusely hydrophobic . Unlike effects of these modifications on interaction with the E . coli enzyme, substitution of the carboxylate by hydrophobic groups leads to large losses in affinity for the rat liver enzyme . Although both enzymes bind D-phenylalanine very poorly, their relative affinities for the D isomers of phenylalanine analogues vary greatly . The most dramatic difference is observed with N-benzyl-D-amphetamine, which binds 24 000-fold tighter to E . coli phenylalanyl-tRNA synthetase than the rat liver enzyme . The affinity of rat liver phenylalanyl-tRNA synthetase for naturally occurring adenine compounds is similar to that of the E . coli enzyme, suggesting that the binding of ATP occurs via similar interactions . Adenine provides a major protion of the free energy of binding of ATP . The remainder may be viewed as the sum of detrimental interactions with the phosphate groups of ATP and a favorable contribution by the ribose moiety.

J Med Chem, 1976 Nov, 19(11), 1270 - 5
Phenylalanyl transfer ribonucleic acid synthetase from Escherichia coli B . Potent inhibition by analogues of N-benzyl-2-phenylethylamine; Anderson RT Jr et al.; A potent new class of inhibitors of phenylalanyl-tRNA synthetase from Escherichia coli B is described . N-Benzyl-2-phenylethylamine is a competitive inhibitor with respect to L-phenylalanine and appears to possess the structural features required for near-optimal binding . Hydrophobic substituents at the ortho position of either ring appear to be well tolerated, but substituents on both rings lead to large losses in binding . Poor noncompetitive inhibitors resllt from alkylation of the secondary nitrogen, further separation of the N-benzyl group from the nitrogen, or alkylation at the alpha position of the N-benzyl moiety . In contrast, placement of a methyl group at the 1 position of the 2-phenylethylamine moiety to give N-benzyl-D-amphetamine results in the most potent inhibitor yet described for this enzyme.

Genetics, 1976 Nov, 84(3), 403 - 21
Deletion and amber mutants of fla loci in Escherichia coli K-12; Kondoh H et al.; A rapid screening method for amber fla mutants of E . coli was devised and many mutants were obtained . In addition, strains with deletions of the fla genes in the his-uvrC region were isolated from high-temperature survivors of a lambda cI857 lysogen in which the prophage is located between his and fla . Utilizing these mutants, eleven fla genes (I--XI) and one hag gene were identified in the his-uvrc region, in the following order: his-supD-I-II-(III, IV)-V-(VI, VII)-VIII-IX-hag-(X, XI)-uvrC . The fla genes X and XI and hag are located at about 42.5 min and the other fla genes at about 43.0 min on the E . coli genetic map (Bachmann, Low and Taylor 1976) . Mutants of fla gene X showed a slight sensitivity to chi phage, although they lack the flagellar filament.

Can J Biochem, 1976 Nov, 54(11), 935 - 40
Synthesis of guanosine 5'-triphosphate,3'-diphosphate in a spo T strain of Escherichia coli; Chaloner-Larsson G et al.; A spo T stringent strain of Escherichia coli rapidly accumulates guanosine 5'-triphosphate,3'-diphosphate (pppGpp) immediately after the onset of isoleucine starvation . Subsequently, its level rapidly falls, as guanosine 5'-diphosphate,3'-diphosphate (ppGpp) continues to rise to the maximum value, which is abnormally high compared with that in the spo T+ strain . The ppGpp level in the spo T strain never reaches a steady state as it does in the spo T+ strain . Immediately after starvation, pppGpp and ppGpp are labeled with {3H}guanosine at a similar differential rate in both the spo T and spo T+ strains, suggesting that the two strains synthesize these nucleotides by the same pathway . However, by 15 min after starvation, the synthesis of these nucleotides is nearly halted in the spo T strain, and is greatly reduced in the spo T+ strain . Since ppGpp is labeled with {3H}guanosine more slowly than pppGpp in the starved spo T+ strain, ppGpp cannot be a precursor of pppGpp . The kinetics of the GTP level during starvation suggests that GTP is a precursor of pppGpp . The observed differences between the spo T and spo T+ strains can be explained by postulating, firstly, that ppGpp negatively controls the conversion of GTP to pppGpp, which is subsequently converted to ppGpp; secondly, that a catabolite of ppGpp negatively controls the conversion of pppGpp to ppGpp; and thirdly, that the spo T mutation primarily reduces the rate of ppGpp catabolism.

Biochem J, 1976 Nov, 159(2), 259 - 65
The construction and analysis of sucrose gradients for use with zonal rotors; Hirst W et al.; The rate of sedimentation of a particle in a sucrose solution depends on the viscosity and density of the medium . These two variables are related to the sucrose concentration and the temperature of the medium by new simple equations . These equations were used in a rapid iterative procedure that relates the distance moved by a zone in a continuous sucrose gradient to its sedimentation coefficient . It is shown by comparison with experiment that this iterative method allows the distance moved by a zone to be calculated rapidly . The method may therefore be used to optimize the separation of particles in a sucrose-gradient-centrifugation experiment . The method also allows the unknown sedimentation coefficients of several zones to be measured from a single sucrose-gradient-centrifugation experiment.

Proc Natl Acad Sci U S A, 1976 Nov, 73(11), 3979 - 83
Identification and radiochemical purification of the recA protein of Escherichia coli K-12; McEntee K et al.; The product of the recA gene of E . coli has been identified by labeling proteins synthesized in UV-treated cells after infection with specialized transducing phages carrying the recA gene . Following infection of UV-treated cells by lambda precA, which carries the recA+ gene, a major protein with a molecular weight of 43,000 is detected on polyacrylamide gels containing sodium dodecyl sulfate . This protein is also made after infection of suppressing hosts by lambda precA99, which carries an amber recA- mutation, but is not synthesized after infection of nonsuppressing hosts by this transducing phage . A spontaneous recatrevertant of lambda preca99 induces synthesis of this protein after infection of a nonsuppressing host . The product of the recA gene is a soluble protein found in a complex with a molecular weight of approximately 150,000 after mild detergent lysis of cells.

Proc Natl Acad Sci U S A, 1976 Nov, 73(11), 3961 - 5
Purification and characterization of a putative sigma factor from Chalamydomonas reinhardi; Surzycki SJ et al.; Two proteins with sigma-like activity have been isolated from the alga, Chlamydomonas reinhardi . One protein, sigma 2, has been partially purified and appears to have a molecular weight of 51,000 . The interaction of this protein with a heterologous (Escherichia coli) and homologous (Chlamydomonas, chloroplast rifampicin-sensitive) core RNA-polymerase (RNA nucleotidyltransferase, nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) was studied . Sigma 2 protein appears to stimulate the formation of open (rapid starting) binary complexes by both of the core enzymes . Stimulation of transcription by sigma 2 on chloroplast DNA was greater when Chlamydomonas core enzyme was used . Moreover, in vitro transcription on a variety of templates using RNA polymerases I and II from Chlamydomonas was not stimulated by this protein.

Proc Natl Acad Sci U S A, 1976 Nov, 73(11), 3910 - 4
Recombination promoted by superhelical DNA and the recA gene of Escherichia coli; Holloman WK et al.; When a mixture of superhelical DNA (RFI) of phage phiX174 am3 and fragments of single-stranded DNA from wild-type phiX174 was added to spheroplasts of E . coli carrying an amber suppressor, several percent of the progeny phage were recombinant . The yield of wild-type progeny was 10(3) to 10(4) times lower when the fragments came from phiX174 am3 or phage G4 am+, or when fragments were absent . Fewer recombinants were produced in proportion to the decrease in the fraction of RFI in samples treated with S1 nuclease, whereas the total yield of phage did not decrease . Transfection by fragments and superhelical DNA produced 20 to 100 times more recombinants than transfection by fragments and either nicked circular DNA or relaxed closed circular DNA . Transfection of a recA- strain by RFI DNA and fragments yielded 5-10% as many recombinants as transfection of a rec+ strain . This partial requirement for recA was bypassed by transfection with complexes of RFI AM3 DNA and am+ fragments made in vitro.

J Gen Microbiol, 1976 Nov, 97(1), 73 - 82
Location of the Aspartase Gene (aspA) on the linkage map of Escherichia coli K12; Spencer ME et al.; The aspartase (L-asparatate ammonia-lyase, EC . 4.3.I.I) structural gene, aspA, was mapped by two-factor and three-factor transductional crosses using phage PI . Cotransduction frequencies between aspA and other markers were: ampA (69%); frdA (48 TO 67%) mel (35%); purA (17%); fdp (I-6%) . The sequence of markers within the corresponding segment (91 to 95 min) of the Escherichia coli linkage map was shown to be mel-aspA-ampA-frdA-purA-fdp.

Am J Surg, 1976 Nov, 132(5), 653 - 6
Intramesenteric perforation of sigmoid diverticulitis with nonfatal venous intravasation; Juler GL et al.; Complications from barium enema are rare (0.035 per cent) . A patient with venous intravasation during barium enema complicated by pylephlebitis and portal vein obstruction is the tenth to be reported on, the fourth to survive . This accident was associated with colon disease in eight of the patients studied, five of whom had diverticular disease.

Surgery, 1976 Nov, 80(5), 544 - 9
Infected aortic bifurcation grafts: experience with fourteen patients; Becker RM et al.; The experience with 14 patients with infected aortic bifurcation grafts has been reviewed . Factors which appeared to predispose to infection in 11 patients included "re-do" operations, concomitant cholecystectomy or gastrostomy, and ruptured abdominal aneurysm . A mixture of gastrointestinal organisms was responsible for the infections . The pathogenesis, presentation, and treatment varied according to whether the proximal or distal anastomosis was involved or not . Aortoduodenal communications were present in five patients; they presented with gastrointestinal bleeding or septicemia . One patient survived as a result of early, aggressive surgical therapy . Infection presented at the distal anastomosis in nine patients, either as groin abscess or false aneurysm . Conservative therapy failed in the majority of patients but apparently was successful in three of five patients in whom infection did not involve the intra-abdominal portion of the graft . When infection does involve the intra-abdominal portion of the graft, then the graft must be excised also . Revascularization often can be accomplished with extra-anatomic bypasses of prosthesis or autogenous material, depending on the characteristics of the individual patient . Regardless of the mode of presentation or the site of infection, the early institution of judicious surgical management offers the best chance of success in these patients, and temporization usually leads to failure.

Surg Gynecol Obstet, 1976 Nov, 143(5), 738 - 40
Animal models of peritonitis; Browne MK et al.; A reproducible animal model of peritonitis has been developed . It uses standard animals, standardized cultures of bacteria and simple techniques . Use of this model would facilitate comparison of data from workers in this field . The use of small rodents permits the use of comparatively large populations for statistical evaluation . The method is cheap and economic of laboratory space and does not require surgical expertise.

J Urol, 1976 Nov, 116(5), 544 - 6
The urographic findings in acute pyelonephritis: non-obstructive hydronephrosis; Kass EJ et al.; Excretory urography was performed on 4 patients early in the course of acute pyelonephritis . These studies showed dilatation of the involved collecting system without obstruction . The dilatation resolved after therapy in 3 patients and was not present on a previous excretory urogram in the fourth . Although previous studies have shown that the excretory urogram is usually normal in cases of acute pyleonephritis these cases demonstrate that this disease can produce dilatation of the collecting system and mimic obstruction.

J Bacteriol, 1976 Nov, 128(2), 681 - 2
New types of Escherichia coli recombination-deficient mutants; Freifelder D; A set of Escherichia coli mutants deficient in intramolecular recombination and different from those previously found is described . All have temperature-sensitive lethal mutations . The mutants have been characterized with respect to the following properties: the Pap phenotype, deoxyribonucleic acid synthesis, sensitivity to ultraviolet light, ability to support the growth of phage lambda, filament formation, and mutation frequency.

J Bacteriol, 1976 Nov, 128(2), 671 - 2
Escherichia coli mutant lacking 4-thiouridine in its transfer ribonucleic acid; Ramabhadran TV et al.; A mutant of Escherichia coli has been isolated that lacks 4-thiouridine, a rare base in transfer ribonucleic acid . The mutant grows at the same rate as wild-type cells . It shows little near-ultraviolet-induced growth delay, thus supporting earlier hypotheses that 4-thiouridine in transfer ribonucleic acid is the chromophore for this growth delay.

J Bacteriol, 1976 Nov, 128(2), 604 - 8
TolF locus in Escherichia coli: chromosomal location and relationship to loci cmlB and tolD; Foulds J; The tentative map position on the Escherichia coli chromosome of the tolF locus, determining tolerance to colicins A, E2, E3, K, and L, has been confirmed by three-point transduction . It lies between the aroA and pyrD loci at about 21 min on the linkage map of Bachmann et al . (1976) . The cmlB locus, determining increased resistance to the antibiotics chloramphenicol and tetracycline, also lies in this region (Reeve, 1966) . Phenotypic and genetic comparison of isogenic strains that carry a mutation in either the tolF or cmlB locus makes it likely that these loci are closely related or identical . The tolD locus determining tolerance to colicins E2 and E3 as well as increased resistance to antibiotics has been reported to be located close to the aroA locus as a result of conjugation experiments (Eriksson-Grennberg et al . 1965) . However, tolD did not cotransduce with any of several loci in this region, indicating that the mutation is not located within the region of the genetic map corresponding to approximately 19 to 22.5 min.

J Bacteriol, 1976 Nov, 128(2), 557 - 72
Isolation and characterization of mutations creating high-efficiency transcription initiation signals within the trp operon of Escherichia coli; McPartland A et al.; Two different mutational events generate promoter-active deoxyribonucleic acid sequences within the trp operon of Escherichia coli, probably through single base-pair changes . The mutations, obtained after ethyl methane sulfonate mutagenesis by selecting for elevated lac gene expression in a trp-lac fusion, are cis dominant and trans recessive with respect to their effects on the synthesis of downstream enzymes . One of the mutants (trpD11), obtained repeatedly under the selective conditions employed, prevents the formation of active phosphoribosyltransferase . The second mutation, trp3B, has no effects on any trp enzyme . By deletion mapping, trpD11 was localized near the operator-distal end of trpD, outside the segment of deoxyribonucleic acid that contains the low-efficiency internal promoter trpP2 . Reversion to prototrophy of trpD11 was greatly stimulated by 2-aminopurine and ethyl methane sulfonate . Tests with suppressors indicated that trpD11 is a UAA (ochre) nonsense mutation . Under repression conditions, strains harboring either lesion in a normal trp operon synthesize the tryptophan biosynthetic enzymes in a noncoordinate fashion . The products of the operator-distal structural genes trpC, trpB, and trpA are formed at rates approximately 15-fold higher than those of wild type . The enzymes encoded by operator-proximal genes trpE and trpD are low or not detectable . Under derepression conditions, coordinate expression of the operon was observed.

J Bacteriol, 1976 Nov, 128(2), 522 - 7
Enzyme secretion in Escherichia coli: synthesis of alkaline phosphatase and acid hexose phosphatase in the absence of phospholipid synthesis; Beacham IR et al.; De novo synthesis of two periplasmic enzymes in Escherichia coli, alkaline phosphatase and acid hexose phosphatase, have been studied in the presence and absence of new phospholipid synthesis . Alkaline phosphatase synthesis was initiated by a temperature shift in a strain carrying a phoA amber mutation and a temperature-sensitive suppressor mutation; acid hexose phosphatase was studied after relief of catabolite repression . Glycerol auxotrophs (gpsA) were used to control phospholipid synthesis . Synthesis of both enzymes proceeded at a normal rate for 0.5 to 1.0 generation of growth, although it was then curtailed . It is concluded that secretion of these enzymes is not obligatorily coupled to new net phospholipid synthesis.

Cancer Res, 1976 Nov, 36(11 Pt 1), 4099 - 101
Mutagenicity of cyclic nitrosamines in Escherichia coli following activation with rat liver microsomes; Elespuru RK et al.; Ten cyclic nitrosamines were tested for mutagenicity in Escherichia coli after incubation in vitro with 9000 X g microsomal supernatants prepared from rat liver, and the results were compared with carcinogenicity data from the same species . None of the compounds was mutagenic in the absence of microsomes . Seven carcinogenic compounds, nitrosopyrrolidine, nitrosopiperidine, nitrosohexamethyleneimine, nitrosoheptamethyleneimine, nitrosomorpholine, dinitrosopiperazine, and dinitrosohomopiperzine, were mutagenic after microsomal activation . One compound, nitrosohepamethyleneimine, was toxic to the bacteria . Two noncarcinogens, 1-nitrosopiperazine and 1-methyl-4-nitrosopiperazine, and 1 strong carcinogen, 2,6-dimethyldinitrosopiperazine, were not mutagenic with or without microsomal incubation . The liver microsome preparation activated equally well those compounds that are liver carcinogens in Sprague-Dawley rats, and compounds for which the liver is not a target organ.

Blood, 1976 Nov, 48(5), 731 - 42
A method for the recognition and separation of human blood monocytes on density gradients; Loos H et al.; The density distribution of human mononuclear blood leukocytes was studied in order to define the optimal conditions for the separation of monocytes and lymphocytes by isopycnic centrifugation . Under standardized conditions, two populations of cells with partially overlapping, normally distributed densities were consistently found . The cells with the lowest density were recognized as monocytes, using phagocytosis and size distribution analysis as criteria . Since the density of monocytes continuously increased during the centrifugation, optimal separation of monocytes and lymphocytes could only be achieved by limiting the time of centrifugation to 10 min at 2200 g and 4 degrees C . The separation on discontinuous density gradients decreased when the load exceeded 8 X 10(6) mononuclear cells per sq cm . Analysis of the composition of the two cell populations obtained after separation on a three-layer discontinuous gradient revealed that the contamination of the monocytes with lymphocytes was due to the partial overlapping density distributions of both cell types . A small and a large scale method for isolation of monocytes from blood on discontinuous density gradients are presented . Under the described conditions, a preparation of functionally intact monocytes can be obtained which is comparable, both in yield and purity, to those obtained by methods based on surface adherence without the drawbacks of the latter methods.

Mol Biol Rep, 1976 Nov, 3(2), 157 - 65
The existence of triphosphorylated 5'-ends in virus-specific RNA isolated from SV-40 transformed cells; Georgiev GP et al.; The question about the nature of promoters in the transcriptional units containing SV-40 sequences in transformed cells was analyzed . It was found that the pulse-labeled RNA hybridizing to SV-40 DNA contains small but significant amounts of triphosphorylated 5'-ends detected as pppGp in alkaline hydrolyzates of this RNA . In another series of experiments the fragments of RNA containing triphosphorylated 5'-ends about 100 nucleotides in length have been isolated by hydroxyapatite chromatography . Some of them form hybrids with SV-40 DNA . The conclusion is drawn that at least some of SV-40 promoters are used for transcription initiation in SV-40 transformed cells.

Nucleic Acids Res, 1976 Nov, 3(11), 3227 - 33
Excision in vitro of the DNA bound carcinogen, 4-nitroquinoline 1-oxide; Yoshida S et al.; It has been shown that 4-hydroxyaminoquinoline 1-oxide, the proximate form of the carcinogen 4-nitroquinoline 1-oxide, binds covalently to the purine bases of DNA . Here we report that carcinogen-bound nucleotides can be excised from DNA by a 5' leads to 3' exonuclease associated with DNA polymerase I of E . coli in the forms of either mononucleotides or oligonucleotides . Beef spleen phosphodiesterase II (5' leads to 3') also split carcinogen-bound nucleotides, while a 3' leads to 5' exonuclease of DNA polymerase I and E . coli exonuclease III (3' leads to 5') could not excise the modified nucleotide.

Nucleic Acids Res, 1976 Nov, 3(11), 3133 - 41
Partial characterization of an endonuclease activity which appears in nuclease free T4 polynucleotide kinase; Loewen PC; A nuclease activity has been found to appear in preparations of T4 induced polynucleotide kinase which had originally been nuclease free . The nuclease introduced random nicks into T7 DNA suggesting that it was an endonuclease . Destabilization of the kinase molecule by osmotic shock or by the removal of reducing agents, ATP or salts was shown to stimulate the endonuclease appearance . The molecular weight was found to be 32,000 +/- 10% by gel filtration on G100 Sephadex . The nuclease was active over a wide pH range from pH 5.0 to pH 9.2 in a number of buffer systems and required MgCl2 and reducing agent for maximum activity . Sodium azide did not affect the nuclease appearance.

Am J Physiol, 1976 Nov, 231(5 Pt . 1), 1330 - 6
Phosphorylation of cardiac regulatory proteins by cyclic AMP-dependent protein kinase; Reddy YS; Cardiac myofibrils were purified from canine myocardium, and the regulatory proteins (troponin + tropomyosin) were extracted and shown to contain endogenous cyclic AMP-dependent protein kinase activity . Other cyclic nucleotide stimulated the protein kinase activity but only at higher concentrations . The enzyme was able to catalyze phosphorylation of conventional substrates such as histones and casein as well as a component of the regulatory protein fraction with a molecular weight of 28,000 daltons . Endogenous phosphorylation required the presence of Mg2+ and was inhibited by Ca2+ . A protein kinase inhibitor obtained from skeletal muscle inhibited the cyclicAMP-dependent phosphorylation . Escherichia coli alkaline phosphatase dephosphorylated the endogenous substrates . The level of phosphorylation found is severalfold higher than we have previously reported . A protein kinase, with its close association with the regulatory proteins, seems to be well suited to transmitting the message from the cyclic AMP to the regulatory proteins, a phenomenon that may influence the cardiac contractility via the troponin phosphorylation . The inhibitory effect of troponin on actomyosin might be changed by its state of phosphorylation.

Proc Natl Acad Sci U S A, 1976 Nov, 73(11), 3872 - 6
DNA gyrase: an enzyme that introduces superhelical turns into DNA; Gellert M et al.; Relaxed closed-circular DNA is converted to negatively supercoiled DNA by DNA gyrase . This enzyme has been purified from Escherichia coli cells . The reaction requires ATP and Mg++ and is stimulated by spermidine . The enzyme acts equally well on relaxed closed-circular colicin E1, phage lambda, and simian virus 40 DNA . The final superhelix density of the DNA can be considerably greater than that found in intracellularly supercoiled DNA.

Infect Immun, 1976 Nov, 14(5), 1130 - 7
Mechanism of fever induction in rabbits; Siegert R et al.; Three exogenous pyrogens (Escherichia coli lipopolysaccharide, synthetic double-stranded ribonucleic acid . Newcastle disease virus) were compared with respect to their mechanisms of fever induction in rabbits . All inducers stimulated the production of an endogenous pyrogen demonstrated in the blood as well as prostaglandins of the E group, and of cyclic adenosine 3',5'-monophosphate in the cerebrospinal fluid . The concentrations of these compounds were elevated approximately twofold as compared to the controls . Independently of the mode of induction, the fever reaction could be prevented by pretreatment with 5 mg of cycloheximide per kg, although the three fever mediators were induced as in febrile animals . Consequently, at least one additional fever mediator that is sensitive to a 30 to 50% inhibition of protein synthesis by cycloheximide has to be postulated . The comparable reactions of the rabbits after administration of different pyrogens argues for a similar fever mechanism . In contrast to fever induction there was no stimulation of endogenous pyrogen, prostaglandins of the E group, and cyclic adenosine 3',5'-monophosphate in hyperthermia as a consequence of exposure of the animals to exogenous overheating . Furthermore, hyperthermia could not be prevented by cycloheximide.

Can J Microbiol, 1976 Nov, 22(11), 1595 - 602
A comparison of the prophylactic and therapeutic effects of poly I:C and endotoxin in mice infected with Mengo virus; Campbell JB et al.; We have compared the protective effect in AKR mice of poly I:C and bacterial endotoxins against lethal doses of Mengo virus . Administered intravenously or intraperitoneally, both interferon inducers protected mice to about the same extent from virus challenges of 2-3 LD50's . Endotoxin, however, was unable to protect the mice effectively against higher challenge doses of virus . Evidence is presented that the level of protection afforded by both inducers is related to the level of circulating interferon produced . We have also shown that a single intravenous dose of poly I:C results in the appearance of two distinct bursts of interferon activity, with maxima at about 2 h and 9 h post injection . Endotoxin, on the other hand, produced only one peak of activity, at 2 h post injection.

Nucleic Acids Res, 1976 Nov, 3(11), 3101 - 8
Physical map of Neurospora crassa mitochondrial DNA and its transcription unit for ribosomal RNA; Bernard U et al.; A circular denaturation and restriction map of mitochondrial DNA from Neurospora crassa is presented . The map shows the position of all twelve fragments produced by restriction endonuclease Eco R I and the position of the largest Hin III fragment along the previously established map of AT-rich sequences . The two wild type strains Em 5256 and 7A differ in the lengths of two Eco R I fragments . No difference was found between the mitochondrial mutant "poky" and its parent strain . The position of the DNA segment carrying the transcription unit for the two ribosomal RNA molecules has been determined by molecular hybridization.

J Bacteriol, 1976 Nov, 128(2), 677 - 80
Glycolytic and tricarboxylic acid cycle enzyme activities during intraperiplasmic growth of Bdellovibrio bacteriovorus on Escherichia coli; Hespell RB; Selected enzyme activities were measured in extracts of the total cell pellets obtained at various times during aerobic intraperiplasmic growth of Bdellovibrio bacteriovorus 109J on anaerobically grown Escherichia coli substrate cells . Initially, the glycolytic enzyme activities were associated with the input of E . coli and the tricarboxylic acid cycle enzyme activities with the input of bdellovibrios . During the first 90 min of Bdellovibrio development, the glycolytic activities declined about 25 to 60%, whereas the tricarboxylic acid cycle activities increased about 10% . Between 110 and 180 min, the glycolytic activities decreased to trace levels and tricarboxylic acid cycle activities increased about 50 to 90% . Both bdellovibrio cell extracts and the cell-free growth menstruum (obtained after bdellovibrio growth on E . coli) caused the inactivation of glycolytic enzymes in E . coli extracts.

Mol Biol Rep, 1976 Nov, 3(2), 167 - 73
Gene specific priming of complementary DNA synthesis; Crawford RJ et al.; DNA, complementary to chicken globin mRNA was synthesized using either Avian Myeloblastosis virus reverse transcriptase, or E . coli DNA polymerase I . Transcriptase cDNA sediments at 9 S on sucrose gradients, and is 620 nucleotides in length, representing a complete copy of globin mRNA template . In contrast, Polymerase I cDNA sediments at 4 S, is 100 to 200 nucleotides in length, and is a copy of a small region at the 3'(poly A) end of globin mRNA . Similarly, Transcriptase cDNA and Polymerase I cDNA hybridize to globin mRNA template with characteristic, individual Crot1/2 values . The Crot1/2 value for Transcriptase cDNA hybridization is 7 X 10(-4) mol s 1(-1), and that for Polymerase I cDNA is 5 X 10(-3) . This work shows that Avian Myeloblastosis virus reverse transcriptase can use Polymerase I cDNA to prime further cDNA synthesis along the mRNA template . The product of extended cDNA synthesis is identical in length and hybridization properties to oligo (dT) primed transcriptase cDNA.

J Bacteriol, 1976 Nov, 128(2), 580 - 6
Evidence for a role of N-acetylmuramyl-L-alanine amidase in septum separation in Escherichia coli; Wolf-Watz H et al.; Septum formation and septum separation have been studied in a chain-forming mutant of Escherichia coli K-12 bearing the envA mutation and its parental strain . In comparison to the wild type, the mutant showed a sixfold reduction in the specific activity of the enzyme, N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28), part of which was associated to the outer membrane . Genetic as well as physiological suppression of chain formation resulted in an increase in amidase activity . The addition of N-acetylmuramyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid to growing wild-type cells and to cells bearing the envA mutation caused an inhibition of cell separation and an increased frequency of visible septa . The kinetics of septum formation and separation was followed in chains by the use of ampicillin and nalidixic acid . The latter drug inhibited initiation of new septa but allowed preformed ones to go to cell separation at a rate corresponding to that of steady-state growing cells . Ampicillin treatment, on the other hand, resulted in a more rapid decrease in the frequency of septa . The disparate effects of ampicillin and nalidixic acid were not explained by a difference in amidase activity but could be due to an inhibitory effect of ampicillin on a septal peptidoglycan fusing activity.

Nucleic Acids Res, 1976 Nov, 3(11), 2971 - 84
polA6, A mutation affecting the DNA binding capacity of DNA polymerase I; Kelly WS et al.; The polA6 mutation is an allele of the polA gene of Escherichia coli which produces a DNA polymerase I species readily distinguishable from that produced by the wild type allele . Experiments described here show that this enzyme has an altered pH optimum for polymerization and a lower binding affinity for DNA . The defect clearly lies within the carboxyl-terminal large fragment of the enzyme produced by in vivo or in vitro proteolysis since the fragment has the same pH optimum for polymerization as the intact enzyme . The polA6 enzyme and its fragment are more sensitive to phosphate ions than the wild type polymerase, and the large fragment is less efficient at binding poly d(AT) in in vitro binding assays . Although the specific nucleolytic activity of the polA6 enzyme is higher than that of the wild type, there is no apparent alteration in pH optimum for the hydrolysis of eigher double or single stranded DNA.

Nature, 1976 Oct 28, 263(5580), 744 - 8
Cloned synthetic lac operator DNA is biologically active; Marians KJ et al.; A chemically synthesised duplex DNA fragment containing the sequence of the lac operator was cloned in E . coli using the vehicle pMB9 . Clones containing lac-pMB9 hybrid DNA produced beta-galactosidase constitutively and the hybrid DNA bound the lac repressor specifically.

J Biol Chem, 1976 Oct 25, 251(20), 6259 - 66
Isolation and identification of the messenger ribonucleic acid for a structural lipoprotein of the Escherichia coli outer membrane; Takeishi K et al.; The cells of Escherichia coli strain CP 78 were labeled with {32P}orthophosphate and the total radioactive RNA was prepared from the cells . The mRNA that codes for a structural lipoprotein in the outer membrane was purified from the total RNA by three successive electrophoreses on polyacrylamide slab gels, twice at pH 8.3 and once at pH 3.5 in 7 M urea . Approximately 0.002% of the total radioactive phosphate used was incorporated into the fraction containing the most purified mRNA . The two-dimensional fingerprint of the T1 ribonuclease digest of the 32P-labeled mRNA showed that the purity of the mRNA was as high as 90% . A preliminary sequence analysis was carried out on the T1 ribonuclease oligonucleotides which had been separated by the fingerprinting procedure . By using the established amino acid sequence of the lipoprotein and the genetic code, three relatively long oligonucleotides were assigned to code for three different parts of the lipoprotein . From these data, the present RNA fraction was identified as the lipoprotein mRNA . From the analysis of the T1 ribonuclease oligonucleotides, the mRNA was estimated to be 360 +/- 10 nucleotides in length . Although the length of the mRNA was enough to code for 2 lipoprotein molecules, T1 ribonuclease digestion of the mRNA yielded only 1 mol/mol of mRNA of the individual oligonucleotides assigned to parts of the amino acid sequence of the lipoprotein . This suggests that the mRNA codes for only 1 molecule of the lipoprotein . It was also found that the mRNA has no polyadenylate sequence at the 3' end.

J Biol Chem, 1976 Oct 25, 251(20), 6274 - 86
Further studies on the isolation and properties of polyriboadenylate polymerase from Escherichia coli PR7 (RNase I-, pnp); Ramanarayanan M et al.; Polyriboadenylate polymerase was isolated from Escherichia coli PR7 (RNase I-, pnp) in good yield and high purity . The enzyme catalyzes the polymerization of ATP and ADP . These polymerizations show an initial lag which can be removed by the addition of poly(A) . However, poly(A) does not function as a primer . UDP and CDP can also serve as substrates but with decreased efficiency . The polymerization of CDP is enhanced by the presence of an oligonucleotide which again does not function as a primer . Polymerization of {gamma-32P}ATP or {beta-32P}ADP result in products with no radioactivity . The product formed from {alpha-32P}ATP on hydrolysis with alkali yields labeled pAp and 2',3'-AMP; thus the enzyme synthesizes poly(A) chains de novo . During the polymerization of ATP, no burst of free ADP can be detected and the time course of phosphate release from ATP ro ADP follows very closely the kinetics of polymerization . dATP and dADP are effective inhibitors of poly(A) synthesis from either ATP or ADP . Sulfhydryl reagents inhibit only the polymerization of ATP and the inhibition is fully reversed by dithiothreitol . However, the enzyme can be protected from sulfhydryl reagents by preincubation with either ATP or ADP in the absence of Mg2+ which is required for polymerization . Studies using acrylamide gel electrophoresis indicate that the polymerization activity with either ATP or nucleoside diphosphates resides in the same protein . The enzyme catalyzes the following exchanges: 32Pi into ADP, 32Pi into ATP, and {14C} ADP into ATP in the presence of phosphate . While the enzyme catalyzes the phosphorolysis of its own product, (pAp-(Ap)nA), it fails to cleave the dephosphorylated product, (Ap(Ap)nA), or ribosomal RNA or tRNA in the presence of inorganic phosphate . The differences and similarities between poly(A) polymerase and polynucleotide phosphorylase are discussed . Based on the 32P exchange studies and other properties of poly(A) polymerase, a plausible mechanism for its action is proposed.

J Biol Chem, 1976 Oct 25, 251(20), 6179 - 82
D-Serine dehydratase from Escherichia coli . Essential arginine residue at the pyridoxal 5'-phosphate binding site; Kazarinoff MN et al.; D-Serine apodehydratase from Escherichia coli is rapidly inactivated by butanedione in K+ borate buffer or by phenylglyoxal in K+ phosphate buffer at pH 8, 25 degrees . Pyridoxal-P protects against the inactivation . Modification of the apoenzyme abolishes its ability to bind the cofactor, pyridoxal-P, but the apparent Km for the substrate, D-serine, is not altered . The concentration dependence of the rate of butanedione inactivation in K+ borate buffer indicates that it is a two-step process with one butanedione bound per molecule of apoenzyme to give an inactive complex; half-maximal rate of inactivation is obtained at 37 mM butanedione . Butanedione inactivation is fully reversed following removal of excess reagent and borate . Similar studies with {14C}phenylglyoxal show that in the presence of pyridoxal-P at least 2 arginine residues may be modified without loss of activity . In the absence of pyridoxal-P modification of a single additional arginine residue results in loss of activity . Results with both inactivating reagents thus demonstrate that a critical arginine residue participates in binding of the coenzyme, pyridoxal-P . The stoichiometry of phenylglyoxal incorporation into the enzyme is different in the presence and absence of borate . Under both conditions incorporated phenylglyoxal is slowly lost on dialysis at neutral pH . A possible explanation of these effects is discussed.

Biochim Biophys Acta, 1976 Oct 19, 448(3), 474 - 91
Mutational change of membrane architecture . Mutants of Escherichia coli K12 missing major proteins of the outer cell envelope membrane; Schweizer M et al.; Mutants of Escherichia coli have been analyzed which miss two of the major proteins of the outer cell envelope membrane . The two proteins I and II, normally are present at high concentrations (about 10(5) copies per cell) . In such mutants, as compared with wild type, the phospholipid-to-protein ratio in the outer membrane has increased by a factor of 2.3 causing a considerable difference in density between wild type and mutant membranes . The concentrations of two other major components of the outer membrane, lipopolysaccharide and Braun's lipoprotein, did not change . The protein-deficient mutants do not exhibit gross functional defects in vitro . An increased sensitivity to EDTA and a slight such increase to dodecyl sulfate (but not to deoxycholate or Triton X-100) was observed, loss of so-called periplasmic enzymes was not found, and other differences to wild type are marginal . The mutants can grow with normal morphology . It is not possible, however, to prepare "ghosts" (particles of size and shape of the cell without murein, surrounded by a derivative of the outer membrane, and possessing the major proteins of this membrane) from them . This fact confirms our earlier suggestion that the proteins in question are required for the shape maintenance phenomenon in ghosts, and the mutants reject the speculation that these proteins are involved in the expression of the genetic information specifying cellular shape . Freeze-fracturing showed that in mutant cells, and in sharp contrast to wild type, the far predominant fracture plane is within the outer membrane . The concentration of the well known densely packed particles at the outer, concave leaflet of this fracture plane is greatly reduced . It was not possible, however, to clearly establish that one or the other protein is part of these particles because these ultrastructural differences were not apparent in mutants missing either one of the proteins only . The biochemical and ultrastructural data allow the conclusion that the loss of two major proteins and the concomitant increase of phospholipid concentration has changed the architecture of the outer membrane from a highly oriented structure, with a large fraction of protein-protein interaction, to one predominantly exhibiting planar lipid bilayer characteristics . E . coli thus can assemble rather different outer membranes, a fact excluding that outer membrane formation constitutes a highly ordered or strictly sequential assembly-line process.

Biochemistry, 1976 Oct 19, 15(21), 4742 - 8
Nuclear magnetic resonance relaxation time studies on the manganese(II) ion complex with succinyl coenzyme A synthetase from Escherichia coli; Lam YF et al.; Nuclear magnetic resonance, as well as electron paramagnetic resonance, experiments were carried out in a study of the Mn(II) ion complex with phosphorylated succinyl-CoA synthetase . For high specific activity enzyme samples, there are 3.5 +/- 0.7 metal ion binding sites per enzyme molecule with indistinguishable dissociation constants (KD = 6.9 X 10(-4) M) . However, for enzyme samples with a lower specific activity yet equivalent purity, there are 1.6 strong metal ion binding sites (KD = 6.6 X 10(-4) M) and 2.0 weak metal ion binding sites (KD = 4.0 X 10(-3) M), a result that is easily reconciled with the alpha2beta2 subunit structure of the enzyme . Water proton relaxation rate measurements indicate that each strongly bound Mn(II) ion is coordinated to three water molecules . The present results strongly suggest that the existence of nearly 4 high-affinity metal binding sites per enzyme molecule is related to the integrity or configuration of that portion of the molecule which interacts with substrates.

Biochemistry, 1976 Oct 19, 15(21), 4712 - 7
Cooperative and noncooperative binding of pyridoxal 5'-phosphate to tryptophan synthase from Escherichia coli; Bartholmes P et al.; An improved purification procedure for the beta2 subunit of tryptophan synthase from from Escherichia coli has led to an essentially pure and stable preparation with a specific enzymatic activity that is 30% higher than the previously reported maximum value . Sedimentation analysis shows that the apo-beta2 subunit is monodisperse and dimeric down to a concentration of 0.02 mg of protein/ml . The binding of pyridoxal 5'-phosphate (pyridoxal-P) to the apo-beta2 subunit and to the alpha2-apo-beta2 complex was studied by equilibrium dialysis and spectroscopic titration . Both the beta2 subunit and the alpha2beta2 complex bind 2 mol of pyridoxal-P with no unspecific binding observable at higher concentrations of pyridoxal-P . The binding of pyridoxal-P to the apo-beta2 subunit is cooperative (Hill coefficient nH = 1.7) . The data have been fitted to the Adair equation, yielding the apparent microscopic dissociation constants for the complexes with one and two bound ligand molecules . They differ by a factor of 38, suggesting that the apo- and holo-beta2 subunits have distinct conformations . The binding of pyridoxal-P to the alpha2-apo-beta2 complex is noncooperative with a value of the dissociation constant intermediate between the two values of the beta2 subunit . This finding suggests that the alpha subunit may stabilize a third conformational state of the beta2 subunit.

Biochemistry, 1976 Oct 19, 15(21), 4570 - 4
Equilibrium measurements of the interactions of guanine nucleotides with Escherichia coli elongation factor G and the ribosome; Baca OG et al.; The interactions among Escherichia coli elongation factor G (EF-G), guanine nucleotides, ribosomes, and fusidic acid were investigated by a number of physical techniques . Equilibrium dialysis studies demonstrated the existence of a binary EF-G-GDP complex . This complex forms with a stoichiometry of ca 1:1 and an apparent Ka of 2.5 X 10(5) M-1 . While no evidence was obtained for the formation of a ribosome-GDP complex, in the presence of ribosomes, the apparent Ka for guanosine diphosphate (GDP) increased 40-fold over that for binding to EF-G alone . Although the apparent Ka increased, the stoichiometry remained ca . 1 mol of GDP/mol of EF-G . An upper limit of 1.3 X 10(7) M-1 was calculated for the Ka for binding of ribosomes to the EF-G-GDP complex . Fusidic acid had no effect on the apparent Ka's for either the EF-G-GDP or EF-G-beta, gamma-methyleneguanosine triphosphate (GMP-P(CH2)P)=ribosome complexes, but markedly increased the Ka for GDP in the EF-G-GDP-ribosome complex without altering the stoichiometry . The apparent Ka for GDP was shown to be dependent upon the fusidic acid concentration . In addition, the rate of GDP exchange into the quaternary EF-G-GDP-ribosome-fusidic acid complex was inversely related to the fusidic acid concentration . All of the data obtained in these studies suggest that the formation and dissociation of complexes involving EF-G and guanine nucleotides is ordered . GDP is the first component to bind to EF-G, followed by the ribosome, and, finally, fusidic acid . This conclusion is consistent with the kinetic mechanism for the hydrolysis of GTP by EF-G and the ribosome proposed in the preceding paper of this issue (Rohrbach and Bodley (1976b) . In addition to these binding studies, guanine nucleotides have also been shown to protect EF-G against both limited trypsinolysis and chemical modification by N-ethylmaleimide . These observations offer additional evidence for the existence of a guanine nucleotide binding site on EF-G.

Biochemistry, 1976 Oct 19, 15(21), 4565 - 9
Steady state kinetic analysis of the mechanism of guanosine triphosphate hydrolysis catalyzed by Escherichia coli elongation factor G and the ribosome; Rohrback MS et al.; The mechanism of guanosine triphosphate (GTP) hydrolysis catalyzed by elongation factor G and the ribosome in the absence of other participants in protein synthesis was examined by steady-state kinetic analysis . Optimal hydrolytic conditions were determined to be approximately pH 8.0, 20 mM Mg2+, and 80 mM NH4+ . Kinetic analyses were performed under these conditions at constant elongation factor G concentrations and variable ribosome and GTP concentrations . The resulting double-reciprocal plots in conjunction with the inhibition patterns obtained with GDP indicated that the reaction occurs by an ordered mechanism in which GTP is the leading obligatory substrate . Dissociation constants for GTP and guanosine diphosphate (GDP), as well as limiting Michaelis constants for GTP and ribosomes, were calculated from the double-reciprocal plots . These values are: KSGTP = 37.0 muM, KSGDP = 16.5 muKMGTP = 8.0 muM, KMR = 0.22 muM . Inhibition was also observed at high ribosomal concentrations and suggests that inhibition was due both to the decreased breakdown of the tertiary elongation factor G-GDP-ribosome posthydrolytic complex and to the formation of a nonproductive elongation factor G-ribosome complex . A sequential mechanism with a dead-end elongation factor G-ribosome complex has been constructed to describe the hydrolysis of GTP catalyzed by elongation factor G and the ribosome.

Mol Gen Genet, 1976 Oct 18, 148(1), 99 - 104
Catabolite translational effects on the lac messenger RNA of Escherichia coli K12; Sanchez de Rivas C et al.; The transcriptional and translational events occurring during the induction of the lac operon, were separated by blocking the translational step, either by aminoacid starvation or by addition of chloramphenicol . It was found that the carbon source used during the subsequent translation, affected the rate of beta-galactosidase synthesis . A decoordination effect on the production of enzymes of the lac system was also observed in high catabolite repression media, as well as in nitrogen limiting conditions . These findings suggested a similarity with the polarity phenomenon . In order to test this similarity, polarity suppressors of a Z- polar mutant were isolated . In one of these mutants, probably suA like, no carbon source effect was observed during the translational step . The induction kinetics in different media, after distinct pregrowth conditions, supported the idea that this mutant could be considered catabolite repression resistant only in certain restrictive conditions.

Mol Gen Genet, 1976 Oct 18, 148(1), 25 - 9
Host genes involved in the replication of single-stranded DNA phage phiK; Taketo A; Using various replication mutants of E . coli, the host genes that participate in the replication of some K12-specific single-stranded DNA phages have been determined . Functional products of dnaE, -F, -G and -Z genes are required for the multiplication of phiK, whereas dnaA, -B, -C(D), H, -I and -P are dispensable for viral replication . In contrast with polB, recA, B, C, or xth functions, host rep activity is essential for phiK . At the restrictive temperature, the yield of phiK was markedly reduced in the ligts7 mutant and partially decreased in a polAts strain . The phage phiK is thus less dependent on the host cells than phiX174 and phiA which require additionally the dnaB, -C(D) and -H functions . Replication of phage St-1 depends on dnaG and -Z gene products, but not on dnaP function . Although not much affected in polAts host, growth of St-1 was significantly diminished in dnaF or ligts7 mutants.

Mol Gen Genet, 1976 Oct 18, 148(2), 221 - 3
Genetic mapping of chromosomal mutations affecting the replication of the F-factor of Escherichia coli; Jamieson AF et al.; A genetic study has been performed on a set of mutations which prevent the replication of F-factors in Escherichia coli K-12 . The gene affected is designated seg and has been located by transduction in the serB-thr segment of the chromosome . The seg gene does not appear to be related to the dnaC locus.

Mol Gen Genet, 1976 Oct 18, 148(2), 111 - 24
Polarity and the regulation of the ilv gene cluster in Escherichia coli strain K-12; Smith JM et al.; A series of Mu-1 induced isoleucine and valine auxotrophs derived from the wild type K-12 strain of Escherichia coli and from a valine resistant (ilvO-) mutant were examined . It was concluded that the genes ilvE, ilvD and ilvA constitute a single operon and are transcribed from E to A . Furthermore, the ilvG gene, expressed only in ilvO- strains, does not lie between ilvE and ilvD as previously assumed . A mutation in rho was examined for its effect on the ilvEDA operon . One effect of the rho- mutation was a mimicking of an isoleucine limitation signal . A model for the regulation of the ilvEDA operon is discussed . The model involves multiple attentuation sites and a possible role for the ilvO locus which lies at the distal end of the ilvEDA operon (but is not part of it) . Supportive evidence for the proposed direction of transcription was obtained by examination of a series of gammailv transducing phages.

Biochim Biophys Acta, 1976 Oct 18, 447(3), 249 - 59
DNA repair and recovery in Escherichia coli after psoralen and angelicin photosensitization; Bordin F et al.; The correlation between DNA repair and recovery of biological functions was studied using three wild type strains of Escherichia coli and two skinphotosensitizing furocoumarins, psoralen and angelicin, which are well known specific reagents of the pyrimidine bases of DNA . In addition to mono-adducts psoralen is able to form a high number of inter-strand cross-links, while angelicin forms only mono-adducts . Both of these damages were repaired, in a short time, in the following way: at first DNA was cut into small pieces that were then rejoined into molecules of normal size, free from cross-links, while the furocoumarin residue was split from DNA almost quantitatively . Recovery of biological functions was studied performing photosensitization experiments in such a manner that the same amounts of psoralen or of angelicin were linked to bacterial DNA . DNA synthesis, tested just after the damage, was inhibited in a similar extent by both drugs . The same bacteria, however, showed a very different colony-forming capacity; angelicin was much less effective than psoralen with a D37 dose about 2.7 times higher . A similar picture was obtained studying DNA synthesis at different times after photosensitization: in the bacteria damaged by angelicin it was restored while no recovery was observed in cells photosensitized by psoralen . These results suggest that both mono-adducts and cross-links can be chemically repaired more or less in a quantitative measure, but that repair of cross-links in much less effective on cell recovery; this behaviour is very probably connected with the different repair mechanisms of mono-adducts and of cross-links.

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