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 be