Escherichia coli

Cancer Res, 1977 May, 37(5), 1384 - 8
Inhibition of the lethality of bleomycin A5 in L-cells by hirudonine; Lapi L et al.; The lethality of several individual bleomycin derivatives, i.e., the spermidine derivative (A5), The dimethyl sulfonium aminopropyl derivative (A2), and the agmatine derivative (B2), was compared on mouse fibroblasts . The spermidine derivative, bleomycin A5, was the most toxic, producing more than a 2-log drop in clonability in 50 hr at 20 microng/ml . 6-Azauridine, a relatively nonlethal inhibitor of RNA synthesis and cell multiplication, produced a 60% decrease of adenine incorporation into nucleic acids without inhibiting the lethal action of A5 . This result differed from the effects of inhibition of RNA synthesis on the lethality of A5 in Escherichia coli . Hirudonine (1,8-diamidino-spermidine) markedly and specifically inhibited the lethal effects of A5 in L-cells but not in E . coli . However, hirudonine did not affect the toxicity of A2 and B2, separately or together, as it did in the mixture used clinically . Nor did arcaine (diamindinoputrescine) reduce the lethality of the agmatine (monoamidinoputrescine) derivative, B2.

J Bacteriol, 1977 May, 130(2), 839 - 45
Enzymatic methyl esterification of Escherichia coli ribosomal proteins; Kim S et al.; Enzymatic methyl ester formation in Escherichia coli ribosomal proteins was observed . Alkali lability of the methylated proteins and derivatization of the methyl groups as methyl esters of 3,5-dinitrobenzoate indicate the presence of protein methyl esters . The esterification reaction occurs predominantly on the 30S ribosomal subunit, with protein S3 as the major esterified protein . When the purified 30S subunit was used as the methyl acceptor, protein S9 was also found to be esterified . The enzyme responsible for the esterification of free carboxyl groups in proteins, protein methylase II (S-adenosyl-L-methionine:protein carboxyl methyltransferase, EC 2.1.1.24), was identified in E . coli Q13 . This enzyme is extremely unstable when compared with that from mammalian origin . By molecular sieve chromatography, E . coli protein methylase II showed multiple peaks, with a major broad peak around 120,000 daltons and several minor peaks in the lower-molecular-weight region . Rechromatography of the major enzyme peak showed activities in several fractions that are much lower in molecular weight . The substrate specificity of the E . coli enzyme is similar to that of the mammalian enzyme . The Km value for S-adenosyl-L-methionine is 1.96 X 10(-6) M, and S-adenosyl-L-homocysteine was found to be a competitive inhibitor, with a Ki value of 1.75 X 10(-6) M.

Mol Gen Genet, 1977 Apr 29, 152(3), 253 - 7
A new nucleic acid-protein cross-linking reagent; Oste C et al.; A new photoactivable reagent is described, which allows the formation of RNA-protein cross-links via disulfide bridges in combination with mercaptobutyrimidate . The reconstituted L24 protein-23S RNA complex from the large subunit of E . coli ribosomes has been used as a model system for the cross-linking . The main advantages of the reagent are the absence of U.V . generated cross-links, since photoactivation is carried out at 360 nm, on one hand and the ease of cleavage of the cross-link by mild reduction (beta-mercaptoethanol) on the other.

Mol Gen Genet, 1977 Apr 29, 152(3), 331 - 6
Cold-sensitive growth of a mutant of Escherichia coli with an altered ribosomal protein S8: analysis of revertants; Geyl D et al.; 26 cold-resistant revertants of a cold-sensitive Escherichia coli mutant with an altered ribosomal protein S8 were analyzed for their ribosomal protein pattern by two-dimensional polyacrylamide gel electrophoresis . It was found that 16 of them had acquired the apparent wild-type form of protein S8, one exhibits a more strongly altered SC than the original mutant and two revertants regained the wildtype form of S8 and, in addition, possess alterations in protein L30 . The ribosomes of the residual revertants showed no detectable difference from those of the parental S8 mutant . The mutation leading to the more strongly altered S8 was genetically not separable from the primary S8 mutation; this indicates that both mutations are very close to each other or at the same site . The structural gene for ribosomal protein L30 was mapped relative to two other ribosomal protein genes (for proteins S5 and S8) by the aid of one of the L30 mutants: The relative order obtained is: aroE....rpmD(L30)....rpsE(S5)....rpsH(S8)....rpsL(S12) . The L30 mutation impairs growth and ribosomal assembly at 20 degrees C and is therefore the first example of a mutant with defined 50S alteration that has (partial) cold-sensitive ribosome assembly . A double mutant was constructed which possesses both the S8 and the L30 mutations . It was found that the L30 mutation had a slight antagonistic effect on the growth inhibition caused by the S8 mutation . Thus the L30 mutants might have possibly arisen from the original S8 mutant first as S8/L30 double mutants which was followed by the loss of the original S8 lesion.

Mol Gen Genet, 1977 Apr 29, 152(3), 259 - 66
Kinetics of ribosome synthesis during a nutritional shift-up in Escherischia coli K-12; Champney WS; The rates of total protein synthesis, polyribosome formation and 70S ribosome accumulation were measured following a nutritional shift-up of Escherichia coli K-12 . Changes in ribosome content and distribution during the shift-up were measured by examining the total cellular content of free and polysome-associated ribosomes using a sensitive double isotope labeling method . The kinetics of ribosomal subunit formation and the biosynthesis of subunit protein and RNA species were also defined . The results indicated that a pre-shift population of ribosomal subunits was utilized for the immediate post shift increase in both total and ribosomal-specific protein synthesis . An assembly time for new subunits of about 3 min was observed . The formation of certain ribosomal proteins during the shift suggested that new subunit assembly was limited by the rate of synthesis of particular ribosomal proteins during this growth transition.

Mol Gen Genet, 1977 Apr 29, 152(3), 239 - 43
Further temperature-sensitive mutants of Escherichia coli with altered ribosomal proteins; Isono K et al.; Various alterations in ribosomal proteins were detected in forty-one mutants of E . coli isolated as temperature-sensitive mutants . Out of these, six are new classes of mutants harboring mutations in proteins S3, L5, L7 (L12), L29, L30 and L33 . One of them apparently lacks protein L7 of the large subunit . These mutants together with those reported previously (Isono et al., 1976) total one hundred and one ribosomal mutants in thirty different proteins.

Mol Gen Genet, 1977 Apr 29, 152(3), 231 - 7
Properties of hybrid plasmids, consisting of parts of the mini-R1 factor Rsc11 and ColE1; Boidol W et al.; In vitro joining of the two small multicopy plasmids Rsc11 and ColE1 by a poly dAdT linker resulted in hybrid plasmids, which determine resistance to ampicillin and immunity to colicin E1 . Isolation of the plasmid DNA from single colonies revealed that a variety of hybrid plasmids was formed . Cleavage of these plasmids with restriction endonucleases HinII, HindIII, EcoRI, SmaI and BamI and hybridization with ColE1 demonstrated that they contain different parts of the parent plasmids, Rsc11 and ColE1 . Their copy number in the cell is between 6 and 15 per chromosome depending on the plasmid . None of these plasmids can replicate in polA mutants . Replication continues in the presence of chloramphenicol . This suggests that replication can only occur from the ColE1 origin and that the replication function of the Rsc11 part is lost . The hybrid plasmids are compatible with Rsc11 but not with ColE1 . The comparison of the physical maps of these Rsc11--ColE1 hybrids with their functions allows a partial determination of the location of ampicillin resistance, replication and incompatibility on the Rsc11 genome.

Mol Gen Genet, 1977 Apr 29, 152(3), 211 - 7
Replication of Fpoh+ plasmid in mafA mutants of Escherichia coli defective in plasmid maintenance; Wada C et al.; A class of F' plasmids, designated Fpoh+, was previously shown to be able to replicate extra-chromosomally on Hfr strains by virtue of carrying the specific site or region poh+ (permissive on Hfr) of the E . coli chromosome (Hiraga, 1975, 1976a) . These plasmids were now found to replicate on E . coli mafA mutants (mafA1 and mafA23) that cannot support vegetative replication of F and some other F-like plasmids . The derivatives of Fpoh+ that have lost the poh+ site, on the other hand, failed to replicate on mafA mutants . These mutants harboring Fpoh+ (but not Poh- derivatives thereof) exhibit abnormal cell division and form elongated cells, presumably due to competition between Fpoh+ and the host chromosome for some factor(s) essential for the initiation of DNA replication of the both replicons . It is tentatively concluded that the poh+ site is required for F' plasmids to replicate on mafA mutants as well as on Hfr strains . In view of the fact that the mechanism of inhibition of autonomous F DNA replication in mafA mutants and in Hfr strains are clearly different, the present data seem to provide strong support to the notion that the poh+ region contains the replication origin of the E . coli chromosome.

Mol Gen Genet, 1977 Apr 29, 152(3), 205 - 10
A specialized transducing lambda phage carrying the Escherichia coli genes for phenylalanyl-tRNA synthetase; Hennecke H et al.; A lambda phage has been isolated which specifically transduces the Escherichia coli pheS and pheT genes coding for the alpha and beta subunits of the phenylalanyl-tRNA synthetase (PRS) . This phage transduces with high frequency (i) several temperature-sensitive PRS mutants to thermoresistance and (ii) a p-fluorophenylalanine resistant PRS mutant to sensitivity against this amino-acid analog . The in vitro PRS activities of such lysogens suggest that the alpha and beta subunits coded by the transducing phage complement the mutant host PRS-subunits in vivo by means of formation of hybrid enzymes.The transducing lambda phages were also used to infect UV light irradiated cells . The SDS-gel electrophoretic analysis of the proteins synthesized in such cells revealed that the phage codes at least for four different E . coli proteins . Two proteins with molecular weights of 94,000 and 38,000 daltons cross-reacted with an anti PRS serum and were thus identified as the beta and alpha subunits of PRS, respectively . A third protein with a molecular weight of 22,000 daltons is identical with the ribosomal initiation factor IF3 (Springer et al., 1977b) . The other protein (Mr 78,000) is still unidentified.

Mol Gen Genet, 1977 Apr 29, 152(3), 161 - 5
Control of ribosomal RNA synthesis in Escherichia coli . III . Cytoplasmic factors for ribosomal RNA synthesis; Muto A; The ribosomal RNA synthesis in a cell-free system containing the nucleoids and the cytoplasmic fraction prepared from Escherichia coli cells has been investigated . The addition of the "4S" fraction from the cytoplasm to the isolated nucleoids induces RNA synthesis by a new chain initiation . In this system a preferential initiation or rRNA chains occurs . The experimental results suggest that the 4S fraction contains at least two activities, one for releasing RNA-polymerases from the nucleoids, and another for the frequent initiation of rRNA chains . No restriction of the rRNA synthesis has been observed in the nucleoids and the 4S fraction from the amino acid-starved rel+ cells . The rRNA synthesized in the above system is detected at about 23S and 16S rRNA regions.

Mol Gen Genet, 1977 Apr 29, 152(3), 153 - 9
Control of ribosomal RNA synthesis in Escherichia coli . II . Ribosomal RNA synthesis in isolated nucleoids; Muto A; The effect of amino acid-starvation on the transcription in vitro of overall RNA and ribosomal RNA was investigated using nucleoids prepared from the exponentially growing and the amino acid-starved cells of rel+ and rel- strains of Escherichia coli . In this system, the synthesis of RNA is exclusively due to elongation of the chains which have been initiated in vivo . The amounts of overall and ribosomal RNA synthesized per unit of DNA in the nucleoids were analyzed for each preparation . The following observations have been made . (1) The total RNA synthesis per unit of DNA in the nucleoids from the amino acid-starved rel+ and rel- cells was not significantly different from each other . (2) The preferential ribosomal RNA synthesis occurred in the nucleoids from the growing cells; the ribosomal RNA synthesis was restricted in the nucleoids from the starved rel+ cells, while no restriction was observed in the nucleoids from the starved rel- cells . The results suggest that the ribosomal RNA synthesis is regulated at the initiation or less likely elongation level of the transcription . (3) A ribosomal RNA of a discrete size of about 30S was synthesized in the nucleoids . No mature ribosomal RNA species was produced in this system . The 30S RNA is probably a primary transcript of ribosomal RNA genes containing 23S, 16S and 5S mature ribosomal RNA sequences.

Mol Gen Genet, 1977 Apr 29, 152(3), 145 - 52
Replication of the mini-R1 plasmid Rsc11 and Rsc11 hybrid plasmids; Mayer H et al.; Replication of the multicopy mini-R1 plasmid, Rsc11, is dependent on host replication functions dna A, B, C, E and G but independent of polA1 . Chloramphenicol immediately stops its replication . A stable relaxation complex is not formed . Composite plasmids were constructed with Rsc11 and other small replicons like pSC101, ColE1 and mini-ColE1 . In all combinations the amount of hybrid plasmid DNA in the cell never exceeds the amount of Rsc11 DNA itself . This leads to varying copy numbers of the hybrid plasmids depending on the size of the second plasmid . Replication of the composite plasmids proceeds probably always under the control of the Rsc11 part although the second replicon is still functional . The composite plasmids are incompatible with both the parent replicons.

Mol Gen Genet, 1977 Apr 29, 152(3), 129 - 35
Physical and genetic studies with restriction endonucleases on the broad host-range plasmid RK2; Meyer R et al.; The cleavage map of the plasmid RK2 was determined for the five restriction endonucleases EcoRI, HindIII, BamH-I, SalI and HpaI . DNA has been inserted into several of these sites and cloned in Escherichia coli . Efforts to obtain derivatives of RK2 reduced in size by restriction endonuclease digestion of the plasmid were not successful and indicated that genes required for the maintenance of this plasmid in E . coli are not tightly clustered . An RK2 derivative possessing an internal molecular rearrangement was obtained by transformation with restriction endonuclease digests of the plasmid.

Biochim Biophys Acta, 1977 Apr 27, 497(2), 548 - 57
Production of enterochelin by Escherichia coli 0111; Rogers HJ et al.; The major neutral iron-transporting compound produced by Escherichia coli 0111/K58/H2 has been isolated from iron-deficient cultures of the organism and compared with the corresponding compound, enterochelin, produced by E . coli K12 . The product contained serine and 2,3-dihydroxybenzoic acid and formed a complex with Fe3+ . Since the PMR spectra of the products from the two strains were identical, it was concluded that E . coli 0111 also secreted enterochelin under iron-deficient conditions . Although it was not possible to establish the optical configuration of the serine residues in the molecule, the CD spectra of the metal free and Fe3+, complexes were found to be of the same sign and magnitude . The spectra show that metal binding results in considerable conformational changes in the enterochelin molecule . The biological properties of the two compounds appear to be identical as judged by their ability to abolish the bacteriostatic effect of serum on E . coli 0111.

J Biol Chem, 1977 Apr 25, 252(8), 2698 - 701
Evidence for single mechanism for aminoacyl-tRNA synthetases including aminoacyl adenylates as intermediates; Kim JJ et al.; The rate of transfer of amino acid from enzyme-bound aminoacyl adenylate to tRNA has been compared with the rate of esterification of free amino acid . The approach of Lovgren et al . (Lovgren, T . N . E., Heinonen, J., and Loftfield, R . B . (1975) J . Biol . Chem . 250, 3854-3860) was used, with 14C in the aminoacyl adenylate and 3H in the free amino acid and with both the lysine and isoleucine systems of Escherichia coli . In both systems kinetic analyses show more rapid transfer from the preformed enzyme complex when interference by the back reaction with inorganic pyrophosphate was eliminated . Parallel experiments, in which the amount of enzyme complex was measured, confirmed that aminoacyl adenylate is an intermediate in both systems . No evidence was found for an alternative mechanism.

J Biol Chem, 1977 Apr 25, 252(8), 2534 - 44
Selective inhibition of phiX RFII compared with fd RFII DNA synthesis in vitro . II . Resolution of discrimination reaction into multiple steps; Vicuna R et al.; In the presence of RNA polymerase, RNase H, discriminatory factors alpha and beta, Escherichia coli binding protein, DNA elongation factor I, DNA elongation factor II preparation, DNA polymerase III, and ATP, UTP, GTP, CTP, dATP, dTTP, dGTP, and dCTP, fd viral DNA can be quantitatively converted to RFII containing a unique gap in the linear minus strand . This gap, mapped with the aid of restriction endonucleases HinII and HpaII, is located within Fragment Hpa-H of the fd genome . The discrimination reaction has been resolved into two steps: Step A, fd viral DNA, E . coli binding protein, and discriminatory factors alpha and beta form a protein DNA complex; Step B, the complex isolated by agarose gel filtration selectively forms fd RFII when supplemented with RNase H, RNA polymerase, and the DNA elongation proteins . The omission of any of the proteins described above during the first reaction resulted in either no discrimination or a decrease in discrimination when the missing protein was added during the second step . Results are presented which indicate that E . coli binding protein, discriminatory factors alpha and beta, and RNase H must be present during the time RNA synthesis occurs in order to selectively form RFII from fd DNA and not phiX RFII . The amount of fd and phiX174 RNA-DNA hybrid formed in vitro is directly related to the DNA synthesis observed . Thus, under discriminatory conditions, only fd viral DNA leads to fd RNA-DNA complexes and no phiX RNA-DNA hybrid is formed . Under nondiscriminatory conditions, both DNAs yield RNA-DNA hybrids and DNA synthesis . In the absence of discriminatory factor alpha, no RNA-DNA hybrid is formed with either DNA, and in turn, no DNA synthesis is detected with either DNA template.

J Biol Chem, 1977 Apr 25, 252(8), 2492 - 7
Uridine and cytidine transport in Escherichia coli B and transport-deficient mutants; Leung KK et al.; Three mutants of Escherichia coli B which are defective in components of the transport system for uridine and uracil were isolated and utilized to study the mechanism of uridine transport . Mutant U- was isolated from a culture resistant to 77 micronM 5-fluorouracil . Mutant U-UR-, isolated from a culture of mutant U-, is resistant to 770 micronM 5-fluorouracil and 750 micronM adenosine . Mutant NUC- is resistant to 80 micronM showdomycin and has been reported previously . The characteristics of uridine transport by E . coli B and the mutants provide data supporting the following conclusions . The transport of adenosine, deoxyadenosine, guanosine, deoxyguanosine, adenine, or guanine by mutant U- and mutant U-UR- is identical with that in the parental strain . Uridine is transported by E . coli B as intact uridine . In addition, extracellular uridine is also rapidly cleaved to uracil and the ribose moiety . The latter is transported into the cells, whereas uracil appears in the medium and is transported by a separate uracil transport system . The entry of the ribose moiety of uridine is fast relative to the uracil and uridine transport processes . The Km values and the inhibitory effects of heterologous nucleosides for the transport of uridine and the ribose moiety of uridine are similar . Studies of cytidine uptake in the parental and mutant strains provide evidence that cytidine is transported by two independent systems, one of which is the same as that involved in the transport of intact uridine . Uridine inhibits but is not transported by the other system for cytidine transport . Evidence for the above conclusions was based on comparisons of the characteristics of {2-14C}uridine, {U-14C}uridine, and {2-14C}cytidine transport using E . coli B and the three transport mutants under conditions which measure initial rates . The nature of the inhibitory effects of heterologous nucleosides on the uridine transport processes and identification of extracellular components from radioactive uridine provides supportive data for the conclusions.

J Biol Chem, 1977 Apr 25, 252(8), 2524 - 33
Selective inhibition of in vitro DNA synthesis dependent on phiX174 compared with fd DNA . I . Protein requirements for selective inhibition; Vicuna R et al.; Crude extracts of Escherichia coli selectively convert fd viral DNA and not phiX174 DNA to duplex DNA via a complex series of reactions one of which involves RNA polymerase . Reactions leading to formation of fd duplex-replicative (RFII) structures have been reconstituted with purified proteins from E . coli . Maximal synthesis requires the combined action of E . coli binding protein, DNA elongation factor I, DNA elongation factor II preparations (which are a mixture of dna Z and DNA elongation factor III), DNA polymerase III, DNA-dependent RNA polymerase, Mg2+, dATP, dGTP, dCTP, dTTP, and ATP, GTP, CTP, and UTP . In contrast to crude extracts of E . coli, purified protein fractions do not distinguish between fd DNA and phiX174 DNA in duplex DNA formation . The addition of crude fractions of E . coli to the purified components listed above selectively permits fd RFII formation and prevents phiX RFII formation . This selective inhibition was used as an assay to isolate proteins essential for this phenomenon; they include RNase H, discriminatory factor alpha, and discriminatory factor beta.

Biochemistry, 1977 Apr 19, 16(8), 1684 - 9
Initial rate kinetic analysis of the mechanism of initiation complex formation and the role of initiation factor IF-3; Gualerzi C et al.; Initial rate kinetics of the formation of ternary complexes of Escherichia coli 30S ribosomal subunits, poly(uridylic acid), and N-acetylphenylalanyl transfer ribonucleic acid in the presence and in the absence of IF-3 are consistent with the hypothesis that the ternary complex is formed through a random order of addition of polynucleotide and aminoacyl-tRNA to separate and independent binding sites on the 30S ribosomes . The transformation of an intermediate into a stable ternary complex which probably entails a rearrangement of the ribosome structure leading to a codon-anticodon interaction represents the rate-limiting step in the formation of the ternary complex . The rate constant of this transformation, as well as the association constants for the formation of the 30S-poly(U) and 30S-N-AcPhe-tRNA binary complexes, are enhanced by the presence of IF-3 which acts as a kinetic effector on reactions which are intrinsic properties of the 30S ribosome . The IF-3-induced modification of these kinetic parameters of the 30S ribosomal subunit can per se explain the effect of IF-3 on protein synthesis without invoking a specific action at the level of the mRNA-ribosome interaction . This seems to be confirmed by the finding that IF-3 can stimulate several-fold the formation of a ternary complex even if one by-passes the ribosome-template binding step by starting with a covalent 30S-polynucleotide binary complex . Furthermore, the above-mentioned changes induced by IF-3 appear to be compatible with the previously proposed idea that the binding of the factor modifies the conformation of the 30S subunit . The random order of addition of substrates determined for the 30S-N-AcPhe-tRNA-poly(U) model system was found to be valid also for the more physiological 30S initiation complex containing poly(A,U.G) and (fMet-tRNA formed at low Mg2+ concentration in the presence of GTP and all three initiation factors.

Biochemistry, 1977 Apr 19, 16(8), 1677 - 83
Purification and characterization of covalently closed replicative intermediates of ColEl DNA from Escherichia coli; Katz L et al.; Pulse-labeled ColEl DNA molecules, undergoing replication in Escherichia coli cells either in the absence or presence of chloramphenicol, were extracted and purified by neutral sucrose density gradient sedimentation and equilibrium centrifugation in an ethidium bromide-cesium chloride gradient . In the dye-buoyant density gradient, the replicating molecules were found in regions between the supercoiled and open-circular nonreplicating plasmid DNA, as well as in the open-circular region . In a neutral sucrose gradient, peaks of pulse label were found in the region of 26 to 38 S as well as at the 23 and 17 S positions corresponding to the positions of supercoiled and open-circular ColEl DNA . In alkaline sucrose gradient, nascent ColEl DNA was found to sediment as discrete peaks corresponding to 5-6, 7-9, and 14-16 S, indicating that at least one growing strand of the replicating molecule is produced discontinuously . In the electron microscope, many of the molecules appeared as partially supercoiled structures containing two open-circular branches of equal length, of less than 20% to more than 90% replicated . Branched open-circular molecules were not observed to any significant extent without prior treatment to induce single-strand scissions . The parental strands of the replicating molecules were determined to be covalently closed, but the superhelical density of the DNA was shown to be progressively decreased as replication proceeded.

Biochemistry, 1977 Apr 19, 16(8), 1590 - 6
Studies on the mechanism of Escherichia coli glucosamine-6-phosphate isomerase; Midelfort CF et al.; Escherichia coli glucosamine-6-phosphate isomerase is specific for removal of the 1-pro-R hydrogen of fructose 6-phosphate (fructose-6-P) . The conversion of {2-3H}glucosamine-6-P to fructose-6-P plus ammonia is accompanied by 99% exchange of tritium with water and 0.6% transfer to C-1 of fructose-6-P . The enzyme is active toward alpha-glucosamine-6-P and apparently inactive toward the beta anomer . The combination of the above results supports a cisenolamine intermediate for the reaction . The labeling of substrate and product pools in tritiated water shows that the two halves of the reaction are each freely reversible . No single step appears to be rate determining . 2-Amino-2-deoxyglucitol-6-P is an unusually strong competitive inhibitor (K1 = 2 X 10(-7) M, compared with the Km = 4 X 10(-4) M for glucosamine-6-P), suggesting the enzyme has a strong affinity for the open-chain form of glucosamine-6-P.

Biochemistry, 1977 Apr 19, 16(8), 1621 - 5
A confirmation of the phase behavior of Escherichia coli cytoplasmic membrane lipids by X-ray diffraction; Linden CD et al.; The lipid fatty acid composition of the cytoplasmic membranes of Escherichia coli can be varied by growing an unsaturated fatty acid auxotroph in the presence of different fatty acid supplements . Electron spin resonance (ESR) studies of spin-label partitioning into the cytoplasmic membranes of different lipid fatty acid compositions as a function of temperature have been interpreted as indicating a broad order-to-disorder transition in the membrane lipids, the end points of the transition depending upon the fatty acid composition . We have utilized x-ray diffraction to confirm the ESR studies for three different fatty acid supplements (oleic, elaidic, and bromostearic) . We found that the characteristic end-point temperatures detected by ESR were indeed the end-point temperatures of a broad order-to-disorder transition of the cytoplasmic membrane lipids . In addition, Patterson functions calculated from lamellar x-ray diffraction from partially oriented cytoplasmic membranes indicate a decrease in average membrane thickness upon fatty acid chain melting.

Biochemistry, 1977 Apr 19, 16(8), 1548 - 54
Fluorescence energy transfer between heterologous active sites of affinity-labeled aspartokinase of Escherichia coli; Wright K et al.; The distance between aspartokinase and homoserine dehydrogenase active sites was determined using fluorescence energy transfer between modified substrates . The fluorescent 1,N(6)-ethenoadenosine 5'-triphosphate was bound at the kinase active site by Co(III) affinity labeling . Reduced thionicotinamide adenine dinucleotide phosphate quenched the fluorescence of bound nucleotide . Fluorescence depolarization measurements led to a delimitation of the value of the dipolar orientation factor to the range 0.3 to 2.8 . The distance between the fluorescent probe and the quencher was 29 +/- 4 A . In the presence of threonine, this distance increased to 36 +/- 5 A . Threonine binding either increased the intersite distance by ca . 7 A or caused a reorientation of the probe at the dehydrogenase site.

Biochemistry, 1977 Apr 19, 16(8), 1541 - 8
Interaction of substrates and inhibitors with the homoserine dehydrogenase of kinase-inactivated aspartokinase I; Wright JK et al.; The aspartokinase activity of the aspartokinase-homoserine dehydrogenase complex of Escherichia coli was affinity labeled with substrates ATP, aspartate, and feedback inhibitor threonine . Exchange-inert ternary adducts of Co(III)-aspartokinase and either ATP, aspartate or threonine were formed by oxidation of corresponding Co(II) ternary complexes with H2O2 . The ternary enzyme-Co(III)-threonine adduct (I) had 3.8 threonine binding sites per tetramer, one-half that of the native enzyme . The binding of threonine to I was still cooperative as determined by equilibrium dialysis (nH = 2.2) or by studying inhibition of residual dehydrogenase activity (nH = 2.7) . Threonine still protected the SH groups of I against 5,5'-dithiobis(2-nitrobenzoate) (DTNB) reaction but the number of SH groups reacting with thiol reagents (DTNB) was reduced by 1-2 per subunit in the absence of threonine . This suggests either that Co(III) is bound to the enzyme via sulfhydryl groups or that 1-2SH groups are buried or rendered inaccessible in I . The binding of threonine to sites not blocked by the affinity labeling produced changes in the circular dichroism of the complex comparable to changes produced by threonine binding to native enzyme and also protected against proteolytic digestion . The major conformational changes produced by threonine are thus ascribable to binding at this one class of regulatory sites . The interactions of kinase substrates with various aspartokinase-Co(III) complexes containing ATP, aspartate, or threonine and a threonine-insensitive homoserine dehydrogenase produced by mild proteolysis were studied . The inhibition of homoserine dehydrogenase by kinase substrates is not due to binding of these inhibitors at the kinase active site but was shown to be due to binding to sites within the dehydrogenase domain of the enzyme . L-alpha-Aminobutyrate, a presumed threonine analogue, also inhibits the dehydrogenase by binding at the same or similar sites in the dehydrogenase domain and not at threonine regulatory site.

Biochim Biophys Acta, 1977 Apr 19, 475(4), 571 - 88
A complete cleavage map of Neurospora crassa mtDNA obtained with endonucleases Eco RI and Bam HI; Terpstra P et al.; A physical map of Neurospora crassa mitochondrial DNA has been constructed using specific fragments obtained with restriction endonucleases . The DNA has 5 cleavage sites for endonuclease Bam HI, 12 for endonuclease Eco RI and more than 30 for endonuclease Hind III . The sequence of the Eco RI and Bam HI fragments has been established by analysis of partial fragments . By digestion of the Eco RI fragments with Bam HI, a complete overlapping map has been constructed . The position of the largest Hind III fragment on this map has also been determined . The map is circular and the added molecular weight of the fragments is 40 - 10(6), which is in good agreement with earlier measurements on intact DNA, using the electron microscope.

Biochim Biophys Acta, 1977 Apr 18, 466(2), 269 - 82
Architecture of the outer membrane of Escherichia coli K12 . II . Freeze fracture morphology of wild type and mutant strains; Verkleij A et al.; Freeze fracturing electron microscopy of Escherichia coli K12 cells showed that the outer fracture face of the outer membrane is densily occupied with particles . On the inner fracture face of the outer membrane, pits are visible, which are probably complementary to the particles at opposite fracture face . This observation suggests that the particles are micelle-like . In some mutants which lack one or more major outer membrane proteins the density of particles is reduced . The loss of protein d appeared to a prerequisite for this phenomenon . However, mutants which lack all glucose and heptose-bound phosphate in their lipopolysaccharide also have a reduction in particle density whereas, the amount of protein d is normal . Moreover, loss of lipopolysaccharide by EDTA treatment also caused a reduction in the density of particles . From these results it is hypothesized that the particles consist of lipopolysaccharide aggregates stabilized by divalent cations and probably complexed with protein and/or phospholipid.

Biochim Biophys Acta, 1977 Apr 18, 466(2), 257 - 68
Architecture of the outer membrane of Escherichia coli K12 . I . Action of phospholipases A2 and C on wild type strains and outer membrane mutants; van Alphen L et al.; Phospholipids in whole cells of wild type Escherichia coli K12 are not degraded by exogenous phospholipases, whereas those of isolated outer membranes are completely degraded . It is concluded that the resistance of phospholipids in whole cells is due to shielding by one or more other outer membrane components . The nature of the shielding component(s) was investigated by testing the sensitivity of whole cells of a number of outer membrane mutants . Mutants lacking both major outer membrane proteins b and d or the heptose-bound glucose of their lipopolysaccharide, are sensitive to exogenous exogenous phospholipases . Moreover, cells of a mutant which lacks protein d can be sensitized by pretreatment of the cells with EDTA . From these results and from data on the chemical composition of the outer membranes, it is concluded that proteins b and d, the heptose-bound glucose of lipopolysaccharide and divalent cations are responsible for the inaccessibility of phospholipids to to exogenous phospholipases.

Biochim Biophys Acta, 1977 Apr 18, 466(2), 245 - 56
Cross-linking of the proteins in the outer membrane of Escherichia coli; Reithmeier RA et al.; 1 . The organization of the proteins in the outer membrane of Escherichia coli was examined by the use of cross-linking agents and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Treatment of protein A-peptidoglycan complexes with dithiobis(succinimidyl propionate) or glutaraldehyde produced the dimer, trimer, and higher oligomers of protein A . Both forms of this protein, proteins A1 and A2, produced similar cross-linking products . No cross-linking of protein A to the peptidoglycan was detected . 2 . The proteins of the isolated outer membrane varied in their ease of cross-linking . The heat-modifiable protein, protein B, was readily cross-linked to give high molecular weight oligomers, while protein A formed mainly the dimer and trimer under the same conditions . The pronase resistant fragment, protein Bp, derived from protein B was not readily cross-linked . No linkage of protein A to protein B was detected . 3 . Cross-linking of cell wall preparations, consisting of the outer membrane and peptidoglycan, showed that protein B and the free form of the lipoprotein, protein F, could be linked to the peptidoglycan . A dimer of protein F, and protein F linked to protein B, were detected . 4 . These results suggest that specific protein-protein interactions occur in the outer membrane.

Eur J Biochem, 1977 Apr 15, 74(3), 481 - 93
Methionyl-tRNA synthetase from Escherichia coli: substituting magnesium by manganese in the L-methionine activating reaction; Hyafil F et al.; While Mg2+ can be efficiently replaced by Ni2+, Co2+ and Mn2+ in the ATP-PPi isotopic exchange reaction catalysed by methionyl-tRNA synthetase from Escherichia coli, the latter ion was selected for detailed analysis of the L-methionine activation reaction . In order to avoid artefactual results due to the slow aggregation of Mn2+ with pyrophosphate, this process was investigated by electron paramagnetic resonance and conditions were determined where it does not interfere with enzymic experiments . The thermodynamic parameters derived from steady-state (ATP-PPi isotopic exchange, fluorescence at equilibrium) or prestationary (fluorescence stopped-flow) experiments are compared to those obtained in the presence of Mg2+ {Hyafil et al . (1976) Biochemistry, 15, 3678-3685} . While the standard deltaG for the reaction (E-Met-ATP-Me2+equilibriumE-Met approximately AMP-PPi-Me2+) is close to zero in the case of Mg2+, Mn2+ slows down the rate of adenylate reversion and thus shifts the reaction towards the latter species . The deltaG for the formation of the E-Met approximately AMP complex does not depend on the metal used, suggesting that the divalent ion does not participate in the structuration of this complex . Substituting Mn2+ for Mg2+ decreases notably the dissociation constant of PPi-Me2+ from the E-Met approximately AMP-PPi-Me2+ species and from its abortive analog E-Met-Ado-PPi-Me2+ . Similarly the dissociation constant of ATP-Me2+ from another dead-end analog E-methioninol-ATP-Me2+ is decreased by Mn2+ . Involvement of the purine N7 atom in the binding of the metal ion to the active site of methionyl-tRNA synthetase is ruled out by the use of 7-deaza-adenosine . The role of the metal in the catalytic process of methionine activation and its relevance to the specificity of the reaction is then discussed in the light of the results obtained without metal and with Mg2+ and Mn2+.

Eur J Biochem, 1977 Apr 15, 74(3), 447 - 56
Poly(4-thiouridylic acid) as messenger RNA and its application for photoaffinity labelling of the ribosomal mRNA binding site; Fiser I et al.; Poly(4-thiouridylic acid) {poly(s4U)} synthesized by polymerization of 4-thiouridine 5'-diphosphate with Escherichia coli polynucleotide phosphorylase (EC 2.7.7.8) acts as messenger RNA in vitro in a protein-synthesizing system from E . coli . It stimulates binding of Phe-tRNA to ribosomes both in the presence of EF-Tu-Ts at 5 mM Mg2+ concentration and nonenzymatically at 20 mM Mg2+ concentration . It codes for the synthesis of polyphenylalanine . Poly(s4U) competes with poly(U) for binding to E . coli ribosomes . Light of 330 nm photoactivates poly(s4U) thus making it a useful photoaffinity label for the ribosomal mRNA binding site . Upon irradiation of 70-S ribosomal complexes, photoreaction occurs with ribosomal proteins as well as 16-S RNA . Ribosomes pre-incubated with R17 RNA are protected against the photoaffinity reaction . The labelling of 16-S RNA can be reduced by treatment of ribosomes with colicin E3.

Eur J Biochem, 1977 Apr 15, 74(3), 425 - 33
Aminopeptidase N from Escherichia coli . Unusual interactions with the cell surface; Murgier M et al.; The subcellular localization of aminopeptidase N (previously called aminoendopeptidase) has been investigated . This enzyme was found to be partially released (30-40%) by osmotic shock or by converting Escherichia coli K10 cells to spheroplasts . However, in all other E . coli strains (K12, B/r, MRE 600, ML 308) tested, this enzyme is not released at all by these procedures and thus behaves like a cytoplasmic enzyme . The crypticity of aminopeptidase N is surprisingly low, 75-85% of the enzyme activity is directly assayable in intact cells of any E . coli strain . Various inhibitors of transport systems do not interfer with this assay . Aminopeptidase activity could also be assayed in spheroplasts, even when an insolubilized substrate was used, which suggests a surface location of this enzyme . As well, N-ethylmaleimide (0.4 mM), under conditions which do not allow penetration in the cytoplasm, caused 70% inhibition of aminopeptidase N . Binding of 125I-labeled antiaminopeptidase N antibody to spheroplasts (from K12 strain) was used to assay the orientation of aminopeptidase N in the membrane . This enzyme is exposed on the outer surface of the cytoplasmic membrane . Confirmation of this orientation was obtained by comparing the accessibility of aminopeptidase, alkaline phosphatase and beta-galactosidase to fluorescamine in intact cells . Only 16% of the total beta-galactosidase was labeled with this fluorescent reagent whereas 44-45% of the aminopeptidase N and 59% of the alkaline phosphatase were labeled . Electron microscopic visualization of insolubilized reaction products of aminopeptidase N within the cells showed that these products are located at the poles of the cells . Neither mutant cells which were devoid of aminopeptidase N activity nor parental strains with the enzyme activity inhibited with phenylmercuric chloride contained the characteristic black caps . Thus, it appears that the periplasm is enlarged at the poles of the cells and that the reaction product is mainly located in these places . Investigation of the type of interactions of aminopeptidase N with the plasma membrane only revealed that aminopeptidase N has mainly an electrostatic interaction with the outer surface, probably mediated by magnesium ion bridges . Additional interactions are involved since disruption of the integrity of the cytoplasmic membrane is required to totally release this enzyme.

Eur J Biochem, 1977 Apr 15, 74(3), 567 - 74
Role of divalent ions in folding of tRNA; Leroy JL et al.; The native structure of tRNA is not achieved in low salt (4.5 mM Na+, 25 degrees C), but can be restored by addition of divalent ions . We have explored the structure of the central region in Escherichia coli tRNAfMet by absorption and emission spectroscopy of 4-thiouracil, and the structure of the anticodon loop in yeast tRNAPhe by fluorescence of the 'Y' base, versus the number of manganese ions bound to tRNA, which was derived from electron spin resonance . The fluorescence of the reduced 8-13 photoproduct (in which 4-thiouracil at position 8 is crosslinked to cytosine at position 13) was also analysed . In low salt (e.g . 4.5 mM Na+), the region of 4-thiouracil is affected strongly as the first eight Mn2+ bind to tRNA, whereas the fluorescence of the 'Y' base is affected only after four Mn2+ are bound . Considering the structural similarities of the two tRNAs, this suggests that the reorganisation brought about by divalent ions starts in the central region, the anticodon loop being affected later . The binding of divalent ions to each region starts together with its restructuration . Monovalent ions can substitute for divalent ions in this process, a 15 mM sodium concentration being equivalent to the binding of the first five Mn2+ . If divalent ions are then added, even the first ones distribute themselves between both the central and the anticodon region . Alternatively, the renaturation may be achieved by monovalent ions only, implying that no sites exist whose occupancy by divalent ions is crucial for the native structure . These observations suggest that the role and means of divalent ion binding to tRNA are largely explainable in terms of a simple maganese-phosphate binding supplemented by electrostatic interaction with distant phosphates.

Science, 1977 Apr 15, 196(4287), 303 - 5
A phospholipid derivative of cytosine arabinoside and its conversion to phosphatidylinositol by animal tissue; Raetz CR et al.; We have synthesized an analog (ara-CDP-DL-dipalmitin) of cytidine diphosphate diglyceride (CDP-diglyceride) in which the antitumor drug, cytosine arabinoside, is substituted for the cytidine moiety . Enzymes in rat and human liver convert this analog to phosphatidylinositol, thereby releasing cytosine arabinoside-5'-monophosphate, an obligatory intermediate in the activation of cytosine arabinoside . Unlike cytidine diphosphate diglyceride, however, ara-CDP-DL-diapalmitin is not an efficient substrate for phosphatidylglycerophosphate synthesis in liver or phosphatidylserine in Escherichia coli . The antitumor activity of ara-CDP-DL-dipalmitin in mice bearing L5178Y leukemia is described.

Biochem J, 1977 Apr 15, 164(1), 193 - 8
A mutation affecting a second component of the F0 portion of the magnesium ion-stimulated adenosine triphosphatase of Escherichia coli K12 . The uncC424 allele; Gibson F et al.; A new mutant strain of Escherichia coli in which phosphorylation is uncoupled from electron transport was isolated . The new mutant strain has a similar phenotype to the uncB mutant described previously; results from reconstitution experiments in vitro indicate that the new mutation also affects a component of the F0 portion of the Mg2+-stimulated adenosine triphosphatase . A method was developed to incorporate mutant unc alleles into plasmids . Partial diploid strains were prepared in which the uncB402 allele was incorporated into the plasmid and the new unc mutation into the chromosome, or vice versa . Complementation between the mutant unc alleles was indicated by growth on succinate, growth yields on glucose, ATP-dependent transhydrogenase activities, ATP-induced atebrin-fluorescence quenching and oxidative-phosphorylation measurements . The gene in which the new mutation occurs is therefore distinct from the uncB gene, and the mutant allele was designated uncC424.

Eur J Biochem, 1977 Apr 15, 74(3), 533 - 7
Are the aerobic and anaerobic phosphofructokinases of Escherichia coli different?
Babul J, Robinson JP, Fraenkel DG.
Phosphofructokinase has been purified from Escherichia coli strain K-12 grown in a glucose-limited chemostat, both aerobically and anaerobically . The enzymes migrated together in polyacrylamide gel electrophoresis, had the same subunit size in denaturing (dodecylsulfate) gels (Mr approx . 34000) and the same kinetic characteristics as described earlier for E . coli phosphofructokinase {e.g . Blangy et al . (1968) J . Mol . Biol . 31, 13-35}: a sigmoid curve of velocity vs . fructose 6-phosphate concentration, activation by ADP, and inhibition by phosphoenolpyruvate . Findings {e.g . Doelle (1975) Eur . J . Biochem, 50, 335-342} of quite different enzymes in aerobic and anaerobic cells were not confirmed.

Biochem J, 1977 Apr 15, 164(1), 265 - 7
Proton translocation coupled to electron flow from endogenous substrates to fumarate in anaerobically grown Escherichia coli K12; Gutowski SJ et al.; Observed leads to H+/2e- values for proton translocation during the reduction of fumarate by endogenous substrates in anaerobic cells of Escherichia coli K12 varied with fumarate concentration . This variation was probably due mainly to incomplete fumarate utilization . Under optimum conditions a minimum value for leads to H+/2e- of 1.04+/-0.20 was obtained.

Mol Cell Biochem, 1977 Apr 12, 15(2), 145 - 8
Kinetic properties of soluble adenosine triphosphatase of Escherichia coli; Ahlers J; Bound and solubilized ATPase from Escherichia coli show similar kinetic properties . The saturation curves for MgATP are hyperbolic with both preparations . The straight lines in the Line-weaver-Burk plot indicate that MgATP is the true substrate, that one molecule MgATP is bound per enzyme molecule, and that there is no cooperativity . Presence of EDTA leads to sigmoidal saturation curves . This effect could be reversed by adding MgCl2 stoichiometrically to EDTA . Different results in other publications, especially in that of CARREIRA and MUNOZ1 can be explained as being primarily the consequence of complexing agent contaminations in the assay.

Biochim Biophys Acta, 1977 Apr 12, 481(2), 340 - 7
Purification and crystallization of NADP+-specific isocitrate dehydrogenase from Escherichia coli using polyethylene glycol; Hackert ML et al.; A simple and rapid method is presented for purifying the NADP+-dependent isocitrate dehydrogenase (threo-DS-isocitrate:NADP+ oxidoreductase (decarboxylating), from Escherichia coli, which relies on fractionation of the enzyme with polyethylene glycol . The shortened preparation results in a 32% relative recovery of purified enzyme at a specific activity of 127 micronmol/min per mg of protein . The Km values for threo-DS-isocitrate, NADP+, NAD+, Mg2+ and Mn2+ are 6.4, 36, 3000, 19.7 and 2.0 micronM, respectively . The stability of the enzyme as a function of dilution and temperature are also reported . Recrystallization of the purified enzyme under different conditions readily produces a variety of single crystals . Crystals grown from ammonium sulfate solutions belong to monoclinic space group C2 with a = 125 A, b = 111 A, c = 83.5 A and beta = 108degrees 45' . Density measurements of these crystals indicate there are two 80 000-dalton dimers per asymmetric unit.

J Biol Chem, 1977 Apr 10, 252(7), 2396 - 404
Effect of estrogen on gene expression in the chick oviduct; Towle HC et al.; Mercurated UTP was used as a substrate for RNA polymerases in the in vitro transcription of chromatin so that newly synthesized RNA could be efficiently separated from endogenous chromatin RNA by means of sulfhydryl-Sepharose affinity chromatography . Utilizing this technique, it was possible to examine the effect of varying enzyme to DNA ratios on the transcription of specific genes from chromatin . For both Escherichia coli RNA polymerase and wheat germ RNA polymerase II, lowering the enzyme to DNA ration resulted in an increase in the percentage of ovalbumin mRNA sequences transcribed from chick oviduct chromatin . Similar results were also obtained for the transcription of the globin gene from chick reticulocyte chromatin . On the other hand, transcription of the globin gene from oviduct chromatin or the ovalbumin gene from reticulocyte chromatin or deproteinized chick DNA was not significantly affected by varying enzyme to DNA ratios . These results indicate that preferential transcription of certain chromatin genes relative to total RNA synthesis can occur and that this process is dependent on the presence of chromosomal proteins . Utilizing a cDNA probe complementary to the anticoding strand of the ovalbumin gene, the degree of asymmetry of the in vitro transcription of this gene was also examined . The percentage of ovalbumin RNA sequences homologous to the anticoding strand was not significantly affected when the enzyme to DNA ratio was lowered 16-fold . Since the percentage of coding ovalbumin mRNA sequences increased more than 6-fold over the same range, the percentage of asymmetric transcription of this gene increased . At the lowest enzyme to DNA ratio tested, the transcription of the ovalbumin gene from oviduct chromatin was almost totally asymmetric and, thus, closely resembled the pattern of gene transcription characteristic of the in vivo state.

J Biol Chem, 1977 Apr 10, 252(7), 2324 - 30
A new form of structural lipoprotein of outer membrane of Escherichia coli; Halegoua S et al.; Among the membrane proteins synthesized in toluene-treated cells of Escherichia coli were two distinct membrane proteins of different molecular weights, which were cross-reactive with antiserum against a structural lipoprotein of the outer membrane . One was thought to be the known membrane lipoprotein since it migrated to the same position as that of the lipoprotein (Mr = 7,200) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis . However, the other protein migrated slower than the lipoprotein . No protein corresponding to the slower-migrating species was detected in the membrane proteins synthesized in vivo . The apparent molecular weight of the protein at the new peak was estimated to be between 10,000 and 15,000 . Both the new protein and the lipoprotein were found to be synthesized from stable mRNA(s) in the toluene-treated cells . The synthesis of the new protein as well as the lipoprotein was sensitive to chloramphenicol, indicating that both proteins were synthesized on ribosomes . Peptides mapping of the new protein revealed the same COOH-terminal sequence as in the lipoprotein . This indicates that the new protein has an extra sequence at the NH2-terminal end . This hypothesis is supported by the finding that the NH2 terminus of the new lipoprotein is methionine, while that of the lipoprotein is a substituted cysteine . From double label experiments with each of 17 different amino acids and arginine, the amino acid composition of the extra region was deduced . The new protein was found to contain at least 18 to 19 extra amino acid residues over the lipoprotein, if it is assumed that the new protein has no extra arginine residues . It was found that 4 out of the 5 amino acids which were deficient in the lipoprotein (phenylalanine, tryptophan, proline, and histidine) were also deficient in the new protein, but the fifth one, glycine, was present in the new protein . From these results, it seems possible that this new form of the lipoprotine is a precursor of the lipoprotein (prolipoprotein) in the process of biosynthesis and assembly of the lipoprotein in the outer membrane.

J Biol Chem, 1977 Apr 10, 252(7), 2311 - 8
Isolation of Escherichia coli precursor tRNAs containing modified nucleoside Q; Vogeli G et al.; Affinity chromatography based on the complex formation of the modified nucleoside Q with boronic acid has been applied to the isolation of specific tRNA precursors containing this modified nucleoside . When {32P}RNA isolated from an Escherichia coli strain containing a thermolabile ribonuclease P was chromatographed on dihydroxyboryl-substituted cellulose, the precursors for asparagine, aspartate, histidine, and tyrosine tRNA were specifically retained . All precursors were monomeric . The nucleotide sequences of four asparagine tRNA precursors were determined.

J Biol Chem, 1977 Apr 10, 252(7), 2209 - 17
Cloning and amplification of rabbit alpha- and beta-globin gene sequences into Escherichia coli plasmids; Rougeon F et al.; cDNA, synthesized from rabbit globin mRNA, was used in a self-priming reaction, with avian myeloblastosis virus DNA polymerase, for the synthesis of double-stranded DNA . Globin DNA ranging from about 400 to 650 base pairs was elongated with dG tails using deoxypolynucleotide transferase and was annealed to linear Escherichia coli plasmic pCR1, elongated with dC tails . Preparation of the plasmid DNA involved an enzymatic reconstruction of one EcoRI-specific site on each side of the molecule . After transformation of E . coli cells to kanamycin resistance with the hybrid molecules, bacterial clones harboring recombinant plasmids were studied for the presence of globin-specific DNA . Plasmids containing either alpha or beta rabbit globin gene sequences were obtained . There was a 4-fold excess of recombinant plasmids containing beta-globin sequences over those with alpha-globin DNA . The longest beta-globin sequences found in plasmids were about 550 to 600 pairs long, and correspond therefore to the entire beta-globin structural gene and to some of the untranslated regions . The alpha-globin sequences were 400 to 450 base pairs long . Treatment of clone pCR1betarG 19 with EcoRI endonuclease released two DNA fragments (410 and 210 base pairs) resulting from cleavage at two reconstructed external EcoRI sites and at one internal EcoRI site within the rabbit globin gene . The same treatment of pCR1alpharG 11 released one fragment . In most other recombinant plasmids studied however, no fragment was released by EcoRI digestion.

J Biol Chem, 1977 Apr 10, 252(7), 2304 - 10
Initiation of synthesis of messenger RNA of deoxynucleotide kinase by oligoribonucleotides; Natale PJ et al.; The effects of nucleoside triphosphates and oligoribonucleotides on the initiation of synthesis of messenger RNA of the T4 phage-specific enzyme, deoxynucleotide kinase, have been studied . The procedure involved incubation of T4 DNA, purified RNA polymerase from Escherichia coli, and selected nucleotide compounds during a brief period to permit initiation of RNA synthesis . Further initiation was arrested by the addition of ribampicin, and completion of the transcription of the newly initiated RNA was permitted to take place in the presence of the full complement of nucleoside triphosphates . After translation of the messenger RNA into phage-specific enzymes, the measured activities of the latter whe first incubation period . The effectiveness of individual nucleoside triphosphates, when present singly or in combination during the initiation period, was compared to that when all four nucleoside triphosphates were available . ATP alone was extremely effective as an initiator of the synthesis of the messenger RNA for deoxynucleotide kinase . The addition of UTP to ATP not only enhanced the magnitude of initiation but also affected the kinetics of ATP interaction with T4 DNA and RNA polymerase during the initiation period . Several oligoribonucleotides including a series ApA to ApApApA, UpU to UpUpUpU, and the heteropolymers, Ap1pU and ApApApU, were tested as initiators of kinase mRNA synthesis . A sequence of nucleotides in the promoter region of T4 DNA for the deoxynucleotide kinase gene has been proposed as a result of these experiments.

J Biol Chem, 1977 Apr 10, 252(7), 2262 - 70
Proton magnetic relaxation of aspartate transcarbamylase - succinate complexes; Ireland CB et al.; Nuclear magnetic relaxation methods were used to investigate the interaction of the inhibitor succinate with aspartate transcarbamylase from Escherichia coli . Over the pH range 7 to 9, the dissociation constant for succinate remains less than the inhibitor concentration used for most of this work (0.05 M) . As a result, the enzyme predominantly exists in a single "gross" conformational state . Succinate binding to this enzyme state (generally known as the R form) parallels the behavior seen previously with the isolated catalytic subunit (Beard, C . B., and Schmidt, P.G . (1973) Biochemistry 12, 2255-2264) . The pH and temperature dependence of succinate proton relaxation rates, 1/T2 - 1/T1, in the presence of carbamyl phosphate, is interpreted in terms of a binding mechanism involving three forms of the enzyme, differing by their states of protonation . The least protonated form of the enzyme does not interact with succinate, the singly protonated species binds succinate to form a rapidly dissociating complex, and the doubly protonated species undergoes a conformational isomerization upon succinate binding, yielding a slow exchange complex . Relaxation data provide sufficient information to determine pKa values of 7.2 and 8.9 for two ionizing groups, as well as the dissociation constant for succinate in the fast exchange complex, Kd =1.6 X 10(-2) M . Rate constants for the forward and reverse steps of the isomerization, 1.3 X 10(3) s-1 and 33 s-1, respectively, indicate a significantly slower reverse rate from that obtained in the earlier NMR study of the isolated catalytic subunit . In experiments where the succinate concentration was varied, the relaxation rates showed sigmoidal binding of that ligand to the fast exchange complex above pH 9.1, (a) indicating cooperative binding of succinate, and (b) suggesting that above pH 9.1, the system cannot be characterized by a single dissociation constant, ionization constant, or relaxation effect . CTP and ATP were tested for their ability to affect succinate binding to the fast exchange complex . Heterotropic interactions were observed for CTP but not for ATP . Addition of low concentrations of the transition state analog N-(phosphonacetyl)-L-aspartate to the enzyme-carbamyl phosphate-succinate complex sharply decreased the relaxation rate, indicating that the measurements are sensitive only to succinate bound specifically to the active site.

Science, 1977 Apr 8, 196(4286), 200 - 2
Human globin messenger RNA: importance of cloning for structural analysis; Wilson JT et al.; The sequence of most of the human beta globin messenger RNA and large sections of the alpha globin messenger RNA has been determined . Partly because of genetic polymorphism, it was necessary to clone globin complementary DNA in order to extend the analysis . Purified human fetal globin messenger RNA was isolated and used as a template by reverse transcriptase to produce duplex complementary DNA molecules . These molecules were linked in vitro to plasmid DNA by use of T4 ligase in the presence of Escherichia coli Pol 1 . Several colonies transformed by these molecules have been shown to hybridize with labeled human globin complementary RNA.

Science, 1977 Apr 8, 196(4286), 220 - 1
Interbacterial transfer of Escherichia coli--Drosophila melanogaster Recombinant plasmids; Hamer DH; Recombinants were constructed between various Escherichia coli plasmids and fragments of Drosophila melanogaster DNA . These recombinant plasmids are nonconjugative, but can be mobilized from one cell to another by conjugative sex factors . Of 47 recombinants studied, 46 were mobilized at approximately the same or slightly lower frequencies than the parental plasmids, whereas one was mobilized 1000 times less efficiently.

Science, 1977 Apr 8, 196(4286), 202 - 5
Cloned ribosomal RNA genes from chloroplasts of Euglena gracilis; Lomax MI et al.; Fragments of Euglena chloroplast DNA generated by endonuclease R-Eco RI were separated by agarose-gel electrophoresis into 24 distinct bands . At least five fragments contain sequences complementary to chloroplast ribosomal RNA, Most of the Eco RI fragments have been cloned in a plasmid of Escherichia coli . Three of the cloned fragments were shown to contain chloroplast ribosomal RNA sequences by DNA-RNA hybridization.

Science, 1977 Apr 8, 196(4286), 216 - 8
Degradation of DNA by nucleases in intestinal tract of rats; Maturin L Sr et al.; Strains of Escherichia coli K12 have been constructed as safer hosts for use in recombinant DNA research, These strains are unable to survive passage through the intestinal tracts of rats because of a constellation of mutations conferring bile sensitivity and requirements for diaminopimelic acid and thymine . Since death caused by diaminopimelic acid deprivation could release recombinant DNA before DNA is degraded because of thymine starvation, it is important to determine the "survival potential" of the released DNA's . Bacterial and plasmid DNA's extracted from bacterial cells are rapidly degraded when added to low dilutions of rat intestinal contents . This observation, coupled with the stringent requirements necessary for in vitro transformation or transfection, make in vivo transmission of naked recombinant DNA in the rat intestinal tract highly improbable.

Science, 1977 Apr 8, 196(4286), 205 - 8
Cloning of yeast transfer RNA genes in Escherichia coli; Beckmann JS et al.; Four thousand Escherichia coli clones containing yeast DNA inserted into the plasmid pBR313 have been isolated . Of these, 175 clones were identified as carrying yeast transfer RNA genes . The initial analysis of the inserted transfer RNA genes via the colony hybridization technique with individual radioactive transfer RNA species is reported . The data indicate that yeast transfer RNA genes are not highly clustered, although some clustering exists . In addition, it was observed that the reiteration number of different transfer RNA genes may vary extensively.

Science, 1977 Apr 8, 196(4286), 188 - 9
Five hundredfold overproduction of DNA ligase after induction of a hybrid lambda lysogen constructed in vitro; Panasenko SM et al.; A lambda vector that contains the gene for Escherichia coli DNA ligase (lambdagt4-lop-11 lig+) has been modified to achieve overproduction of this enzyme . The third Eco RI site in the lambda chromosome has been altered by mutation, and the left-hand Eco RI fragment has been shortened . The new vector, lambdagt4-lop-11 lig+, forms a stable lysogen which, upon induction, produces a 100-fold increase in DNA ligase activity . Introduction of a phage mutation (S7) that prevents cell lysis results in an even greater increase (500-fold).

Science, 1977 Apr 8, 196(4286), 186 - 7
Increase in conjugational transmission frequency of nonconjugative plasmids; Crisona NJ et al.; Addition of Eco RI fragment 6 of the Escherichia coli sex factor F to pSC101 increases the frequency of its transmission by RI-19 and ColVB . Transmission frequencies of pSC101 and two pSC101 chimeras are also increased after the putative transposition of drug resistance element Tn3 from RI-19 . These increases may result from addition of an origin of conjugatinal transfer to the plasmids.

Science, 1977 Apr 8, 196(4286), 172 - 4
Gene cloning and containment properties of plasmid Col E1 and its derivatives; Armstrong KA et al.; Colicinogenic plasmid E1 (Col E1) and Col E1 derivatives offer advantages as plasmid cloning vehicles with regard to both utility and biological containment . The Col E1 derivative pCR1 does not alter those essential characteristics of the enfeebled Escherichia coli strain x1776 that make this strain particularly useful as a host-vehicle system for recombinant DNA research.

Biochemistry, 1977 Apr 5, 16(7), 1504 - 12
Positions of sea urchin (Strongylocentrotus purpuratus) histone genes relative to restriction endonuclease sites on the chimeric plasmids pSp2 and pSp17; Holmes DS et al.; The positions of the several sea urchin histone genes on the eukaryotic fragments of the chimeric plasmids pSp2 and pSp17 have been mapped relative to the Eco RI and Hind III restriction endonuclease sites on the plasmids . Two principal mapping methods using the electron microscope have been used: (a) the R-loop procedure and a new modification thereof to map the genes on duplex DNA; (b) the gene 32-ethidium bromide technique to visualize RNA-DNA hybrids on single strands of DNA . It is known that there are two histone genes, H3 and H2A, on pSp17 . There are two Eco RI sites at the two junctions of the procaryotic segment with the eucaryotic segment on the plasmid . We show, by an electron microscope method, that for H2A, with a length of 0.52 kilobases (kb), one end of the gene is situated 0.02 to 0.03 kb from one RI site, and that there is a Hind III site within this gene at about 0.13 kb from the end phe other RI site of this plasmid . The H4 gene lies between H2B and H1 . The ms the incubation temperature is raised up to a temperature just below that at which strand dissociation of the duplex DNA occurs.

Biochemistry, 1977 Apr 5, 16(7), 1391 - 8
Evidence for a precursor-product relationship in the biosynthesis of ribosomal protein S20; Mackie GA; The kinetics of labeling ribosomal protein S20 of Escherichia coli strains H882 and H882 groE44 have been examined using partial reconstitution as a means of binding this and some other 30S subunit proteins selectively to 16S RNA from crude extracts prepared by acetic acid extraction of pulse-labeled whole cells . The rate of labeling of S20 during short pulses at 44 degrees C is less than 20% of that observed at 28 degrees C . S20 can be recovered from the cells labeled at the higher temperature if they are chased at 28 degrees C, but not at 44 degrees C, in the presence of excess sulfate prior to their extraction . These observations suggest that S20 is derived from a precursor whose processing is blocked at 44 degrees C . Among the proteins extracted from cells labeled at 44 degrees C capable of binding to 16S RNA is a novel polypeptide, p2, which is not normally present on the 30S subunit . The kinetics of its appearance at 44 degrees C, and its chasing at 28 degrees C, suggest a precursor-product relationship with S20 . p2 contains a tryptic peptide with the chromatographic properties of the peptide Ser-Met-Met-Arg at position 25-28 in S20 . A second methionine-containing peptide at positions 49-59 of S20 is missing from p2 . In addition, the apparent molecular weight of p2 (8600) is less than that of S20 (9500) . p2 may represent the product of degradation of a precursor to S20, yet retains the ability to bind to 16S RNA . It is much less likely that p2 is a bona fide precursor which is converted into S20 by fusion to some other polypeptide.

Biochemistry, 1977 Apr 5, 16(7), 1283 - 90
Mechanistic interpretation of the influence of lipid phase transitions on transport functions; Thilo L et al.; In an attempt to understand the mechanism by which a structural change of membrane lipids affects transport functions, the temperature dependence of transport rate has been measured to below the low temperature end of the fluid in equilibrium ordered phase transition of the membrane lipids . The unsaturated fatty acid requiring Escherichia coli strain T105 was supplemented with either trans-delta9-octadecenoate or trans-delta9-hexadecenoate or supplemented with and subsequently starved for cis-delta9-octadecenoate . Fluid in equilibrium ordered phase transitions measured in whole cells using the fluorescence probe N-phenyl-1-naphthylamine were compared with the temperature dependence of beta-glucoside and beta-galactoside transport . In addition to the previously observed downward "break" in the Arrhenius plot of transport rate which occurred near the middle of the phase transition temperature range, a second upward "break" was observed which could be correlated with the low-temperature end of the phase transition . These experiments are interpreted in terms of a partitioning of transport proteins between ordered and fluid domains which is described by a lateral distribution coefficient, k . This distribution coefficient varies with the membrane lipid composition as well as with the transport system . Values for k suggest a 2-20-fold preference for the partitioning of transport proteins into the fluid parts of the membrane.

Biochemistry, 1977 Apr 5, 16(7), 1278 - 83
Stabilization by the 30S ribosomal subunit of the interaction of 50S subunits with elongation factor G and guanine nucleotide; Marsh RC et al.; The role of the 30S ribosomal subunit in the formation of the complex ribosome-guanine nucleotide-elongation factor G (EF-G) has been examined in a great variety of experimental conditions . Our results show that at a large molar excess of EF-G or high concentrations of GTP or GDP, 50S ribosomal subunits are as active alone as with 30S subunits in the formation of the complex, while at lower concentrations of nucleotide or lower amounts of EF-G, addition of the 30S subunit stimulates greatly the reaction . The presence of the 30S ribosomal subunit can also moderate the inhibition of the 50S subunit activity that occurs by increasing moderately the concentrations of K+ and NH4+, and extends upward the concentration range of these monovalent cations in which complex formation is at maximum . The Mg2+ requirement for complex formation with the 50S subunit appears to be slightly less than that needed for association of the 30S and 50S ribosomal subunits . Measurement of the reaction rate constants of the complex formation shows that the 30S ribosomal subunit has only little effect on the initial association of EF-G and guanine nucleotide with the 50S subunit; but once this complex is formed, the 30S subunit increases its stability from 10- to 18-fold . It is concluded that stabilization of the interaction between EF-G and ribosome is a major function of the 30S subunit in the ribosome-EF-G GTPase reaction.

Biochemistry, 1977 Apr 5, 16(7), 1321 - 6
Electron spin resonance evidence for vertical asymmetry in animal cell membranes; Wisnieski BJ et al.; Two electron spin resonance (ESR) spin labels were used to monitor the physical state of bacterial and animal cell membranes: 5N10, a nitroxide derivative of decane, and 12NS-GA, a glucosamine derivative of 12-nitroxide stearic acid . Spectra were recorded at 1 degrees C intervals from approximately 5 to 45 degrees C . Arrhenius plots of log hH/hP vs . 1/K were obtained by measuring the amplitudes of the hydrocarbon and water signals, hH and hP, respectively . Two discontinuities in the Arrhenius plot (at characteristic temperatures t1 and th) were observed with bacterial cell membranes independent of the spin label employed . Analysis of sealed animal cell membrane samples revealed four characteristic temperatures when the hydrophobic spin lable 5N10 was used, but only two when the amphiphilic spin label 12NS-GA was used . The specific set of characteristic temperatures revealed with 12NS-GA depended on whether the membrane preparation was inside out (ISO) or right side out (RSO) . Analysis of Newcastle disease virus, a source of RSO plasma membrane derived from host, revealed two characteristic temperatures at approximately 14 and 33 degrees C . Analysis of phagosomes, a source of ISO plasma membrane derived from LM cells, revealed two characteristic temperatures at approximately 23 and 38 degrees C . When unsealed or disrupted membrane preparations were spin labeled with 12NS-GA, both sets (RSO and ISO) of characteristic temperatures were revealed . The results indicate that the inner and outer monolayers of animal cell membranes are physically distinct and that the glycosylated spin label, 12NS-GA, is apparently restricted in its ability to flip across the membrane bilayer . In this study, characteristic temperatures were pinpointed by computer analysis of the ESR spectral data.

Biochemistry, 1977 Apr 5, 16(7), 1290 - 5
Recognition of different pools of phosphatidylglycerol in intact cells and isolated membranes of Acholeplasma laidlawii by phospholipase A2; Bevers EM et al.; Phospholipase A2 (EC 3.1.1.4) from pig pancreas hydrolyzes phosphatidylglycerol in intact cells and isolated membranes of Acholeplasma laidlawii . Complete degradation of phosphatidylglycerol in intact cells at 37 degrees C does not result in lysis as shown by the retention of intracellular K+ ions and the cytoplasmic glucose-6-phosphatase, as well as the inability to detect activity of membrane-bound intracellular NADH-oxidase . A . laidlawii was grown on linoleic acid . Phospholipase A2 treatment of these cells at 5 degrees C, at which temperature the lipids are still in the liquid-crystalline state, results in a rapid breakdown of 50% of the phosphatidylglycerol . The residual phosphatidylglycerol can be hydrolyzed only at elevated temperatures and at much smaller rates, depending strongly on the incubation temperature . When membranes isolated from these cells are incubated at 5 degrees C, 70% of the phosphatidylglycerol is hydrolyzed immediately . The hydrolysis of the residual 30% is again strongly temperature dependent . Cells were grown on palmitate, elaidate, or oleate to investigate possible effects of the lipid phase transition on the accessibility of phosphatidylglycerol for phospholipase A2 . Under conditions in which all the lipid is in the solid state, no hydrolysis occurs . When solid and liquid-crystalline lipid phases coexist, a limited hydrolysis of phosphatidylglycerol can be observed . The results demonstrate the disposition of phosphatidylglycerol in three different pools in the membrane of A . laidlawii . Phospholipase A2 has been used to discriminate between these pools and to estimate the amount of phosphatidylglycerol which is present in the liquid-crystalline phase . The present data, however, do not allow a definite localization of the phosphatidylglycerol pools.

Biochemistry, 1977 Apr 5, 16(7), 1360 - 3
Selective chemical modification of Escherichia coli elongation factor G: butanedione modification of an arginine essential for nucleotide binding; Rohrbach MS et al.; Treatment of Escherichia coli elongation factor G with the arginine reagent, 2,3-butanedione, leads to the inactivation of the enzyme when performed in sodium borate buffers . The inhibition follows pseudo-first-order kinetics until 95% of the activity has been lost and further incubation results in complete inhibiton . Removal of the borate by exhaustive dialysis results in the restoration of approximately 85% of the original activity . The pH dependence of the reaction suggests that the ionization of a group in the protein with a pKa of approximately 8.8 facilitates the reaction with butanedione . A reaction order of 1.01 +/- 0.13 was calculated for the inhibition reaction, indicating that the incorporation of one butanedione per elongation factor G results in the inactivation of the enzyme . The kinetics of inhibition in the presence of GTP indicate that the elongation factor G-GTP complex is refractory to butanedione inhibiton . Elongation factor G which has been partially inactivated by butanedione has the same apparent Km for GTP as does the native enzyme . These results indicate that elongation factor G contains only one essential arginine residue which is reactive with butanedione and that this residue is located at its nucleotide binding site.

C R Acad Sci Hebd Seances Acad Sci D, 1977 Apr 4, 284(14), 1345 - 7
{Mutants of Escherichia coli deficient in 4-thiouridine in which growth is insensitive to illumination at 365 nm}; Thomas G et al.; Thirteen different mutants of E coli K12 selected for a reduced near ultra-violet induced growth delay have been isolated . The t-RNAs extracted from these clones have all a depressed content of 4-thiouridine . One of these mutants, called Nop has an almost negligible growth delay and completely lacks 4-thiouridine in its t-RNAs . Thus we provide genetic proofs that 4-thiouridine is the chromophore for growth delay.

Biochim Biophys Acta, 1977 Apr 4, 475(3), 501 - 13
Release of template restriction in chromatin by nuclear 4.5s RNA; Kanehisa T et al.; The stimulatory mechanism of RNA synthesis of calf-thymus chromatin by nuclear 4.5 S RNA from the homologous tissue was investigated by using exogenously added Escherichia coli RNA polymerase . The RNA synthesis was initiated at low concentration of salt, and then the chain elongation was achieved at high concentration of ammonium sulfate in the presence of polyvinyl sulfate . Under these conditions the number of binding sites of RNA polymerase on chromatin which were capable of initiating RNA chain was increased by the addition of the 4.5 S RNA . This stimulation was presumed to result from the release of template restriction in chromatin . The polyvinyl salt minimized ribonuclease activity without changing the RNA polymerase activity bound to the template . Neither rearrangement nor release of chromatin proteins affected the amount or size of RNA produced . Preliminary analysis suggested that the molecular species of RNA produced upon the addition of the 4.5 S RNA from various tissues seemed to be heterologous.

Biochim Biophys Acta, 1977 Apr 4, 475(3), 424 - 36
Ribonucleic acid from the higher plant Matthiola incana . Molecular weight measurements and DNA-RNA hybridisation studies; Grierson D et al.; The percentage of DNA from the crucifer Matthiola incana coding for different types of RNA was measured by filter saturation hybridisation experiments using RNA labelled in vivo . In addition, the melting curves of the various DNA - RNA hybrids formed and the buoyant densities of the DNA sequences complementary to different types of RNA were measured . 1 . The RNA preparations used were 25, 18, and 5 S rRNA and 4 S RNA, purified by gel electrophoresis, and poly(A)-containing RNA purified by oligo-(dT)-cellulose chromatography . The molecular weights of the 25 S and 18 S rRNAs, calculated from the mobility in formamide-acrylamide gels relative to Escherichia coli RNA, are 1.25 - 10(6) and 0.64 - 10(6) . The rRNA precursor has a molecular weight of approx . 2.1 - 10(6) and the average molecular weight of the poly(A)-containing RNA from both cotyledons and roots is 4 - 10(5) . 2 . The percentage of the genome, calculated on the basis of double-stranded DNA, coding for these RNAs and the estimated number of genes per haploid DNA amount are approximately 0.46% and 1100 for 25 S plus 18 S rRNA, 0.032% and 3600 for 5 S rRNA and 0.072% and 13 000 for 4 S RNA . In filter hybridisation experiments very little hybridisation of poly(A)-containing RNA was found . A rapidly-hybridising component is attributed to small amounts of contaminating rRNA . 3 . M . incana DNA has a main band at 1.697 g - ml-1 in CsCl and a satellite constituting approximately 3% of the DNA, at 1.708 g - ml-1 - 25 and 18 S rRNA hybridise to DNA with a buoyant density of 1.701--2 g - ml-1 . The buoyant density of 5 S DNA is slightly less at 1.700--1 g - ml-1 . 4 . S RNA hybridises to at least two separate regions, one within the main-band DNA and a second lighter component . None of the RNAs tested hybridised to the satellite DNA . The Tm of the DNA - RNA hybrids in 1 X SSC is 89 degrees C for 25 S rRNA, 85 degrees C for 5 S rRNA and 82 degrees C for 4 S RNA . 4 . 5 and 4 S RNA preparations contain fragments which hybridise to sequences complementary to high-molecular-weight rRNA . This spurious hybridisation can be eliminated by competition with unlabelled high-molecular-weight RNA.

Chem Phys Lipids, 1977 Apr, 18(3-4), 367 - 73
A method for 13C enrichment of phospholipid acyl chains using Tetrahymena; Nwanze CE et al.; A method has been devised for obtaining highly 13C enriched (20-25%) phospholipids and membranes from the ciliate Tetrahymena, the enrichment occurring in the acyl chains . This method provides both phosphatidylcholine and phosphatidylethanolamine with enriched chains . The central feature of the method is the monoaxenic growth of Tetrahymena on an E . coli mutant which has a high incorporation of exogenous acetate into fatty acid chains . The 13C NMR spectra of the phospholipids are sufficiently enriched to permit NMR relaxation studies.

Infect Immun, 1977 Apr, 16(1), 12 - 9
Bacteriocin typing by leakage of ultraviolet light-absorbing material; Farkas-Himsley H et al.; A rapid and reproducible method of bacteriocin typing is described based on leakage of ultraviolet light-absorbing material (UVAM), detectable in supernatants of bacteriocin-sensitive cultures, by means of a spectrophotometer . The prerequisites for reproducible results, with nonsignificant fluctuations in standard error of the mean, are: a set of standardized bacteriocins, produced under defined conditions and of determined strength . These must interact with the unknown bacterial culture in suspension and at a given ratio in order to achieve an optimal multiplicity of interaction . Pyocin and colicin typing by the "scrape and streak" technique of Gillies (J . Hyg . 62:1-10, 1963) was compared with the UVAM leakage method in 275 tests; the two tests were found to be in good agreement for the strains tested.

Eur J Biochem, 1977 Apr 1, 74(2), 343 - 52
Analysis of the alpha-satellite DNA from African green monkey cells by restriction nucleases; Fittler F; By the use of restriction endonucleases the organization of the alpha-satellite DNA from African green monkey cells (Cercopithecus aethiops) has been analyzed . With endo R-HindIII, endo R-AluI and with endo R-EcoRI at conditions of low salt and high pH (endo R-EcoRI) all of the satellite was digested while only a part of the satellite was cleaved with endo R-Bsu and endo R-EcoRI under standard conditions . With each of the four nucleases a series of fragments was formed which were multiplies in size of a basic repeat unit linked in tandem arrays in the intact satellite . The quantitative evaluation of the digestion with each nuclease as well as with combinations of two nucleases yielded information about the distribution of the cleavage sites . While the arrangement of the endo R-HindIII cleavage sites conforms to a random distribution across the entire satellite, the results from the endo R-Bsu and endo R-EcoRI cleavage patterns are consistent with a picture where the cleavage sites are clustered in fractions of the satellite . Since endo R-AluI recognizes the central four nucleotide pairs of the endo R-HindIII cleavage site, the redigestion of the endo R-HindIII dimer with endo R-AluI gave information about the distribution of mutations in the satellite . The results of these experiments together with the comparison of the sequence divergence determined from digestion with endo R-HindIII and endo R-EcoRI lend support to the hypothesis that mutations have affected all bases in the satellite evenly . The gamma-satellite, another fraction of the African green monkey DNA, could be separated by Ag+/CsSO4 density gradient centrifugation into two components . With the three restriction nucleases used both components gave a background of fragments of heterogenous length on gel electrophoresis with some faint bands of no apparent regularity in one case.

Chem Phys Lipids, 1977 Apr, 18(3-4), 285 - 303
Conformational difference in the polar groups of phosphatidylcholine and phosphatidylethanolamine in aqueous phase; Akutsu H et al.; Proton and phosphorus nuclear magnetic resonance was used to investigate conformations of o-phosphorylcholine(OPC), o-phosphorylethanolamine(OPE) and L-alpha-glycerophosphorylethanolamine in aqueous solution, and the conformations of dipalmitoyl-3-sn-phosphatidylcholine and phosphatidylethanolamine from E . coli in methanol and chloroform solutions . It has been shown that in every case the O-C-C-N system prefers a gauche conformations, but in the choline moiety the dihedral angle around the C-C bond is distorted from the usual gauche angle, 60 degrees, to a larger one . The dihedral angle of OPC is shown to be more variable than that of OPE . This may be due to the curvature of its potential curve, i.e . asymmetrical curvature around the gauche minima . This property of the phosphatidylcholine molecule may be partly responsible for the flexibility of the phosphatidylcholine bilayer . The coupling is dominant in the P-O-C-C systems of the 5 compounds examined . The results also indicated that the two hydrocarbon chains in phosphatidylcholine or phosphatidylethanolamine are apt to take nearly parallel orientation in methanol solution . This characteristic is favourable for the formation of the bilayer structure.

Immunology, 1977 Apr, 32(4), 491 - 7
Quantitative phagocytosis by human polymorphonuclear leucocytes . Use of radiolabelled emulsions to measure thae rate of phagocytosis; Forsgren A et al.; A new micro-method for the quantitative measurement of phagocytosis by neutrophils is described . The material used for phagocytosis consists of a radioactive oil emulsion coated with E . coli lipopolysaccharide . Uptake of radioactive material is a function of cell number, duration of incubation, dilution of serum used for opsonization, content of lipopolysaccharide and concentration of emulsion . This method can be used to quantify rapidly and precisely phagocytosis rates of as few as 5 x 10(4)-10(6) polymorphonuclear leucocytes and the opsonic activity of 10 microliter serum.

Immunology, 1977 Apr, 32(4), 435 - 43
Sequential IgM and IgG2 anti-DNP antibody responses against DNP-E . coli and DNP-lipopolysacchardies in guinea-pigs; Furuichi K et al.; When 2,4-dinitrophenylated cells (DNP-E . coli) and lipopolysaccharides (DNP-LPS) of Escherichia coli were injected i.p . into guinea-pigs, they were capable of inducing sequential production of IgM and IgG2 anti-DNP antibodies, both of which were substantially thymus-independent, but only a trace of IgG1 anti-DNP antibody was produced . On the other hand, thymus-dependent DNP-bovine serum albumin (DNP-BSA) induced concomitantly both the IgG1 and IgG2 antibody responses in the presence of LPS . Therefore, the preferential IgG2 antibody response against DNP-LPS seems to be elicited with DNP-LPS itself and not by a combination of mitogenic stimulation with LPS and haptenic stimulation with other contaminating substances carrying DNP residues . Furthermore, it may not be related to affinity of the antibodies produced since there was no significant difference in the affinity for DNP residue between the IgG2 and IgG1 anti-DNP antibodies produced with DNP-E . coli and DNP-BSA, respectively.

Ultramicroscopy, 1977 Apr, 2(2-3), 199 - 203
Advantages of megavolt electron microscopy in biological research; Dupouy G; New electron microscopes, operating within the megavolt range have opened important prospects for scientific research . These microscopes were, at first, mainly used by physicists and metallurgists; but nowadays more and more biologists are interested in high voltage electron microscopy: they have obtained important and significant results . The present paper gives more information concerning experiments that have been achieved in Toulouse with our two big instruments working at 1 million volts (1 MV) and three million volts (3 MV).

J Biochem (Tokyo), 1977 Apr, 81(4), 871 - 7
Effects of detergents on the conformation of Escherichia coli tRNA as measured by circular dichroism; Okabe N et al.; The effect of a cationic detergent, lauryl pyridiniumchloride (LPC), and an anionic one, sodium n-octylbenzenesulfonate (SOBS), on the conformation of unfractionated Escherichia coli tRNA was investigated at various molar ratios of detergent to tRNA (D/R) in the presence and absence of Mg2+ and Na+ ions by measuring the circular dichroism (CD) at 265 nm and 340 nm, which reflects conformational change involving base pairs and/or base stacking, and the disymmetry in the vicinity of 4-thiouridylate (4-TU), respectively . In the presence of Mg2+ and Na+ ions, the tRNA retains its native structure even in the presence of high molar ratios of detergent to tRNA (D/R congruent to 40 at 265 nm and D/R congruent to 20 at 340 nm) . However, in the absence of these metal ions, the ellipticity at 340 nm was very sensitive to LPC concentration and decreased from 5,600 to nearly--1,600 at 25 degrees C with the increase of D/R ratios up to 20, and a similar decrease in the ellipticity at 340 nm was observed on thermal denaturation . This result suggests that the local environment involving the 4-TU region might be readily influenced by LPC, reflecting a large conformational change . However, no effect was observed in the case of the SOBS: tRNA system . On the other hand, secondary base pairing and/or base stacking structure was virtually invariant on adding both LPC and SOBS even at high D/R ratios in the absence of Mg2+ and Na+ ions.

Biophys Chem, 1977 Apr, 6(3), 291 - 8
An NMR approach to tRNA tertiary structure in solution; Robillard GT et al.; Atomic coordinates of E . Coli tRNA1Val have been generated from the X-ray crystal structure of Yeast tRNAPhe by base substitution followed by idealization...

J Gen Microbiol, 1977 Apr, 99(2), 369 - 77
The influence of growth substrate and capacity for oxidative phosphorylation on respiratory oscillations in synchronous cultures of Escherichia coli K12; Poole RK; Fluctuations in cell volume during exponential growth of Escherichia coli K12 changed the effectiveness of the continuous-flow centrifugation method for preparing synchronous cultures . Rates of oxygen uptake in synchronous cultures were measured using an electrode system open to the atmosphere . In synchronous cultures of both the parental strain and an adenosine triphosphatase-deficient mutant, which was incapable of oxidative phosphorylation, respiration rates doubled during the cell cycle but oscillated with a periodicity of approximately half a cycle . Synchronous cultures of the parental strain growing on glycerol and Casamino acids showed a stepwise pattern of oxygen consumption . Continuous flow centrifugation did not markedly affect the increases in the numbers and respiration rates of cells in syndhronous cultures . Respiratory oscillations also occurred on inoculation of a late-stationary phase culture into fresh medium, although synchronous division was not observed . The possible mechanisms underlying respiratory fluctuations under different growth conditions are discussed.

J Gen Microbiol, 1977 Apr, 99(2), 353 - 7
Preliminary characterization of cell-free K99 antigen isolated from Escherichia coli B41; Morris JA et al.; The K99 antigen of Escherichia coli B41 was isolated by isoelectric precipitation from heated bacterial suspensions . Chromatography and immunoabsorption experiments suggested that the mannose-resistant haemagglutinating activity of partially purified preparations of antigen was K99 . The antigen was partially susceptible to bacterial proteases and was inactivated by periodate oxidation . Haemagglutination inhibition experiments with sugars and absorption of K99 with antisera to human blood groups A and B substances suggested that K99 contains a terminal alpha-linked N-acetylgalactosamine moiety, which is involved in the haemagglutination reaction, and an adjacent terminal alpha-linked galactose moiety, which plays no part in the reaction.

J Gen Microbiol, 1977 Apr, 99(2), 283 - 90
Induction of cell division in a temperature-sensitive division mutant of Escherichia coli by inhibition of protein synthesis; de Pedro MA et al.; Synchronous cells of the thermosensitive division-defective Escherichia coli strain MACI (divA) divided at the restrictive temperature (42 degrees C) if they were allowed to grow at 42 degrees C for a certain period before protein synthesis was inhibited by adding chloramphenicol (CAP) or rifampicin . The completion of chromosome replication was not required for such divA-independent division . Synchronous cells of strain MACI divided in the presence of an inhibitor of DNA synthesis, nalidixic acid, if they were shifted to 42 degrees C and CAP or rifampicin was added after some time; cells of the parent strain MC6 (div A+) treated in the same way did not divide . These data suggest that coupling of cell division to DNA synthesis depends on the divA function . The ability to divide at 42 degrees C, whether or not chromosome termination was allowed, was directly proportional to the mean cell volume of cultures at the time of CAP addition, suggesting that cells have to be a certain size to divide under these conditions . The period of growth required for CAP-induced division had to be at the restrictive temperature; when cells were grown at 30 degrees C, in the presence of nalidixic acid to prevent normal division, they did not divide on subsequent transfer to 42 degrees C followed, after a period, by protein synthesis inhibition . A model is proposed in which the role of divA as a septation initiator gene is to differentiate surface growth sites by converting a primary unregulated structure, with the capacity to make both peripheral wall and septum, to a secondary structure committed to septum formation.

J Gen Microbiol, 1977 Apr, 99(2), 263 - 76
Biochemical genetics of the alpha-keto acid dehydrogenase complexes of Escherichia coli K12: isolation and biochemical properties of deletion mutants; Langley D et al.; Mutants of Escherichia coli K12 with deletions in the nadC-lpd region of the chromosome were obtained for use in studies on the expression of the ace (pyruvate dehydrogenase complex, specific components) and lpd (lipomide dehydrogenase) genes . These were isolated by selecting spontaneous aroP mutants (lacking the general aromatic amino-acid permease and thus resistant to inhibitory aromatic amino-acid analogues) and screening for auxotrophy due to deletions extending into neighbouring genes . From 2892 isolates tested, the AroP- phenotypes of 2322 were confirmed and, of these, 28 stable and independently-derived auxotrophos were designated as deletion mutants . Six nutritionally-distinct categories were recognized: Nad- (8 strains); Nad-Ace-(7): Nad-'Ace-' (3); Ace- (8); 'Ace-' (I); Lpd-(I) . The Ace- phenotypes of four isolates designated 'Ace-' were leaky and enzymological studies confirmed that they had less than 7% of parental pyruvate dehydrogenase complex activity . Enzymological studies showed that the 15 Ace- or Nad-Ace- strains all lacked the pyruvate dehydrogenase complex and pyruvate dehydrogenase (EIp) activities and only three retained detectable dihydrolipoamide acetyltransferase (E2p) . The one Lpd- strain lacked pyruvate dehydrogenase, dihydrolipoamide acetyltransferase and lipoamide dehydrogenase (E3) activities as well as the activities of the pyruvate and alpha-ketoglutarate dehydrogenase complexes . The results confirmed the gene order nadC-aroP-aceE-aceF-lpd and indicated that no other essential functions are determined by genes within the nadC-lpd region . Resistance to lactate during growth of pps mutants on acetate was directly related to the specific activity of the pyruvate dehydrogenase complex . None of the deletions promoted the high degree of resistance characteristically associated with constitutive expression of the dehydrogenase complex . Six pps mutants having Ace+ or 'Ace-' phenotypes were more sensitive than the parental strains and expression of their ace operons appeared to be affected; most sensitive were the Ace- strains which lacked pyruvate dehydrogenase complex and phosphoenolpyruvate synthetase activities . The lipoamide dehydrogenase activities of the deletion strains (Lpd+) ranged between 30% and 100% of parental levels indicating that expression of their ace operons appeared to be affected; most sensitive were the Ace- strains which lacked pyruvate dehydrogenase complex and phosphoenolpyruvate synthetase activities . The lipoamide dehydrogenase activities of the deletion strains (Lpd+) ranged between 30% and 100% of parental levels indicating that expression of the lpd gene may be affected by the ace operon but can be independent.

Infect Immun, 1977 Apr, 16(1), 26 - 31
Interactions of radio-detoxified Escherichia coli endotoxin preparations with the complement system; Fust G et al.; Escherichia coli O89 lipopolysaccharide (LPS) was treated with different doses of gamma irradiation (5, 10, 15, and 20 Mrad) . Various biological activities such as lethal effect, decrease in arterial blood pressure in dogs, and interaction with the complement system were determined for the parent and irradiated preparations . Irradiation of LPS significantly and in a dose-dependent manner decreased its lethal and blood pressure-depressing effects along with its ability to activate the complement system . In contrast, radio-detoxified LPS fixed more strongly the isolated human C1 than did the parent LPS . The possible connection between the toxicity of endotoxin and endotoxin-induced complement activation is discussed.

Biochem J, 1977 Apr 1, 163(1), 177 - 9
The dissociation of sigma-factor from ribonucleic acid polymerase; Campbell AM et al.; The sigma-factor of Escherichia coli RNA polymerase was shown to dissociate from the core enzyme as a function of absolute concentration . The association constant is in the range 10(6)-10(8) litre/mol . This implies that the amount of holoenzyme, core enzyme and sigma-factor in RNA polymerase assays may vary according to the absolute concentration of the enzyme.

Biochem Genet, 1977 Apr, 15(3-4), 287 - 96
Proline excretion in Escherichia coli: a comparison of an argD+ strain and a proline-excreting argD- derivative; Rossi JJ et al.; In an attempt to deduce the physiological basis of proline excretion in argD- strains of Escherichia coli K12, several properties of an argD+ (nonexcreting) and an argD- (excreting) derivative were compared . No difference was found in the transport or in the utilization of either proline or its immediate precursor, delta1-pyrroline-5-carboxylate (PCA) . Furthermore, no differences were found in the physical or kinetic properties of partially purified preparations of the enzyme mediating the final step in proline biosynthesis, PCA reductase . The specific activity of PCA reductase was, however, consistently higher in crude extracts prepared from the argD- mutant.

Z Immunitatsforsch Immunobiol, 1977 Apr, 153(1), 11 - 22
Induction of lymphocyte proliferation and membrane changes by lipopeptide derivatives of the lipoprotein from the outer membrane of Escherichia coli; Bessler W et al.; Lipoprotein from the outer membrane of E . coli, a potent novel mitogen, was digested by pronase treatment resulting in lipopeptide fragments containing 2-5 amino acids bound to diacylglyceryl-N-acylcysteinthioether . The lipopeptides were characterized by amino acid analysis and gas chromatography and were checked for mitogenicity . We found that all lipopeptide fragments were able to stimulate the uptake of 3H-uridine into RNA and 3H-thymidine into DNA in mouse spleen cells of several strains . The response of splenocytes of congenitally athymic mice was comparable to that of normal animals . A weak stimulation of DNA synthesis was also observed in thymocytes . The mitogenicity of the products was abolished by mild alkali hydrolysis which removes the ester-bound fatty acids . We conclude that the N-terminal lipopeptide region of lipoprotein is responsible for the mitogenic activity of the molecule . Lipopeptide as well as lipoprotein were found to cause early membrane changes in lymphocyte plasma membranes . After 4 hours we found an increased incorporation of 14C-oleate and 14C-acetate into lecithin . The membrane changes observed are similar to those brought about by mitogenic lectins, which suggests a similar mechanism for the induction of lymphocyte activation for both types of mitogens.

Nucleic Acids Res, 1977 Apr, 4(4), 1097 - 1110
Specific cleavage of ribosomal RNA caused by alpha sarcin; Schindler DG et al.; Alpha sarcin causes the specific cleavage of RNA from 80S ribosomes and 60S subunits of yeast, but not from the 40S subunits to produce a small RNA fragment . The fragment was also produced on treatment of the 60S subunits of wheat germ ribosomes . The fragment has a molecular weight of 100,000 and is a cleavage product of the large RNA species in the 60S subunits . The fragment is not derived from the 5'end of the yeast 25S RNA nor does it bind to 5.8S RNA and we propose that the fragment represents the 3' terminal 320 nucleotides of 25S rRNA . The ability to produce fragment could not be separated from the ability of alpha sarcin to inhibit protein synthesis . Alpha sarcin also causes the specific cleavage of the 23S RNA of the E . coli subunit to produce a smaller fragment of RNA than that produced from eukaryote ribosomes.

Nucleic Acids Res, 1977 Apr, 4(4), 1047 - 63
Synthesis of oligonucleotides with sequences identical with or analogous to the 3'-end of 16S ribosomal RNA of Escherichia coli: preparation of A-C-C-U-C-C via the modified phosphotriester method; van Boom JH et al.; A combination of two different methods for the synthesis of oligoribonucleotides, i.e . the two-step phosphotriester method with 2-chlorophenyl phosphate as bifunctional phosphate source and the modified triester method with 2,2,2-trichloroethyl 2-chlorophenyl phosphorochloridate as monofunctional phosphate source, is applied for the synthesis of the fully-protected hexaribonucleotide A-C-C-U-C-C . The two-step method is used for the synthesis of the required dinucleotide monophosphates 9, 10 and 11 . Application of the modified triester method for the coupling of the oligonucleotide blocks results in the formation of the fully-protected hexamer 15 . Furthermore, attention is paid to 2,4,6-triisopropylbenzenesulphonyl 4-nitroimidazolide as a new condensing agent for the coupling of larger oligonucleotide blocks.

Nucleic Acids Res, 1977 Apr, 4(4), 1039 - 45
The sequence specificity of a mammalian DNA methylase; Browne MJ et al.; The sequence specificity of an extensively purified DNA methylase preparation from Krebs II mouse ascites cells has been examined . The enzyme appears to be highly sequence dependent . Moreover the sequence distribution of cytosine residues that are methylated, bears a very close resemblance to the sequence distribution of 5'-methyl cytosine found in vivo in a wide range of vertebrate cells and is consistent with methylation of cytosines in the sequence R-Yn-C-R.

Mutat Res, 1977 Apr, 43(1), 11 - 24
Physiological modification of alkylating agent induced mutagenesis . II . Influence of the numbers of chromosome replicating forks and gene copies on the frequency of mutations induced in Escherichia coli; Hince TA et al.; The frequency of reversions induced in Escherichia coli K-12 trpA58 by any of five different monofunctional alkylating agents increased as the growth rate of the organism was raised prior to mutagen treatment . The increase in mutation frequency did not correlate with growth rate-dependent changes in cell area or total cellular protein and DNA . After treatment of cells with N-methyl-N-nitrosourea (MNUA), no growth rate-dependent change was observed in the total DNA alkylation or percentage of O6-methylguanine present in the DNA extracted . The frequency of reversions induced by one mutagen, methyl methanesulphonate (MMS), increased in proportion to the average number of trpA gene copies per cell, whereas the frequency of reversions induced by the other compounds was dependent on the average number of chromosome replicating forks per cell . This difference was attributed to the different ratios of DNA base alkylation products observed, formed after treatment with MMS, an SN2-type reagent, or after treatment with the SN1-type reagents ethyl methanesulphonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), MNUA and N-ethyl-N-nitrosourea (ENUA) . Possible reasons for the dependence of mutation frequency on the number of replicating forks per cell are discussed.

Hoppe Seylers Z Physiol Chem, 1977 Apr, 358(4), 475 - 90
Inhibition of ribonucleases by ribonucleotides and transition state analogs in cell-free extracts from Ehrlich ascites tumor cells; Egberts E et al.; We investigated the ribonucleolytic breakdown of poly(U), poly(A), RNA trascribed from calf thymus DNA with E . coli RNA polymerase, ribosomal RNA, tRNA and mengovirus RNA by an enzyme fraction obrained from a postribosomal supernatant of Ehrlich ascites tumor cells . The single-stranded homopolyribonucleotides are preferentially degraded by the enzyme fraction with the production of ribonucleoside 5'-monophosphates . The RNase activity is completely dependent on the presence of Mg2+ ions and is highest at Mg2+ and K+ concentrations optimal for cell-free protein synthesis . Ribonucleoside 5'-monophosphates, ribonucleoside 2'(3')-monophosphates, ribonucleoside 2'(3'),5'-bisphosphates and transition state analogs consisting of vanadyl sulfate and either ribonucleosides or ribonucleoside 5'-monophosphates in a molar ratio 1:1 inhibit the ribonucleolytic activity of the enzyme fraction . The ribonucleoside 2'(3'),5'-bisphosphates and the transition state analogs are the most effective inhibitors . However, only in the presence of ribonucleoside 2'(3'),5'-bisphosphates a concomitant stimulation by 50 to 60% of poly(U)-directed polyphenylalanine synthesis is observed; all the other RNase inhibitors tested also inhibit polypeptide synthesis . The results of preliminary experiments show that poly(U) and ribonucleoside 2'(3'),5'-bisphosphates are well suited as ligands for affinity chromatography of ribonucleases from Ehrlich ascites tumor cells.

Proc Natl Acad Sci U S A, 1977 Apr, 74(4), 1561 - 4
Hydrogen-bonded protons in the tertiary structure of yeast tRNAPhe in solution; Romer R et al.; Temperature-dependent lowfield proton magnetic resonance spectra of yeast tRNAPhe were recorded between 10 and 15 parts per million . Seven resonances of hydrogen-bonded protons disappeared reversibly under two sets of conditions where the selective broadening of tertiary structure resonances were predicted by temperature jump experiments . The seven resonances were assigned to the seven tertiary hydrogen bonds expected between 10 and 15 parts per million from the crystal structure of yeast tRNAPhe . Some of the non-Watson-Crick base pairs have unusual unshifted standard chemical shifts after the ring current contributions calculated from the crystal coordinates were subtracted . The differences of the chemical shifts of homologous tertiary structure base pairs in Escherichia coli tRNAfMet and yeast tRNAPhe give experimental evidence for details of the conformational differences postulated by model building on the basis of the x-ray coordinates of yeast tRNAPhe and sequence homologies.

Proc Natl Acad Sci U S A, 1977 Apr, 74(4), 1507 - 10
The amino acid sequence of beta-galactosidase of Escherichia coli; Fowler AV et al.; The amino acid sequence of beta-galactosidase was determined . The protein contains 1021 amino acid residues in a single polypeptide chain . The subunit molecular weight calculated from the sequence is 116,248 . The sequence determination, carried out mainly by conventional methods, was aided by complementation tests, by the use of termination mutant strains, and by a new immunochemical method . The five residue sequence Thr-Pro-His-Pro-Ala appears twice within the polypeptide chain, but no other striking homologous features are evident.

Proc Natl Acad Sci U S A, 1977 Apr, 74(4), 1468 - 72
Ribosome structure: localization of N6,N6-dimethyladenosine by electron microscopy of a ribosome-antibody complex; Politz SM et al.; Antibodies to the minor nucleoside N6,N6-dimethyladenosine have been used to map a unique location of the nucleoside in the small subunit of the Escherichia coli ribosome . Antibodies were induced in rabbits by a nucleoside-bovine albumin conjugate and shown to be highly specific for the dimethyladenosine hapten . The antibodies were shown to interact with 30S ribosomal subunits from strain PR7, but not with subunits from its mutant strain TPR201, which is resistant to kasugamycin and lacks the two successive residues of dimethyladenosine normally found near the 3'-end of E . coli 16S ribosomal RNA . Electron micrographs of strain PR7 subunits, crosslinked by single IgG molecules, show a single binding site on the surface of the ribosome . This binding site is consistent with observations relating the 3'-end of the ribosomal RNA, binding of initiation factor IF-3 and messenger RNA, and mapping of specific ribosomal proteins.

Proc Natl Acad Sci U S A, 1977 Apr, 74(4), 1440 - 4
Synthesis and processing of an Escherichia coli alkaline phosphatase precursor in vitro; Inouye H et al.; Alkaline phosphatase {orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1} of E . coli was synthesized in a cell-free system, and the size of the direct translation product was analyzed . The product has a higher molecular weight than the mature alkaline phosphatase found in the periplasm . The direct translation product can be processed to the mature size by an E . coli membrane fraction; the processing activity copurifies with the outer-membrane fraction . The presumed precursor can dimerize to form active enzyme without being processed, and the resultant enzyme appears to be more hydrophobic than the mature enzyme . These findings are discussed in connection with the "signal hypothesis" proposed for the excretion of proteins across membranes.

Proc Natl Acad Sci U S A, 1977 Apr, 74(4), 1407 - 11
Rotational relaxation of 70S ribosomes by a depolarization method using triplet probes; Lavalette D et al.; Rotational relaxation on the microsecond time scale has been followed by a depolarization technique using the properties of the long-lived triplet state of covalently bound labels . Two triplet probes, which efficiently bind to ribosomal proteins, are described . The rotational correlation time of 70S ribosomes of Escherichia coli has been measured . The average hydrodynamic radius of the functionally active 70S particle in solution has been estimated to 147 A . A concentration dependence of the correlation time has been observed, which may result from an association of the 70S ribosomes to form 100S dimers.

Proc Natl Acad Sci U S A, 1977 Apr, 74(4), 1393 - 7
Differential utilization of leucyl-tRNAs by Escherichia coli; Holmes WM et al.; The utilization of the isoaccepting species of leucyl-tRNA in protein synthesis has been examined in E . coli . Two minor leucyl-tRNA species, isoacceptors tRNA3Leu and tRNA4Leu, are the predominant species found bound to ribosomes during exponential growth in minimal medium . In rich medium, an increased proportion of tRNA1Leu is found bound to ribosomes . One species, tRNA5Leu, is always absent from ribosomes . In a mutant strain in which normal tRNA3Leu and tRNA5Leu are reduced or absent, tRNA1Leu is a major ribosome-bound species in minimal medium . Protein synthesis in vitro with RNA from E . coli further suggests that tRNA5Leu is rarely used for total protein synthesis; however, this species is active with RNA from MS2 phage . We propose that tRNA1Leu can substitute for tRNA3Leu under rapid growth conditions, and that tRNA5Leu is used minimally in total protein synthesis.

Proc Natl Acad Sci U S A, 1977 Apr, 74(4), 1388 - 92
Biochemical characterization of a mutant lipoprotein of Escherichia coli; Wu HC et al.; A lipoprotein mutant of E . coli K-12 has been characterized . The mutant lipoprotein was found to differ from the wild-type lipoprotein in the following respects: (i) it is present in an appreciable amount in the soluble fraction (275,000 X g supernatant); (ii) it lacks the covalently-linked diglyceride; (iii) it contains an unmodified cysteine which can be carboxymethylated in vitro; (iv) it undergoes dimerization and the dimer can be converted into monomeric form by reduction with 2-mercaptoethanol; (v) both the monomeric form and especially the dimeric form of the mutant lipoprotein migrate more slowly than the corresponding forms of wild-type lipoprotein in sodium dodecyl sulfate/urea polyacrylamide gel electrophoresis; and (vi) the mutant lipoprotein is not assembled into the murein sacculi, and this results in a greatly reduced amount of bound-form lipoprotein in the mutant . These data strongly suggest that the mutation has affected the primary structure of lipoprotein, in such a way that it is not modified normally, leading to the production of a structurally-altered lipoprotein deficient in covalently-linked lipid as well as a defective assembly of the altered lipoprotein into the rigid layer of the cell envelope.

J Infect Dis, 1977 Apr, 135(4), 531 - 9
Colonization of porcine small intestine by Escherichia coli: colonization and adhesion factors of pig enteropathogens that lack K88; Isaacson RE et al.; The colonizing and adhesive attributes of enterotoxigenic acapsular and/or nonpiliated mutants from K88-negative enteropathogenic Escherichia coli strains were compared with their capsulated and piliated parents (parents were piliated when grown in vitro and in vivo) . Acapsular, nonpiliated mutants from three different colonizing strains of enteropathogenic E . coli lost their ability to colonize the ileum of newborn pigs . Acapsular, piliated and capsular, nonpiliated mutants were derived from one of the parental strains (987), and both mutants lacked the ability to colonize the ileum of pigs . The only mutants available from a fourth strain (431) were acapsular and piliated, and they colonized as well as their parents . These data indicate that both capsule and pili are involved in colonization by strain 987 . In contrast, capsule is not required for colonization by strain 431, but pili may be.

J Bacteriol, 1977 Apr, 130(1), 57 - 61
Unstable mutations that relieve catabolite repression of tryptophanase synthesis by Escherichia coli; Yudkin MD; From strains of Escherichia coli that carry deletions of the trp region, five different mutants were isolated that were capable of synthesizing tryptophanase at unusually high rates in conditions of severe catabolite repression . Notwithstanding the comparative insensitivity to catabolite repression, the rates of tryptophanase synthesis in the mutants were greatly diminished by the introduction of a defective gene for adenyl cyclase . Each of the mutants segregated variants of the parental type . The results of genetic analysis appear to be consistent with the mutants arose by duplication of the tryptophanase gene.

J Bacteriol, 1977 Apr, 130(1), 566 - 8
Escherichia coli K-12 mutant with alternate requirements for vitamin B6 or branched-chain amino acids and lacking transaminase C activity; Falkinham JO 3rd; An Escherichia coli K-12 auxotrophic derivative that grows if supplemented with pyridoxine, isoleucine, leucine, or alanine is described.

J Bacteriol, 1977 Apr, 130(1), 506 - 11
Genetic complementation analysis of Escherichia coli type 1 somatic pilus mutants; Swaney LM et al.; A genetic complementation analysis of 75 stable nonpiliated mutants of a type 1 piliated strain of Escherichia coli K-12, AW405, was performed . Strains containing pairs of pil mutations were constructed by the infectious transfer of an F101 plasmid containing one pil mutation into E . coli K-12 AW 405 containing another pil mutation . The presence or absence of type 1 pili on the merodiploid strains was determined by agglutination with type 1 pilus antiserum . All 75 mutants fell into one of four complementation groups . The pattern of complementation defined three cistrons involved in pilus formation, pilA, pilB, and pilC . The fourth complementation group was composed of a large number of mutants defective in both pilA and pilB functions.

J Bacteriol, 1977 Apr, 130(1), 495 - 505
Isolation and characterization of Escherichia coli phase variants and mutants deficient in type 1 pilus production; Swaney LM et al.; Type 1 pili of Escherichia coli are the prototype of the somatic class of pili found on many strains of bacteria . As a first step in the genetic analysis of type 1 piliation, an extensive series of nonpiliated derivatives of E . coli K-12 strain AW405, was characterized to produce attached or free pili when examined in the antiserum or appeared to produce attached or free pili when examined in the electron microscope . The derivatives fell into two classes; phase variants and mutants . Phase variants that formed colonies of two distinctive types, one associated with a predominantly piliated (P+), and the other associated with a nonpiliated (P-) phase, were obtained . Each phase could give rise to the other at a relatively high rate, which was greater in the P- to P+ direction during culture in unshaken liquid medium . In addition, 77 Pil- mutants were selected on the basis of a subtle difference in colonial morphology . The mutants reverted, if at all, at a much lower rate than that of the P- to P+ change . The stability of Pil- derivatives grown in unshaken liquid medium was used as a criterion for distinguishing between phase variants and mutants, Phase variation also effected colonial morphology and chemotactic swarming . These properties did not directly depend upon piliation since Pil- mutants were only slightly altered in colonial form and unaltered in chemotactic swarming . Piliation of Pil+ bacteria was quantitatively affected by growth conditions.

J Bacteriol, 1977 Apr, 130(1), 441 - 4
Mapping of the aspartate and aromatic amino acid aminotransferase genes tyrB and aspC; Gelfand DH et al.; Co-transduction experiments using P1-mediated reciprocal and three-factor crosses have been used to map two mutations affecting the aspartate and aromatic amino acid aminotransferases of Escherichia coli . tyrB-, which inactivates the tyrosine-repressible component of these activities is co-transducible with metA and malB; the gene order is metA-malB-tyrB . aspC-, which inactivates the nonrepressible aminotransferase with high activity for aspartate, maps between and is co-transducible with serC and pyrD.

J Bacteriol, 1977 Apr, 130(1), 4 - 10
Biochemical and regulatory properties of Escherichia coli K-12 hisT mutants; Bruni CB et al.; Escherichia coli K-12 hisT mutants were isolated, and their properties were studied . These mutants are derepressed for the histidine operon, map close to the purF locus at about 49.5 min on the E . coli linkage map, and lack pseudouridylate synthetase activity . The defect in this enzyme leads to the absence of pseudouridines in the anticodon loop of several transfer ribonucleic acid species, as evidenced by the altered elution profile on reversed-phase chromatography and resistance to amino acid analogues . Finally, the hisT mutants studied have a reduced growth rate that appears to be linked to hisT, although it is not known whether it is due to the same mutation . The normal generation time can be restored by supplementing the medium with adenine, uracil, and isoleucine.

J Bacteriol, 1977 Apr, 130(1), 384 - 92
Escherichia coli transport mutants lacking binding protein and other components of the branched-chain amino acid transport systems; Anderson JJ et al.; Further evidence on the role of binding proteins in branched-chain amino acid transport in Escherichia coli was obtained by selecting mutants with altered expression of the binding proteins . The mutator phage Mu was used to induce E . coli L-valine-resistant mutants defective in branched-chain amino acid transport . By making use of mild selective conditions and strain backgrounds with derepressed high-affinity, binding protein-mediated transport systems, we were able to isolate a new class of transport mutants defective in these systems . Mutant strains AE84084 (livK::Mucts) and AE840102 (livJ) were found to be defective in leucine-specific and LIV binding proteins, respectively, by transport assay, in vitro binding activity, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Mutant strain AE4107 (livH::Mu), although lacking high-affinity, branched-chain amino acid transport, retained functional binding proteins and therefore evidently codes for an additional component of high-affinity transport . The livH, livJ, and livK mutations were mapped by transduction and shown to be closely linked to each other in the malT region (min 74) of the E . coli genetic map.

J Bacteriol, 1977 Apr, 130(1), 329 - 38
Immunocytological investigation of protein synthesis in Escherichia coli; MacAlister TJ et al.; Ferritin-conjugated specific antibodies have been used to localize beta-galactosidase and both the monomer and active dimer of alkaline phosphatase in frozen thin sections of cells of Escherichia coli O8 strain F515 . The even distribution of the ferritin marker throughout cells that had been induced for beta-galactosidase synthesis, frozen, sectioned, and exposed to ferritin-anti-beta-galactosidase conjugate showed that this enzyme was present throughout the cytoplasm of these cells . Frozen thin sections of cells that had been derepressed for the synthesis of alkaline phosphatase were exposed to both ferritin-anti-alkaline phosphatase monomer and ferritin-anti-alkaline phosphatase dimer conjugates, and the ferritin markers showed a peripheral distribution of both the monomer and the dimer of this enzyme . This indicates that alkaline phosphatase is present only in the peripheral regions of the cell and argues against the existence of a cytoplasmic pool of inactive monomers of this enzyme . This peripheral location of both the monomers and dimers of alkaline phosphatase supports the developing concensus that this enzyme is, like other wall-associated enzymes, synthesized in association with the cytoplasmic membrane and vectorially transported to the periplasmic area, where it assumes its tertiary and quaternary structure and acquires its enzymatic activity.

J Bacteriol, 1977 Apr, 130(1), 318 - 28
Cell surface-localized alkaline phosphatase of Escherichia coli as visualized by reaction product deposition and ferritin-labeled antibodies; MacaAlister TJ et al.; When cells of a wild-type Eschericia coli O8 strain bearing a complete lipopolysaccharide were incubated for alkaline phosphatase reaction product and examined by electron microscopy, the depostion of lead salts was to be observed primarily within the periplasmic space . A similar treatment of cells derived from this strain, which bears a highly abbreviated lipopolysaccharide, showed a mixed cell surface and periplasmic localization of reaction product, suggesting a surface association of a portion of the enzyme . To further explore this possibility, ferritin-antibody conjugates against the active enzyme and its irreversibly dissociated subunits were prepared and allowed to react with cells of both strains . The results obtained from these experiments revealed the presence of both the active enzyme and inactive subunits of the enzyme at the cell surface of the mutant strain . The evidence obtained offers further proof of the validity of the reaction product deposition technique and indicates that alkaline phosphatase may be associated with some component of the outer membrane in this organism . The observation of enzyme subunits at the cell surface further suggests that an association of these subunits with structural components of the cell envelope may provide a locus at which they may dimerize to form active enzyme.

J Bacteriol, 1977 Apr, 130(1), 303 - 11
Suppression of an Escherichia coli dnaA mutation by the integrated R factor R100.1: generation of small plasmids after integration; Chandler M et al.; We have observed that integration of the R plasmid R100.1 into the chromosome of Escherichia coli is associated with the formation of small, covalently closed circular elements . Contour length measurements, partial denaturation mapping, and analysis of the deoxyribonucleic acid fragments produced by digestion of one of these, pLC1, with the restriction endonuclease EcoRI indicate that it is the r-determinant element of R100.1.

J Bacteriol, 1977 Apr, 130(1), 212 - 22
Physiological regulation of a decontrolled lac operon; Wanner BL et al.; The expression of the lac operon was studied under a variety of growth conditions in induced and in constitutive cells of Escherichia coli that carried different catabolite-insensitive lac promoters . Use of such "decontrolled" lac operons permitted a study of the expression of an operon that was presumably subject only to passive control . Since the use of toluenized cells was demonstrated not to be completely reliable, all enzyme assays were performed on sonic supernatant fluids . The cells contained different catabolite-insensitive promoters, which included the L1 and UV5 lac promoters, as well as others isolated in this study . There were three major observations . First, small but real carbon source effects were seen . Second, there was only a small change in beta-galactosidase specific activity with changes in the growth rate . This result implies a limited transcription and/or translation capacity within the cell . Third, at rapid growth rates, most promoters exhibited a decreased expression . The UV5 promoter, which was the "strongest" promoter, was an exception . A mechanism to explain this promoter-dependent control is discussed.

J Bacteriol, 1977 Apr, 130(1), 187 - 91
Repair of hydrogen peroxide-induced single-strand breaks in Escherichia coli deoxyribonucleic acid; Ananthaswamy HN et al.; Near-ultraviolet (300 to 400 nm) irradiation of L-tryptophan yielded H2O2 (a toxic photoproduct) that was selectively lethal for rec and polA1 Escherichia coli mutants . H2O2 treatment of cells resulted in the induction of single-strand deoxyribonucleic acid breaks . These breaks were repaired to only a small extent in polA1, recA recB, and recA mutants, but were efficiently repaired in wild-type strains . We conclude that H2O2 deoxyribonucleic acid lesions require both the polA+ and recA+ pathways for repair.

J Bacteriol, 1977 Apr, 130(1), 167 - 72
Method for the isolation of Escherichia coli mutants with enhanced recombination between chromosomal duplications; Konrad EB; A method is described for the isolation of Escherichia coli mutants that show increased recombination between a pair of chromosomal duplications . These "hyper-rec" mutants display a variety of secondary phenotypes . I have isolated a large number of hyper-rec mutants and found them useful in screening for mutants that accumulate labeled DNA fragments after short pulses with {3H}thymidine . The mutants so recovered include ones that are defective in deoxyribonucleic acid ligase, deoxyribonucleic acid polymerase I and its associated 5' yields 3' exonuclease, and a group of mutants, dnaS, that accumulate abnormally short Okazaki fragments . Evidence is presented that suggests that the lac-att80 segment of the chromosome cannot be inverted.

J Bacteriol, 1977 Apr, 130(1), 136 - 43
Regulation of PRPP and nucleoside tri and tetraphosphate pools in Escherichia coli under conditions of nitrogen starvation; Villadsen IS et al.; The ribonucleoside triphosphate, deoxyribonucleoside triphosphate, 3' -diphosphate guanosine 5' -diphosphate (ppGpp), and 5-phosphoribosyl 1-pyrophosphate (PRPP) pools in Escherichia coli B were determined by thin-layer chromatography during changing conditions to ammonium starvation . The intracellular concentrations of all nucleotides were found to change in a well-defined order several minutes before andy observed change in the optical density of the culture . The levels of purine nucleoside triphosphates (adenosine 5' -triphosphate {CTP}, dCTP) and uridine nucleotides (uridine 5' -triphosphate, deoxythymidine 5'-triphosphate) . The deoxyribonucleotides thus behaved as the ribonucleotides . The levels of ppGpp increased 11-fold after the decrease in uridine nucleotides, when the accumulation of stable ribonucleic acid (RNA) stopped . The level of the nucleotide pool did not stabilize until 30 min after the change in optical density . The pool of dGTP dropped concomitantly with the pool of CTP . The nucleotide precursor PRPP exhibited a transient increase, wtih maximum value of four times the exponential levels at the onset of starvation . Apparently the cell adjusts early to starvation by reducing either the phosphorylating activity or the nucleotide biosynthetic activity . As in other downshift systems, the accumulation of stable RNA stopped before the break in optical density and before the stop in protein accumulation . Cell divisions were quite insensitive to the control mechanisms operating on RNA and protein accumulation under ammonium starvation, since the cells continued to divide for 21 min without any net accumulation of RNA.

J Bacteriol, 1977 Apr, 130(1), 118 - 27
Medium-dependent variation of deoxyribonucleic acid segregation in Escherichia coli; Cooper S et al.; The degree to which deoxyribonucleic acid segregates nonrandomly has been investigated for Escherichia coli B/r growing in different media . The degree of nonrandom segregation observed is dependent on the medium, with segregation becoming less random as the growth rate decreases . This indicates that there must be some varying probabilistic component to the segregation process . A probabilistic modification of the Pierucci-Zuchowski model is proposed as well as a probabilistic model, in which it is proposed that deoxyribonucleic acid strands segregate, with a probability greater than 0.5, in the same direction (toward the same pole) as at the previous cell division.

J Bacteriol, 1977 Apr, 130(1), 114 - 7
relA gene control of the synthesis of lipid A fatty acyl moieties; Spencer A et al.; The incorporation of {14C}acetate into the fatty acid moieties of lipid A was measured during amino acid starvation of rel+ and relA strains of Escherichia coli K-12 . The synthesis of the beta-hydroxymyristate and other fatty acid moieties was inhibited two- to fourfold in rel+ strains, whereas no inhibition was observed in relA strains . The fatty acid compositions of the phospholipids synthesized after amino acid starvation or rel+ and relA strains were also determined.

J Bacteriol, 1977 Apr, 130(1), 100 - 6
Pseudoreversion of lactose operator-constitutive mutants; Norwood WI et al.; A set of pseudorevertants of lactose operator-constitutive (lacOc) mutant has been obtained . Analysis of a subset of these pseudorevertants indicates that, in some cases, the secondary mutation alters the lactose repressor (lacl gene product), whereas in others it seems to have occurred in the lactose operator (lacO) itself . Of the lacl gene mutations, the lacl8 mutation, already known to suppress all lacOc mutations nonspecifically, was recovered by a selection technique developed for this study . However, two additional lacl gene mutants were selected which appear to suppress lacOc sequences in a more-or-less specific fashion; repressor interaction with some operator sequences is facilitated, whereas the binding with lacO+ and others is attenuated concomitantly.

Eur J Biochem, 1977 Apr 1, 74(2), 253 - 61
A nuclear elongation factor of transcription from Physarum polycephalum in vitro; Ernst GH et al.; Homogenates of Physarum plasmodia contain a factor which stimulates UMP incorporation on native DNA by solubilized homologous RNA polymerases in vitro . The factor is a heat-sensitive protein and has been located in nuclei . It does not alter the template activity of DNA nor the initiation frequency of transcription . The factor interacts with free or bound RNA polymerase molecules (only 37 degrees C and at low ionic strength) and yields larger transcripts in vitro . The level of the factor in vitro fluctuates: it is gradually reduced during spherulation and reaches its maximum in mid S phase of the cell cycle of Physarum.

Can J Biochem, 1977 Apr, 55(4), 267 - 81
The Ayerst Award Lecture 1976 . Novel factors in protein biosynthesis; Ganoza MC; Comparison of nucleotide sequences surrounding the initiation sites of a number of mRNAs reveals few common features . These may be the presence of in- or out-of-phase nonsense codons and (or) polypurine bases complementary to the 16S RNA of the 30S subunit of ribosomes . Since the bases which precede or follow an initiation site vary in length and composition we have examined whether they play a role as spacers between cistrons or whether they have an active function in the termination and initiation of translation . In vitro we have observed that some sequences 5' terminal to AUG are preferred over others in forming an initiation complex . The same bases have much less effect when present at the 3' terminal end of an AUG codon . When the 5' terminal codon is the termination codon UAA, absolutely no initiation complex can be detected . This suggests that spacing may be needed between a stop and a start codon . Conversely, the hexamer AUGUAA failed to elicit chain termination . This was so in systems that terminated when free UAA was added or when a sense triplet was present between the initiation and termination triplets . These results suggest that ribosomes may recognize the stop triplet . Hence ribosomes may not obey simple A and P site models in the termination reaction.

J Med Chem, 1977 Apr, 20(4), 583 - 4
Correction of prior estimate of the biological activity of an N-trifluoroacetyl analogue of D-threo-chloramphenicol relative to chloramphenicol; Garrett ER; The inhibitory rate constants, k, for the inhibition of the rate of Escherichia coli generation at various concentrations, C, of chloramphenicol nad its N-trifluoroacetyl analogue were determined in vitro in accordance with kapp = k0 - kC where kapp is the apparent first-order generation rate constant at a drug concentration C and k0 is that constant in the absence of drug . The activity of the analogue was one-tenth that of chloramphenicol in contrast to a previously reported value.

J Med Chem, 1977 Apr, 20(4), 463 - 9
A manual method for applying the Hansch approach to drug design; Topliss JG; A procedure is described in which an initial small group of compounds is selected, tested, and ordered according to potency . The potency order in the group is then compared to the tabulated potency order calculated for various parameter dependencies relating to hydrophobic, electronic, and steric effects . From this activity pattern analysis the probable operative parameters can be deduced and a new substituent selection made for the synthesis of potentially more potent analogues . Application of the method is illustrated with a series of examples . It differs from a previously described decision tree, single compound stepwise approach in that it involves the batchwise analysis of small groups of compounds, usually the preferred procedure for logistical reasons if the compounds are relatively easy to synthesize.

J Lab Clin Med, 1977 Apr, 89(4), 910 - 8
An improved in vitro pyrogen test: to detect picograms of endotoxin contamination in intravenous fluids using limulus amoebocyte lysate; Nandan R et al.; A method for in vitro pyrogen testing using Limulus amoebocyte lysate (LAL) has been described . The method is based upon the measurement of endotoxin-precipitable protein and can be used to measure picogram quantities equivalent to E . coli endotoxin in unknown solutions . When increasing concentrations of E . coli endotoxin are added to a constant amount of LAL and the reaction is allowed to proceed to completion, there is a proportional increase in the protein precipitated by endotoxin . Therefore, by measuring the amount of protein precipitated from LAL, it is possible to determine the equivalent E . coli endotoxin concentration in unknown solutions, when samples of the unknowns are run simultaneously with E . coli endotoxin standards and negative controls . The endotoxin proportional precipitation of protein occurs in reaction mixture showing gelation as well as in reaction mixture where the levels of endotoxin are lower than required for gelation . Determination of precipitated protein provides greater sensitivity for endotoxin detection than the gelation methods currently in use.

J Hyg (Lond), 1977 Apr, 78(2), 235 - 42
Bacteriostasis of Escherichia coli by milk . II . Effect of bicarbonate and transferrin on the activity of infant feeds; Dolby JM et al.; Fresh human and bovine milk are bacteriostatic in vitro for only some (milk-sensitive) strains of E . coli . The addition of bicarbonate to the test system potentiates the bacteriostasis so that otherwise milk-resistant strains are inhibited . By titration of the bicarbonate in the milk, it is possible to determine the minimum concentration that will activate milk aganinst a milk resistant strain but be ineffective in boiled milk, i.e . it potentiates a heat-labile system in milk and does not merely exert a direct toxic effect . This concentration is lower for human milk than for cow's milk and can be reduced even further by the addition of more iron-binding protein . Lactoferrin and bicarbonate may be present in the gut of the newborn . In an attempt to imitate conditions in the infant gut, we therefore reinvestigated, in vitro and in the presence of added bicarbonate and transferrin, the bacteriostatic activity against E . coli of fresh breast-milk, commercial bottle-milk, and mixtures of these as fed to infants in the study . The results, and information about events in vivo deduced from the ratio of milk-sensitive to milk-resistant strains of E . coli isolated from babies' stool suggest that neonatal intestinal secretions may contribute to the bacteriostatic activity of their feeds so that (1) in fully breastfed babies all strains of E . coli are inhibited to the same extent; there is no selection on the basis of milk sensitivity and equal numbers of strains resistant and sensitive to milk are found in the stools; (2) in fully bottle-fed babies E . coli is not inhibited since the milk is non-bacteriostatic and again there is no selection; (3) in babies fed at the breast but bottle-milk supplemented, only milk-sensitive strains are inhibited; milk-resistant strains are not, and preferentially colonize the large intestines.

Gastroenterology, 1977 Apr, 72(4 Pt 1), 630 - 3
Effects of diet on glucaric acid concentration in bile and the formation of calcium bilirubinate gallstones; Matsushiro T et al.; The authors reported previously that beta-glucuronidase in bile, especially during biliary infection with Escherichia coli, plays a substantial role in producing cium bilirubinate gallstones . In the present study, bile levels of glucaro-1:4-lactone (measured as glucaric acid) the leading inhibitor of beta-glucuronidase, were measured in both man and in rats fed high, medium, and low protein-fat diets . Glucaric acid and total bilirubin in bile correlated well in human controls but not in gallstone patients . In animal experiments, the levels of these substances in bile were high in rats on high protein-high fat and low in those on low protein-low diets . These data suggest that when bile is infected with E . coli, calcium bilirubinate gallstones seemed to form more easily in patients on low protein-low fat diet than in those consuming food rich in protein and fat . On the other hand, the ratio of lecithin to cholesterol was higher in low protein-low fat rats than in high protein-high fat rats, suggesting that cholesterol gallstones were more likely to form on the latter diet . The animal, clinical, epidemiological, and dietary data are consistent with the known trend to a decreased incidence of calcium bilirubinate and an increased incidence of cholesterol gallstones in Japan.

Gann, 1977 Apr, 68(2), 175 - 82
Suggestive evidence for relationship between sensitivity and repair capability in rat ascites hepatoma cells treated with the antitumor agent, 1-(gamma-chloropropyl)-2-chloromethylpiperidine hydrobromide; Fukuda S et al.; Antitumor activity of 1-(gamma-chloropropyl)-2-chloromethylpiperidine hydrobromide (CAP-2) was studied in vivo and in vitro, using various rat ascites hepatoma cell lines . Among eight ascites hepatoma cell lines, AH-13 was extremely sensitive both to in vivo antitumor and to in vitro lethal action of the agent, whereas AH-44 was resistant in both cases . The sensitivity of ascites hepatoma cell lines to CAP-2, nitrogen mustard N-oxide, 4-nitroquinoline 1-oxide, and ultraviolet ray in vitro was widely different but their relative sensitivities were very similar against these agents . For all the agents, AH-13 was inactivated very rapidly and AH-109A moderately, whereas AH-44 was relatively resistant . These results indicate that the sensitivity of the cells to CAP-2 may be closely related to their repair-capability of damaged DNA . Similar experiments using various DNA repair-deficient mutants of Escherichia coli B strain demonstrated that the repair-deficient mutants were several times more sensitive to CAP-2 than the wild type strain . From these results, it may be concluded that CAP-2 induces DNA lesions repairable by the same repair mechanisms that work on pyrimidine dimers.

Nucleic Acids Res, 1977 Apr, 4(4), 1001 - 14
Nucleotide sequence of the restriction fragment Hind F-Eco RI2 of SV40 DNA; Contreras R et al.; The nucleotide sequence of the SV40 genome region between the Hind K fragment and the Eco RI cleavage site has been determined by a combination of three different approaches : analysis of RNA products obtained by transcription with Escherichia coli DNA dependent RNA polymerase, partial degradations with snake venom exonuclease and base-specific chemical degradation of 5'-terminal labeled restriction fragments . This nucleotide sequence shows only one open reading frame and allows the deduction of a small segment of the amino acid sequence of VP1, the major structural protein.

Proc Natl Acad Sci U S A, 1977 Apr, 74(4), 1412 - 6
Gene cloning for the isolation of enzymes of membrane lipid synthesis: phosphatidylserine synthase overproduction in Escherichia coli; Raetz CR et al.; We have screened a bank of 2000 E . coli strains carrying hybrid ColE1 plasmids {Clarke, L . & Carbon, J . (1976) Cell 9, 91-99} for those that correct the temperature sensitivity of a mutant in CDP-1,2-diacyl sn-glycerol:L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthase) . Two hybrid plasmids of this kind (pLC34-44 and pLC34-46) were identified and characterized . Strains carrying these plasmids overproduce the synthase by 6- to 15-fold, as demonstrated by assays of extracts and purification to homogeneity of the overproduced enzyme . The overproduced synthase, like the wild-type enzyme, is found associated predominately with the ribosomal fraction of crude cell extracts . Because the membrane phospholipid composition of these overproducers is not greatly altered, we suggest that the synthase is normally present in excess.

J Gen Virol, 1977 Apr, 35(1), 99 - 106
Requirement of the bacteriopahge T7 0.7 gene for phage growth in the presence of the Col 1b factor; Gomez B et al.; A variety of colicinogenic factors were examined for their effect on the growth of phages related to T7 . Col B1 and Col B4 inhibited the growth of every phage tested . Col B2, however, did not interfere with T3 and H . Col E2 interfered only with growth of T3, while Col Ib produced abortive infection with some T7 mutants . The abortive infection associated with Col Ib seems to be correlated with the absence of the virus phosphokinase; mixed infection with T7 wild type and a phosphokinase deficient mutant was productive but the phage yield was depressed . The abortively infected cells lysed.

J Bacteriol, 1977 Apr, 130(1), 563 - 5
Differential binding of cyclic adenosine 3' ,5'-monophosphate to the cyclic adenosine 3' ,5'-monophosphate receptor protein in Escherichia coli; Danley DE et al.; Binding of cyclic adenosine 3' ,5'-monophosphate (cAMP) by the cAMP receptor protein in crude cell-free extracts of Escherichia coli was characterized . When cell were grown in glucose, binding was inhibited 50% relative to extracts from cells grown with succinate as carbon source . This inhibition could be relieved by dialysis.

J Bacteriol, 1977 Apr, 130(1), 552 - 7
Biochemical characterization of an Escherichia coli hisT strain; Lawther RP et al.; An Escherichia coli hisT strain was characterized biochemically and shown to contain altered transfer ribonucleic acid and to be altered in the regulation of amino acid biosynthesis.

J Biochem (Tokyo), 1977 Apr, 81(4), 1071 - 7
Membrane-bound adenosine triphosphatase of Escherichia coli . III . Effects of sodium azide on the enzyme functions; Kobayashi H et al.; 1) Sodium azide and diphenyl phosphorazidate (DPPA) inhibited purified membrane-bound ATPase {coupling factor of oxidative phosphorylation; EC 3.6.1.3} of Escherichia coli non-competitively with Ki values of 39 and 51 micrometer, respectively . 2) Sodium azide and DPPA inhibited the activity of ATPase bound to the membrane as effectively as that of the purified enzyme . 3) The effects of sodium azide on succinate-dependent ATP synthesis, Pi-ATP exchange, and ATP hydrolysis reactions by the membrane vesicles were compared under the same conditions . At concentrations below 1.0 mM, sodium azide inhibited ATP hydrolysis, but Pi-ATP exchange and ATP synthesis were almost unaffected . At 10 mM sodium azide, both Pi-ATP exchange and ATP synthesis reactions were completely inhibited, probably because at this concentration, sodium azide acted as a proton-conducting uncoupler.

J Bacteriol, 1977 Apr, 130(1), 26 - 36
Uptake of ferrienterochelin by Escherichia coli: energy dependent stage of uptake; Pugsley AP et al.; The uptake of the siderophore-iron complex ferrienterochelin was found to be strongly dependent upon an energized membrane state, as demonstrated by its sensitivity to dinitrophenol, azide, and cyanide . Ferrienterochelin uptake may also be dependent upon phosphate bond energy, as indicated by sensitivity to arsenate and iodoacetic acid . Although the adenosine triphosphatase does not appear to be involved in this energy coupling mechanism, ferrienterochelin uptake was shown to be less dependent upon phosphate bond energy than was glutamine uptake . Sensitivity of ferrienterochelin uptake to osmotic shock was shown to be due to the release of a ferrienterochelin binding compound located in the outer membrane of the cells and probably identical to the colicin B receptor protein.

J Virol, 1977 Apr, 22(1), 118 - 29
New procedure for the direct analysis of in vitro reverse transcription of Rous sarcoma virus RNA; Darlix JL et al.; Based on the observation that in vitro transcription of Rous sarcoma virus (RSV) RNA by avian myeloblastosis virus DNA polymerase renders the RNA PROGRESSIVELY MORE SENSITIVE TO Escherichia coli RNase H digestion, a new procedure for the in situ analysis of this process has been developed . In vitro transcription products of 32P-labeled RSV RNA are first treated with RNase H, the resistant fraction is then digested to completion with RNase T1, and the oligonucleotides are analyzed by a fingerprint technique . By using the established order of these oligonucleotides along the RNA molecule, a comparison of the yields of each oligonucleotide, before and after transcription, allows qualitative and quantitative in situ analyses of the transcription process . Using this new procedure, we find that upon transcription of purified RSV RNA, DNA synthesis occurs mainly at three sites, one near the 5' end and two near the center of the subunit RNA molecule, and that most of these RNA molecules are competent templates for limited transcription at these specific sites . We also show that purified RSV 70S RNA contains a low-molecular-weight DNA hybridized to a nucleotide sequence near the center of the subunit molecule . Furthermore , we find that the low-molecular-weight nucleic acid fraction extracted from purified RSV virions contains DNA that can hybridize to RSV 70S RNA and that the virion DNA in such hybrids can function as a primer for an extensive in vitro reverse transcription.

J Bacteriol, 1977 Apr, 130(1), 107 - 13
Regulation of ribonucleoside diphosphate reductase synthesis in Escherichia coli: increased enzyme synthesis as a result of inhibition of deoxyribonucleic acid synthesis; Filpula D et al.; Inhibition of deoxyribonucleic acid (DNA) synthesis in Escherichia coli by chemical inhibitors or by shifting cultures of temperature-sensitive elongation (dnaE and dnaB) or initiation (dnaA) mutants to nonpermissive conditions led to greatly increased synthesis of the enzyme ribonucleoside diphosphate reductase, which catalyzes the first reaction unique to the pathway leading to DNA replication . In contrast to the Gudas and Pardee proposed model for control of the synthesis of DNA repair enzymes, in which both DNA inhibition and DNA degradation are involved, DNA synthesis inhibition in recA, recB, recC, or lex strains results in increased synthesis of ribonucleotide reductase, which suggests that DNA degradation is not required . We propose that inhibition of DNA synthesis causes a cell to accumulate an unknown compound that stimulates the initiation of a new round of DNA replication, and that this same signal is used to induce ribonucleotide reductase synthesis.

J Bacteriol, 1977 Apr, 130(1), 429 - 40
Escherichia coli mutants deficient in the aspartate and aromatic amino acid aminotransferases; Gelfand DH et al.; Two new mutations are described which, together, eliminate essentially all the aminotransferase activity required for de novo biosynthesis of tyrosine, phenylalanine, and aspartic acid in a K-12 strain of Escherichia coli . One mutation, designated tyrB, lies at about 80 min on the E . coli map and inactivates the "tyrosine-repressible" tyrosine/phenylalanine aminotransferase . The second mutation, aspC, maps at about 20 min and inactivates a nonrespressible aspartate aminotransferase that also has activity on the aromatic amino acids . In ilvE- strains, which lack the branched-chain amino acid aminotransferase, the presence of either the tyrosine-repressible aminotransferase or the aspartate aminotransferase is sufficient for growth in the absence of exogenous tyrosine, phenylalanine, or aspartate; the tyrosine-repressible enzyme is also active in leucine biosynthesis . The ilvE gene product alone can reverse a phenylalanine requirement . Biochemical studies on extracts of strains carrying combinations of these aminotransferase mutations confirm the existence of two distinct enzymes with overlapping specificities for the alpha-keto acid analogues of tyrosine, phenylalanine, and aspartate . These enzymes can be distinguished by electrophoretic mobilities, by kinetic parameters using various substrates, and by a difference in tyrosine repressibility . In extracts of an ilvE- tyrB- aspC- triple mutant, no aminotransferase activity for the alpha-keto acids of tyrosine, phenylalanine, or aspartate could be detected.

J Bacteriol, 1977 Apr, 130(1), 339 - 46
Cell envelope protection of alkaline phosphatase against acid denaturation in Escherichia coli; MacAlister TJ et al.; The effects of a highly acidic environment on the cell-associated alkaline phosphatase activities of a smooth and a rough strain of Escherichia coli O8 have been examined . The observation that cell-associated enzyme is denatured to a lesser degree than purified enzyme suggests that the association of the enzyme with the cell envelope affords it some degree of protection from potentially disruptive agents in the environment . The degree of protection afforded the enzyme from pH denaturation appears to be dependent upon the presence of a complete lipopolysaccharide in the outer membrane of these strains . An abbreviation of the chemical structure of this cell envelope component produces a change in the outer membrane, resulting in increased susceptibility of the cells to a battery of antibiotics and to lysozyme and in a small, but significant, change in the sensitivity of the cell envelope-associated alkaline phosphatase to the denaturing effect of an acidic environment.

Naunyn Schmiedebergs Arch Pharmacol, 1977 Mar 31, 297 Suppl 1, S95 - 7
Mediation of hyperthermia by prostaglandin E2: a new hypothesis; Splawinski JA; In rat hypothalamus prostaglandin (PG) E2, unlike PGF2 or arachidonic acid shared the site of hyperthermic action with E . coli endotoxin . The in vitro catabolism of PGE2 in the hypothalami of endotoxin-treated rats was significantly suppressed . It is proposed that endotoxin fever in rats is due to the inhibition of PGE2 breakdown in the hypothalamus.

Biochim Biophys Acta, 1977 Mar 29, 497(1), 1 - 13
Selectively deuterated amino acid analogues . Synthesis, incorporation into proteins and NMR properties; Matthews HR et al.; Selectively deuterated analogues of histidine, tyrosine, phenylalanine and tryptophan have been synthesized by chemical exchange . These analogues have been characterized by NMR spectrometry and used for growth of bacteria . Active lactose repressor protein has been isolated from cells grown on the deuterated amino acids, and denatured 1H and 2H NMR spectra have been determined for the protein.

Mol Gen Genet, 1977 Mar 28, 152(1), 71 - 6
Replication of prophage P1 during the cell cycle of Escherichia coli; Prentki P et al.; We have followed, by DNA-DNA hybridization, the variation in the number of copies of prophage P1 relative to two chromosomal markers when the doubling time of the host cells is modified by a change in carbon source . The ratio of P1/chromosome terminus undergoes a twofold decrease when the cell doubling time increases from 24 to 215 min, whereas the ratio of P1/chromosome origin increases 1.4 fold; both ratios tend towards unity at slow growth rates . This suggests that the replication of prophage P1 is not simultaneous with chromosome initiation or chromosome termination . The chromosome replication time is unaffected by the presence of P1, and remains constant over the range of doubling times studied, with a value of about 4o min . Following amino acid starvation, the P1/chromosome origin ratio increases from 0.7 to 0.9, suggesting that P1 retains the ability to replicate after chromosome initiation has stopped and in the absence of essential amino acids . The results are discussed with reference to similar studies done on F and R1.

Mol Gen Genet, 1977 Mar 28, 152(1), 37 - 41
The involvement of polynucleotide ligase in the repair of UV-induced DNA damage in Escherichia coli K-12 cells; Youngs DA et al.; The effect of the ligts-7 mutation on cell survival and the extent of DNA repair after UV (254 nm) irradiation was determined for wild-type and uvrB5 cells of E . coli K-12 at 30 degrees and 42 degrees C . At the restrictive temperature (42 degrees C) the ligts-7 mutation resulted in (i) a decrease in the extent of repair of DNA incision breaks arising during the excision repair process, and (ii) a decrease in the extent of post-replicational repair of gaps in newly-synthesized DNA . These deficiencies in DNA repair correlated with increases in cellular sensitivity to killing by UV radiation . Thus, DNA lagase plays an important role in vivo in both the excision and post-replicational repair processes.

Mol Gen Genet, 1977 Mar 28, 152(1), 29 - 36
Mutagenesis of lambda phage: 5-bromouracil and hydroxylamine; Hutchinson F et al.; Mutagenesis by 5-bromouracil of lambda phage to clear plaque formers does not depend on the recA function of the host E . coli cell or on the red function of the phage . Pretreatment of the host cells with ultraviolet light does not affect bromouracil mutagenesis of the adsorbed phage . Mutagenesis by hydroxlamine to clear plaque formers takes place at a high level in recA- host cells, and is not changed by preirradiation of of rec+ (wild type) hosts with ultraviolet light . Thus, bromouracil and hydroxylamine appear to mutate lambda phage by a process which differs from that responsible for ultraviolet mutagenesis . Two characteristics of bromouracil mutagenesis--the nonlinear dependence of the number of mutants on bromouracil incorporation, and a high frequency of heterozygotes--fit in with Rydberg's (1977) picture of bromouracil mutagenesis as a consequence of base mispairing, with mismatch repair removing the mutations at low incorporation of the analog.

Mol Gen Genet, 1977 Mar 28, 152(1), 109 - 10
Antimutagenic effects of spermine and guanosine in continuous cultures of Escherichia coli mutator strain mutH; Nestmann ER; In E . coli strain RH531 containing mutator gene mutH-, whose mechanism of enhanced mutability depends upon DNA replication, spermine and guanosine reduce the rates of mutation to T5R and to increased fitness.

Mol Gen Genet, 1977 Mar 28, 152(1), 105 - 8
Chromosomal location of gene governing the trehalose utilization in Escherichia coli K12; de Lares LB et al.; Several mutants unable to utilize trehalose were isolated from Escherichia coli, Their genetic analysis led to determine the following gene order on the chromosomal map: pur B-dad R-tre-hem a-trp . Furthermore, the tre gene belongs to the inversion of the trp chromosomal region between E . coli and S . typhimurium.

Vet Rec, 1977 Mar 26, 100(13), 259 - 62
Prophylactic use of hyperimmune serum in experimental colisepticaemia in calves; McBeath DG; Evidence is presented to demonstrate the efficacy of an intravenously administered polyvalent hyperimmune serum prepared from bovine serum, in the prevention of experimental colisepticaemia produced by oral challenge with E coli O78: K80(B) in colostrum deprived calves . It is suggested that the protection afforded by this hyperimmune serum is due to high levels of specific antibody demonstrable in the serum of calves post treatment and that protection is given in this instance by antibodies contained in the IgG class of immunoglobulins.

J Biol Chem, 1977 Mar 25, 252(6), 2154 - 7
The elongation factor Tu coded by the tufA gene of Escherichia coli K-12 is almost identical to that coded by the tufB gene; Furano AV; Radioactive elongation factor Tu coded by either the tufA or the tufB gene of Escherichia coli K-12 was isolated from cells incubated with a mixture of radioactive amino acids after infection with the defective lambda phage particles that carry either of these genes . Two-dimensional chromatographic analyses of tryptic digests of the tufB gene product revealed about 50 radioactive spots . These same spots plus an additional one were also found in tryptic digests of the tufA gene product . Furthermore, these peptide maps are qualitatively the same as those of the elongation factor Tu obtained from two separate isolates of uninfected E . coli K-12 or from rel+ and relA strains of E . coli B . Because the number of spots recovered is consistent with the number of trypsin-sensitive sites, these analyses indicate that the tufA and tufB genes have not significantly diverged from each other.

J Biol Chem, 1977 Mar 25, 252(6), 2072 - 6
Structure of peptide from active site region of Escherichia coli L-asparaginase; Peterson RG et al.; The L-asparagine analogue 5-diazo-4-oxo-L-{5-14C}norvaline binds irreversibly to the active site of Escherichia coli L-asparaginase . Conditions for optimal labeling in buffers containing 50% dimethylsulfoxide have been developed and kinetic parameters of the inactivation have been determined . After reduction, alkylation and subsequent degradation of the modified enzyme with alpha-chymotrypsin, the principal radioactive decapeptide of sequence Val-Gly-Ala-Met-Arg-Pro-Ser-Thr-Ser-Met was isolated . A second radioactive hexapeptide Arg-Pro-Ser-Thr-Ser-Met resulting from chymotryptic digestion of the decapeptide was also isolated . Evidence is presented for the attachment of the 5-diazo-4-oxo-L-norvaline residue to serine-9 in the decapeptide via an acid-labile linkage.

J Biol Chem, 1977 Mar 25, 252(6), 1957 - 64
Interactions of phospho- and dephosphosuccinyl coenzyme A synthetase with manganous ion and substrates . Studies of manganese complexes by NMR relaxation rates of water protons; Buttlaire DH et al.; The interactions of substrates with succinyl-CoA synthetase were investigated by measuring the enhancement of the longitudinal water proton relaxation rate (PRR) due to Mn(II) to the enzyme substrate complexes . The binding of Mn(II) to the enzyme was investigated by EPR . The effects of phosphorylating the enzyme on its interactions with Mn(II) and substrates were also examined . Mn(II) binds weakly to dephosphosuccinyl-CoA synthetase (E) at approximately four sites with a KD value of 0.14 mM, and the PRR enhancement of the complex, epsilonb, at 24.3 MHZ and 25 degree is 18.8 . The phosphoenzyme (E-P) binds Mn(II) more strongly at approximately four sites with a KD value of 0.74 mM, and only a small change in epsilonb to 18.1 . Mm ADP binds to E at one or two sites with K2 = 0.5 muM, the values of epsilont for the ternary E-Mn-ADP complex is 17.0 . Free ADP binds about 126 times more weakly to the enzyme than does Mn-ADP . PRR titrations indicated that the values of epsilont for the ternary E-Mn-ADP and (E-P)-Mn-ADP complexes are about the same . Mn-ATP binds very weakly or not at all to (E-P)-Mn.Formation of the ternary complexes of CoA with E-Mn or (E-P)-Mn could be followed by small but significant increases in the PRR enhancement . No ternary complex with succinate could be detected since the addition of succinate had no effect on the PRR enhancement . However, a large decrease in enhancement, at least 2-fold, was observed upon addition of both succinate and CoA . An increase in the PRR enhancement was produced by the interaction of succinyl-CoA with the E-Mn complex . Upper limits of the dissociation constants for CoA from the quaternary E-Mn-ADP-succinate-CoA complex and for succinyl-CoA from the quaternary E-Mn-ADP-succinyl-CoA complex are 390 and 560 muM, respectively . The epsilon values for the quaternary and quinary complexes are 6.4 and 3.1, respectively . The successive occupation of substrate binding sites of succinyl-CoA synthetase produces alterations in the molecular dynamics or in the conformation of the active site (or both), which are accompanied by progressive decreases in the values of epsilon . Thus, the physical parameter used in these studies relects the previously observed catalytic properties of the enzyme system inasmuch as the catalytic function of succinyl-CoA synthetase is potentiated by substrate binding, and catalytic avtivity in partial reactions is maximized as binding sites are successively occupied.

J Biol Chem, 1977 Mar 25, 252(6), 1852 - 7
Monovalent cation activation of tryptophanase; Suelter CH et al.; The interaction of monovalent cations with holotryptophanase has been examined by spectral and kinetic methods . Using S-orthonitrophenyl-L-cysteine as a substrate, activation by the following monovalent cations was demonstrated; values of KA (mM, in italics) and Vmax (mumol min-1 mg) aare given in parentheses: Li+ (54 +/- 11.6, 4.3 +/- 0.28), Na+ (40 +/- 0.03, 18) K+ (1.44 +/- 0.06, 41.1 +/- 3.5), Tl+ (0.95 +/- 0.1, 39 +/- 4.4), NH4+ (0.23 +/- 0.01, 57.9 +/- 2.6), Rb+ (3.5 +/- 0.3, 33.5 +/- 1.8), Cs+ (14.6 +/- 2.6, 21 +/- 2.3) . It was demonstrated by circular dichroic spectra that the competitive inhibitor, ethionine, interacts with the holoenzyme in the absence of activating monovalent cations, although it does not undergo labilization of the alpha proton . On addition of monovalent cation to the holoenzyme-ethionine complex, a marked increase occurs in absorption of 508 nm resulting from labilization of the alpha proton with formation of the quinoid form of the pyridoxal phosphate moiety of the enzyme-substrate complex at the catalytic center (Morino, Y., and Snell, E.E . (1967) J . Biol . Chem; 242, 2800-2809 . The extent of formation of this quinoid intermediate was linearly related to the maximum velocity observed with each cation except NH4+, which was anomalously active . When measured at 500 nm, the change in absorption ranged from deltaA = 0.45 mg-1 of tryptophanase for NH4+ to 0.06 mg-1 for Li+ . Two moles of thallium (I) were bound per mole of subunit . The data are most consistent with the interaction of monovalent cation at or near the catalytic center in such a way that it either participates directly in the reaction or is required for the critical alignment of one or more functional groups necessary for catalysis.

Biochemistry, 1977 Mar 22, 16(6), 1031 - 8
Transcription and translation of cloned Drosophila DNA fragments in Escherichia coli; Miller DL et al.; The expression of three unique DNA fragments from Drosophila melanogaster which have been inserted into Escherichia coli (E . coli) via the plasmid, pSC 101, was studied . The hybrid plasmid DNA molecules containing Drosophila DNA were transformed into the minicell producing strain of E . coli, X1411 . Drosophila DNA-directed RNA synthesis was studied by hybridizing newly synthesized RNA isolated from the minicells with various DNA fragments which were immobilized on nitrocellulose filters . RNA was synthesized as readily from the inserted Drosophila DNA as from the original bacterial plasmid, pSC 101 . In one case, transcription appeared to be initiated preferentially on one of the two strands of a Drosophila DNA fragment regardless of the orientation of that Drosophila DNA fragment with respect to the pSC 101 sequences . Two of the three Drosophila DNA fragments did not induce the synthesis of new polypeptides in minicells as detected by autoradiography of {35S}methionine-labeled polypeptides on polyacrylamide gels . The third Drosophila DNA fragment caused the synthesis of one additional polypeptide of 29 000 daltons . When an 8200 base pair portion of the third inserted Drosophila DNA (63% OF THE TOTAL Drosophila insertion) was removed by digestion with the restriction enzyme, Eco R1, this new polypeptide was no longer synthesized by minicells containing the remaining Drosophila DNA . When the 8200 base pair fragment was placed back into its parent plasmid as an inversion, the new polypeptide did not reappear . In addition, the presence of some, but not all, of the Drosophila DNA insertions affected the relative synthesis of the six polypeptides coded for by the parent plasmid, pSC 101.

Biochemistry, 1977 Mar 22, 16(6), 1111 - 6
A study of the quaternary structure of Escherichia coli RNA polymerase using bis(imido esters); Coggins JR et al.; The quaternary structure of Escherichia coli RNA polymerase has been studied by cross-linking with a periodate-cleavable bis(imido ester), N,N'-bis(2-carboximidoethyl)tartaramide dimethyl ester dihydrochloride (CETD) . The cross-linked holoenzyme gives a characteristic five-band pattern after electrophoresis on sodium dodecyl sulfate-polyacrylamide gels . The components of each band have been unambiguously identified by (a) molecular-weight measurements, (b) comparisons of cross-linking patterns of holoenzyme and core enzyme, and (c) periodate cleavage of cross-links followed by a second dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The bands are (1) alphabeta and alphabeta', (2) sigmabeta and sigmabeta', (3) alphasigmabeta', (4) betabeta', and (5) sigmabetabeta' . Bands 2 and 4 are the most prominent at low reagent concentrations (up to 2.5 mM) but band 5 becomes the most prominent at higher concentrations . There are no bands corresponding to alphaalpha and alphasigma; a faint band has been tentatively identified as alphabetabeta' . Shorter bis(imido esters) are much less effective cross-linking reagents than CETD and they do not give rise to any other cross-linked species . On the basis of these observations, a model for the subunit arrangement of RNA polymerase is proposed.

Biochemistry, 1977 Mar 22, 16(6), 1077 - 81
Fatty acid synthesis in Escherichia coli is indirectly inhibited by phenethyl alcohol; Nunn WD; Experiments were performed to determine how phenethyl alcohol inhibits phospholipid synthesis in E . coli . At a nonbacteriostatic concentration, the drug reduces the rate of de novo fatty acid and phospholipid synthesis by 60 to 70% . The inhibition of fatty acid synthesis was found to be a secondary consequence of the inhibition of phospholipid synthesis . Phenethyl alcohol reduces the rate of incorporation of exogenous fatty acids into the phospholipids of a fatty acid auxotroph by 60% . These results indicate that this drug controls phospholipid synthesis beyond the level of fatty acid synthesis . Phenethyl alcohol inhibits the synthesis of phospholipids containing saturated fatty acids to a greater extent than it does the synthesis of phospholipids containing unsaturated fatty acids . It controls the synthesis of phospholipids containing saturated fatty acids at both the level of fatty acid synthesis and the level of incorporation of the saturated fatty acids into phospholipids . The synthesis of phospholipids containing unsaturated fatty acids is inhibited at the level of incorporation of the fatty acids into phospholipids.

C R Acad Sci Hebd Seances Acad Sci D, 1977 Mar 21, 284(12), 1115 - 8
{Attempt to express the spontaneous global bacteriolytic power of water}; Schwartzbrod L et al.; The utilization of a technique for the determination of spontaneous bacteriolytic power in surface waters has led us to propose a new way of expressing the spontaneous bacteriolytic power of water: PBSG . PBSG allows us to evaluate the intensity of global spontaneous bacteriolysis and to compare the global spontaneous bacteriolytic powers of different water samples.

Biochim Biophys Acta, 1977 Mar 18, 475(2), 383 - 92
Isolation and comparison of ribothymidine-lacking tRNAs of fetal, newborn and adult bovine tissues; Reszelbach R et al.; Although ribothymidine (rT) is the most common methylated nucleoside in tRNA, a wide variety of bovine tissues have now been found to contain a class of tRNAs which totally lack rT and have an unmodified uridine in its place . The tissues studied include bovine brain, kidney, liver, thymus and testicles from adult, newborn and fetal stages . The class of tRNA was detected by its ability to be methylated with Escherichia coli rT-forming uracil methylase with radioactive S-adenosyl-L-methionine as the methyl donor . In each case rT was shown to account for at least 95% of the methylated products produced . In vitro methylated tRNA populations were compared by fractionation of double-labeled tRNAs on RPC-5 columns . Three major methyl-accepting tRNA peaks were found for all mammalian tissues studied . The level of methyl acceptance in these peaks was found to vary considerably between tRNAs of different tissues . A major difference in the methyl-accepting tRNA populations of bovine liver and calf thymus was observed . Little similarity was found in the rT-lacking class of tRNAs of bovine liver and wheat germ . Three members of the rT-lacking class of bovine liver tRNA were isolated and found to be two species of valine tRNA and one species of threonine tRNA . All three tRNA's completely lacked rT and could be quantitatively methylated with E . coli uracil methylase.

Biochim Biophys Acta, 1977 Mar 18, 475(2), 323 - 36
Structure of animal mitochondrial DNA (base composition, pyrimidine clusters, character of methylation); Vanyushin BF et al.; Base composition, content of pyrimidine isopliths and methylation degree of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) from various vertebrates and protozoon Crithidia oncopelti have been studied . mtDNAs from mammals (ox, rat) do not differ in fact in the G + C content from the respective nDNA . The G + C content in mtDNA from fishes (sheat-fish) and birds (duck, chicken) is 1.5--2.5 mol % higher than in the respective nDNA . Kinetoplast DNA (kDNA) from Crithidia oncopelti (G + C = 42.9 mol %) differs significantly in base composition from nDNA (G + C 51.3 mol%) . All the mtDNA and kDNA studied differ from the respective nDNA by a lower degree of pyrimidine clustering . The amount of mono- and dipyrimidine fragments in mtDNA is more than 30 mol %, whereas in nDNA it does not exceed 23 mol % . The quantity of long pyrimidine clusters (hexa- and others) is 2--4 times lower in mtDNA than in nDNA . The lower degree of clustering of pyrimidine nucleotides seems to be a specific feature of all the mtDNA studied . This may be indicative of common traits in the organization and origin of mtDNA . All mtDNA of vertebrates contain 5-methylcytosine as "minor" base (1.5--3.15 mol %) and surpass by 1.5--2 times the respective nDNA in the methylation degree . It has been found that in animals mtDNA is species specific as far as the 5-methylcytosine content is concerned . mtDNA of beef heart differs significantly from nDNA in the mode of 5-methylcytosine distribution in pyrimidine isopliths, which may indicate that methylation specificity of nuclear and mitochondrial DNA is not the same . In mitochondria and nuclei of rat liver certain DNA-methylase activity has been detected, which provides in vitro the methylation of cytosine residues both in homologbous DNA and various heterologous DNAs . Specificity of methylation in vitro of cytosine residues in one and the same heterologous DNA from Escherichia coli B with nuclear and mitochondrial enzymes is different . Mitochondrial enzyme methylates cytosine residues chiefly in mono-, whereas nuclear enzyme, in di- and tripyrimidine fragments.

Biochim Biophys Acta, 1977 Mar 18, 475(2), 403 - 7
Inactivation of infectivity of phiX174 DNA by menadione and reduced menadione; Morita J et al.; Interaction of menadione and reduced menadione with phage phiX174 DNA was investigated . A concentration of 2-10(-4) M menadione inactivated 60% of the infectivity of phiX174 DNA to spheroplasts of Escherichia coli, while reduced menadione inactivated 97% of the infectivity of phiX174 DNA at the same concentration . Alkaline sucrose gradient centrifugation revealed 2-10(-5) M reduced menadione caused approximately 24% of phiX174 DNA to produce strand break under the condition of 80% lethanlity . DNA strand break was not observed even at 4 - 10(-4) M menadione . These results indicated that there were different mechanisms for inactivation of phiX174 DNA between menadione and reduced menadione.

Biochim Biophys Acta, 1977 Mar 18, 475(2), 228 - 40
On the control of ribosomal protein biosynthesis in Escherichia coli . II . Studies during recovery from amino acid starvation; Marvaldi J et al.; The rate of synthesis of ribosomal proteins relative to that of total protein was measured at various times during recovery from arginine starvation in isogenic re+ and rel- strains of Escherichia coli K 12 . Total ribosomal proteins are preferentially synthesized early during recovery . Higher rates of synthesis are obtained in the rel+ strain than in the rel- strain . Differential rates of synthesis of individual ribosomal proteins are observed at the various times studied . The rate of synthesis of individual proteins increases with time up to maximum values then the rates come down to values similar to those found in exponentially growing cells . The time of restart of synthesis of each protein has been estimated (1) by the time at which the maximum value is reached, and (2) by measuring the rate of synthesis at early time (3 min) . Most ribosomal proteins behave similarlly in rel- and rel+ strains . Proteins have been listed from highly labelled (early proteins) to poorly labelled (late proteins) . The significance of the order of restart is considered.

Biochim Biophys Acta, 1977 Mar 18, 475(2), 217 - 27
On the control of ribosomal protein biosynthesis in Escherichia coli . I . Studies on ribosomal protein biosynthesis in amino acid-starved cells; Pichon J et al.; The rate of individual ribosomal protein synthesis relative to total protein synthesis has been determined in Escherichia coli rel+ and rel- cells, under valyltRNA deprivation . These strains have a temperature-sensitive valyl-tRNA synthetase . Starvation was obtained following transfer to the cells to non-permissive temperature . Ribosomal proteins were obtained by treatment of either total lysates of freeze-thawed lysozyme spheroplasts or ammonium sulphate precipitate of ribosomes, with acetic acid . Differential labelling of the ribosomal proteins was observed in both strains: proteins from the rel+ strain appear more labelled than those from the rel- strain, the rate of labelling of individual proteins being about the same in both strains . Moreover ribosomal proteins were found as stable during starvation as total protein . It is thus concluded that in starving cells individual ribosomal proteins are not synthesized at equal rates . This indicates that the synthesis of ribosomal proteins is not only under the control of the rel gene.

Biochim Biophys Acta, 1977 Mar 17, 465(3), 650 - 6
Optical properties of an outer membrane lipoprotein from Escherichia coli; Lee N et al.; The infrared spectrum of a structural lipoprotein from the Escherichia coli outer membrane indicated the lipoprotein had an alpha-helical conformation but no sign for the existence of beta-structures . From circular dichroism spectra of the lipoprotein, the alpha-helical content of the protein was found to be as high as 88% in 0.01-0.03% sodium dodecyl sulfate in the presence of 10(-5) M Mg2+ at pH 7.1 and 23 degrees C . When sodium dodecyl sulfate concentration increased higher than 0.1%, the alpha-helical content of the lipoprotein decreased to about 57% . Divalent cations, such as Mg2+ and Mn2+, were found to increase the helical content of the lipoprotein . The high alpha-helical content of the lipoprotein was observed in a wide range of temperatures (23 to 55 degrees C) . The significance of the high alpha-helical content of the lipoprotein is discussed in light of the three-dimensional molecular models of the lipoprotein proposed previously.

Mol Gen Genet, 1977 Mar 16, 151(3), 327 - 31
Altered tetracycline resistance in pSC101 recombinant plasmids; Tait RC et al.; Investigation of tetracycline resistance genetically determined by the plasmid pSC101 and several recombinants of pSC101 containing EcoRI generated DNA fragments inserted at the EcoRI site has revealed significant differences in the phenotypic expression of that resistance . The altered phenotypes of the recombinant plasmids may be the result of the location of the EcoRI site of pSC101, which has been determined to be near the genetic elements involved with tetracycline resistance.

Mol Gen Genet, 1977 Mar 16, 151(3), 261 - 7
A spectinomycin dependent mutant of Escherichia coli; Dabbs ER; A mutant of Escherichia coli B has been isolated which shows a novel phenotype of spectinomycin dependence . The mutant, termed RD, needs spectinomycin to grow at temperatures of 37 degrees or below; it is unable to grow at 42 degrees in either the presence or absence of spectinomycin . Secondary mutants which grow well in the absence of spectinomycin can be isolated spontaneously at a frequency of about 10(-6) . Two-dimensional gel electrophoresis of ribosomal proteins from 25 of these revertants showed that two revertants had an alteration in S4; one other showed an alteration in L5, and one showed an apparent absence of L1 . Mutant RD itself had an altered less basic S5, which was maintained in all the revertants that were checked . Genetic analysis indicated that RD was a double mutant: one mutation, which alone conferred a spectinomycin resistant phenotype on the strain, was located in the strA region of the E . coli chromosome and was represented by the mutation in S5 . The other mutation, which conferred the dependence on spectinomycin, mapped close to the rif locus.

Mol Gen Genet, 1977 Mar 16, 151(3), 245 - 52
Accessibility of proteins in 50S ribosomal subunits of Escherichia coli to antibodies: an ultracentrifugation study; Morrison CA et al.; The accessibility of each of the proteins on the 50S ribosomal subunit of Escherichia coli was investigated by establishing whether immunoglobulins (IgG), specific for each of the 34 proteins from the 50S subunit, were able to bind to the 50S subunit . The main criterion for accessibility was the formation of specific antibody-50S subunit complexes that could be detected by means of analytical ultracentrifugation . The proteins fell into two main groups . Immunoglobulins against proteins L1, L2, L3, L4, L5, L6, L7/L12, L8, L9, L10, L11, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L25, L26, L27 and L30 gave large amounts of complex (20-100%) and, therefore, these proteins were considered to be accessible on the surface of the 50S ribosomal subunit . The antibodies against the remaining proteins L13, L24, L28, L29 and L31 to L34 produced small amounts of complexes (10-20%) . Since their effects were unequivocably stronger than those obtained with IgG's from sera of non-immunized animals, the results indicate that these proteins are probably also accessible . Nonetheless, from the ultracentrifugation studies alone definite conclusions about the exposure of the latter group of proteins could not be drawn.

Biochem J, 1977 Mar 15, 162(3), 527 - 37
The effects of hyperphenylalaninaemia on the concentrations of aminoacyl-transfer ribonucleic acid in vivo . A mechanism for the inhibition of neural protein synthesis by phenylalanine; Hughes JV et al.; An acute administration of phenylalanine to neonatal animals has been reported to result in large decreases in the intracellular concentrations of several essential amino acids in neural tissue, as well as an inhibition of neural protein synthesis . The present report evaluates the effects of the loss of amino acids on the concentrations of aminoacyl-tRNA in vivo, with the view that an alteration in the concentrations of specific aminoacyl-tRNA molecules could be the rate-limiting step in brain protein metabolism during hyperphenylalaninaemia . tRNA was isolated from saline- and phenylalanine-injected mice 30-45 min after injection, by using a procedure designed to maintain the concentrations of aminoacyl-tRNA present in vivo . Periodate oxidation of the non-acylated tRNA and aminoacylation with radioactively labelled amino acids was used to determine the proportion of tRNA that was present in vivo as aminoacyl-tRNA . Although decreases in the intracellular concentrations of alanine, lysine and leucine were observed after phenylalanine administration, the concentrations of alanyl-tRNA, lysyl-tRNA and leucyl-tRNA actually increased by 15% . Although tryptophan has been suggested to be rate-limiting during hyperphenylalaninaemia, the proportion of tryptophan tRNA that was acylated was maximal in both normal and hyperphenylalaninaemic animals . This unexpected increase in aminoacyl-tRNA concentration is discussed as perhaps a secondary effect resulting from the phenylalanine-induced inhibition of protein synthesis . In contrast, the proportion of methionine tRNA that was acylated in vivo after phenylalanine administration was demonstrated to be decreased by approx . 17% . When the isoaccepting species of methionine tRNA were separated by reverse-phase column chromatography, three species were separated, one of which was demonstrated to be the initiator species, tRNAfMet, by the selective aminoacylation and formylation with Escherichia coli enzymes . After the administration of phenylalanine, the acylation of each of the three methionine tRNA species was decreased, with the initiator species being lowered by 10% . This effect on aminoacylation of tRNAfMet may be the primary step by which phenylalanine affects neural protein synthesis, and this is consistent with previous reports that re-initiation may be inhibited during hyperphenylalaninaemia.

Eur J Biochem, 1977 Mar 15, 74(1), 155 - 70
RNA sequences in ribonucleoprotein fragments of the complex formed from ribosomal 23-S RNA and ribosomal protein L24 of Escherichia coli; Branlant C et al.; Upon digestion of the complex formed from the 23-S ribosomal RNA and the 50-S ribosomal protein L24 of Escherichia coli, two fragments resistant to ribonuclease were recovered; these fragments contained RNA sections belonging to the 480 nucleotides at the 5' end of 23-S RNA . By determining the sequence of 70% of this latter region we were able to localise the sections which, in the presence of the protein, are resistant to ribonuclease . Our results suggest that the region encompassing the 480 nucleotides starting at the 9th nucleotide from the 5' end of 23-S RNA has a compact tertiary structure, which is stabilised by protein L24.

Eur J Biochem, 1977 Mar 15, 74(1), 199 - 202
Interacting binding sites of isoleucyl-tRNA synthetase from Escherichia coli studied by equilibrium partition; Hustedt H et al.; The binding of tRNAIIe to isoleucyl-tRNA synthetase in the presence of isoleucine or ATP was investigated using the equilibrium partition method . Isoleucine decreased the affinity of tRNAIIe for the enzyme by a factor of about 5 . For the free standard energy of interaction a value of about 1 kcal/mol (4.2 kJ/mol) was calculated . ATP exhibits qualitatively the same effect as isoleucine . A binding of two molecules isoleucine per molecule of enzyme could not be demonstrated even in the presence of ATP and pyrophosphatase.

Eur J Biochem, 1977 Mar 15, 74(1), 191 - 8
Studies of the interaction between aminoacyl-tRNA synthetase and transfer ribonucleic acid by equilibrium partition; Hustedt H et al.; The partition behavior of isoleucyl-tRNA synthetase, leucyl-tRNA synthetase and tRNA in aqueous two-phase systems composed of the polymers poly(ethyleneglycol) and dextran was investigated . From the results of this investigation a two-phase system could be derived which can be employed for the study of the interactions between synthetases and their cognate tRNAs by equilibrium partition . These measurements show that in each case one molecule of cognate tRNA is bound per molecule of enzyme . The binding constants were in the range 1-5micronM-1 . It could be demonstrated that equilibrium partition is a useful method for the study of interactions between macromolecules.

Biochim Biophys Acta, 1977 Mar 15, 481(1), 80 - 5
Regulation of aspartate carbamoyltransferase of Escherichia coli by the interrelationship of magnesium and nucleotides; Christopherson RI et al.; Purified aspartate carbamoyltransferase from Escherichia coli K12 (carbamoylphosphate: L-aspartate carbamyltransferase, EC 2.1.3.2) shows greater activity with nucleotide effectors as the magnesium nucleotide complex than with similar amounts of the sodium nucleotide . Regulation of aspartate carbamoyltransferase activity in vivo may occur by changes in the total concentration of regulatory nucleotides or, under conditions of magnesium-limited growth, by variation of the saturation of the nucleotides with magnesium.

Biochim Biophys Acta, 1977 Mar 15, 481(1), 42 - 9
Quasi-elastic light scattering studies on pyruvate oxidase; Raj T et al.; Quasi-elastic or dynamic light scattering has been used to examine the translational diffusion properties of the enzyme pyruvate oxidase (pyruvate: ferricytochrome beta 1 oxidoreductase, EC 1.2.2.2.) . Controlled proteolysis of the enzyme converts the native form of the enzyme to a protease-activated form which has a specific activity about 20-fold greater than the native oxidase . Light scattering studies indicate no significant change in the size or shape of pyruvate oxidase as a result of this proteolytic activation . In both cases the enzyme may be characterized as a hydrated sphere with a Stokes radius of about 53A . The sedimentation velocity-diffusion technique was used to obtain the molecular weight of this tetrameric enzyme, about 252 000 with a value of f/f0 of 1.25.

Biochem J, 1977 Mar 15, 162(3), 665 - 70
Partial diploids of Escherichia coli carrying normal and mutant alleles affecting oxidative phosphorylation; Gibson F et al.; A plasmid was isolated which included the region of the Escherichia coli chromosome carrying the known genes concerned with oxidative phosphorylation (unc genes) . This plasmid was used to prepare partial diploids carrying normal unc alleles on the episome and one of the three mutant alleles (unc A401, uncB402 or unc-405) on the chromosome . These strains were compared with segregants from which the plasmid had been lost . Dominance of either normal ormutant unc alleles was determined by growth on succinate, growth yields on glucose, Mg-ATPase (Mg2+-stimulated adenosine triphosphatase) activity, atebrin-fluorescence quenching, ATP-dependent transhydrogenase activity and oxidative phosphorylation . In all the above tests, dominance of the normal allele was observed . However, in membranes from the diploid strains which carried a normal allele and either of the mutant alleles affecting Mg-ATPase activity (uncA401 or unc-405), the energy-linked functions were only partially restored.

Biochim Biophys Acta, 1977 Mar 11, 459(3), 596 - 604
Isolation and characterization of an inhibitory subunit of the Mg2+--Ca2+-ATPase of Escherichia coli; Nieuwenhuis FJ et al.; 1 . Stimulation of the Escherichia coli ATPase activity by urea and trypsin shows that the ATPase activity both in the membrane-bound and the solubilized form is partly masked . 2 . A protein, inhibiting the ATPase activity of Escherichia coli, can be isolated by sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified ATPase . The inhibitor was identified with the smallest of the subunits of E . coli ATPase . 3 . The molecular weight of the ATPase inhibitor is about 10,000, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and deduced from the amino acid composition . 4 . The inhibitory action is independent of pH, ionic strength or the presence of Mg2+ or ATP . 5 . The ATPase inhibitor is heat-stable, insensitive to urea but very sensitive to trypsin degradation . 6 . The Escherichia coli ATPase inhibitor does not inhibit the mitochondrial or the chloroplast ATPase.

J Biol Chem, 1977 Mar 10, 252(5), 1739 - 44
Function of initiation factor 1 in the binding and release of initiation factor 2 from ribosomal initiation complexes in Escherichia coli; Stringer EA et al.; 1 . Studies on the function of initiation factor 1 (IF-1) in the formation of 30 S initiation complexes have been carried out . IF-1 appears to prevent the dissociation of initiation factor 2 (IF-2) from the 30 S initiation complex . The factor has no effect on either the initial binding of IF-2 nor does it increase the amount of IF-2 dependent fMet-tRNA and GTP bound to the 30 S subunit . Bound fMet-tRNA remains stable to sucrose gradient centrifugation even in the absence of IF-1 . 2 . It is postulated that the presence of IF-2 on the 30 S complex is necessary so that at the time of junction with the 50 S subunit to form a 70 S complex, the 70 S-dependent GTPase activity of IF-2 can hydrolyze GTP . This hydrolysis provides a means by which GTP can be removed to facilitate formation of a 70 S initiation complex active in peptidyl transfer . In support of this postulate, it was observed that 30 S initiation complexes formed in the absence of IF-1 could be depleted of their complexes were still able to accept 50 S subunits to form 70 S complexes which could still donate fMet-tRNA into peptide linkages . These results indicate that 30 S complexes lacking GTP do not require IF-2 for formation of active 70 S complexes . 3 . IF-1, which is required to prevent dissociation of IF-2 from the 30 S initiation complex, is also required for release of IF-2 from ribosomes following 70 S initiation complex formation . The mechanisms of the release of IF-2 has been studied in greater detail . Evidence is presented which rules out the presence of a stable IF-2 GDP complex on the surface of the 70 S ribosome following GTP hydrolysis and of any exchange reactions between IF-1 and guanine nucleotides necessary for effecting the release of IF-2 . IF-2 remains on the 70 S initiation complexes after release of guanine nucleotides and can be liberated solely by addition of IF-1.

J Biol Chem, 1977 Mar 10, 252(5), 1696 - 701
Limited proteolysis of nitrate reductase purified from membranes of Escherichia coli; DeMoss JA; The heterogeneous form of nitrate reductase released from the membrane fraction of Escherichia coli by heat treatment was converted to a new electrophoretic form by incubation with trypsin . As a result of the trypsin treatment, the heat-released enzyme was converted from an associating-dissociating system to a nonassociating monomer (Mr approximately 200,000) which retained full enzymatic activity . Several distinct subunits in the 47,000- to 59,000-dalton range were converted to a single 43,000-dalton subunit during the trypsin treatment, while the other major subunit (155,000 daltons) was unaffected . Nitrate reductase extracted from the membrane fraction with deoxycholate and ammonium sulfate was composed of two apparently homogeneous subunits (155,000 and 59,000 daltons) . The detergent-extracted enzyme preparation was converted by trypsin to an electrophoretic form very similar to the product of trypsin treatment of the heat-released enzyme with an identical subunit composition (155,000 and 43,000 daltons) . These results demonstrate that the heterogeneous subunits present in the heat-released enzyme are produced during heat treatment by proteolytic cleavage of a single 59,000-dalton subunit . The fragments removed by trypsin treatment are implicated in the self-associating properties of the heat-released enzyme.

J Biol Chem, 1977 Mar 10, 252(5), 1527 - 38
Transient kinetic studies on the allosteric transition of phosphoglycerate dehydrogenase; Dubrow R et al.; Stopped flow spectrophotometry was used to investigate the kinetics of the transition of the phosphoglycerate dehydrogenase (3-phosphoglycerate: NAD oxidoreductase, EC 1.1.1.95) reaction from the active to the inhibited rate upon the addition of the physiological inhibitor serine . The transition was characterized by a single first order rate constant (kobs,i) which was independent of enzyme concentration . At pH 8.5, kobs,i increased in a hyperbolic manner with serine concentration from 2 to 8 s-1 . The increase in kobs,i occurred at serine concentrations where the steady state inhibition was virtually complete . These results indicate that serine inhibition is an allosteric process involving a conformational change in the enzyme . A model is presented in which serine at low concentrations binds exclusively to the inhibited state of the enzyme and shifts the equilibrium toward that state; at high serine concentrations, serine binds to the active state, facilitating its conversion to the inhibited state . An alternative model, which we favor, proposes two classes of inhibitor binding sites . The kinetics of the fluorescence quenching of enzyme-bound NADH by serine (Sugimoto, E., and Pizer, L.I . (1968) J . Biol . Chem . 243, 2090-2098), measured by stopped flow fluorimetry, was also characterized by a single first order rate constant (kobs,f.q.) which was independent of enzyme concentration . At pH 8.5, kobs,f.q . ranged from 0.4 s-1 at low serine concentrations to 1.1 s-1 at high serine concentrations . These results indicate that the fluorescence quenching induced by serine is a manifestation of a structural change in the enzyme . Enzyme and excess NADH were mixed with substrate and serine in the stopped flow instrument, and enzyme-bound NADH fluorescence was monitored by exciting through the protein at 285 nm . A rapid fluorescence quenching process, which occurred within the mixing time, was followed by a slower fluorescence enhancement process which terminated in a steady state level corresponding to the quenched fluorescence of the enzyme NADH serine complex . The rapid quenching was the result of substrate binding (Dubrow, R., and Pizer, L.I . (1977) J . Biol . Chem . 252, 1539-1551) . The fluorescence enhancement was characterized by a single first order rate constant whose value for a given serine concentration corresponded with Kobs,j . This data shows that the quenched state of the enzyme-NADH-complex is the state which is directly responsible for the inhibition of enzyme activity . During catalysis the quenched state is achieved from a different initial conformation, and consequently at a different rate, than in the absence of substrate . kobs,j and kobs,f.q . were also measured using glycine, another inhibitor . The ultraviolet difference spectrum between enzyme and enzyme plus serine was determined and proposed to be the result of the same structural change which is responsible for the fluorescence quenching by serine.

J Biol Chem, 1977 Mar 10, 252(5), 1539 - 51
Transient kinetic and deuterium isotope effect studies on the catalytic mechanism of phosphoglycerate dehydrogenase; Dubrow R et al.; The catalytic mechanism of the phosphoglycerate dehydrogenase reaction in both directions was investigated by studying: (a) pre-steady state transients in reduced coenzyme appearance or disappearance or disappearance and in protein fluorescence; (b) deuterium isotope effects on the transients and on the steady state reactions; and (c) the partial reaction between the enzyme-NADH complex and hydroxypyruvate-P . These studies led to the scheme below for the ternary complex interconversion . E1-NADH-hydroxypyruvate-P(1)equilibriumE2-NADH-hydroxypyruvate-P(2)equilibriumE3-NADH-hydroxypyruvate-P + H+(3)equilibriumE3-NAD+-3-phosphoglycerate(4)equilibriumE4-NAD+-3-phosphoglycerate Steps 1,2, and 4 are ternary complex isomerizations . Step 3 is the hydride transfer . Under steady state conditions isomerization 2 is the rate-determining step in the direction of hydroxypyruvate-P reduction at higher pH values . At lower pH values, the hydride transfer step is also partially rate-determining . The rate-determining step in the direction of 3-phosphoglycerate oxidation occurs subsequent to the hydride transfer step at higher pH values . At lower pH values the rate is determined by both isomerization 4 and the hydride transfer step . Isomerizations 1, 2, and 4 were inhibited by serine, an allosteric inhibitor, indicating that the inactive conformation of the enzyme is incapable of performing any of the steps of the ternary complex interconversion . Phosphoglycerate dehydrogenase corresponds to a V-type allosteric enzyme . When the enzyme-NADH complex was mixed with hydroxypyruvate-P at pH 8.5, a rapid quenching of enzymebound NADH fluorescence occurred . This process was studied under pseudo-first order conditions and shown to be the result of hydroxypyruvate-P binding.

Biochemistry, 1977 Mar 8, 16(5), 964 - 71
Visualization of ribosome--single-stranded DNA complexes in the electron microscope; Calame K et al.; A procedure is described which allows ribosomes bound to single-stranded DNA to be visualized in the electron microscope . The number of bound ribosomes may be determined and the position of the bound ribosomes may be readily measured along the DNA . The distribution of ribosomes bound to separated l and r strands of lambda DNA was shown to conform to the pattern predicted for binding at specific sites . The procedure should allow mapping of ribosome binding sites for the determination of genetic maps and may also be useful for studying translational control and relative binding affinities for ribosomes.

Biochemistry, 1977 Mar 8, 16(5), 938 - 43
Isolation of amino-terminal fragment of lactose repressor necessary for DNA binding; Geisler N et al.; lac repressor can be dissected by trypsin into a homogenous tetrameric core (accounting for residues 60 to 347), carrying inducer binding activity, and the monomeric amino-terminal peptides ("headpieces") accounting for residues 1 to 59 and 1 to 51, respectively . This restriction of the action of trypsin on lac repressor is obtained in 1 M Tris-HCl (pH 7.5)-30% in glycerol at 25 degrees C since only the peptide bonds at lysine-59 and to a lesser extent after at arginine-51 are cleaved under these conditions . The headpieces can be purified by gel filtration . They have ordered secondary structure as revealed by circular dichroism studies . The monomeric headpieces show the relatively weak binding to nonoperator DNA but not the highly specific and strong binding to operator DNA typical for tetrameric lac repressor.

Biochemistry, 1977 Mar 8, 16(5), 921 - 4
Photoreactivating enzyme from Escherichia coli: appearance of new absorption on binding to ultraviolet irradiated DNA; Wun KL et al.; The photoreactivating enzyme, PRE, monomerizes pyrimidine dimers in DNA in a light requiring reaction (lambda greater than 300 nm) . However, the purified PRE from E . coli has no well-defined absorption band for lambda greater than 300 nm . Using absorption difference spectroscopy, we show that when PRE is mixed with ultraviolet-irradiated DNA, new absorption appears in the spectral region required for catalysis . There is a concomitant decrease in the absorption of the mixture for wavelength less than 300 nm . The hyperchromicity for lambda greater than 300 nm is true absorption, not an artifact due to light scattering . Both the hyperchromicity (lambda greater than 300 nm) and hypochromicity (lambda less than 300 nm) can be reversed by irradiation of 365 nm with identical first-order kinetics . We estimate the molar extinction coefficient of the new absorption to be 6900 +/- 1400 at 350 nm . We conclude that the PRE from E . coli does not possess a distinct "chromophore" which by itself is entirely responsible for the absorption of photoreactivating light . Instead, new absorption results when PRE binds its substrate, dimer-containing DNA.

Biochemistry, 1977 Mar 8, 16(5), 829 - 35
Conjugated polyene fatty acids as fluorescent probes: biosynthetic incorporation of parinaric acid by Escherichia coli and studies of phase transitions; Tecoma ES et al.; The use of the fluorescent fatty acid, parinaric acid (9, 11, 13, 15-octadecatetraenoic acid) (PnA), was studied in cells of an unsaturated fatty acid auxotroph of Escherichia coli . Growth conditions were found that permitted biosynthetic incorporation of PnA (up to 3%) into membrane phospholipids during growth on oleic or elaidic acid . Fluorescence measurements of incorporated PnA revealed phase transitions in cells, membranes, and phospholipids at temperatures that reflected the fatty acid composition of the sample . Transitions had a well-defined onset from high temperature, while the lower and end point was less well defined . cis- and trans-PnA (cis, trnas, trans, cis, and all trans, respectively) gave comparable results . Similar phase transitions were detected with PnA, which was not biosynthetically incorporated . Fluorescence of tryptophan was measured in E . coli membranes as a function of concentration of PnA . Significant quenching of tryptophan fluorescence by PnA was observed.

Biochemistry, 1977 Mar 8, 16(5), 1025 - 30
Editing mechanisms in protein synthesis . Rejection of valine by the isoleucyl-tRNA synthetase; Fersht AR; Although the isoleucyl-tRNA synthetase from Escherichia coli (IRS) does not catalyze the overall mischarging of tRNAIle with valine, it does undergo the first step of the reaction, the formation of an IRS-Val-AMP complex . The addition of tRNAIle to this complex leads to its quantitative hydrolysis and the IRS acts as an ATP pyrophosphate in the presence of valine and tRNAIle (Baldwin, A.N., and Berg, P . (1966), J . Biol . Chem . 241, 839) . It is shown that during the ATP pyrophosphatase reaction: (a) IRS forms an IRS-Val-AMP complex; (b) the turnover number of the ATP pyrophosphatase reaction is the same at the rate constant for the transfer of isoleucine from IRS-Ile-AMP to tRNAIle over a wide range of temperature and pH; (c) mischarged Val-tRNAIle is hydrolyzed by IRS with a turnover number of 10 s-1 at pH 7.78 and 25 degrees C, compared with a value of 1.2 s-1 for the transfer of isoleucine from IRS-Ile-AMP to tRNA or for the ATP pyrophosphatase reaction . Although this appears to be consistent with an editing mechanism in which there is a slow transfer of the valine from the IRS-Val-AMP to tRNAIle follwed by the rapid hydrolytic step, as recently found for the rejection of threonine by the valyl-tRNA synthetase, there is an inconsistency . This scheme predicts that on mixing IRS.{14C}Val-AMP with tRNAIle there should be a transient misacylation of the tRNA such that about 10% of the {14C}Val is present as {14C}Val-tRNAIle at the peak . But 0.8% or less is found . This could possibly be caused by the IRS having a higher hydrolytic activity during the mischarging reaction than is measured on mixing the unligated enzyme with performed Val-tRNAIle . Alternatively, a two-stage editing mechanism must be considered in which the majority of the Val-AMP is destroyed before the transfer to tRNA in the major editing step, while the hydrolytic activity of the IRS towards Val-tRNAIle is a second editing step to mop up any mischarged tRNA formed by the Val-AMP escaping the first editing step . It is shown that the "kinetic proofreading" mechanism of Hopfield is not consistent with the experimental data.

Biochemistry, 1977 Mar 8, 16(5), 1010 - 6
Galactose-1-phosphate uridylyltransferase: isolation and properties of a uridylyl-enzyme intermediate; Wong LJ et al.; Galactose-1-P uridylyltransferase catalyzes the interconversion of UDP-galactose and galactose-1-P with UDP-galactose and glucose-1-P by a double displacement pathway involving a uridylyl-enzyme intermediate . The amount of radioactivity incorporated into the protein by uracil-labeled UDP-glucose is decreased by the presence of UDP-galactose, which completes with UDP-glucose for uridylylating the enzyme . The amount of glucose-1-P released upon reaction of the enzyme with UDP-glucose indicates that the dimeric enzyme contains more than one active site per molecule, 1.7 on the average for the most active preparation obtained . This suggests that there is one uridylylation site per subunit and that the subunits are similar or identical . The ureidylyl-enzyme is stable to mild alkaline conditions, 0.10 M NaOH at 60 degrees C for 1 h, but is very sensitive to acid, being largely hydrolyzed after 12 h at pH 3.5 and 4 degrees C . The principal radioactive product resulting from hydrolysis of {uracil-2-14C}uridylyl-ens of the uridylyl-enzyme under the latter conditions is {l}ump . The hydrolytic properties of the uridylyl-enzyme show that the uridylyl moiety is bonded to the protein through a phosphoramidate linkage . Complementary studies on the effects of group selective reagents on the activity of the enzyme suggest that the active site nucleophile to which the uridylyl group is bonded may be a histidine residue . The enzyme is rapidly inactivated by diethyl pyrocarbonate at pH 6 and 0 degrees C and reactivated by NH2OH . UDP-glucose at 0.5 mM fully protects the enzyme against diethyl pyrocarbonate while 70 mM galactose-1-P has only a slight protective effect . Uridylyl-enzyme in inactivated by diethyl pyrocarbonate at no more than 2% of the rate for free enzyme . The enzyme is not inactivated by NaBH4 or by NaBH4 in the presence of UDP-glucose . It is not inhibited by 1 mM pyridoxal phosphate or by 0.5 mM 5-nitrosalicylaldehyde at pH 8.6 and it is not inactivated by NaBH4 in the presence of pyridoxal phosphate . The enzyme is inactivated by 5 to 50 muM p-hydroxymercuribenzoate at pH 8.5, but substrates exert no detectable protective effect against this reagent . It is concluded that the enzyme contains at least one essential sulfhydryl group which is not located in the active site in such a way as to be shielded by substrates.

J Membr Biol, 1977 Mar 8, 31(3), 233 - 55
A protonmotive force as the source of energy for galactoside transport in energy depleted Escherichia coli; Flagg JL et al.; An artificially produced electrochemical potential difference for protons (portonmotive force) provided the energy for the transport of galactosides in Escherichia coli cells which were depleted of their endogenous energy reserves . The driving force for the entry of protons was provided by either a transmembrane pH gradient or a membrane potential . The pH gradient across the membrane was created by acidifying the external medium . The membrane potential (inside negative) was established by the outward diffusion of potassium (in the presence of valinomycin) or by the inward diffusion of the permeant thiocyanate ion . The magnitude of the electrochemical potential difference for protons agreed well with magnitude of the chemical potential difference of the lactose analog, thiomethylgalactoside . The observations are consistent with the view that the carrier-mediated entry of each galactoside molecule is accompanied by the entry of one proton.

Biochemistry, 1977 Mar 8, 16(5), 854 - 9
The relationship between the electrochemical proton gradient and active transport in Escherichia coli membrane vesicles; Ramos S et al.; In the previous paper {ramos, S., and Kaback, H.R . (1977), Biochemistry 16 (preceding paper in this issue)}, it was demonstrated that Escherichia coli membrane vesicles generate a large electrochemical proton gradient (delta-muH+) under appropriate conditions, and some of the properties of delta-muH+ and its component forces {i.e., the membrane potential (delta psi) and the chemical gradient of protons (deltapH)} were described . In this paper, the relationship between delta-muH+, delta psi, and deltapH and the active transport of specific solutes is examined . Addition of lactose or glucose 6-phosphate to membrane vesicles containing the appropriate transport systems results in partial collapse of deltapH, providing direct evidence for the suggestion that respiratory energy can drive active transport via the pH gradient across the membrane . Titration studies with valinomycin and nigericin lead to the conclusion that, at pH 5.5, there are two general classes of transport systems: those that are driven primarily by delta-muH+ (lactose, proline, serine, glycine, tyrosine, glutamate, leucine, lysine, cysteine, and succinate) and those that are driven primarily by deltapH (glucose 6-phosphate, D-lactate, glucuronate, and gluconate) . Importantly, however, it is also demonstrated that at pH 7.5, all of these transport systems are driven by delta psi which comprises the only component of delta-muH+ at this external pH . In addition, the effect of external pH on the steady-state levels of accumulation of different solutes is examined, and it is shown that none of the pH profiles correspond to those observed for delta-muH+, delta psi, or deltapH . Moreover, at external pH values above 6.0-6.5, delta-muH+ is insufficient to account for the concentration gradients established for each substrate unless the stoichiometry between protons and accumulated solutes is greater than unity . The results confirm many facets of the chemiosmotic hypothesis, but they also extend the concept in certain important respects and allow explanations for some earlier observations which seemed to preclude the involvement of chemiosmotic phenomena in active transport.

Biochemistry, 1977 Mar 8, 16(5), 848 - 54
The electrochemical proton gradient in Escherichia coli membrane vesicles; Ramos S et al.; Membrane vesicles isolated from Escherichia coli grown under various conditions generate a transmembrane pH gradient (delta pH) of about 2 pH units (interior alkaline) under appropriate conditions when assayed by flow dialysis . Using the distribution of weak acids to measure delta pH and the distribution of the lipophilic cation triphenylmethylphosphonium to measure the electrical potential (delta psi) across the membrane, the vesicles are demonstrated to develop an electrochemical proton gradient (delta-muH+) of almost - 200 mV (interior negative and alkaline) at pH 5.5 in the presence of reduced phenazine methosulfate or D-lactate, the major component of which is a deltapH of about - 120 mV . As external pH is increased, deltapH decreases, reaching 0 at about pH 7.5 and above, while delta psi remains at about - 75 mV and internal pH remains at pH 7.5-7.8 . The variations in deltapH correlate with changes in the oxidation of reduced phenazine methosulfate or D-lactate, both of which vary with external pH in a manner similar to that described for deltapH . Finally, deltapH and delta psi can be varied reciprocally in the presence of valinomycin and nigericin with little change in delta-muH+ and no change in respiratory activity . These data and those presented in the following paper (Ramos and Kaback 1976) provide strong support for the role of chemiosmotic phenomena in active transport and extend certain aspects of the chemiosmotic hypothesis.

Mol Gen Genet, 1977 Mar 7, 151(2), 215 - 9
Altered dihydrofolate reductase in fol regulatroy mutants of Escherichia coli K12; Sheldon R; Three spontaneous fol regulatory mutants contain dihydrofolate reductase molecules which differ in physical properties from enzymes of their parent strains . The enzymes were purified over 100-fold by affinity chromatography and were shown to differ in vitro in thermolability and in affinity for trimethoprim, a competitive inhibitor of the enzyme . These results indicate that some fol regulatroy mutations occur in the structural gene for dihydrofolate reductase.

Mol Gen Genet, 1977 Mar 7, 151(2), 197 - 201
Complementation of transfer deficient ColE1 mutants; Warren G et al.; The transfer defect of some ColE1 mutants is complemented by ColE1 or ColK, but not by ColE2 . This implies that at least one ColE1-specified protein or RNA is normally needed for ColE1 conjugal transfer . The gene(s) postulated for this function lies within a region whose length is at most 50% of the genome.

Biochim Biophys Acta, 1977 Mar 2, 475(1), 176 - 83
Mapping and length measurements of restriction enzyme fragments by electron microscopy; Keegstra W et al.; 1 . We have mapped by electron microscopy the DNA-fragments formed by the action of the restriction endonuclease from Arthrobacter luteus of phi X 174 replicative form DNA . These fragments were separated by polyacrylamide gel electrophoresis and hybridized to phiX 174 single stranded DNA . The partial duplex molecules were inspected in the electron microscope . In this way the relative order of eleven fragments ranging in size from approximately 100 to 1000 nucleotide pairs has been established and compared with that deduced from reciprocal digestion studies . 2 . The measured lengths of the fragments agreed well with the lengths found by gel electrophoresis . 3 . The purity of the isolated fragments was checked . Most of the contaminating fragments derive from nearest neighbours in the preparative polyacrylamide gels.

Can J Microbiol, 1977 Mar, 23(3), 311 - 8
Outer-membrane damage in sublethally heated Escherichia coli K-12; Hitchener BJ et al.; Exponentially grown cells of Escherichia coli K-12 heated at 48 degrees C in potassium phosphate buffer at pH 7.0 were structrually injured before death . During heating for 60 min about 20% of the cellular lipopolysaccharide (LPS) was released from the outer membrane into the heating medium . Removal of 30% of the cellular LPS, by washing the cells in buffer containing ethylenediaminetetraacetic acid (EDTA), caused no significant increase in the rate of death and structural injury produced by heating . The addition of EDTA to the heating medium produced only a slight increase in the rate of thermal death but a large increase in the rate of structural injury . By a combination of heating at 48 degrees C and washing with EDTA, a maximum of 50% of the LPS was released from cells . These results taken together suggest that structural injury and loss of LPS are not the direct causes of death . The addition of 5 m M Mg2+ to the heating medium protected the cells from death and structural injury caused by heating at 48 degrees C.

Blut, 1977 Mar, 34(3), 223 - 6
Determination of the bacteriostatic capacity of neutrophils and monocytes in mixed cell populations; Meuret G et al.; Cline {1, 2} described a method of determining the phagocytic and bacteriostatic activity of individual types of leukocytes within mixed cell populations . We tried to improve the applicability of this method for the investigation of clinical problems.--Bacteria in the log-phase of growth were incubated in test tubes with leukocytes separated from venous blood . After a short period of phagocytosis 3H-thymidine was added to label DNA-synthesizing organisms . Smears were prepared and processed by autoradiography . The labeling indices of extracellular bacteria and of those phagocytized by neutrophils and monocytes were determined microscopically . The intracellular inhibition of DNA-synthesis was taken as indicative of the bacteriostatic activity of the leukocytes . The proposed modification of Cline's assay is suited to investigate clinical problems of phagocyte dysfunction.

Mol Biol (Mosk), 1977 Mar-Apr, 11(2), 418 - 22
{Conformational transition of DNA within the B-family as revealed by anisotropy circular dichroism}; Zavriev SK et al.; The native T2 DNA in solutions with different LiCl concentrations was oriented by pumping through a capillary device similar to that described in rf . 1 . The change of the parallel, deltaepsilon parallel, and perpendicular, 2 delta epsilon perpendicular, components of circular dichroism tensor was studied as a function of salt concentration . Positive (delta epsilon parallel 280) and negative (2 delta epsilon perpendicular 280) components were shown to increase in absolute magnitude, so that there was a monotonous drop in the longwave band magnitude of the isotropic delta epsilon 280 as a result of compensation; the shortwave band magnitude of the isotopic delta 245 and its anisotropic components were constant . It is proposed that while the helix is winding as a result of LiCl rise progressive tilting of base pairs takes place . It was also shown that the glucosylated T2 DNA in solution is wound to a greater extent than the non-glucosylated DNA of the same GC-content.

Inflammation, 1977 Mar, 2(1), 47 - 54
Lipid chemotaxins isolated from culture filtrates of Escherichia coli and from oxidized lipids; Sahu S et al.; Lipid extracts of sterile culture filtrates of Escherichia coli were shown to contain approximately 75% of the chemotactic activity for human polymorphonuclear leukocytes and rabbit alveolar macrophages . Fractionation and purification of these lipids revealed the presence of many unknown lipids of widely different properties, but all were anionic and at very low concentrations, chemotactic . The only one of active molecules that could be identified was an unsaturated ultraviolet-absorbing hydroxy fatty acid, which, following catalytic reduction with hydrogen, was found to be hydroxyeicosanoic acid . This fatty acid's chromatographic behavior was very similar to that of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), which is a potent chemotaxin for polymorphonuclear leukocytes and macrophages . Unknown chemotaxins could be generated by the oxidation of known unsaturated lipids . Prostaglandins A2 and E2 produced potent chemotaxins upon aerobic oxidation . Malonaldehyde, a peroxidation product of unsaturated lipids, when reacted with phosphatidylethanolamine in aerobic conditions, also produced strong chemotactic agents . The chemotactic activity of these products could be destroyed by catalytic reduction with hydrogen and by methylation with dry methanolic HCl . These data indicate that the nonenzymatic oxidation of unsaturated lipids generates some products that are potent chemotaxins for mammalian inflammatory cells.

J Pathol, 1977 Mar, 121(3), 149 - 58
Diverse effects of triamicinolone on the ocular response to circulating endotoxin; Howes EL Jr et al.; The ocular response to circulating bacterial endotoxin (E coli 055:B5, 100 microgram/kg) can be either significantly reduced or made more servere by pretreatment with the synthetic glucocorticoid triamcinolone . A single injection (25 mg/kg) 3 hr prior to endotoxin sharply curtails the response . Daily injections for 3 days preceding endotoxin produces an enhanced response . With this regimen, an enhanced alteration in ocular vascular permeability is produced 4 hr following endotoxin if 5 mg of triamcinolone is injected daily; if larger quantities of steroid are employed (25 mg), there is a slight reduction in ocular vascular permeability, but an enhanced intravascular fibrin accumulation in ocular blood vessels as well as in capillaries of renal glomeruli (generalised Shwartzman reaction) . Once the enhancement of the ocular response to endotoxin has been established by prior treatment with steroids, additional triamcinolone given 2--3 hr before endotoxin is no longer effective in protecting against ocular or renal changes . These divergent effects of steroid could be produced by either subcutaneous or intra-orbital injection, and the response was equal in both eyes following intra-orbital injection, suggesting that steroids probably do not exert their effects locally in this situation . Although different mechanisms of action of corticosteroids may be responsible for these diverse effects, it is suggested that a loss of responsiveness to steroids may be important in their ability to enhance the effects of endotoxinemia.

Nucleic Acids Res, 1977 Mar, 4(3), 761 - 9
DNA-synthesis with methylated poly(dA-dT) templates: possible role of O4-methylthymine as a pro-mutagenic base; Abbott PJ et al.; The alternating copolymer poly(dA-dT) has been methylated with either dimethyl sulphate (DMS) or N-methyl-N-nitrosourea (MNU) and the levels of the various methylation products determined . In addition to the methylated adenines formed by both methylating agents, MNU resulted also in the formation of 3-methylthymine, O4-methylthymine and phosphotriesters . The methylated polymers have been ution of complementary and non-complementary nucleotides determined . With the DMS methylated template no wrong nucleotide incorporation was detectable, but with the MNU methylated polymer the incorporation of dGMP was observed . The amount of dGMP incorporated correlated with the level of O4-methylthymine in the template over the range of methylation studied . The results indicate that O4-methylthymine is capable of miscoding on a one-to-one basis while the products of DMS methylation (1-, 3- and 7-methyladenines), and also possibly the phosphotriesters, do not lead to any misincorporation.

Nucleic Acids Res, 1977 Mar, 4(3), 747 - 59
Synthesis of oligonucleotides with sequences identical with or analogous to the 3'-end of 16S ribosomal RNA of Escherichia coli: preparation of m-6-2-A-C-C-U-C-C and A-C-C-U-C-m-4-2C via phosphotriester intermediates; van Boom JH et al.; The synthesis of two fully-protected hexanucleotides (11a and 11b) via a phosphotriester approach, which is based on the use of two types of protecting groups for the internucleotide linkages, i.e . one 2,2,2-tribromo-ethyl at the 5'-terminus and four 2-chlorophenyl groups for the remaining linkages, is reported . The hexanucleotides 11a and 11b, assembled via a block-wise two-step phosphotriester method, can be deblocked conveniently to give the two hexamers 12a and 12b containing only 3'leads to5' internucleotide linkages.

Nucleic Acids Res, 1977 Mar, 4(3), 673 - 83
On the binding of aminoalkyl adenylates to isoleucyl-tRNA synthetase from Escherichia coli MRE 600; Flossdorf J et al.; The binding of nine aminoalkyl adenylates to isoleucyl-tRNA synthetase from Escherichia coli MRE 600 was measured and compared with the binding of the cognate amino acids . It was found that they bind rather tightly to the enzyme, the Kd's ranging from 3.1.10(-4) M with glycinol-AMP ester to 3.7.10(-9) M with L-isoleucinol-AMP ester . The binding is not affected by magnesium . It is shown that the free energies of binding of the esters can be calculated adding a constant contribution of the AMP-moiety of about - 4.1 (- 17) kcal/mole (kJ/mole) to the free energies of binding of the cognate amino acids, which we have reported earlier (19, 25, 26).

J Gen Microbiol, 1977 Mar, 99(1), 99 - 105
Timing of cold-sensitive stages in the cell division cycle of Escherichia coli K12; Wolosker HB et al.; Synchornous cultures obtained by selection at division were used to investigate the occurrence of cold-sensitive stages during the division cycle of Escherichia coli (lambdaind--) . There are two such stages within the 50 min cycle: one (early) at 10 to 20 min and the other (late) at 40 to 45 min . Similar results were obtained from calculations based both on the age frequency distribution of cells in exponential growth and on the size of the populations which accumulate as a result of a single change of temperature . Times of about 17 and 44 min were found for the early and the late stages, respectively . It is concluded that the two-step doubling of E . coli K12 cultures synchronized by a single cold shock is due to two cold-sensitive stages in the division cycle.

J Gen Microbiol, 1977 Mar, 99(1), 157 - 69
Location of the gene specifying hexose phosphate transport (uhp) on the chromosome of Escherichia coli; Essenberg RC et al.; The uhp gene, which specifies the uptake of hexose phosphates, and several other genes in the vicinity of minute 81 on the E . coli linkage map have been located by phage-mediated transductions . The order found is mtl-gpsA-pyre-gltc-uhp-tna-dnaa . Alleles specifying the Uhp- and Uhp+ characters were separated from that specifying constitutivity of hexose phosphate uptake (Uhpc) . Although cotransduction frequencies between gltC and uhp as high as 90%, and between uhp and tna as high as 80%, were observed, these frequencies were unusually strongly dependent on which marker was selected . This may be due to the proximity of the uhp region to the point of origin of chromosome replication.

J Gen Microbiol, 1977 Mar, 99(1), 13 - 8
Role of ionic strength in colicin K adsorption; Cavard D; The rate of colicin K adsorption to Escherichia coli, and consequent death of the bacteria, is progressively inhibited with increasing ionic strength of the medium . Comparison of the kinetics of colicin adsorption with the kinetics of colicin killing suggests that the lethal event provoked by colicin occurs soon after irreversible colicin adsorption . Factors, such as salts, which protect bacteria against the lethal action of colicin act by preventing colicin adsorption.

J Biochem (Tokyo), 1977 Mar, 81(3), 665 - 71
Structural specificity of the allosteric inhibitor of phosphoenolpyruvate carboxylase of Escherichia coli; Yoshinaga T; The structural specificity of the allosteric inhibitor of phosphoenolpyruvate carboxylas {EC 4.1.1.31} of Escherichia coli W was investigated using native enzyme and photooxidized enzyme which was desensitized to L-aspartate . Inhibitory activity was expressed in terms of the concentration of the compound required for 50% inhibition (I0.5) . For the native enzyme, L-aspartate and L-malate were the strongest inhibitors with I0.5 values of about 0.10-0.15 mM among about 20 componds tested . For the photooxidized enzyme, oxaloacetate and L-malate were relatively strong inhibitors wiht I0.5 values of about 11-16 mM . The results obtained suggest that the inhibition of the native enzyme mainly reflects allosteric inhibition.

J Antibiot (Tokyo), 1977 Mar, 30(3), 252 - 61
In vitro development of resistance to fosfomycin; Dulaney EL et al.; Resistant colonies develop with an apparent high frequency in zones of inhibition around fosfomycin filter discs on nutrient agar . The resistance is due, primarily, to loss of the fosfomycin transport system . The resistant colony type usually observed in the inhibition zones seldom arise directly by mutation from a cell sown in the area of the zone of inhibition . Instead, small translucent colonies develop first in the inhibition zone . Within these small translucent colonies, mutational events occur which give rise to the normal resistant type colonies.

Biokhimiia, 1977 Mar, 42(3), 540 - 3
{Catabolyte inhibition of lactate transport in Escherichia coli}; Garaev MM et al.; E . coli cells growing on the medium containing glucose and lactate do not utilize lactate . One reason of preferential utilization of glucose is catabolite inhibition of lactate transport . It is necessary for glucose to penetrate into the cell to inhibit lactate transport . Besides glucose the inhibition of the lactate transport is also caused by fructose and by non-metabolized analogue of glucose--alpha-methylglucoside.

Infect Immun, 1977 Mar, 15(3), 1002 - 3
Heat-labile enterotoxin produced by Escherichia coli serogroup O149 isolated from diarrheic calves; Ellis RP et al.; Nine strains of Escherichia coli serogroup O149 isolated from diarrheic calves were found to produce a heat-labile enterotoxin . The heat-labile enterotoxin activity was destroyed by heat (85 degrees C/30 min) and was neutralized by rabbit anti-heat-labile enterotoxin . One of the strains also produced a heat-stable enterotoxin.

Can J Microbiol, 1977 Mar, 23(3), 331 - 6
A modified bioassay for heat-stable Escherichia coli enterotoxin1; Stavric S et al.; Infant mice were injected orally with preparations containing Escherichia coli heat-stable enterotoxin (ST) and Evans blue dye, and incubated at 22 degrees C . With enterotoxin-positive samples, the stomach was distended and contained essentially all of the dye . With enterotoxin-negative samples, the stomach remained normal in size and the dye passed freely into the intestines . The time required to obtain the maximum ratio of gut weight to body weight varied from 30 to 90 min and was dependent upon the concentration of enterotoxin . Heat-labile enterotoxin (LT) had no effect during this period . Based on these findings, the mouse incubation time was reduced from 4 h to 90 min, and the heating of test samples was retained only for confirmation of ST . The location of the dye and stomach distention served as an indicator of positive responses to ST . Incubation of the mice at room temperature (22 degrees C) was found satisfactory.

Biull Eksp Biol Med, 1977 Mar, 83(3), 305 - 8
{Effect of somatotropic hormone on autoimmune responses induced in mice of different genotypes by syngenous heated erythrocytes}; Trufakin VA et al.; A single administration of 1 X 10(9) heated erythrocytes to C57BL and BALB/c mice caused on the 13th day the appearance of antierythrocytic autoantibodies, an increase in the weight of the lymphoid organs, and lymphoreticular hyperplasia . These changes were more pronounced in BALB/c mice . During the development of autoimmune reactions the changes in the number of E- and EAC-rosette-forming cells in the thymus and spleen and in the immune response to the sheep erythrocyte immunization and E . coli endotoxin were revealed; distinct strain differences were observed . Daily somatotropic hormone administration (5 mg/kg of body weight) for 10 days decreased the degree of the autoimmune reactions development in mice of both strains . Its action was more expressed in BALB/c mice.

Am J Vet Res, 1977 Mar, 38(3), 297 - 305
Lesions of experimentally induced colibacillosis in neonatal gnotobiotic pigs: a scanning electron microscopic study; Newman LE et al.; Scanning electron and light microscopy were used in studies of stomach, duodenum, cranial, and caudal portions of the jejunum, ileum, cecum, and spiral colon from 18 gnotobiotic pigs . Five pigs were raised as controls and 13 were exposed at 6 days of age by oral administration of 1.6 X 10(6) colony-forming units of Escherichia coli O138:K81:NM . Infiltration of leukocytes into the mucosa of the stomach seen with the light microscope has not been previously reported . The irregular pattern of the mucosal surface of the stomach formed by the gastric pits and the mucosal extensions on the individual rugae revealed with scanning electron microscopy was different than anticipated . Sections of the ileum from control and infected pigs contained collapsed cells around the extrusion zone at the tips of the villi . These collapsed cells were more numerous in infected pigs and appeared to have sloughed from the area of the extrusion zone resulting in exposure of the lamina propria . Cecum and spinal colon were free of changes . Alterations of the mucosa of the intestinal tract of gnotobiotic pigs infected with E coli as visualized by scanning electron microscopy were considered too inconsistent to be of diagnostic significance.

Proc Natl Acad Sci U S A, 1977 Mar, 74(3), 818 - 21
Eukaryote ribosomes possess a binding site for concanavalin A; Howard GA et al.; Ribosomes prepared from chicken liver or rabbit reticulocytes bound concanavalin A in a molar ratio of approximately 1:1 . This binding is to the large subunit of the eukaryote ribosomes with a dissociation constant of 5 X 10(-7) M (0 degrees) . The binding of concanavalin A to Escherichia coli ribosomes was much less . Binding to the RNA or to possible membrane contaminants was ruled out in control experiments . Chicken liver ribosomes were labeled in vivo with 3H-labeled amino acids, purified, and dissociated in sodium dodecyl sulfate . Affinity chromatography of this preparation made it possible to isolate the small proportion of the ribosomal proteins (about 1.5%) containing the concanavalin A binding site . This protein moved as a single band during electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate and showed an apparent molecular weight of 31,000.

Proc Natl Acad Sci U S A, 1977 Mar, 74(3), 1190 - 4
Physical mapping of a DNA sequence common to plasmids of incompatibility group F I; Palchaudhuri S et al.; We have mapped a region of homology within a 2.2 kilobase segment of plasmids of incompatibility group F I, WHICH IS CONTAINED IN AN EcoRI restriction enzyme fragment bearing genes for autonomous replication; The coordinates of the 2.2 kilobase segment are 46.4F-48.6F on the physical map of the F plasmid . The orgin of vegetative replication has been mapped at 42.5F, at which site F has no sequence homology with other group F I plasmids . Plasmids belonging to other incompatibility groups have no homology with the 46.4-48.6 F segment . Possible mechanisms underlying incompatibility are discussed . We think that incompatibility is due to either a limitation in a common equipartitioning mechanism or a limitation in a common replication control . In either case, the common mechanism is specific for plasmids belonging to a given incompatibility group.

Proc Natl Acad Sci U S A, 1977 Mar, 74(3), 1162 - 6
Isolation and characterization of conditional-lethal rho mutants of Escherichia coli; Inoko H et al.; Temperature-sensitive nitA (rho) mutants of E . coli were isolated; one of them was characterized as an amber mutant . These strains show the Nit phenotype (transcription of phage lambda DNA independent of the N gene) at low temperatures and are inviable at high temperatures . The mutated sites appear to be between cya and metE on the chromosome . Temperature-sensitive nitA bacteria not only permit leftward transcription of the lambda genome at a high rate in the absence of the lambda N protein, but also allow lambda growth at low temperatures . At high temperatures, phages lambda and T4 are incapable of normal development in these cells, while growth of T7 is not affected . The production of thermally unstable rho by the nitA temperature-sensitive mutant suggests that nitA is the structural gene for rho.

Proc Natl Acad Sci U S A, 1977 Mar, 74(3), 1157 - 61
Interaction between mutations of ribosomes and RNA polymerase: a pair of strA and rif mutants individually temperature-insensitive but temperature-sensitive in combination; Chakrabarti SL et al.; A temperature-sensitive lethal mutant of Escherichia coli has been constructed by combining two temperature-insensitive mutations: a rif180 mutation that modifies RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) and a strA24 mutation that modifies the ribosomal protein S12 . The temperature sensitivity is a property of the combination of these two particular alleles; replacement of either of the alleles relieves the temperature sensitivity . An isogenic strain containing a different strA mutation (i.e., rif180 strA11) is not temperature sensitive . Evidently ribosomes modified by the particular strA24 polymerase altered by the rif180 mutation, which suggests that in vivo there may exist some interaction between structures of ribosomes and the RNA polymerase.

Proc Natl Acad Sci U S A, 1977 Mar, 74(3), 1152 - 6
A protease inhibitor blocks SOS functions in Escherichia coli: antipain prevents lambda repressor inactivation, ultraviolet mutagenesis, and filamentous growth; Meyn MS et al.; Inhibition of DNA synthesis in E . coli by treatment with carcinogenic and mutagenic agents results in the coordinate expression of a group of diverse functions (SOS functions) including lambda prophage induction, filamentous growth, and an error-prone DNA repair activity (SOS repair) believed to be responsible for ultraviolet mutagenesis . It has been proposed that this SOS induction proceeds via irreversible proteolytic inactivation of repressor(s) for SOS functions . To test this hypothesis, we investigated the effect of a protease inhibitor, antipain {(1-carboxy-2-phenylethyl)carbamoyl-L-arginyl-L-valylargininal}, on SOS induction . We found that 0.5 mM antipain (which has no effect on cell growth, overall RNA and protein synthesis, or induction of beta-galactosidase) drastically decreases mutagenesis . Antipain also blocks expression of thermally induced mutator activity (another manifestation of SOS repair) and filamentous growth in a tif-1 mutant that expresses SOS functions at 42 degrees without inhibition of DNA synthesis or detectable DNA damage . Furthermore, antipain inhibits thermal induction of lambda prophage in the tif-1 mutant without affecting the kinetics of thermal induction of lambdacI857 prophage . This lambda mutant codes a temperature-sensitive repressor that is directly destroyed by heat and does not require the SOS induction pathway for inactivation at 42 degrees . From our results we conclude that antipain inhibits lambda prophage induction by blocking proteolytic inactivation of lambda repressor and that it inhibits the induction or expression of SOS repair and filamentous growth . Our results suggest a role for proteolytic cleavage in the regulation of SOS functions.

Proc Natl Acad Sci U S A, 1977 Mar, 74(3), 1138 - 41
Bidirection replication from a unique origin in a mini-F plasmid; Eichenlaub R et al.; Replicating molecules of the mini F-kanamycin resistance plasmid, pML31, derived from F'lac, have been isolated from Escherichia coli as covalently-closed circular DNA molecules . These molecules were examined in the electron microscope after digestion with either EcoRI or BamHI restriction endonuclease . The structure of the majority of the molecules replicating was consistent with a bidirectional mode of replication starting at a unique origin on the F-fragment . This origin is located approximately 2.3 kilobases from one of the EcoRI sites . Orientation of the F-fragment relative to the physical map of F showed the position of the origin to be at 42.6 kilobases . A small proportion of molecules appeared to be replicating unidirectionally in either direction from this origin . Termination of replication of pML31 apparently occurs in the fragment containing the locus for kanamycin resistance in a unique region opposite the origin in the circular DNA molecule.

Proc Natl Acad Sci U S A, 1977 Mar, 74(3), 1028 - 31
High-resolution proton magnetic resonance study of the secondary structure of the 3'-terminal 49-nucleotide fragment of 16S rRNA from Escherichia coli; Baan RA et al.; The 3' terminus of 16S rRNA has been implicated in the recognition of mRNA's by the ribosome . A fragment containing the 3'-terminal 49 nucleotides cleaved from the rRNA by cloacin DF13 was isolated in a pure form . The secondary structure of this fragment has been studied by measuring the high-resolution proton magnetic resonance spectra . The resonances observed at low field can be assigned to hydrogen-bonded iminoprotons of base-pairs present in the fragment . From the data we conclude that the rRNA fragment, under the conditions used, exists as a hairpin consisting of eight intramolecular base-pairs, the 3'-terminal dodecanucleotide being unpaired . The implications of these findings with respect to the function of the ribosomal protein S1 are discussed.

Proc Natl Acad Sci U S A, 1977 Mar, 74(3), 1004 - 8
Amino acid sequence for the peptide extension on the prolipoprotein of the Escherichia coli outer membrane; Inouye S et al.; The messenger RNA for the lipoprotein of the E . coli outer membrane was found to code for a putative precursor, prolipoprotein, which has 20 additional amino acid residues extending from the amino terminus of the lipoprotein . Using the prolipoprotein synthesized in an E . coli cell-free system directed by purified messenger RNA for the lipoprotein, the complete amino acid sequence of the amino-terminal precursor region was determined to be as follows: (formula: see text) . It was also found that the prolipoprotein that accumulates in toluene-treated cells has the same sequence . The significance of the amino acid sequence is discussed in terms of the mechanism of biosynthesis and assembly of the lipoprotein in the E . coli outer membrane.

Mech Ageing Dev, 1977 Mar-Apr, 6(2), 131 - 142
Ribosome editing and the error catastrophe hypothesis of cellular aging; Menninger JR; Ribosome editing involves the dissociation during protein synthesis of inappropriate peptidyl-tRNA's, ones whose structure does not correctly complement the codon of the mRNA . This process is one of three stages in protein biosynthesis in which the frequency of errors in cellular proteins is controlled . These stages are reviewed and the implications of ribosome editing are described . A model for stability of the translation apparatus is criticized . Calculations using a revision of the model and experimentally reasonable values for the various parameters show varying time courses for error catastrophes.

J Med Chem, 1977 Mar, 20(3), 458 - 60
Synthesis and antitumor activity of 1,2-dihydro-1-(2-deosy-beta-D-erythro-pentofuranosyl)-2-oxo-5-methylpyrazine 4-oxide, a structural analogue of thymidine; Bobek M et al.; 1,2-Dihydro-1-(2-deoxy-beta-D-erythro-pentofuranosyl)-2-oxo-5-methylpyrazine 4-oxide was synthesized by condensation of the silylated pyrazine base with the blocked chloro sugar, followed by removal of the protecting groups . The compound inhibited the growth of leukemia L1210 cells in vitro by 50% at 2 x 10-7 M . At 400 mg/kg/day x 6 it increased the life-span of leukemia L1210 bearing mice by approximately 55%, without apparent toxicity to the host.

J Med Chem, 1977 Mar, 20(3), 452 - 3
Synthesis and biological activity of potential antimetabolites; Glover GI et al.; Several known alpha-amino acid analogues and a new compound, N-chloroacetylphosphoramidate, a carbamyl phosphate analogue, were screened as antitumor agents . All gave 50% growth inhibition of cultures of human epidemeroid carcinoma of the nasopharynx at dosage levels of 2-8 mug/ml while showing no activity against L1210 lymphoid leukemia in vivo in BDFi mice.

J Med Chem, 1977 Mar, 20(3), 439 - 47
P-Aminobenzoic acid derivatives . Mode of action and structure-activity relationships in a cell-free system (Escherichia coli); Seydel JK et al.; The agonistic and antagonistic effects of nuclearly substituted p-aminobenzoic acids (PABA) on the folate-synthesizing system of E . coli have been studied in whole cell and cell-free systems . All studied derivatives form dihydropteroic acid analogues in the presence of a cell-free folate-synthesizing enzyme system . A thin-layer chromatographic system has been elaborated to determine the rate of analogue formation in the cell-free system . Physicochemical parameters of the PABA derivatives, such as pKa, pi, and Rm values, have been determined . These values have been used in a structure-activity analysis which revealed that the rate of analogue formation in the absence of PABA is independent of the lipophilic properties . Ionization seems to be the decisive factor for the incorporation . As all studied PABA derivatives are totally ionized under the experimental conditions, the rates of analogue formation are very similar with the exception of compounds bearing bulky groups in the 2 position . The variance in inhibitory power may therefore either be due to differences in the ability of the analogues to serve as metabolites or to competition with PABA.

J Infect Dis, 1977 Mar, 135(3), 482 - 5
Enterotoxigenic Escherichia coli and diarrheal disease in Mexican children; Donta ST et al.; Enterotoxigenic strains of Escherichia coli were isolated from eight (16%) of 50 Mexican children admitted to the hospital with diarrhea and from one of 50 children hospitalized for nonenteric disorders . All of the toxigenic strains tested elaborated a heat-labile enterotoxin, and in seven of nine patients no E . coli capable of concomitant production of heat-stable enterotoxin were found . None of the strains of E . coli with classical enteropathogenic serotypes isolated from nine patients with diarrhea produced either heat-labile or heat-stable enterotoxin . Although the results of this study strongly suggest that enterotoxigenic strains of E . coli are probably responsible for a significant number of cases of diarrhea in an indigenous Mexican pediatric population, further proof will require demonstration of in vivo production of enterotoxin and/or antitoxin.

J Infect Dis, 1977 Mar, 135(3), 454 - 62
Diarrhea due to Escherichia coli in the rabbit: a novel mechanism; Cantey JR et al.; A strain of Escherichia coli O15 (RDEC-1) isolated from several rabbits with diarrhea was examined to determine (1) whether the strain could produce diarrhea when administered by the orogastric route to other rabbits and (2) whether this strain was invasive or enterotoxigenic . Strain RDEC-1 produced diarrhea in 48 of 62 rabbits when given by the orogastric route in doses that ranged from 1.5 X 10(2) to 4 X 10(10) bacteria . The organism did not give a positive result in the 18-hr ileal loop or Sereny tests and did not invade HeLa cells . Culture supernatants of strain RDEC-1 did not give positive results in the 6- or 18-hr rabbit ileal loop, suckling mouse, Y-1 adrenal cell, or Chinese hamster ovary cell assays . Fluorescent antibody staining of sections of intestine prepared in a cryostat revealed great numbers of E . coli strain RDEC-1 that adhered to the epithelial surface . It is evident that E . coli can produce diarrhea without being able to invade the mucosa or synthesize enterotoxin . Strains of E . coli similar to RDEC-1 may account for some of the E . coli-associated diarrhea that occurs in humans.

J Bacteriol, 1977 Mar, 129(3), 1651 - 2
Action of a major outer cell envelope membrane protein in conjugation of Escherichia coli K-12; Schweizer M et al.; Protein II, a major outer cell envelope membrane protein, was found together with lipopolysaccharide to stoichiometrically inhibit conjugation in Escherichia coli K12.

J Bacteriol, 1977 Mar, 129(3), 1613 - 22
Identification of a membrane protein associated with expression of the surface exclusion region of the F transfer operon; Minkley EG Jr et al.; Membrane preparations from radioactively labeled male and female strains of Escherichia coli K-12 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography . An intensely labeled band corresponding to a protein of molecular weight of 24,000 was readily apparent in preparations from Hfr and F-prime strains but not in those from female strains . When preparations from a series of Hfr strains containing transfer operon deletions were examined, presence of the band was found to be associated with retention of the region of the F transfer operon between ilzA and traD . Thus, the band ("protein S") appears to be the product of an F tra operon activity corresponding to traS (the gene for surface or entry exclusion), or an unknown gene in its vicinity . As predicted, protein S was subject to Fin+ control; only a faint band was detectable if the repressed plasmid R100 was also present in the F lac strain . A 24,000-dalton protein was also found in membrane preparations from strains carrying the derepressed plasmids R100-1 and R1-19 but not in those from strains carrying the repressed plasmids R100 or R1 . Thus, the appearance of protein S in the membrane may be a general phenomenon resulting from transfer operon expression of F-like plasmids.

J Bacteriol, 1977 Mar, 129(3), 1574 - 83
Enzymatic defenses against the toxicity of oxygen and of streptonigrin in Escherichia coli; Hassan HM et al.; Anaerobically grown Escherichia coli K-12 contain only one superoxide dismutase and that is the iron-containing isozyme found in the periplasmic space . Exposure to oxygen caused the induction of a manganese-containing superoxide dismutase and of another, previously undescribed, superoxide dismutase, as well as of catalase and peroxidase . These inductions differed in their responsiveness towards oxygen . Thus the very low levels of oxygen present in deep, static, aerobic cultures were enough for nearly maximal induction of the manganese-superoxide dismutase . In contrast, induction of the new superoxide dismutase, catalase, and peroxidase required the much higher levels of oxygen achieved in vigorously agitated aerobic cultures . Anaerobically grown cells showed a much greater oxygen enhancement of the lethality of streptonigrin than did aerobically grown cells, in accord with the proposal that streptonigrin can serve as an intracellular source of superoxide . Anaerobically grown cells in which enzyme inductions were prevented by puromycin were damaged by exposure to air . This damage was evidenced both as a decline in viable cell count and as structural abnormalities evident under an electron microscope.

J Bacteriol, 1977 Mar, 129(3), 1563 - 73
Effects of lipid phase transition of the freeze-cleaved envelope of Escherichia coli; Bayer ME et al.; We studied the fine structure of the envelope of Escherichia coli auxotroph K1060 after the cells were grown in the presence of one of the following fatty acids; oleic, palmitelaidic, or elidic acid . The cells were freeze-fractured after exposure to temperatures above and below the lipid phase transition range . As judged by freeze-etching methods, we observed that below the transition range the fracture plane of the inner membrane showed the typical aggregation of intramembranous particles (IMP) and concomitant development of areas devoid of IMP . In these areas we found a regular arrangement of equally spaced ridges, often intersected at 90 degrees by arrays of similar ridges . The ridges were composed of spherical particles measuring 4 to 5 nm in diameter . Formation and melting of these arrays took place within 15 to 30s after temperature shift-down or shift-up, respectively . Fixation in glutaraldehyde prevented these changes . The outer-membrane fracture plane revealed ordered areas to a lesser degree; these were discernible only by the regular arrangement of the IMP of the concave fracture plane . We interpret the data by suggesting that the pattern of ridges in E . coli K1060 is analogous to the band patterns described for artificial liposomes, and that the particles, possibly proteins, are lined up or extruded along the ridges during membrane lipid crystallization.

J Bacteriol, 1977 Mar, 129(3), 1524 - 36
Growth of the Escherichia coli cell surface; Begg KJ et al.; Phage T6 was used as a label to follow the growth of the outer membrane in a strain of Escherichia coli temperature sensitive for the production of the T6 receptor . Extension of the surface takes place at the cell poles . Small cells extend at only one pole, whereas larger cells grow from both poles . The change from unipolar to bipolar growth appears to depend on the attainment of a particular cell size and not on completion of chromosome replication.

J Bacteriol, 1977 Mar, 129(3), 1476 - 86
NOVEL Escherichia coli dnaB mutant: direct involvement of the dnaB252 gene product in the synthesis of an origin-ribonucleic acid species during initiaion of a round of deoxyribonucleic acid replication; Zyskind JW et al.; The initiation process of deoxyribonucleic acid (DNA) replication in Escherichia coli has been studied using the thermoreversible dna initiation mutant E . coli HfrHl65/120/6 dna-252 . This dna mutation was incorrectly classed as a dnaA mutation . Biochemical and genetic evidence suggests that the dna-252 mutant is a novel dnaB mutant, possessing phenotypic properties which distinguish it from other dnaB mutants . Sensitivity of reinitiation in the dna-252 mutant to specific inhibitors of protein, ribonucleic acid (RNA), and DNA synthesis was studied . Reinitiation is shown to be sensitive to rifampin and streptolydigin but not to cholramphenicol . Thus, the dna-252 gene product appears to be required during the initiation process for a step occurring either before or during synthesis of an RNA species (origin-RNA) . Using reversible inhibition of RNA synthesis by streptolydigin of a streptolydigin-sensitive derivative of the dna-252 mutant, the dna-252 gene product is shown to be directly involved in the synthesis of an orgin-RNA species . These results are included in a schematic model presented in the accompanying paper of the temporal sequence of events occurring during the initiation process.

J Bacteriol, 1977 Mar, 129(3), 1257 - 65
Role of transport systems in amino acid metabolism: leucine toxicity and the branched-chain amino acid transport systems; Quay SC et al.; The livR locus, which leads to a trans-recessive derepression of branched-chain amino acid transport and periplasmic branched-chain amino acid-binding proteins, is responsible for greatly increased sensitivity toward growth inhibition by leucine, valine, and serine and, as shown previously, for increased sensitivity toward toxicity by branched-chain amino acid analogues, such as 4-azaleucine or 5',5',5'-trifluoroleucine . These phenotypes are similar to those of relA mutants; however, the livR mutants retain the stringent response of ribonucleic acid synthesis . However, an increase in the rate of transport or in the steady-state intracellular level of amino acids in the livR strain cannot completely account for this sensitivity . The ability of the LIV-I transport system to carry out exchange of pool amino acids for extracellular leucine is a major factor in leucine sensitivity . The previous finding that inhibition of threonine deaminase by leucine contributes to growth inhibition is confirmed by simulating the in vivo conditions using a toluene-treated cell preparation with added amino acids at levels corresponding to the internal pool . The relationship between transport systems and corresponding biosynthetic pathways is discussed and the general principle of a coordination in the regulation of transport and biosynthetic pathways is forwarded . The finding that the LIV-I transport system functions well for amino acid exchange in contrast to the LIV-II system provides another feature that distinguishes these systems in addition to previously described differences in regulation and energetics.

J Bacteriol, 1977 Mar, 129(3), 1234 - 8
Oscillations in the synthesis of cell wall components in synchronized cultures of Escherichia coli; Hakenbeck R et al.; The rate of cell wall synthesis with respect to both proteins and lipids was determined in synchronized cultures of Escherichia coli B/r . Whereas the rate of total protein synthesis showed an exponential increase with cell age, the rate of incorporation of proteins and lipids into cell wall had a maximum at a cell age of 30 to 35 min, 15 min before cell division . This oscillation was observed in both the cytoplasmic membrane and in the outer membrane of the cell envelope.

J Bacteriol, 1977 Mar, 129(3), 1208 - 14
Pyruvate formation during the catabolism of simple hexose sugars by Escherichia coli: studies with pyruvate kinase-negative mutants; Pertierra AG et al.; Escherichia coli K-12 mutants lacking the adenosine 5'-monophosphate-activated pyruvate kinase have been isolated accidentally and used to prepare further mutants additionally devoid of the fructose bisphosphate-activated pyruvate kinase . Such double mutants totally devoid of pyruvate kinase activity still grow well under aerobic conditions on sugars that are catabolized by the phosphoenolpyruvate (PEP):sugar phosphotransferase system, but they grow poorly on non-phosphotransferase system sugars . This suggests that although pyruvate kinase plays a major role in the formation of pyruvate from PEP during growth on non-phosphotransferase system sugars, the operation of the PEP:sugar phosphotransferase system can contribute significantly to pyruvate production from PEP . In the absence of pyruvate kinase and an active PEP:sugar phosphotransferase system the methylglyoxal glycolytic bypass may also function to some extent for the formation of pyruvate during the catabolism of simple hexose sugars . No unique physiological role can yet be ascribed to the adenosine 5'-monophosphate-activated pyruvate kinase as a result of these studies.

Eur J Biochem, 1977 Mar 1, 73(2), 591 - 9
The reactivity of one essential cysteine as a conformational probe in Escherichia coli tryptophanase . Application to the study of the structural influence of subunit interactions; Raibaud O et al.; It is first shown that the inactivation of Escherichia coli apotryptophanase byN-ethylmaleimide results from the labeling of a single particularly reactive cysteine per protomer . The reactivity of this cysteine under various conditions is investigated and the results indicate that the protein can exist under two classes of conformation: one, corresponding to inactive protein,in which the cysteine is reactive, and second, corresponding to active enzyme, where the cysteine is masked . The rate of the isomeriation step involved in this change in conformation is measured by the stopped-flow technique(tou = 0.4s) . Finally, the reactivity of the cysteine is used to characterize the conformation of dimeric holotryptophanase (i.e . a dissociated form of the enzyme obtained as a transient species between dimeric apoenzyme and the natural tetrameric holoenzyme) . By this criterion, it is shown that dimeric holotryptophanase falls in the class of 'inactive' conformations . These results are used to discuss the influence of the quaternary structure on the functional and conformational properties of tryptophanase and the nature of the conformational change involved in the activation of the enzyme by its cofactor and specific cations.

Eur J Biochem, 1977 Mar 1, 73(2), 545 - 51
beta-D-Galactoside transport in Escherichia coli . Reversible inhibition by Aprotic Solvents and its Reconstitution in transport-negative membrane vesicles; Altendorf K et al.; At relatively low concentrations (less than 3M) the aprotic solvents: dimethylsulfoxide, N-methylpyrrolidone, tetramethylurea and hexamethylphosphoric triamide, inhibited beta-D-galactoside transport by whole cells, and the derived membrane vesicles of Escherichia coli . Inhibition was not due to gross leakiness of the membrane and could be largely reversed by a simple washing procedure...

Eur J Biochem, 1977 Mar 1, 73(2), 383 - 92
Characterization of distinct segments in mouse satellite DNA by restriction nucleases; Horz W et al.; The long-range periodicity of mouse satellite DNA has been analyzed by digestion with five restriction nucleases . With all nucleases tested, a major repeat unit approximately 245 nucleotide pairs became apparent . Minor registers of shorter length were also detected . The total number of cleavage sites per haploid genome for each restriction enzyme as well as their positions relative to each other were determined . While endo R-EcoRII was known to cleave all of the satellite DNA, the other four restriction enzymes were found to generate only weak degradation patterns . The results taken together with quantitative analyses of codigestion experiments indicate that the recognition sequences for each of these four nucleases are clustered on separate parts of the satellite DNA . It is concluded that the satellite DNA, which appears homogeneous by digestion with endo R-EcRII, contains distinct segments each susceptible to degradation with one of the other nucleases . These results have certain implications for theories on the evolution of mouse satellite DNA . A simple mechanism of multiplication and divergence by mutation is not sufficient to explain the data . Additional and alternative processes which are relevant to the evolutionary considerations are discussed.

Can J Surg, 1977 Mar, 20(2), 162 - 3, 165
Chronic subphrenic abscess: the missed diagnosis; Milne GA et al.; This report of a case of chronic subphrenic abscess, with a brief review and recommendations for diagnosis and prevention, draws attention to this clinical entity as a potential cause of chronic ill health . The clinical presentation and laboratory findings in this case were unremarkable . It is clear that the condition can be easily missed and the patient may be exposed unnecessarily to prolonged illness and to inappropriate investigation and therapy.

J Lab Clin Med, 1977 Mar, 89(3), 471 - 82
Studies on the effects of C . coli endotoxin on canalicular bile formation in the isolated perfused rat liver; Utili R et al.; The impairment of biliary excretory function induced by E . coli endotoxin in the isolated perfused rat liver was studied by investigating its effects on canalicular bile formation . In experiments where no bile salt was infused, endotoxin administration resulted in significant decreases in bile salt-independent bile flow (BSIF) and sulfobromophthalein (BSP) excretion . It also caused dose-dependent decreases in the transport maximum of BSP but did not alter the ability of the liver to concentrate BSP in the bile . At 2 mg./100 ml . it decreased the hepatic removal rate but increased the hepatic storage of the dye . Measurement of 14 C-erythritol clearance and bile salt secretion also revealed that endotoxin significantly reduced the BSIF fraction of bile . The results indicated that the primary effects of endotoxin were on BSIF and BSP excretion.

Gastroenterology, 1977 Mar, 72(3), 434 - 9
In vitro studies of intestinal endotoxin absorption . I . Kinetics of absorption in the isolated everted gut sac; Nolan JP et al.; Previous studies have shown in a qualitative manner that endotoxin can cross gut epithelium, but precise quantitation has not been possible . The present studies were undertaken to measure quantitatively the mucosal to serosal unidirectional flux of endotoxin with the use of an in vitro rat gut sac preparation . 51Cr-Labeled endotoxin was placed in the mucosal bath in concentrations ranging from 0.05 to 2.0 mg per ml . Over a 2-hr period of time, a small amount of endotoxin was transported transmurally, which was shown chromatographically to be similar to the starting material and which retained its toxic and immunogenic properties . It was first shown that the presence of 2.0 mg per ml of endotoxin in the mucosal bath did not significantly alter the tissue's histology or permeability to 3-O-methyl-D-glucose . When unidirectional fluxes were measured, it was found that the flux was not proportional to the endotoxin concentration as would be expected with a passively permeable solute, but rather its transport system became "saturated," displaying a maximum transport rate of 4.72 (mug per cm) per 2 hr and a Km of 0.425 mg per ml . The isolated gut sac provides an excellent model for the precise study of factors involved in endotoxin absorption.

Eur J Biochem, 1977 Mar 1, 73(2), 449 - 59
Nucleotide sequence of a region in 23-S RNA adjacent to peptidyl transferase catalytic center of Escherichia coli ribosomes; Yukioka M et al.; N-Iodoacetylphenylalanyl-tRNAPhe was used as an affinity label to localize the RNA components intimately involved in the catalytic center of Escherichia coli ribosomes . This analogue could alkylate the specific region of 23-S RNA that waslocated within 2000 nucleotides from the 3' terminus of the molecule . Sequence analysis revealed that the alkylation by the active substrate (N-iodoacetylphenylalanyl-tRNAPhe) was directed to 5'-terminal adenosine residue of a heptanucleotide, A-U-U-U-U-A-Gp, which seemed to be derived from a heptadecanucleotide, U-U-A-A-A-A-A-C-A-C-A-U-U-U-U-A-Gp, in the original 23-S RNA . The significance of the unique sequence in the ribosomal functions is discussed.

Z Naturforsch {C}, 1977 Mar-Apr, 32(3-4), 229 - 35
Activation of chromatin-bound DNA-dependent RNA polymerase (E.C . 2.7.7.6.) in plant storage tissue slices; Kahl G et al.; The synthesis of RNA by chromatin-bound RNA polymerase (E.C . 2.7.7.6.) from white potato tubers proceeds at a low rate, which is enhanced after slicing the tissue, however . Concomitantly DNA template availability as measured with saturating amounts of Escherichia coli polymerase is diminished drastically . Nearest neighbor frequency analysis proved that the RNA synthesized on chromatin of intact tubers is different from that synthesized on chromatin of sliced tissue . The RNA polymerase of white potato tubers is dependent on all four ribonucleoside triphosphates and a divalent metal ion such as Mg2+ or Mn2+ and totally inhibited by the presence of pyrophosphate . Actinomycin D blocks the formation of the RNA product, which could be shown to be a heteropolymer by nearest neighbour frequency technique . The Km of the chromatin-bound enzyme with regard to ATP, GTP, CTP and UTP was 5.1 X 10(-5) M, 1.6X10(-5) M, 0.9X10(-5) M and 0.45X10(-5) M/l respectively . alpha-amanitin inhibits the overall activity to about 50%, which indicates the presence of equal amounts of polymerase I and polymerase II.

J Biochem (Tokyo), 1977 Mar, 81(3), 749 - 56
Involvement of outer membrane proteins in enterochelin-mediated iron uptake in Escherichia coli; Ichihara S et al.; Escherichia coli K-12 grown in iron-deficient media contained a large amount of outer membrane proteins O-2a, O-2b, and O-3, while cells grown in iron-supplemented media contained far smaller amounts of these proteins . The iron uptake by the iron-deficient cells was significantly stimulated in the presence of enterochelin, while that by the iron-rich cells was not . The outer membrane isolated from cells grown in the iron-deficient media showed enterochelin-stimulated binding of iron, while the outer membrane from iron-rich cells and cytoplasmic membranes from both types of cells did not show such binding activity . The amount of iron bound by the outer membrane was almost equivalent to the amount of O-2a, O2b, or O-3, irrespective of the amount of these proteins in the outer membrane, which is controlled by the amount of iron in the medium . Small particles rich in these proteins were prepared from cells by EDTA extraction . The particles were active in enterochelin-mediated iron binding and the amount of iron bound was equivalent to the amount of each of these proteins in the particles . Although the outer membrane of E . coli B was as active in iron binding as that of E . coli K-12, it did not possess an appreciable amount of O-2a . Gel electrophoretic analysis revealed that 9-2b and 9-3 were identical with the proteins missing mutants feuB and feuA, respectively.

J Bacteriol, 1977 Mar, 129(3), 1397 - 406
Adenosine 5'-triphosphate-linked transhydrogenase in cytoplasmic membranes of colicin-treated and untreated Escherichia coli; Sabet SF; The adenosine 5'-triphosphate (ATP)-linked transhydrogenase reaction, present in the particulate fractions of Escherichia coli, was previously shown to be inhibited in these fractions when the bacteria were treated with colicins K or El . The purpose of this study was to characterized the ATP-linked transhydrogenase reaction and the colicin-caused inhibition of the reaction in purified cytoplasmic membranes . Particulate fractions from bacteria treated or untreated with colicins were separated on sucrose gradients into cell wall membrane and cytoplasmic membrane fractions . The ATP-linked transhydrogenase reaction was found to be exclusively associated with the cytoplasmic membrane fractions . The reaction was inhibited by carbonylcyanide m-chlorophenlhdrazone, dinitrophenol, N,N'-dicyclohexylcarbodiimide, and trypsin . Although the cytoplasmic membrane fractions were purified from the majoriy of the cell wall membrane and its bound colicins, they showed the inhibitory effects of colicins K and El on the ATP-linked transhydrogenase reaction . The inhibition of ATP-linked transhydrogenase reaction induced by the colicin could not be reversed by subjection the isolated membranes to a variety of physical and chemical treatments . Cytoplasmic membranes depleted of energy-transducing adenosine triphosphatase ATPase) complex (coupling factor) lost the ATP-linked transhydrogenase activity . The ATPase complexes isolated from membranes of bacteria treated or untreated with colicins El or K reconstituted high levels of ATP-linded transhydrogenase activity to depleted membranes of untreated bacteria . The same ATPase complexes reconstituted low levels of activity to depleted membranes of the treated bacteria.

Infect Immun, 1977 Mar, 15(3), 781 - 8
Nutrition and enterotoxin synthesis by enterotoxigenic strains of Escherichia coli: defined medium for production of heat-stable enterotoxin; Alderete JF et al.; A defined medium has been developed which supports synthesis of heat-stable enterotoxin (ST) by porcine and bovine strains of enterotoxigenic (ENT+) Escherichia coli in levels equivalent or better than a complex Casamino Acids-salts medium . The medium components did not support production of heat-labile enterotoxin (LT) but were similar for ST synthesis by ENT+ strains producting only ST and those which produced ST in addition to LT . The amino acids in Casamino Acids found to be necessary for growth and enterotoxin synthesis were proline, serine, aspartic acid, and alanine . Maximal growth and toxin levels were obtained after 8 h of incubation . Improved growth, but not an increase in synthesis of ST, was observed in the presence of Mg2+, Mn2+ and Fe3+ compared with Mg2+ alone . A chelator, tricine, was necessary for maximal cell densities,, probably to solubilize trace ions and make them more available to the bacteria . Increased growth was observed upon addition of glucose to both complex and defined media; however, glucose as well as gluconate and pyruvate appeared to cause repression of toxin synthesis . Addition of vitamins, oleic acid, or DL-lactic acid to the defined medium slightly increased levels of ST.

Proc Natl Acad Sci U S A, 1977 Mar, 74(3), 888 - 91
High-resolution 31P nuclear magnetic resonance studies of metabolism in aerobic Escherichia coli cells; Navon G et al.; 31P nuclear magnetic resonance spectra at 145.7 MHZ were obtained of concentrated suspensions of E . coli cells . The position of the Pi resonance was used to determine the pH, and in most experiments it was possible to distinguish the intracellular (pHin) and extracellular (pHex) values . During respiration pHin approached 7.55, while pHex varied from 6.0 to 8.0 . With succinate as a carbon source and in a N2 environment, pHin - pHex . Upon addition of glucose, pHin greater than pHex . In the presence of an ATPase (adenosinetriphosphatase; ATP phosphohydrolase; EC 3.6.1.3) inhibitor dicyclohexylcarbodiimide, pHin remained equal to pHex even in the presence of glucose . In other experiments, oxygenation brought pHin above pHex even in the presence of dicyclohexylcarbodiimide . These experiments are consistent with Mitchell's hypothesis that, first, delta pH can be created by the reversal of the ATPase reaction and, second, that protons are pumped outward during respiration . In addition to Pi, about 10 more resonances were resolved, several of which were assigned to different phosphate metabolites.

J Immunol, 1977 Mar, 118(3), 789 - 96
Lipopolysaccharide-induced suppression of the primary immune response to a thymus-dependent antigen; Persson U; The immune response to a thymus-dependent antigen was depressed in vivo and in vitro in spleen cells from mice injected with LPS i.p . a few days before challenge with the antigen . Spleen cells from LPS-injected mice could, however, respond with increase DNA synthesis after activation with polyclonal B and T cell activators in vitro . The LPS-activated spleen cells could actively suppress normal cells in their response to the antigen sheep red blood cells . The suppressor cells contained in the LPS-activated spleens were most likely B lymphocytes, and the possible mechanism for their inhibitory function is discussed.

Eur J Biochem, 1977 Mar 1, 73(2), 461 - 8
A mutant ATP synthetase of Escherichia coli with an altered sensitivity to N,N' -dicyclohexylcarbodiimide: characterization in native membranes and reconstituted proteoliposomes; Friedl P et al.; Dicyclohexylcarbodiimide-resistant mutants of Escherichia coli were isolated and characterized In one mutant the unc genes and affects the membrane-integrated part of the ATP synthetase . The sensitivity of ATP synthetase functions to N,N' -dicyclohexylcarbodiimide was compared in wild-type and mutant membranes . The membrane-integrated part of the wild-type ATP synthetase is highly sensitive to ATP-dependent membrane energization and restoration of lactate-dependent energization of ATPase-depleted membranes . In mutant membranes this concentration has only a slight effect on these activities whereas a severe inhibition is obtained at 200 muM.Using the highly water-soluble 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide theactivities of wild-type and mutant membranes are inhibited to the same extent . TheATP synthetase of wild-type and mutant was partially purified and incorporated muM . Uinto liposomes . These showed an uncoupler-sensitive ATP-32Pi exchange and ATP-dependent quenching of acridine-dye fluorescence . The activities of mutant and wild-type proteoliposomes exhibit the same pattern of sensitivity to dicyclohexylcarbodiimide as the corresponding membranes.

Mol Gen Genet, 1977 Feb 28, 151(1), 89 - 94
Translational fidelity in Escherichia coli: contrasting role of neaA and ramA gene products in the ribosome functioning; Topisirovic L et al.; Strains carrying both the ramA1 and the neaA301 mutations do not exhibit the restriction of informational suppressors normally associated with resistance to neamine . Furthermore, ribosomes from such strains exhibit increased misreading in vitro with respect to particles from the neaA strain . These properties suggest that translational fidelity may be cooperatively controlled by ribosomal proteins S4 and S17, coded by ramA (rpsd) and neaA (rpsq) genes respectively.

Mol Gen Genet, 1977 Feb 28, 151(1), 83 - 8
Identification of an amber fragment of the beta subunit of Escherichia coli RNA polymerase: a yardstick for measuring controls on RNA polymerase subunit synthesis; Glass RE; An amber fragment of the beta subunit of Escherichia coli RNA polymerase has been recovered from strains carrying the rpoB12 amber mutation, indicating that the B12 mutation resides in the structural gene for the beta subunit . The fragment is readily assayed and can be used to determine the degree of expression of a single rpoB cistron in strains haploid or diploid for this region . These studies confirm that the bacterial mechanism, which can compensate for reduced translation of the beta message, operates by the co-ordinate induction of rpoB and rpoC . Furthermore, I show that rpo control depends upon cistron(s) located on the F' factor, KLF10, whose product(s) can act negatively in trans on rpoBC expression.

Mol Gen Genet, 1977 Feb 28, 151(1), 57 - 67
Induction of protein synthesis in Escherichia coli following UV- or gamma-irradiation, mitomycin C treatment or tif Expression; West SC et al.; The rate of synthesis of total cellular proteins has been studied by pulse labelling cells at various periods after irradiation with UV or gamma-rays, after treatment with mitomycin C (MMC) or after expression of the temperature sensitive mutation tif . Subsequent gel electrophoresis and autoradiography reveals changes in the rate of synthesis of several proteins . The most striking change is in a protein of molecular weight 40,000, protein X, which has been previously most extensively studied in cells treated with nalidixic acid (Gudas, 1976) . Synthesis of large quantities of protein X is induced by UV, gamma-rays, MMC treatment or tif expression in rec+ but not recA cells . A feature of recA cells is that they break down their DNA excessively after irradiation or MMC treatment . However, if protein synthesis following irradiation is prohibited by chloramphenicol, post-irradiation degradation becomes excessive in recA+ cells . This inverse relationship between DNA degradation and new protein synthesis consistent with the hypothesis that an induced protein such as X is responsible for controlling DNA degradation following irradiation . Protein X is not induced in a lexB mutant following MMC treatment . In this respect the lexB mutant behaves like lexA and recA mutants in that the ability to induce protein X can be correlated with excessive DNA degradation . Studies on the induction of proteins in inf, tif and tif sfi mutants fail to reveal any correlation between induction of protein X and either the induction of prophage lambda or septation.

Mol Gen Genet, 1977 Feb 28, 151(1), 27 - 34
A new host gene (groPC) necessary for lambda DNA replication; Sunshine M et al.; The isolation of a bacterial mutation in a gene, designated groPC, which affects the growth of phages lambda and P2 is described . Lambda replication is severely limited in the strain, and some lambda pi mutations, which map in (or near) the P gene, allow growth . The gro mutation, groPC259, is recessive to wild type and maps between threonine (thr) and diaminopimelate (dapB) on the E . coli chromosome . The possibility that the groPC gene is concerned with host DNA replication is discussed.

Mol Gen Genet, 1977 Feb 28, 151(1), 11 - 6
DNA synthesis in an Escherichia coli dna B dnaC mutant; Schuster H et al.; An Escherichia coli K12 dnaB dnaC mutant was constructed by P1 transduction of the dnaC allele into a dnaB recipient stain . dnaB dnaC transductant were discriminated from dnaB mutants by their inability to grow at 40 degree C after lysogenization with phage P1bac . The dnaB dnaC mutant character was verified by 1 . P1 transduction, and 2 . by in vitro complementation with dnaB and dnaC wild type protein fractions . DNA synthesis was studied in strains containing dnaB, dnaB dnaC alleles in an otherwise uniform genetic background with the dnaB character either unsuppressed or suppressed by P1bac prophage . Degradation at 42 degree C of {3H}-thymidine pulse-labeled DNA in dnaB and dnaB dnaC mutants is suppressed by P1bac . However, unlike the dnaC mutant, the P1bac lysogen of the dnaB dnaC mutant exhibits an abrupt cessation of DNA synthesis and less residual cell divisions at 42 degree C indicating an inhibition of DNA chain elongation rather than a defect in DNA initiation . It is suggested that denaturation of the dnaB protein effects the dnaC function.

Biochim Biophys Acta, 1977 Feb 28, 496(2), 349 - 58
Glucocorticoid receptor-steroid complex binding to DNA . Competition between DNA and DNA-cellulose; Simons SS Jr; The binding of the glucocorticoid receptor-steroid complex from a line of rat hepatoma tissue culture (HTC) cells to DNA has been examined . An equilibrium competition assay involving a constant, low total amount of double-stranded DNA was developed to compare the complex binding ability of DNA free in solution and bound to cellulose . This binding ability is lowered by a factor of five when DNA is associated with cellulose . Similar studies with HTC cell, calf-thymus, and Escherichia coli DNA revealed no difference in the relative number or affinity of binding sites for receptor-steroid complex in each DNA . The synthetic DNA molecules poly{d(A-T)-d(A-T)} and poly{d(G-C)-d(G-C)} bound complexes equally well but less than the three "natural" DNA molecules . This appears to be due to differences in acceptor site affinity and suggests that nucleotide complexity and/or sequence influences the affinity of HTC cell receptor-glucocorticoid complexes for DNA.

J Biol Chem, 1977 Feb 25, 252(4), 1394 - 401
Energy coupling to net K+ transport in Escherichia coli K-12; Rhoads DB et al.; Energy coupling for three K+ transport systems of Escherichia coli K-12 was studied by examining effects of selected energy sources and inhibitors in strains with either a wild type or a defective (Ca2+, Mg2+)-stimulated ATPase . This approach allows discrimination between transport systems coupled to the proton motive force from those coupled to the hydrolysis of a high energy phosphate compound (ATP-driven) . The three K+ transport systems here studied are: (a) the Kdp system, a repressible high affinity (Km=2 muM) system probably coded for by four linked Kdp genes; (b) the Trka system, a constitutive system with high rate and modest affinity (Km=1.5 mM) defined by mutations in the single trkA gene; and (c) the TrkF system, a nonsaturable system with a low rate of uptake (Rhoads, D.B., Waters, F.B., and Epstein, W . (1976) J . Gen . Physiol . 67, 325-341) . Each of these systems has a different mode of energy coupling: (a) the Kdp system is ATP-driven and has a periplasmic protein component; (b) the TrkF system is proton motive force-driven; and (c) the TrkA system is unique among bacterial transport systems described to date in requiring both the proton motive force and ATP for activity . We suggest that this dual requirement represents energy fueling by ATP and regulation by the proton motive force . Absence of ATP-driven systems in membrane vesicles is usually attributed to the requirement of such systems for a periplasmic protein . This cannot explain the failure to demonstrate the TrkA system in vesicles, since this system does not require a periplasmic protein . Our findings indicate that membrane vesicles cannot couple energy to ATP-driven transport systems . Since vesicles can generate a proton motive force, the inability of vesicles to generate ATP or couple ATP to transport (or both) must be invoked to explain the absence of TrkA in vesicles . The TrkF system should function in vesicles, but its very low rate may make it difficult to identify.

J Biol Chem, 1977 Feb 25, 252(4), 1148 - 55
Inhibition of DNA replication in Escherichia coli by dibromophenol and other uncouplers; Weigel PH et al.; DNA replication in Escherichia coli is inhibited by uncouplers such as 2,4-dibromophenol and 3,3'4',5-tetrachlorosalicylanilide . Inhibition occurs in either aerobically or anaerobically growing cells or in cells made permeable by toluene . With anaerobically growing cells, inhibition by dibromophenol is reversible and occurs under conditions in which there is no change in pools of ATP or deoxynucleoside triphosphates . With toluenized cells, inhibition is not due to breakdown of deoxynucleoside triphosphates . The rates of protein and RNA synthesis are not inhibited either in vivo or in toluenized cells by concentrations of dibromophenol or tetrachlorosalicylanilide which inhibit replication . It is generally believed that uncouplers inhibit many other cellular processes by collapsing a proton gradient across a membrane . However, the relative effectiveness of eight uncouplers and related compounds to inhibit replication did not parallel their ability to transport protons into E . coli cells . Therefore, the inhibition by uncouplers does not suggest that replication depends on a chemiosmotic process . A possible explanation for the uncoupler sensitivity is provided by the finding that many of the purified enzymes tested, including DNA polymerases II and III, are inhibited by dibromophenol and tetrachlorosalicylanilide.

J Biol Chem, 1977 Feb 25, 252(4), 1381 - 5
Characterization of the nucleoside triphosphate phosphohydrolase (ATPase) activity of RNA synthesis termination factor p . II . Influence of synthetic RNA homopolymers and random copolymers on the reaction; Lowery C et al.; The ability of various kinds of RNA molecules to activate the ATP hydrolysis reaction catalyzed by the p transcription termination factor from Escherichia coli has been studied . The most active RNA polymers are those containing cytidylate residues and very little ordered structure . Free poly(C) is the most active homopolymer; it is 45 times more active than poly(U), which is the only non-cytidine containing RNA that has detectable activity . Poly(C) has no activity when complexed with poly(I) or when the chain lengths are shorter than 22 nucleotides long . Although cytidylate residues are important they need not be frequent; a random copolymer of uridine and cytidine nucleotides with as few as 1 cytidylate residue out of 20 is as active as poly(C) . The extent of activation with poly(C) depends on the ratio of p to poly(C) . Poly(C) becomes saturated with p at a ratio of 1.8 ng of p/pmol poly(C), which is equivalent to one p monomer/27 nucleotides . A further increase in this ratio leads to a reduction in p activity . Decreasing the length of the poly(C) does not alter the observed saturation value but does decrease the rate of ATP hydrolysis when the RNA is in excess . The possible relevance of these results to p termination activity is discussed.

J Biol Chem, 1977 Feb 25, 252(4), 1375 - 80
Characterization of the nucleoside triphosphate phosphohydrolase (ATPase) activity of RNA synthesi termination factor p . I . Enzymatic properties and effects of inhibitors; Lowery C et al.; The purification of p protein to homogeneity from Escherichia coli has shown that its RNA-dependent ATPase activity is physically inseparable from its termination activity . The biochemical properties of pATPase have been studied using poly(C) as the activating RNA . This reaction is stimulated by Mg2+ ions and Mn2+ ions and is prevented by excess EDTA; it is not stimulated by Ca2+ ions . The reaction is not affected by a Zn2+ ion chelator and is inhibited by 1 mM Zn2+ . With Mg2+ present, the activity is essentially constant from pH 7 to pH 9.7 . pATPase is sensitive to p-hydroxymercuribenzoate and to N-ethylmaleimide . All four ribonucleoside triphosphates are hydrolyzed by p action . ATP has the lowest Km (0.009 mM), while CTP has the highest Vmax . In a mixture containing all four nucleoside triphosphates at a concentration of 0.4 mM, p shows no strong preference for any one of the substrates . The response of p ATPase to a variety of inhibitors of other ATPases and GTPases and of transcription has been studied . Of the compounds tested, aurintricarboxylic acid, an inhibitor of protein-nucleic acid interactions, was found to be a potent inhibitor of p ATPase, while rifampicin and heparin had no effect . pATPase showed partial sensitivity to thiostrepton, fusidic acid, Dio 9, and sodium azide.

Biochemistry, 1977 Feb 22, 16(4), 665 - 72
Secondary-structure predictions of calcium-binding proteins; Argos P; The known tertiary structure of carp muscle parvalbumin is consistent with an "EF-hand" architecture (helix-loop-helix) for each calcium-ion binding site . Primary-sequence alignments have indicated four EF hands in rabbit skeletal muscle troponin C and in rabbit myosin alkali light chains . Five secondary-structure prediction methods, based on amino acid sequence only, have been fully computerized and used to calculate joint prediction histograms for several calcium-binding proteins . The joint histogram can suggest directly the extent and sequence of the helical- and loop-structural elements, as well as any secondary structural distortions or evolutionary developments . Since the histogram predicted well the length and sequence of secondary structural elements in carp muscle parvalbumin, it seemed reasonable to calculate the joint distribution for other proteins that might bind calcium through the EF-hand configuration . The histograms indicated the four EF-hand regions speculated fro rabbit skeletal muscle troponin C but suggested only three such hands in bovine cardiac muscle troponin C and with a distorted fourth hand . Considerable secondary structural distortion is postulated for the alkali light chains . Possible EF configurations consistent with the histogram results are speculated for Escherichia coli acyl-carrier protein and bovine prothrombin fragment 1, which have been shown to bind calcium . The secondary-structure-prediction algorithms appear to be a useful adjunct to sequence-alignment techniques, especially in cases where the primary sequence homology is weak or the evolutionary distance is large.

Biochemistry, 1977 Feb 22, 16(4), 776 - 81
Expression of the chloroplast ribosomal RNA genes of Euglena gracilis during chloroplast development; Chelm BK et al.; The cellular content and transcription program of the chloroplast ribosomal RNA genes of Euglena gracilis Z have been determined during the light-induced development of chloroplasts by hybridization of total cell DNA or RNA to purified 3H-labeled chloroplast ribosomal DNA ({3H}ctrDNA) . Pancreatic DNase activated, partially purified chloroplast rDNA was enzymatically labeled in vitro by E . coli DNA polymerase I with {3H}TTP as a substrate . The {3H} DNA was denatured and hybridized with a vast excess of purified chloroplast 16 and 23S rRNA . The rRNA-{3H}ct rCNA hybrid was isolated by chromatography on hydroxylapatite . The {3H}ct rDNA was purified and characterized by the kinetics of its renaturation with chloroplast DNA and rRNA, and by the thermal stability of {3H}DNA-DNA and {3H}DNA-RNA hybrids . {3H}ct rDNA was hybridized in trace amounts to cellular RNA or DNA isolated from Euglena cells 0,4,8,12,24,48, and 72 h after the onset of chloroplast development . From a comparison of the kinetics of hybridization with hybridization of standards of known kinetic complexity quantitative estimates of the cellular rRNA and rDNA gene content were made . Chloroplast rRNA increases from 2 to 26% of the cellular RNA during development, while the percentage of cellular DNA represented by ct rDNA increases two- to threefold . Correcting for the change in cellular RNA and DNA content during development, the number of copies of the rRNA gene increases less than twofold, while the number of copies of rRNA per cell increases sixfold . The results are consistent with either a transcriptional activation of the ribosomal genes or an increased rRNA stability during developmental.

Biochemistry, 1977 Feb 22, 16(4), 756 - 65
Preparation of Escherichia coli tRNAs terminating of modified nucleosides by the use of CTP(ATP):tRNA nucleotidyltransferase and polynucleotide phosphorylase; Chinault AC et al.; Two procedures were investigated for the modification of tRNAs at the 3'-terminal nucleoside . The first involved the incubation of an enzymatically abreviated tRNA (tRNA-C-COH) with appropriate nucleoside triphosphates in the presence of CTP(ATP):tRNA nucleotidyltransferase from Escherichia coli and yeast . The E . coli enzyme did not utilize 2'- or 3'-deoxyadenosine 5'-triphosphate as substrates, but affected incorporation of the 2'- and 3'-O-methyladenosine triphosphates onto tRNA-C-Cou to the extent of 30 and 37%, respectively . Although incorporation of the deoxynucleotides could not be effected using the E . coli enzyme, yeast CTP(ATP:tRNA nucleotidyltransferase produced the desired tRNAs in yields of 45-65% . The second modification procedure involved incubation of tRNA-C-COH with (appropriately blocked) nucleoside diphosphates in the presence of polynucleotide phosphorylase . This procedure afforded the tRNAs terminating in 2'- and 3'-deoxyadenosine in yields of 4% (and the yield of the former was increased to 36% when the incubation was carried out in the presence of 20% methanol) . The yields of tRNAs terminating in 2'- and 3'-O-methyladenosing produced by this procedure were 55 and 17%, respectively . Because only single isomers of most of the tRNAs terminating in 2'- and 3'-deoxy- and O-methyladenosine are aminoacylated, attempts were made to obtain the other isomericaminoacyl-tRNA by enzymatic introduction of chemically preaminoacylated nucleotides onto tRNA-C-COH . Although incubation of tRNA-C-COH with three aminoacylated nucleoside 5'-triphosphates and E . coli CTP(ATP):tRNA nucleotidyltransferase did not result in production of the desired tRNAs to a detectable extent, incubation with 2'-deoxy-3'-O-L-phenylalanyladenosine 5'-diphosphate and polynucleotide phosphorylase afforded E . coli tRNA terminating with the corresponding aminoacylated deoxynucleoside.

Biochemistry, 1977 Feb 22, 16(4), 587 - 93
Binding of allosteric effectors to carbamyl-phosphate synthetase from Escherichia coli; Anderson PM; The binding of ornithine and inosine 5'-monophosphate (IMP), positive allosteric effectors, and of uridine 5'-monophosphate (UMP), a negative allosteric effector, to carbamyl-phosphate synthetase from Escherichia coli was studied by the technique of equilibrium dialysis . The monomeric form of the enzyme has one binding site for each of the three allosteric ligands . The binding of UMP is inhibited by ornithine, IMP, MgATP, and ammonia (also a positive allosteric effector) . Bicarbonate, L-glutamine, and adenosine 5'-triphosphate (ATP) (Mg2+ absent) had no effect on the binding of UMP . The affinity of the enzyme for UMP was increased if phosphate buffer was replaced by 2-amino-2-hydroxymethyl-1,3-propanediol (Tris) buffer . The binding of ornithine was inhibited by UMP and ammonia, enhanced by MgATP, MgADP, and IMP, and not affected by bicarbonate, L-glutamine, or ATP (Mg2+ absent) . Ornithine and ammonia probably bind to the same site on the enzyme . The binding of IMP is facilitated by ornithine and ammonia, but is inhibited by MgATP or ATP, indicating that adenine nucleotides can also bind to the IMP binding site . The results of these binding studies are consistent with a scheme previously proposed in which the allosteric effectors function by stabilizing one or the other of two different conformational states of the enzyme which are in equilibrium with each other (Anderson, P.M., and Marvin, S.V . (1970), Biochemistry 9, 171) . According to this scheme, binding of the substrate MgATP is greatly facilitated when the enzyme exists in the conformational state stabilized by the positive allosteric effectors.

Biochemistry, 1977 Feb 22, 16(4), 583 - 6
Evidence that the catalytic and regulatory functions of carbamylphosphate synthetase from Escherichia coli are not dependent on oligomer formation; Anderson PM; Carbamyl-phosphate synthetase from Escherichia coli is an allosteric enzyme which undergoes reversible association reactions in phosphate buffer . The positive allosteric effectors, ornithine, inosine 5'-monophosphate (IMP), and ammonia, facilitate oligomer formation, whereas uridine 5'-monophosphate (UMP), a negative effector, prevents or decreases oligomer formation . When the enzyme is immobilized by reaction with activated Sepharose, under conditions where the enzyme exists only as a monomer, nearly full catalytic activity is retained and the effects of ornithine, IMP, and UMP on the catalytic activity as a function of MgATP concentration are not significantly altered . Gel-filtration chromatography on Sephadex G-200 of catalytic quantities of the enzyme in the presence of all substrates showed that the elution volume was the same as that measured for the enzyme under conditions where it is known to exist in the monomer form . The specific activity of the enzyme does not increase when the concentration of the enzyme is increased 100-fold from a concentration at which the enzyme exists as monomer to a level at which the enzyme exists predominantly as oligomer . These results indicate that the monomer form of the enzyme is the principle active species and that oligomer formation is not directly related to enzyme activity or enzyme regulation.

Biochemistry, 1977 Feb 22, 16(4), 610 - 4
Effect of magnesium on the properties of zinc alkaline phosphatase; Bosron WF et al.; Alkaline phosphatase of Escherichia coli, isolated by procedures which do not alter its intrinsic metal content, contains 4.0 +/- 0.3 g-atoms of tightly bound zinc per mole (Kd less than 1 muM) and 1.3 +/- 0.2 g-atoms of magnesium per mole (Bosron, W.F., Kennedy, F.S., and Vallee, B.L . (1975), Biochemistry 14, 2275-2282) . Importantly, the binding of magnesium is dependent both upon pH and zinc content . Hence, the failure to assign the maximal magnesium stoichiometry to enzyme isolated by conventional procedures may be considered a consequence of the conditions chosen for optimal bacterial growth and purification of the enzyme which are not the conditions for optimal binding of magnesium to alkaline phosphatase . Under the conditions employed for the present experimental studies, a maximum of six metal sites are available to bind zinc and magnesium, i.e., four for zinc and two for magnesium . Magnesium alone does not activate the apoenzyme, but it regulates the nature of the zinc-dependent restoration of catalytic activity to apophosphatase, increasing the activity of enzyme containing 2-g-atoms of zinc five-fold and that of enzyme containing 4-g-atoms of zinc 1.4-fold . Moreover, hydrogen-tritium exchange reveals the stabilizing effects of magnesium on the structural properties of phosphatase . However, neither the KM for substrate nor the phosphate binding stoichiometry and Ki are significantly altered by magnesium . Hence, magnesium, which is specificially bound to the enzyme, both stabilizes the dynamic protein structure and regulates the expression of catalytic activity by zinc in alkaline phosphatase.

C R Acad Sci Hebd Seances Acad Sci D, 1977 Feb 21, 284(8), 705 - 8
{Reactivity in vitro of the leukocytes of the earthworm Eisenia foetida Sav . to several mitogenic substances}; Roch P; Earthworm leukocytes were stimulated by various mitogens including LPS, PHA and Con A . Con A could stimulate leukocytes when they were cultivated in totality . After separation into sub-populations, small-non-adherent-leukocytes and large-adherent ones showed specific reactions with mitogens . Both PHA and LPS reacted with small leukocytes but LPS had no action on large adherent leukocytes.

Biochim Biophys Acta, 1977 Feb 16, 474(4), 639 - 45
Interaction of DNA with DNA binding proteins . III . Infectivity of protein-complexed phage fd DNA in Escherichia coli spheroplasts; Uhlmann A et al.; Complex formation of circular, single-stranded phage fd DNA with Escherichia coli DNA binding protein HD or phage fd gene 5 protein keeps infection of E . coli spheroplasts at the level of free phage DNA, whereas complexes of this DNA with E . coli DNA unwinding protein show a strongly reduced efficiency of transfection . Displacement of the unwinding protein by HD protein or gene 5 protein also maintains the poor adsorption of the complexes to spheroplasts . Free E . coli DNA unwinding protein and residual amounts of this protein bound to the DNA may interfere with the adsorption and the uptake of the phage genome.

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