J Biol Chem, 1981 Aug 10, 256(15), 8039 - 44 Function of nucleoside triphosphate and polynucleotide in Escherichia coli recA protein-directed cleavage of phage lambda repressor; Craig NL et al.; Escherichia coli recA protein catalyzes a specific proteolytic cleavage of repressors in vitro when it is activated by interaction with a single-stranded polynucleotide and nucleoside triphosphate . The ATP analogue adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) satisfies the NTP requirement . We show here that despite its activity in repressor cleavage, ATP gamma S is hydrolyzed at a negligible rate by the recA protein DNA-dependent nucleoside triphosphatase activity . In the presence of DNA, ATP gamma S binds tightly to recA protein in a complex that can be detected because it is trapped by a nitrocellulose filter . One ATP gamma S molecule is bound per recA monomer . These results suggest that a ternary complex of recA protein, DNA, and nucleoside triphosphate is the species active in repressor cleavage . The activation of recA protein by small, defined oligonucleotides in place of DNA is described and characterized. J Biol Chem, 1981 Aug 10, 256(15), 8177 - 82 Nucleotide sequence of the gene for ribosomal protein S20 and its flanking regions; Mackie GA; The sequence of almost 700 nucleotides encompassing the gene for ribosomal protein S20 of Escherichia coli has been determined using the chemical technique of Maxam and Gilbert (Maxam, A . M., and Gilbert, W . (1977) Proc . Natl . Acad . Sci . U . S . A . 74, 560-564) . Comparison of this sequence with that of known bacterial promoters reveals two promoter-like sequences whose putative initiation sites for transcription lie 132 ("site 1") and 42 ("site 2") base pairs to the 5' side of the coding sequence . Partial digestion experiments demonstrate that RNA polymerase is capable of binding to and protecting both of these sites from DNase 1 digestion, consistent with their functioning as promoters . Interestingly, site 1 is more compact that any previously described bacterial promoter . A second feature of the sequence is the presence of UUG as the translational initiation codon . Finally, the nucleotide sequence supports the hypothesis (Jue, R . A., Woodbury, N . W., and Doolittle, R . F . (1980) J . Mol . Evol . 15, 129-148) that S20 is composed of three tandemly repeated domains. J Biol Chem, 1981 Aug 10, 256(15), 8024 - 9 Permeability properties of chemically modified porin trimers from Escherichia coli B; Tokunaga H et al.; The pore-forming protein of the outer membrane of Escherichia coli, porin, was chemically modified with acetic anhydride, succinic anhydride, and glycinamide . Extensive modification of amino groups of the functional porin trimers caused reduced diffusion rates of the negatively charged solutes such as p-nitrophenyl phosphate and AMP, but did not reduce significantly the diffusion of positively charged molecules carbobenzoxy-glycyl-prolyl-arginine-p-nitranilide and tosyl-glycyl-prolyl-arginine-p-nitranilide . Modification of carboxyl groups of trimers caused decreased diffusion rates of the positively charged solutes more significantly than the diffusion rates of negatively charged solutes . The results suggest that the ionic interactions play an important role for the diffusion of charged solutes through the porin pore . The diffusion of p-nitrophenyl alpha-D-glucoside, an uncharged solute, ws not influenced significantly by modification of either amino or carboxyl groups . This observation suggests that modifications only occurred in areas outside of the narrowest portion of the pore or, alternatively, that amino and carboxyl groups are exclusively located at noncylindrical area of the pore . The structural integrity of the acetylated and the succinylated trimers seemed well preserved . On the other hand, modification of carboxyl groups decreased the thermal stability of trimers and extensive modifications caused the dissociation of trimers into monomers at 37 degrees C. J Biol Chem, 1981 Aug 10, 256(15), 7702 - 4 On the evolutionary relationship of the 4-alpha-helical heme proteins . The comparison of cytochrome b562 and cytochrome c'; Weber PC et al.; The atomic models of the cytochrome b562 and cytochrome c' monomers have been compared . When the respective heme groups are superimposed, the four alpha-helices of each nearly coincide . Four aromatic side chains, including the heme ligands, and a methionine occur in spatially equivalent positions in contact with the heme groups . This structural evidence suggests that the two cytochrome families may have diverged from a common molecular ancestor. J Biol Chem, 1981 Aug 10, 256(15), 7734 - 7 The stereochemical course of the ribosome-dependent GTPase reaction of elongation factor G from Escherichia coli; Webb MR et al.; The stereochemical course of the ribosome-dependent GTPase reaction of elongation factor G from Escherichia coli has been determined . Guanosine 5'-(gamma-thio)triphosphate stereospecifically labeled with 17O and 18O in the gamma-position was hydrolyzed in the presence of the elongation factor and ribosomes . The configuration of the product, inorganic {16O, 17O, 18O}thiophosphate ws analyzed by 31P NMR after its stereospecific incorporation into adenosine 5'-(beta-thio)triphosphate . The analysis showed that the hydrolysis proceeds with inversion of configuration at the transferred phosphorus atom . It is therefore likely that the hydrolysis occurs in a single step by direct, in-line transfer of the phosphorus from GDP to a water oxygen, without a phosphoenzyme intermediate. Biochim Biophys Acta, 1981 Aug 6, 646(1), 119 - 25 Dynamic states of phospholipids in Escherichia coli B membrane . Electron spin resonance studies with biosynthetically generated phospholipid spin labels; Takeuchi Y et al.; Mobility of phospholipid hydrocarbons in the Escherichia coli B membrane fractions was studied by labeling phosphatidylethanolamine or phosphatidylglycerol in situ by biosynthetic incorporation of the spin label . For this purpose, CDP-diacylglycerol spin label was synthesized from phosphatidic acid spin label and cytidine 5'-phosphoromorpholidate and purified by thin-layer chromatography . DCP-diacylglycerol spin label was then incorporated into phospholipids biosynthetically . ESR spectra of these E . coli B membrane fractions showed that phosphatidylglycerol tended to interact with membrane proteins through the mediation of Mg2+, whereas phosphatidylethanolamine had less of this tendency and was more involved in the formation of the bulk of the bilayer continuum of the membrane . These conclusions were also supported by labeling membranes with exogenous spin-labeled phospholipids, although there was some indication that exogenous phospholipids were incorporated into sites different from the sites of incorporation of phospholipids newly synthesized in situ. Biochemistry, 1981 Aug 4, 20(16), 4654 - 62 Zinc(II)-dependent synthesis of diadenosine 5', 5"' -P(1) ,P(4) -tetraphosphate by Escherichia coli and yeast phenylalanyl transfer ribonucleic acid synthetases; Plateau P et al.; A new activity of Escherichia coli and yeast phenylalanyl-tRNA synthetases, the conversion adenosine 5' -triphosphate into diadenosine 5' ,5"' -P(1) ,P(4) -tetraphosphate, is reported . This activity is followed by (31)P NMR and chromatography on poly(ethylenimine)-cellulose . It is revealed by the addition of ZnCl2 to a reaction mixture containing the enzyme, ATP-Mg(2+), L-phenylalanine, and pyrophosphatase It reflects the reaction enzyme-bound phenylalanyl adenylate with ATP instead of PPi and strongly depends on the hydrolysis of pyrophosphate in the assay medium . The zinc dependence of this reaction parallels that of the inhibition of tRNA(phe) aminoacylation which is described in the accompanying paper {Mayaux, J . F., & Blanquet, S . (1981) Biochemistry (preceding paper in this issue)} . In the presence of an unlimiting pyrophosphatase activity, diadenosine tetraphosphate synthesis by E . coli and yeast phenylalanyl-tRNA synthetases occurs at maximal rates of 0.5 and 2 s-1, respectively (37 degrees C, pH 7.8, 150 mM KC1, 5 mM ATP, 10 mM MgCl2, 2 mM L-phenylalanine, and 80 muM ZnCl2) . Under identical experimental conditions, E coli isoleucyl-, methionyl-, and tyrosyl-tRNA synthetases produce small amounts of diadenosine tetraphosphate at rates 2 or 3 orders of magnitude lower than that achieved by phenylalanyl-tRNA synthetase . In the case of E . coli phenylalanyl-tRNA synthetase, it is shown that the diadenosine tetraphosphate synthetase activity is accompanied by a diadenosinetetraphosphatase activity . This activity, actually supported by phenylalanyl-tRNA synthetase, is responsible for the appearance of ADP in the assay medium . It requires also the presence of both ZnCl2 and L-phenylalanine . The formation of ADP from diadenosine tetraphosphate and its reaction with enzyme-bound aminoacyl adenylate account for the appearance in the reaction mixture of diadenosine 5' ,5"' -P(1) ,P(3)-triphosphate, after that of diadenosine tetraphosphate . The significance of these findings in the context of the role of diadenosine tetraphosphate in controlling cellular growth is discussed. Biochemistry, 1981 Aug 4, 20(16), 4638 - 46 Solution behavior of proteins L7/L12 from the 50S ribosomal subunit of Escherichia coli; Kar EG et al.; The behavior of Escherichia coli 50S ribosomal subunit proteins L7/L12 has been investigated in ribosome reconstitution buffer, TMK buffer, by sedimentation equilibrium and analytical gel filtration . Contrary to previous reports that L7/L12 exists in solution solely as dimer species {Moller, W., Groene, A., Terhorst, C., & Amons, R . (1972) Eur J . Biochem . 25, 5}, results presented here indicate that L7/L12 undergoes a monomer-dimer-tetramer self-association, with equal equilibrium constants of 3.5 x 10(4) M-1 obtained for the monomer-dimer and dimer-tetramer steps . These results yield standard Gibbs' free energies of -6.1 +/ 0.6 kcal/mol at 20 degrees C . The observed absence of temperature dependence of this interaction over the range 5-25 degrees C indicated a zero standard enthalpy of self-association . Gel filtration results are presented that confirm the highly elongated shape of the L7/L12 molecule . The data suggest the corresponding Stokes radii for the monomer, dimer, and tetramer are 21-23, 26-28, and 29-32 omicron A, respectively . The significance of these results is discussed. Biochemistry, 1981 Aug 4, 20(16), 4555 - 60 Use of trypsin and lipoamidase to study the role of lipoic acid moieties in the pyruvate and alpha-ketoglutarate dehydrogenase complexes of Escherichia coli; Stepp LR et al.; The relationships between release of (3)H-labeled lipoyl moieties by trypsin and lipoamidase and accompanying loss of overall enzymatic activity of the Escherichia coli pyruvate and alpha-ketoglutarate dehydrogenase complexes were studied . Trypsin releases lipoyl domains together with their covalently attached lipoyl moieties from the "inner" core of the dihydrolipoyl transacetylase and the dihydrolipoyl transsuccinylase whereas lipoamidase releases only the lipoyl moieties . The results show that release of lipoyl domains by trypsin and release of lipoyl moieties by lipoamidase proceeded at faster rates than the accompanying loss of overall activity of the two complexes . Trypsin released about half of the lipoyl domains in the pyruvate dehydrogenase complex without significant effect on the overall activity . A model is presented to explain these and other observations on active-site coupling via lipoyl moieties. Biochemistry, 1981 Aug 4, 20(16), 4774 - 80 Isolation and characterization of the amino and carboxyl proximal fragments of the adenosine cyclic 3' ,5'-phosphate receptor protein of Escherichia coli; Aiba H et al.; The cyclic AMP receptor protein (CRP) is a positive and negative regulatory protein for gene expression in Escherichia coli . The protein has been cleaved proteolytically to determine the relation between CRP structure and function . In the presence of sodium dodecyl sulfate (NaDodSO4), chymotrypsin dissects CRP into two stable fragments of molecular weight 9500 (9.5K) and 13 000 (13K) . After removal of NaDodSO4, the two fragments are resolved by Bio-Rex 70 chromatography in 6 M urea . Analyses of the terminal amino acids released from each fragment and cyanogen bromide cleavage products indicate that the 9.5K fragment is amino proximal in CRP while the 13K fragment is carboxyl proximal . Notable features of amino acid composition are the relatively high amount of arginine and methionine in the 13K fragment and the retention in the 9.5K fragment of the two tryptophans present in the CRP subunit . Following isoelectric focusing in 8 M urea, the 9.5K fragment, 22.5K CRP, and 13K fragment migrate to pH 5.5, 8.3, and 10.3, respectively . While CRP is a cAMP-stimulated DNA binding protein, the 13K fragment binds to DNA in the presence and absence of cAMP . The 9.5K fragment associates to form dimers and decamers . These data are consonant with a model in which the DNA binding domain is present in the carboxyl proximal region of CRP while the amino proximal region contains the subunit-subunit interaction sites and much of the cAMP binding domain. J Cell Sci, 1981 Aug, 50, 199 - 208 Conformational changes induced by salt in complexes of histones and superhelical nuclear DNA; Levin JM et al.; When HeLa cells are lysed in solutions containing a non-ionic detergent and 0.75 M-NaCl, structures are released that retain many of the morphological features of nuclei . These nucleoids contain all the nuclear DNA, RNA and the 'core' histones, but few other proteins characteristic of chromatin . Their DNA is intact . The core histones dissociate on raising the salt concentration . We have probed the structure of nucleoid-histone complexes using the intercalating dye, ethidium, or the RNA polymerase of Escherichia coli . Both have a higher affinity for superhelical DNA than they do for relaxed DNA . The binding of ethidium is measured fluorometrically, and using this probe we find that the DNA of nucleoids containing all the core histones behaves as if it were supercoiled slightly positively . As the salt concentration is increased, free energy characteristic of negative supercoiling appears between 0.92 M and 0.95 M-NaCl . This transition, which is reversible in the presence of the arginine-rich histones, occurs without dissociation of these histones from the DNA and so must reflect a conformational change in the complex . In contrast to the results with ethidium, we find that RNA polymerase can detect the presence of some negative free energy of supercoiling in nucleoids containing the core histones . The transformations of the free energy that can assist the binding of ethidium and RNA polymerase are discussed. Immunology, 1981 Aug, 43(4), 755 - 62 The goldfish immune response . I . Characterization of the humoral response to particulate antigens; Desvaux FX et al.; Anti-red blood cells (RBC) and anti-hapten antibody synthesis were studied in the goldfish, Carassius auratus . Spontaneous haemagglutination titres were found against all the antigens tested . A weak secondary response was observed in RBC-primed fish boosted during the end-phase of the primary antibody production . However, when the second antigenic challenge was performed during the early exponential phase of a primary stimulation, an important amplified response was obtained . The antibody production and immunological memory can be dissociated: no antibody synthesis occurred in glutaraldehyde-fixed RBC (F-RBC) primed was obtained when untreated or F-RBC were given to F-RBC primed animals . The amplified response to sheep red blood cells (SRBC) was significantly inhibited when fish were primed with a mixture of SRBC and Xenopus red blood cells (XRBC), demonstrating an antigenic competition phenomenon . Studies on anti-trinitrobenzene responses confirm the efficiency of E . coli lipopolysaccharide as a carrier for fish anti-hapten immunization . The kinetics and regulation of antibody synthesis in fish are discussed in relation to the described results. Hoppe Seylers Z Physiol Chem, 1981 Aug, 362(8), 1059 - 68 On the cytostatic mechanism of cyclophosphamide . Inhibition of aminoacylation of transfer RNA and induction of stringent control in Escherichia coli by 4-hydroperoxycyclophosphamide; Frank S et al.; 4-Hydroxycyclophosphamide, a highly cytotoxic derivative of cyclophosphamide, has bacteriostatic properties at concentrations higher than 80 microM when given to Escherichia coli growing in glucose/salt medium . The inhibitory effect on growth, protein and RNA syntheses is relieved by the addition of a mixture of amino acids, with leucine being obligatory . In cells treated with the drug, aminoacylation of tRNALeu is drastically decreased, but the content of free leucine is not . It is concluded that 4-hydroxycyclophosphamide interferes with the charging process of tRNALeu in E . coli leading to an inhibition of protein synthesis . The concomitant inhibition of RNA synthesis can adequately by explained by the stringent response phenomena . In a relaxed mutant strain (rel) of E . coli, protein synthesis is also inhibited, but the accumulation of RNA continues after the addition of the drug . Guanosine tetraphosphate (ppGpp), the putative mediator of the stringent control, accumulates in drug-treated stringently controlled E coli but not in the relaxed mutant . An additional direct effect of the drug on RNA synthesis can therefore be ruled out. Genetics, 1981 Aug, 98(4), 677 - 89 High frequency of genetic duplications in the dnaB region of the Escherichia coli K12 chromosome; Sclafani RA et al.; The region that includes the dnaB locus on the E . coli K12 chromosome was shown to be duplicated at high frequency in cell populations . The duplications were shown to be arranged in tandem and segregated at various frequencies . Segregation was dependent on the recA recombination system, but independent of recB,C . Though most of the data was obtained with dnaB::Tn10 insertion mutants, the duplications were shown to occur in the absence of Tn10. Biochem J, 1981 Aug 1, 197(2), 283 - 91 The organization of hydrogenase in the cytoplasmic membrane of Escherichia coli; Graham A; The organization of the membrane-bound hydrogenase from Escherichia coli was studied by using two membrane-impermeant probes, diazotized {125I}di-iodosulphanilic acid and lactoperoxidase-catalysed radioiodination . The labelling pattern of the enzyme obtained from labelled spheroplasts was compared with that from predominantly inside-out membrane vesicles, after recovery of hydrogenase by immunoprecipitation . The labelling pattern of F1-ATPase was used as a control for labelling at the cytoplasmic surface throughout these experiments . Hydrogenase (mol.wt . approx . 63 000) is transmembranous . Crossed immunoelectrophoresis with anti-(membrane vesicle) immunoglobulins, coupled with successive immunoadsorption of the antiserum with spheroplasts, confirmed the location of hydrogenase at the periplasmic surface . Immunoadsorption with sonicated spheroplasts suggests that the enzyme is also exposed at the cytoplasmic surface . Inside-out vesicles were prepared by agglutination of sonicated spheroplasts, and the results of immunoadsorption using these vesicles confirms the location of hydrogenase at the cytoplasmic surface. Biochem J, 1981 Aug 1, 197(2), 269 - 74 Proline biosynthesis in Escherichia coli . Stoichiometry and end-product identification of the reaction catalysed by glutamate semialdehyde dehydrogenase; Hayzer DJ et al.; The stoichiometry of the oxidative phosphorylation of glutamic acid 5-semialdehyde by gamma-glutamyl phosphate reductase (glutamate semialdehyde dehydrogenase) has been established . Equimolar amounts of NADP+ and L-glutamic acid 5-semialdehyde are consumed and equimolar amounts of 5-oxiopyrroilidine-2-carboxylic acid and NADPH are formed . The end-product of the reaction is demonstrated to be 5-oxopyrrolidine-2-carboxylic acid, probably arising from the true end-product gamma-glutamyl phosphate. J Gen Microbiol, 1981 Aug, 125(Pt 2), 431 - 8 The light-reversible binding of carbon monoxide to cytochrome a1 in Escherichia coli K12; Poole RK et al.; Difference spectra at 77 K of intact Escherichia coli K 12 grown under oxygen-limited conditions revealed the presence of cytochrome a1 . In the presence of CO, the band of the reduced form, observed in both the alpha and gamma regions of the spectrum, was decreased . Dual-wavelength scanning spectrophotometry at sub-zero temperatures revealed a flash-dissociable CO-binding pigment with a broad band around 595 nm, identified as cytochrome alpha 1 . Photolysis in the presence of O2 revealed no such band in difference spectra where the reference spectrum was that of the CO-liganded form, a result consistent with the binding of O2 to cytochrome a1 . Repeated cycles of photolysis and recombination of the cytochrome with CO were demonstrated at --46 degrees C . The apparent energy of activation for the reaction with CO was 10.9 kcal mol-1 (45.6 kJ mol-1) . The results are discussed in relation to previous assumptions and results regarding ligand binding to cytochrome a1 and the function of this cytochrome in bacterial respiration. Exp Hematol, 1981 Aug, 9(7), 731 - 41 The effect of osmotic stress on the function of the human granulocyte; Dooley DC et al.; Clinical investigations have shown that the transfusion of human granulocytes can protect the neutropenic patient against acute bacterial infection . The increasing utilization of granulocyte therapy plus an inability to store these cells in liquid media have created a requirement for a cryopreservation technique . However, the cryopreservation of human granulocytes has proven quite difficult to achieve . It is likely that one of the contributing factors is the lability of these cells under osmotic stress . The purpose of this paper was to precisely define the osmotic tolerance limits of the human granulocyte and thereby provide guidelines for the laboratory manipulation of this cell . To accomplish this, cells were exposed to hypertonic or hypotonic media and then tested for viability under isotonic conditions . Function tests included phagocytosis of particles, bacterial killing, migration in the Boyden chamber, and migration under agarose . The experiments revealed that granulocytes will tolerate a narrow range of osmolalities extending from 200 mOsm to 600-700 mOsm . The osmotic damage occurs in less than 30 seconds and appears to be irreversible . The hypertonic limit of the cell cannot be extended by reducing the temperature to 0 degrees C during the osmotic challenge. Gann, 1981 Aug, 72(4), 627 - 30 Effects of anticancer platinum compounds on Escherichia coli strains with normal and defective DNA repair capacity; Konishi H et al.; The effect of anticancer platinum compounds on isogenetic strains of Escherichia coli with normal or defective DNA repair capacity were studied . Cisdichlorodiammineplatinum (II) causes DNA damage that is repairable by the repair systems of bacteria, and the DNA lesion becomes mutational when processed by the excision repair system . Dinitrato (1R, 2S-cyclohexanediamine) platinum (II) exhibits similar killing activity on E . coli strains with both normal and defective DNA repair capacity . Thus, this compound may cause DNA damage that cannot be repaired by bacteria, or its killing activity may be due to effects other than DNA damage. Biochimie, 1981 Aug-Sep, 63(8-9), 685 - 97 Partial deproteinization of ribosomal subunits by treatment with high concentrations of sodium chloride: physical and chemical characterization of protein depleted particles; Guerin MF et al.; Sedimentation of E . coli ribosomal subunits through sucrose gradients containing high concentrations of NaCl (0.1 - 1.0 M) brings about removal of a specific fraction of their proteins . The alterations in the structural properties of the subunits caused by dissociation of these proteins are studied . They appear to be, at least partly the result of removal of constraints on RNA conformation imposed by the released proteins. Proc Natl Acad Sci U S A, 1981 Aug, 78(8), 4699 - 703 Regulation of DNA replication: "target" determinant of the replication control elements of plasmid R6-5 lies within a control element gene; Danbara H et al.; The replication control system of plasmid R6-5 has been investigated by characterization of high-copy-number mutant miniplasmids, development of an in vivo assay for the site of action or "target" of the replication control elements, and sequence analysis of the replication control regions of the wild-type plasmid and two copy-number mutant derivatives . These and other experiments have shown that three plasmid determinants--copA/incA, copB, and copT--are involved in DNA replication control . The products of the copB and copA/incA genes, a 9500-dalton basic polypeptide and either a 7200-dalton basic polypeptide or a short untranslated RNA molecule, respectively, are negative-acting elements that interact with the third element, their target, the copT DNA sequence, or its product to regulate the frequency of initiation of plasmid replication . The location of copT within the copA/incA gene and 1600 base pairs upstream from the origin of replication indicates that regulation is effected at a preinitiation stage of replication, such as the production of a primer or other initiation factor. J Biochem (Tokyo), 1981 Aug, 90(2), 535 - 44 Biological and structural differences between tRNAVal species isolated from rat ascites hepatoma cells and normal rat liver; Shindo-Okada N et al.; On RPC-5 column chromatography, the main valine acceptor activity of tRNA (tRNA2Val) from rat ascites hepatoma cells was eluted later than that of normal rat liver tRNA (tRNA1Val) . The tRNA2Val was aminoacylated by E . coli amino-acyl-tRNA synthetase, while tRNA1Val from normal rat liver was not . Rat fetal liver tRNAVal was also aminoacylated by E . coli aminoacyl-tRNA synthetase . tRNA1Val (rat liver) and tRNA2Val (ascites hepatoma) were each purified to a homogeneous state by RPC-5 column chromatography and two-dimensional polyacrylamide gel electrophoresis, and their sequences were determined by post-labeling techniques . Ascites hepatoma tRNA2Val differed from rat liver tRNA1Val in that Gm18, C32 and an unknown modified nucleoside, N34, in the latter tRNA were mostly replaced by G, Cm, and inosine, respectively . In addition, 3'-terminal adenosine was not present in tRNA1Val (normal rat liver), but was in tRNA2Val (ascites hepatoma) . Other modifications and the primary structures of the two tRNAValS were found to be the same . Thus it was concluded that the new iso-acceptor species of tRNA Val in ascites hepatoma cells is due to a change of post-transcriptional modification, not to a change of tRNA transcription . The unique feature of the change of post-transcriptional modification in tRNA2Val (ascites hepatoma) is that both hypo- and hyper-modification take place simultaneously in the tRNA molecule depending the locations of nucleotide residues. Can J Microbiol, 1981 Aug, 27(8), 835 - 40 The effect of temperature and other growth conditions on the fatty acid composition of Escherichia coli; McGarrity JT et al.; During exponential growth, strain AW405 of Escherichia coli K-12 did not regulate the fatty acid composition of its lipids in response either to temperature or to the addition of NaCl, KCl, or MgCl2 to the medium . Growth was severely restricted at temperatures below 21 degrees C . Differential scanning calorimetry (DSC) of the isolated lipids from a culture with a typical exponential-phase composition yielded a broad transition, extending from approximately 0 to 33 degrees C, with a midpoint at 19 degrees C . During late stages of growth, the fatty acid composition changed . The percentage of palmitic acid increased and cyclopropane fatty acids replaced some of the equivalent unsaturated fatty acids . The increase in palmitate seemed largely independent of growth conditions, whereas the increase in the cyclopropane fatty acids was stimulated by the addition of salts or sucrose . Cultures grown in the presence of sucrose also had higher cyclopropane fatty acid levels during exponential growth . DSC of lipids from a sucrose culture, in which the compositional changes were most pronounced, yielded a much narrower transition with a midpoint at 27 degrees C. Eur J Biochem, 1981 Aug, 118(1), 105 - 11 Lipid-synthesis-dependent biosynthesis (or assembly) of major outer-membrane proteins of Escherichia coli; Bocquet-Pages C et al.; Upon inhibition of fatty acid synthesis in the presence of cerulenin, the uptake of 5' AMP and of other nutrients using similar pore systems can be inhibited as much as 70% . The same effect was observed upon inhibition of phospholipid synthesis after glycerol deprivation in a mutant strain defective in sn-glycerol-3-phosphate acyltransferase . Resumption of both fatty acid synthesis and phospholipid synthesis restores a normal uptake of 5' AMP . The protein composition of the outer membranes, analyzed after pulse radiolabelling by {35S}methionine, was mainly altered in OmpF and OmpC proteins . These proteins are the main porins used by most nutrients like 5' AMP . Whereas the synthesis or the assembly of OmpF protein seems to be more inhibited that that of OmpC protein after inhibition of fatty acid synthesis, the reverse case was observed after inhibition of phospholipid synthesis . No protein produced during inhibition of fatty-acid or phospholipid synthesis is reincorporated into the outer membrane after resumption of these syntheses . These results are discussed with regard to the biosynthesis and assembly of these proteins. Cell, 1981 Aug, 25(2), 347 - 53 Membrane assembly from purified components . II . Assembly of M13 procoat into liposomes reconstituted with purified leader peptidase; Watts C et al.; The major coat protein of coliphage M13 is an integral protein of the E . coli plasma membrane prior to its assembly into new virus particles . It is generated from its precursor, procoat, by a membrane-bound leader peptidase . We now describe the reconstitution of a highly purified preparation of this enzyme into vesicles of E . coli phospholipids . These vesicles bind procoat made in vitro and procoat isolated from in vitro synthesis . Both the crude and the purified substrates were converted post-translationally to coat protein . A significant proportion of the coat protein becomes inserted into the vesicle bilayer, with the N terminus facing the vesicle interior and the C terminus exposed to the external medium . These results strongly suggest that highly purified leader peptidase from E . coli and phospholipids are the only components necessary to mediate the binding, processing and insertion of this integral membrane protein. Cell, 1981 Aug, 25(2), 341 - 5 Membrane assembly from purified components . I . Isolated M13 procoat does not require ribosomes or soluble proteins for processing by membranes; Silver P et al.; The coat protein of coliphage M13 is an integral protein of the host-cell cytoplasmic membrane prior to its assembly into virions . It is initially synthesized as procoat, a soluble precursor with a 23 amino acid leader sequence at its amino terminus . 35S-labeled procoat accumulates during an in vitro translation reaction that contains 35S-methionine and RNA from M13-infected cells . Radiochemically pure procoat has been isolated from in vitro translation reactions by extraction into an organic solvent and gel filtration through Sephadex LH-60 . Radiochemically pure procoat can be used as substrate in rapid and quantitative assays for leader peptidase and for leader peptide hydrolase, an enzyme that degrades the leader peptide after its release from procoat . Procoat solubility, digestion by leader peptidase and processing by membranes are affected by the presence of Mg2+ ion . Isolated procoat is soluble in water at low ionic strength and mildly alkaline pH as well as in detergent solutions . It is cleaved to coat protein by purified E . coli leader peptidase and by inverted E . coli inner-membrane vesicles . These properties of the purified procoat mirror those of the procoat in crude extracts . This suggests that there are no other soluble components that are necessary for the assembly of procoat into the membrane and its conversion to coat; specifically, it provides powerful evidence that protein synthesis is not involved. Antimicrob Agents Chemother, 1981 Aug, 20(2), 155 - 8 Inhibition of aminoglycoside activity in heparin; Nilsson L et al.; Measurements of aminoglycosides by an agar disk diffusion assay are inhibited by heparin in a dose-dependent way . When assayed by a homogeneous immunoassay, this was only evident for tobramycin . This indicates that specimens for aminoglycoside measurement should not be obtained in heparinized tubes . When heparin is used clinically as an anticoagulant, the amount in blood does not reach levels that affect the aminoglycoside activity. Mutat Res, 1981 Aug, 83(1), 49 - 59 Phenotypes of UV-sensitive uvrD3, recL152, and uvrE15 mutants of Escherichia coli; Siegel EC et al.; The mutations uvrD3, uvrE156, and recL152 increase UV sensitivity in Escherichia coli and are closely linked . There is a disagreement as to whether uvrD3 and uvrE strains have distinctive phenotypes . Comparison of strains isogenic except for the uvrD3, uvrE156, and recL152 mutations showed that only the uvrD3 mutation greatly decreased the host cell reactivation of phages T1 and lambda vir and the repair of methyl methanesulfonate (MMS) damage . Only the uvrE156 mutation increased mutation rates . Excessive DNA degradation following UV-irradiation occurred only in the uvrD3 mutant . MMS-resistant mutants of a uvrD3 strain, resulting from mutations closely linked to uvrD3, were still UV-sensitive and fell into two classes: mutator (UvrE--like) and non-mutator . Spontaneous mutator mutants having the UvrE- phenotype were also found in aged uvrD3 cultures . The uvrD3 mutation in heterogenotes was partially dominant. Mutat Res, 1981 Aug, 83(1), 25 - 37 Mutational specificity of the base analogue, 2-aminopurine, in Escherichia coli; Persing DH et al.; 2-Aminopurine (2-AP) is a base analogue of adenine which mispairs with cytosine and causes base-pair substitutions of the transition type . By analyzing the reversion patterns of defined trpA alleles in Escherichia coli we confirm that 2-AP causes both A:T leads to G:C and G:C leads to A:T transitions with the former induced more frequently than the latter . We also find that 2-AP enhances transversions at 3 sites and frameshift mutations at 1 other site . It is unlikely that 2-AP can cause transversions and frameshifts solely by a mispairing mechanism . However, 2-AP-induced transversion and frameshift mutagenesis was not abolished by the presence of an inactive recA allele, indicating this mutagenic activity is not dependent upon recA-directed misrepair. J Bacteriol, 1981 Aug, 147(2), 569 - 77 Identification and characterization of Col plasmids from classical colicin E-producing strains; Watson R et al.; A series of transformants have been derived which carry the Col plasmids from 11 E-colicin-producing strains isolated by Fredericq or Hamon . The ColE plasmids identified included four of type E1, five of type E2, and two of a type designated E4 by Horak (Zentralbl . Bakteriol . Parasitenkd . Infektionskr . Hyg . Abt . 1 Orig . Reihe A 233:58-63, 1975) . Strain K317 was shown to carry a ColE7 plasmid and a 2.7-megadalton (Md) ColE2imm plasmid which confers immunity to colicin E2 but not the ability to produce colicin . Other plasmids identified in the colicinogenic isolates were a 3.4-Md ColN plasmid and a 1.3-Md Col plasmid of unknown type . The ColE1 plasmids all continued replicating in cultures treated with chloramphenicol . Strains carrying the ColE4 or ColE3-CA38 plasmid exhibited partial sensitivity to their own colicins and exhibited an unusual clearing-zone morphology when overlaid on stabs of colicin E2- or E7-producing strains . Except for ColE1-K53, ColE1-K47, ColE2-CA42, and ColE7-K317, all of the ColE plasmids were found to be about 4.3 Md in size . ColE2-CA42 and ColE7-K317 are both about 3.9 Md and are the only two plasmids of the E2, E3, E4, and E7 types that did not yield a 0.39-Md deoxyribonucleic acid fragment as an EcoRI digestion product . The comparisons of the ColE plasmids suggest several structural and functional relationships among them. J Bacteriol, 1981 Aug, 147(2), 410 - 7 Comparative mutagenesis and interaction between near-ultraviolet (313- to 405-nm) and far-ultraviolet (254-nm) radiation in Escherichia coli strains with differing repair capabilities; Turner MA et al.; Comparative mutagenesis and possible synergistic interaction between broad-spectrum (313- to 405-nm) near-ultraviolet (black light bulb {BLB}) radiation and 254-nm radiation were studied in Escherichia coli strains WP2 (wild type), WP2s (uvrA), WP10 (recA), WP6 (polA), WP6s (polA uvrA), WP100 (uvrA recA), and WP5 (lexA) . With BLB radiation, strains WP2s and WP6s demonstrated a high level of mutagenesis, whereas strains WP2, WP5, WP6, WP10, and WP100 did not demonstrate significant mutagenesis . In contrast, 254-nm radiation was mutagenic in strains WP2, WP2s, WP6, and WP6s, but strains WP5, WP10, and WP100 were not significantly mutated . The absence of mutagenesis by BLB radiation in lexA and recA strains WP10, WP5, and WP100 suggests that lex+ rec+ repair may play a major role in mutagenesis by both BLB and 254-nm radiation . The hypothesis that BLB radiation selectively inhibits rec+ lex+ repair was tested by sequential BLB-254-nm radiation . With strain WP2, a fluence of 30 J/m2 at 254 nm induced trp+ revertants at a frequency of 15 X 10(-6) . However, when 10(5) J/m2 or more of BLB radiation preceded the 254-nm exposure, no trp+ revertants could be detected . A similar inhibition of 254-nm mutagenesis was observed with strain WP6 (polA) . However, strains WP2s (uvrA) and wP6s (polA uvrA) showed enhanced 254-nm mutagenesis when a prior exposure to BLB radiation was given. J Bacteriol, 1981 Aug, 147(2), 333 - 9 Analysis of resynthesis tracts in repaired Escherichia coli deoxyribonucleic acid; Kuemmerle N et al.; Excision repair of ultraviolet radiation-induced damage in a wild-type strain of Escherichia coli has been examined, using two methods for characterizing the resynthesis step of the repair process . Comparison of data obtained after both isopycnic analysis of repaired deoxyribonucleic acid and sedimentation velocity analysis of deoxyribonucleic acid after selective photolysis of bromouracil-containing repaired regions has shown that the repaired deoxyribonucleic acid molecules contain a semicontinuous distribution of sizes of repair tracts . Further analysis of our data suggests two major classes of repair patches, one abut 20 to 40 nucleotides in length, and the other containing 1,600 to 2,000 nucleotides . Under the conditions employed, approximately 2 to 10% of the fully repaired regions are long repair patches. J Bacteriol, 1981 Aug, 147(2), 326 - 32 Cytoplasmic membrane fraction that promotes septation in an Escherichia coli lon mutant; Adler HI et al.; A particulate fraction derived from bacterial cells stimulates septation in irradiated Escherichia coli lon mutants when added to postirradiation plating media . It was established that the particles are derived from the cytoplasmic membrane and that they have been partially purified by sucrose density gradient centrifugation . These particles also contain the cytochrome-based respiratory activity of the cell . A variety of experiments established a correlation between the septation-promoting activity of the particles and their ability to remove oxygen from the postirradiation plating medium . It was suggested that the efficient removal of oxygen from the medium allowed the lon cells to repair radiation-induced damage to the septation mechanism. J Bacteriol, 1981 Aug, 147(2), 289 - 96 Selective inhibition of carbohydrate transport by the local anesthetic procaine in Escherichia coli; Granett S et al.; Maltose and lactose transport systems have been used to investigate the action of procaine on insertion and activity of membrane proteins and translocation of exported proteins in Escherichia coli . Procaine mildly inhibited growth on lactose . The level of inhibition was consistent with the small reduction observed in active and facilitated transport functions of the lac permease . However, procaine caused a severe reduction of growth rate on maltose, as well as an inhibition of induction of maltose regulon activities . In both constitutive and inducible strains, the synthesis of both maltose transport activity (malB operon) and amylomaltase activity (malA operon) was inhibited . Coordinate inhibition of soluble and membrane products was not observed with the lac operon . beta-Galactosidase synthesis proceeded normally during growth on procaine, whereas, the appearance of new transport activity was reduced . Regardless of carbon source, procaine specifically inhibited the appearance of ompF protein in the membrane fraction. Surgery, 1981 Aug, 90(2), 221 - 8 Comparison of the effects of saline and homologous plasma infusion on lung fluid balance during endotoxemia in the unanesthetized sheep; Nylander WA Jr et al.; The effects of saline infusion (20 ml/kg/30 minutes) and homologous plasma infusion (20 ml/kg/30 minutes) on the lung fluid balance during increased pulmonary capillary permeability secondary to Escherichia coli endotoxin infusion (1 microgram/kg/15 minutes) were studied in unanesthetized sheep . Saline and homologous plasma infusion increased lung lymph flow by 10.6% and 10.8%, respectively . The bloodless wet-to-dry ratio was 5.1 +/- 0.2 in the saline group and 5.2 +/- 0.2 in the homologous plasma group . The saline infusion decreased the plasma oncotic pressure while the plasma infusion increased plasma oncotic pressure . However, the increase in plasma oncotic pressure was negated by concomitant changes in the lymph oncotic pressure and greater increases in pulmonary microvascular pressure during the plasma infusion . Changes in pulmonary microvascular pressure predominated over changes in the oncotic pressure gradient . Both saline and homologous plasma infusion increase fluid filtration into the interstitial space by the same magnitude . Therefore neither has a clear advantage in the treatment of pulmonary edema during increased permeability. Proc Natl Acad Sci U S A, 1981 Aug, 78(8), 4917 - 21 In vitro reconstruction of the mitochondrial translation system of yeast; Pfisterer J et al.; We have isolated the translation system from yeast mitochondria and have reconstructed it in vitro . This submitochondrial system, composed of mitochondrial ribosomes, tRNA, pH 5 fraction and mRNA, is maximally active at 10 mM Mg2+ and 100 mM KCl or NH4Cl . NH4+ is more stimulatory than K+ . Added Escherichia coli tRNA gives less than half the activity obtained with added mitochondrial tRNA . Activity is enhanced with protease inhibitors but not with Ca2+, spermine, or spermidine . In contrast to heterologous translation systems, the present system produces products with molecular weights similar to those of products synthesized by yeast mitochondria in vivo and by intact yeast mitochondria in vitro . The results support the idea that the unique coding features of the mitochondrial genome require a unique translation system for accurate translation of mitochondrial mRNAs. Can J Microbiol, 1981 Aug, 27(8), 754 - 8 Effect of chemical and pharmacological agents on the secretory activity induced by Escherichia coli heat-stable enterotoxin; Knoop FC et al.; The effect of aspirin (ASP), chlorpromazine (CPZ), diphenoxylate (DP), ethylene glycol tetraacetate (EGTA), hydrocortisone (HC), loperamide (LPA), methylprednisolone (MP), phenotolamine mesylate (PTM), propranolol (PR), and trifluoperazine (TPZ) on the secretory activity induced by Escherichia coli heat-stable (ST) enterotoxin in infant mice was studied . LPA and DP, which are used therapeutically for diarrhea, did not inhibit the effect of ST enterotoxin; MP and HC, known inhibitors of cholera enterotoxin, and two adrenergic agents (PR and PTM) had no effect on ST-induced secretory activity . TPZ, EGTA, ASP, and CPZ caused a significant (P less than 0.05) decrease in the secretory activity induced by ST enterotoxin, CPZ, EGTA, and TPZ inhibited secretory activity induced by 8-bromoguanosine 3'5'-cyclic monophosphoric acid (8-BrcGMP), a cGMP analog. J Bacteriol, 1981 Aug, 147(2), 694 - 7 Position of the lacZX90 mutation and hybridization between complete and incomplete beta-galactosidase; Mandecki W et al.; The position of the termination codon in lacZX90 was determined by isolation of a lac+ revertant . Lysine was found to replace tyrosine at position 1,012 of beta-galactosidase, indicating that X90 protein lacked the carboxyl-terminal 10 residues . A heat- and urea-sensitive hybrid enzyme was formed in vivo when supC, which supplies tyrosine to the position in the polypeptide corresponding to the nonsense codon, was used to suppress lacZX90 . This result shows that suppression that adds back the original amino acid may not lead to the production of the wild-type enzyme if the latter is multimeric, because incomplete chains can be incorporated into the oligomer. Proc Natl Acad Sci U S A, 1981 Aug, 78(8), 4931 - 5 The product of the lon (capR) gene in Escherichia coli is the ATP-dependent protease, protease La; Chung CH et al.; In Escherichia coli, degradation of abnormal proteins is an energy-requiring process; it is decreased in mutants in the lon (capR or deg) gene . We find that the protein encoded by the lon gene is an ATP-dependent protease and is identical to protease La, recently described in E . coli . Both proteins are serine proteases that hydrolyze casein and globin, but not insulin, in the presence of ATP and Mg2+ . Both respond to ATP, less well to other nucleoside triphosphates, and not to nonhydrolyzable ATP analogs . The purified lon protein has an apparent Mr of 450,000 and appears to be composed of four identical subunits . Its size, chromatographic behavior, and sensitivity to various inhibitors and heat are indistinguishable from those of protease La . Moreover, in a strain that carries additional copies of the lon+ allele on a plasmid, the content of protease La, but not of other proteases, is 2- to 10-fold greater than in the lon+ parent strain . Strains carrying the nonsense mutations capR9 and capR- also contain this ATP-dependent proteolytic activity, but it is present in substantially lower amounts and is inactivated by phosphocellulose chromatography, unlike the wild-type enzyme . Degradation of abnormal proteins in these lon- strains, which is slower than in the wild type, still requires ATP . Alterations in the ATP-dependent protease in the lon- mutants can account for the defect in intracellular proteolysis and perhaps also for the other phenotypic effects of this pleiotropic gene. Proc Natl Acad Sci U S A, 1981 Aug, 78(8), 4728 - 32 ATP hydrolysis-dependent protease activity of the lon (capR) protein of Escherichia coli K-12; Charette MF et al.; Mutations in the lon (capR) gene result in multiple phenotypes, one of which is the failure to degrade abnormal and normal proteins (Deg-) . Previous work with partially purified preparations showed that the lon (capR) gene product is a 94,000-dalton polypeptide with an affinity for nucleic acids . The lon (capR) protein has now been highly purified and is demonstrated to have an ATP-dependent protease activity . The enzyme hydrolyzed 3H-labeled alpha-casein into trichloroacetic acid-soluble forms in Tris buffer containing Mg2+ and ATP . The reaction has a pH optimum of 8.5 and ATP was the preferred nucleotide . CTP and UTP could substitute for ATP (75% and 67%, respectively) but GTP, ADP, AMP, cyclic AMP, and PPi could not . Proteolysis by the lon (capR) protein required ATP hydrolysis . Nonhydrolyzable analogs of ATP and CTP did not promote casein cleavage . When low concentrations of ATP were used, proteolysis stopped as the ATP pool was depleted . Casein stimulated lon (capR) ATPase activity, and the products were ADP and inorganic phosphate in equimolar amounts . No protein kinase activity was detected . The DNA-binding activity, present in partially pure preparations, was retained in the purified protein . The gene product purified from a lon nonsense mutant that exhibits the Deg- phenotype (capR9), lacked both the ATP-dependent protease and ATPase activities, though it retained DNA-binding activity . Absence of an ATP-dependent protease activity could account for many of the pleiotropic effects observed in lon mutants. Proc Natl Acad Sci U S A, 1981 Aug, 78(8), 4786 - 90 Polarity of heteroduplex formation promoted by Escherichia coli recA protein; Kahn R et al.; When recA protein pairs circular single strands with linear duplex DNA, the circular strand displaces its homolog from only one end of the duplex molecule and rapidly creates heteroduplex joints that are thousands of base pairs long {DasGupta, C., Shibata, T., Cunningham, R . P . & Radding, C . M . (1980) Cell 22, 437-446} . To examine this apparently polar reaction, we prepared chimeric duplex fragments of DNA that had M13 nucleotide sequences at one end and G4 sequences at the other . Circular single strands homologous to M13 DNA paired with a chimeric fragment when M13 sequences were located at the 3' end of the complementary strand but did not pair when the M13 sequences were located at the 5' end . Likewise circular single-stranded G4 DNA paired with chimeric fragments only when G4 sequences were located at the 3' end of the complementary strand . To confirm these observations, we prepared fd DNA labeled only at the 5' or 3' end of the plus strand, and we examined the susceptibility of these labeled ends to digestion by exonucleases when joint molecules were formed . Eighty percent of the 5' label in joint molecules became sensitive to exonuclease VII . Displacement of that 5' end by recA protein was concerted because it did not occur in the absence of single-stranded DNA or in the presence of heterologous single strands . By contrast, only a small fraction of the 3' label became sensitive to exonuclease VII or exonuclease I . These observations show that recA protein forms heteroduplex joints in a concerted and polarized way. Gene, 1981 Aug, 14(3), 217 - 25 Transposition of a DNA fragment flanked by two inverted Tn1 sequences; Dobritsa AP et al.; The 32 Md fragment (derived from plasmid RP4::Tn1) carrying the Kmr gene and flanked by two inverted Tn1 elements is capable of recA-independent translocation to other plasmids . We designated this new transposon Tn1755 . In various crosses, frequencies of Tn1755 transposition to plasmids Co1B-R3, R15 and F'Co1VBtrp varied from 2.5 to 90% of the frequencies of Tn1 transposition . Tn1755 can integrate into various sites of the recipient plasmids . We failed to observe transposition of another RP4::Tn1 fragment flanked by two opposingly oriented Tn1 transposons and harboring the Tcr gene . Presumably, to form a new transposable structure, other features must also be of importance. Eur J Biochem, 1981 Aug, 118(1), 195 - 201 Localization of the phosphoester bond hydrolyzed by the major apurinic/apyrmidinic endodeoxyribonuclease from rat-liver chromatin; Verly WG et al.; The major apurinic/apyrimidinic (AP) endodeoxyribonuclease from rat liver chromatin, an enzyme specific for AP sites in DNA, cleaves the phosphodiester bridge which is the immediate neighbour of the AP site on its 5' side leaving 3'-hydroxyl and 5'-phosphate ends . In contrast with Escherichia coli endonuclease VI, this chromatin enzyme is inactive on reduced AP sites. Infect Immun, 1981 Aug, 33(2), 459 - 66 Effect of fractions of Ethiopian And Norwegian colostrum on rotavirus and Escherichia coli heat-labile enterotoxin; Otnaess AB et al.; Samples of colostrum from both Ethiopian and Norwegian women contained antirotavirus activities of immunoglobulin and non-immunoglobulin nature . No significant differences in rotavirus immunoglobulin A or in rotavirus-inhibiting activity were found between samples from the two countries . The non-immunoglobulin inhibitory activity was trypsin sensitive and heat stable (100 degrees C for 10 min) . Escherichia coli heat-labile enterotoxin antibodies were measured in the colostrum samples by enzyme-linked immunosorbent assay . No E . coli enterotoxin-specific immunoglobulin A was detected, possibly due to the high background caused by the nonspecific adsorption of immunoglobulin A to the enzyme-linked immunosorbent assay plates in the absence of toxin . A total of 5 of 15 Ethiopian colostrum samples and 0 of 11 Norwegian colostrum samples neutralized the effect of E . coli heat-labile enterotoxin on YI adrenal cells . Both the Ethiopian and the Norwegian colostrum samples contained a non-immunoglobulin enterotoxin-inhibitory activity when the toxin was measured by enzyme-linked immunosorbent assay . This inhibitory activity was not trypsin sensitive, and extraction by chloroform-methanol indicated that the inhibitor was of a lipid nature. Infect Immun, 1981 Aug, 33(2), 401 - 6 Diarrhea in lambs: experimental infections with enterotoxigenic Escherichia coli, rotavirus, and Cryptosporidium sp; Tzipori S et al.; Thirteen gnotobiotic lambs, aged from a few hours to 8 days, were inoculated orally with single infections of enterotoxigenic Escherichia coli (ETEC) (four animals), lamb rotavirus (five animals), and Cryptosporidium (four animals) . Six gnotobiotic and two specific-pathogen-free lambs were co-inoculated with either rotavirus and ETEC (four animals), rotavirus and Cryptosporidium (two animals), or ETEC and Cryptosporidium (two animals) . Lambs 4 days of age and older became only subclinically infected with either rotavirus, ETEC (08:K87:K99 ST+), or both enteropathogens given simultaneously . Six-day-old lambs inoculated with Cryptosporidium became extremely depressed, anorectic, and had intermittent diarrhea . There was no difference in the clinical manifestations, level of disaccharidase activity in the small intestine, or extent of histological damage between lambs inoculated with Cryptosporidium alone or together with either of the other two agents . The results indicate that under the conditions of these experiments, lambs become clinically resistant to infection with ETEC, rotavirus, or both agents together, by 4 days after birth, whereas lambs 2 days old or younger were clinically susceptible to infection by these agents . In contrast, they remained clinically susceptible to infection with Cryptosporidium up to at least 6 days of age . Cryptosporidium infections were not aggravated by coinfection with either ETEC or rotavirus. Biokhimiia, 1981 Aug, 46(8), 1526 - 9 {DNA-methylase from Arthrobacter luteus screens DNA from the action of site-specific endonuclease Alu I}; Kramarov VM et al.; DNA-methylase was isolated from a cell extract of A . luteus and partially purified by chromatography on phosphocellulose . The purified enzyme methylates DNA of phage lambda and plasmids pBr322, thus making them resistant to a subsequent action of endonuclease Alu I . It has been shown that cytosine is the object of methylation within DNA . This modification does not screen DNA from the action of site-specific endonucleases Sal I, Bam HI, EcoR I, EcoR II, Xho I and Xho II . It has been experimentally demonstrated that the isolated methylase is site-specific and identifies in the DNA the nucleotide sequence 5'-AGCT-3', by methylating cytosine in the DNA. J Bacteriol, 1981 Aug, 147(2), 552 - 62 Cloning of genes involved in membrane lipid synthesis: effects of amplification of phosphatidylglycerophosphate synthase in Escherichia coli; Ohta A et al.; The structural gene (pgsA) for the CDP-diacylglycerol:sn-glycero-3-phosphate phosphatidyltransferase (EC 2.7.8.5, phosphatidylglycerophosphate synthase) from Escherichia coli has been cloned, using pSC101 as the vector . The resulting hybrid plasmids not only correct the lack of in vitro synthase activity in pgsA strains but also cause an amplification (6- to 40-fold over wild-type levels) in enzymatic activity in direct proportion to the copy number of the plasmids found in vivo . The cloned gene also corrects the abnormally low level of polyglycerophosphatides found in pgsA strains and actually increases the level of phosphatidylglycerol to above that normally found in E . coli . The degree of alteration in phospholipid composition brought about by these hybrid plasmids is not of the order expected if fluctuations in enzyme levels in vivo were an important regulatory mechanism in phospholipid metabolism . The isolated hybrid plasmids have been mapped by restriction endonuclease analysis . The presence and location of other genetic markers have also been established . The above data, along with analysis of deletion derivatives of these plasmids and subcloning of appropriate restriction fragments, have established the position of the pgsA locus on the hybrid plasmids . From this data, the position of the pgsA locus has been determined to le between flaI and uvrC on the E . coli genetic map. J Bacteriol, 1981 Aug, 147(2), 500 - 8 Cyclic adenosine 3',5'-monophosphate-mediated hyperinduction of araBAD and lacZYA expression in a crp mutant of Escherichia coli K-12; Bankaitis VA et al.; A spontaneous lac+ revertant of an adenylate cyclase deletion strain of Escherichia coli K-12 was isolated and characterized . This revertant, designated strain KC20, exhibited a pleiotropic suppression of the adenylate cyclase defect, with the crp locus being the site of the suppressor mutation . Cyclic adenosine 3',5'-monophosphate at an exogenous concentration of 1 mM severely inhibited the growth of strain KC20 in minimal media . Lower concentrations of the cyclic nucleotide elicited less pronounced effects . Studies on araBAD and lacZYA expression showed that cyclic adenosine 3',5'-monophosphate elicited an initial dose-dependent hyperinduction of these systems . Hyperinduction of araBAD, in L-arabinose grown cultures of strain KC20, resulted in accumulation of inhibitory concentrations of methylglyoxal . Hyperinduction of lacZYA in lactose-grown cultures of strain KC20 did not result in any such methylglyoxal production. J Bacteriol, 1981 Aug, 147(2), 297 - 304 Identification of the tetracycline resistance promoter and repressor in transposon Tn10; Wray LV Jr et al.; The structural and regulatory functions encoding tetracycline resistance in transposon Tn10 lie within a 2,700-base pair region . Using recombinant plasmids with different deoxyribonucleic acid sequences adjacent to a HincII site in this region, we located the promoter controlling the expression of tetracycline resistance . These various sequences conferred altered levels of tetracycline resistance . Plasmids containing deletions of a 695-base pair HincII fragment were constitutive and showed the loss of a 23,000-dalton tetracycline-inducible polypeptide, thus identifying the repressor and the location of its gene. Proc Natl Acad Sci U S A, 1981 Aug, 78(8), 4753 - 7 Site-specific mutagenesis on cloned DNAs: generation of a mutant of Escherichia coli tyrosine suppressor tRNA in which the sequence G-T-T-C corresponding to the universal G-T-pseudouracil-C sequence of tRNAs is changed to G-A-T-C; Kudo I et al.; We have cloned the Escherichia coli tyrosine-inserting amber suppressor tRNA gene into the recombinant single-strand phage M12mp3 . By using the M13mp3SuIII+ recombinant phage DNA as template and an oligonucleotide bearing a mismatch as primer, we have synthesized in vitro an M13mp3SuIII heteroduplex DNA that has a single mismatch at a predetermined site in the tRNA gene . Transformation of E . coli with the heteroduplex DNA yielded M13 recombinant phages carrying a mutant suppressor tRNA gene in which the sequence G-T-T-C, corresponding to the universal G-T-pseudouracil-C sequence in E . coli tRNAs, is changed to G-A-T-C . The mutant DNA has been characterized by restriction mapping and by sequence analysis . In contrast to results with the wild-type suppressor tRNA gene, cells transformed with recombinant plasmids carrying the mutant tRNA gene are phenotypically Su- . Thus, the single nucleotide change introduced has inactivated the function of the tRNA gene . By using E . coli minicells for studying the expression in vivo of cloned tRNA genes, we have found that cells transformed with recombinant plasmids carrying the mutant tRNA gene contain very little, if any, mature mutant suppressor tRNA . In contrast, the predominant low molecular weight RNA in cells transformed with recombinant plasmids carrying the wild-type suppressor tRNA gene is the mature tyrosine suppressor tRNA . Thus, while our results imply an important role for the G-T-pseudouracil-C sequence common to all E . coli tRNAs, whether this sequence is essential for tRNA biosynthesis, tRNA stability in vivo, or tRNA function remains to be determined . The procedures used to generate the mutant should be of general application toward site-specific mutagenesis on cloned DNAs, including regions that possess high degrees of secondary structure . In addition, the frequency of mutants among the progeny is high enough to enable one to identify and isolate site-specific mutants on any cloned DNA without requiring phenotypic selection. J Biochem (Tokyo), 1981 Aug, 90(2), 449 - 61 Conformational studies of Escherichia coli ribosomes with the use of acridine orange as a probe; Horie K et al.; The interaction of E . coli vacant ribosomes with acridine orange (AO) was studied, to obtain conformational information about rRNAs in ribosomes . Acridine orange binds to an RNA in two different modes: cooperative outside binding with stacking of bound AO's and intercalation between nucleotide bases . Free 16S and 23S rRNAs have almost identical affinities to AO . At 1 mM Mg2+, AO can achieve stacking binding on about 40% of rRNA phosphate groups . The number of stacking binding sites falls to about 1/3 in the 30S subunit in comparison with free 16S rRNA . In the 50S subunit, the number of stacking binding sites is only 1/5 in comparison with free 23S rRNA . Mg2+ ions are more inhibitory for the binding of AO to ribosomes than to free rRNAs . The strength of stacking binding appears to be more markedly reduced by Mg2+ in active ribosomes than in rRNAs . "Tight couple" 70S particles are less accessible for stacking binding than free subunits . The 30S subunits that have irreversibly lost the capability for 70S formation under low Mg2+ conditions have an affinity to AO that is very different from that of active 30S but similar to that of free rRNA, though the number of stacking binding sites is little changed by the inactivation . 70S and 30S ribosomes with stacking bound AO's have normal sedimentation constants, but the 50S subunits reversibly form aggregates. Eur J Biochem, 1981 Aug, 118(1), 187 - 94 Size and shape of the alpha subunit of the (Ca,Mg)-dependent adenosinetriphosphate from Escherichia coli in solution in the presence and absence of ATP; Paradies HH; The alpha subunit of the (Ca, Mg)-dependent ATPase from Escherichia coli was studied in solution by means of X-ray scattering experiments at variable contrast and in the presence of ATP . The experiments were carried out on an absolute scale in the range of (45.0 nm-1) less than h less than (1.5 nm-1) at pH 8.0 . The experiments show that this system and the complex of the alpha subunit with ATP can be considered to be ideal and monodisperse so that the following parameters for the alpha subunit and its complex with ATP were determined: the radius of gyration, the volume, the degree of hydration, the maximum particle diameter, and the molecular weight . The molecular weight of the alpha subunit was determined as 58 500 +/- 3000 by means of light scattering measurements, and 57 700 +/- 25 000 by the small-angle X-ray scattering experiments . The radius of gyration of the alpha subunit at pH 8.0 was determined to 2.64 +/- 0.02 nm, the maximum chord in solution to 10.0 +/- 0.5 nm, the volume to 102.4 +/- 2.5 nm3, and the degree of hydration to 0.34 +/- 0.02 mg X g-1 . Binding of ATP to the alpha subunit causes structural changes . These changes are reflected in the radius of gyration, Rg = 2.45 +/- 0.015 nm, in the volume, 117.6 +/- 1.5 nm3, and in the maximum chord length in solution, 7.8 +/- 0.5 nm . These changes imply a decrease of the axial ratio from 2.4 to 1.4, i.e . ATP-binding induced an increase in isometry of the alpha subunit. Biochim Biophys Acta, 1981 Jul 27, 654(2), 187 - 93 Differential effect of N-carboxymethylisatoylation on the DNA polymerase activity, the 5' leads to 3'-exonuclease activity and the 3' leads to 5'-exonuclease activity of DNA polymerase I of Escherichia coli; Klenow H et al.; Treatment with native DNA polymerase I of Escherichia coli with the acylating agent N-carboxymethylisatoic acid anhydride (NCMIA) results under specific conditions in a rapid loss of polymerase activity, an increase in 5' leads to 3'-exonuclease activity and in unchanged 3' leads to 5'-exonuclease activity . When a nucleoside triphosphate and Mg2+ was present the polymerase activity was completely protected against the effect of NCMIA . Treatment with higher concentration of the acylating agent under these conditions led to a loss of 3' leads to 5'-exonuclease activity without any appreciable loss of polymerase activity . Treatment with NCMIA of the two catalytically active fragments of the enzyme led to very similar results . In this case both the polymerase activity and the 3' leads to 5'-exonuclease activity deteriorated more rapidly on treatment with the acylating reagent . The increase in 5' leads to 3'-exonuclease activity as a result of modification of the native enzyme appeared to be due to a change in the optimum conditions with regard to concentration of the assay buffer used . These changes are very similar to those seen when the polymerase is cleaved by limited proteolysis . From the results obtained it is concluded that NCMIA reacts primarily with a site at or near the triphosphate-Mg2+ complex binding site, leading to an almost complete loss of polymerase activity . The acylating reagent reacts also with another group on the native enzyme resulting in a modification of the 5' leads to 3'-exonuclease activity, and at high concentrations with a group leading to a slow loss of 3' leads to 5'-exonuclease activity. S Afr Med J, 1981 Jul 25, 60(4), 129 - 30 Cholestyramine as an aid to management of prolonged diarrhoea in the hospital drip-room . Preliminary findings; Freedman AM et al.; Two groups of children with diarrhoea of more than 7 days' duration were studied . While the length of stay in hospital of the two groups was similar, there was a significant improvement in the frequency and consistency of the stools of the children given cholestyramine . This improvement was also noted in children with positive stool cultures . Cholestyramine also plays an important role in reducing the number of children or intravenous therapy at any time. J Biol Chem, 1981 Jul 25, 256(14), 7565 - 72 Homologous pairing in genetic recombination . The pairing reaction catalyzed by Escherichia coli recA protein; Shibata T et al.; Purified recA protein, which is essential for genetic recombination of Escherichia coli, catalyzed ATP-dependent homologous pairing of double-stranded DNA and single-stranded fragments to form D-loops . When the double-stranded DNA was nicked circular DNA (form II) or linear DNA (form III), the reaction proceeded nearly linearly during 30 min of incubation at 37 degrees C . When the double-stranded DNA was superhelical (form I), anomalous kinetics was observed . This anomaly was suppressed by the addition of spermidine without affecting the final yield of D-loops . The formation of D-loops required stoichiometric amounts of recA protein, which were proportional to the concentration of single-stranded DNA but which were not affected by the concentration of double-stranded DNA . With form II or III DNA as the recipient for the formation of D-loops, the rate of the reaction was greatest when there was one monomer of recA protein/2-3 nucleotide residues of single-stranded DNA; larger amounts of single-stranded DNA inhibited the reaction . The formation of D-loops was half inhibited by 30 mM NaCl and by 0.6 mM ADP, one of the products of the reaction . The thermal stability of D-loops made by recA protein was the same as that of D-loops made by annealing . In addition to pairing linear single strands with duplex DNA, recA protein made joint molecules from single-stranded circular DNA and homologous form II or III DNA . According to these and previous observations (Cunningham, R . P., DasGupta, C., Shibata, T., and Radding, C . M . (1980) Cell 20, 223-235), rcA protein will stably pair two molecules of DNA if one of them is single-stranded or partially single-stranded and if either molecule has a free end. J Biol Chem, 1981 Jul 25, 256(14), 7416 - 23 Inactivation of D-serine dehydratase by alkylamines via a transimination of enzyme-linked cofactor; Federiuk CS et al.; The time-dependent inactivation of D-serine dehydratase by alkylamines was characterized . Evidence is presented indicating that inactivation proceeds via a transimination reaction analogous to the first step in the catalytic pathway . The product of alkylamine attack, an alkylamine pyridoxal 5'-phosphate Schiff base, readily dissociated from D-serine dehydratase to produce inactive apoenzyme . Reaction with alkylamines was shown to be a convenient way of producing apoenzyme which can be reconstituted with pyridoxal 5'-phosphate to fully active holoenzyme . Amino acids such as glycine and alanine, unlike alkylamines, did not resolve D-serine dehydratase but were competitive inhibitors of the enzyme . The inability of amino acids to resolve D-serine dehydratase from its cofactor was attributed to a failure of the amino acid-cofactor Schiff base to dissociate from the enzyme . Transimination of D-serine dehydratase with its substrate D-serine was at least 3.5 X 10(5) times faster than a nonenzymic model transimination reaction and more than 70 million times faster than the reaction of the enzyme with 2-hydroxyethylamine, indicating that the carboxyl group of the substrate is an important structural determinant for catalysis of the transimination step in the catalytic pathway . An analysis of the inhibitory effect of potassium ion on the rate and extent of inactivation of D-serine dehydratase by alkylamines indicated that K+ binding increased the affinity of the enzyme for its cofactor by at least 13-fold. J Biol Chem, 1981 Jul 25, 256(14), 7557 - 64 Homologous pairing in genetic recombination . Purification and characterization of Escherichia coli recA protein; Shibata T et al.; RecA protein, which is essential for genetic recombination in Escherichia coli, was extensively purified from a strain of E . coli which contained the recA gene cloned in a plasmid (Sancar, A., and Rupp, W . D . (1979) Proc . Natl . Acad . Sci . U . S . A . 76, 3144-3148) . Using the DNA-dependent ATPase activity of recA protein as an assay, we obtained about 60 mg of purified recA protein from 100 g of cells . Ten micrograms or 1 microgram of the purified protein exhibited only one detectable band with Mr approximately = 40,000 upon sodium dodecyl sulfate-acrylamide gel electrophoresis . More than 99% of the ATPase activity of purified recA protein was dependent on single-stranded DNA . Purified recA protein had no detectable DNase, topoisomerase, or ligase activities . The enzyme was stable for a least a year when stored at 0-4 degrees C . The half-life of the ATPase activity of 25 microM recA protein was 37 min at 51 degrees C . Purified recA protein binds to single-stranded and double-stranded DNA, unwinds duplex DNA by a mechanism that is stimulated by single-stranded DNA or oligonucleotides, and pairs homologous single strands with duplex DNA. J Biol Chem, 1981 Jul 25, 256(14), 7573 - 82 Escherichia coli recA protein protects single-stranded DNA or gapped duplex DNA from degradation by RecBC DNase; Williams JG et al.; RecA- mutants of Escherichia coli extensively degrade their DNA following UV irradiation . Most of this degradation is due to the recBC DNase, which suggests that the recA gene is involved in the control of recBC DNase in vivo . We have shown that purified recA protein inhibits the endonuclease and exonuclease activities of recBC DNase on single-stranded DNA . The extent of inhibition is dependent on the relative concentration of recA protein, recBC DNase, and the DNA substrate; inhibition is greatest when the concentrations of DNA and recBC DNase are low and the concentrations of recA protein is high . At fixed concentrations of recA protein and recBC DNase, inhibition is eliminated at high concentrations of DNA . In the presence of adenosine 5'-O-(3-thiotriphosphate), an ATP analog which stabilizes the binding of recA protein to both single- and double-stranded DNA, recA protein is a more potent inhibitor of the nuclease activities on single-stranded DNA and is a weak inhibitor of the exonuclease activity on double-stranded DNA . Inhibition of the latter is enhanced by oligodeoxynucleotides, which stimulate the binding of recA protein to double-stranded DNA . In the presence of adenosine 5'-O-(3-thiotriphosphate), recA protein also inhibits the action of exonuclease I on single-stranded DNA and of lambda exonuclease on double-stranded DNA . These observations are most consistent with the idea that recA protein protects DNA from recBC DNase by binding to DNA . RecA protein also blocks the endonucleolytic cleavage of gapped circular DNA by recBC DNase . Since both recA protein and recBC DNase have the ability under certain conditions to unwind duplex DNA and to displace strands, we looked for evidence that their combined action would enlarge gaps but found no extensive enlargement . D-loops, a putative intermediate in genetic recombination, are effectively protected against the action of recBC DNase by the E . coli single strand binding protein and by recA protein in the presence of adenosine 5'-O-(3-thiotriphosphate). J Biol Chem, 1981 Jul 25, 256(14), 7322 - 8 Structure of the metal-nucleotide complex in the acetate kinase reaction . A study with gamma-32P-labeled phosphorothioate analogs of ATP; Romaniuk PJ et al.; The synthesis of the gamma-32P-labeled diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) and the Sp isomer of adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) by a modification of the Glynn and Chappell method (Glynn, I . M., and Chappell, J . T., (1964) Biochem . J . 90, 147-149) is described . These analogs were tested as substrates for acetate kinase in the presence of several divalent metal ions . Both isomers of ATP alpha S are substrates in the presence of Mg2+, Mn2+, Co2+, Zn2+, and Cd2+, the Sp isomer being preferred by a factor of between 4.8 (Mg2+) and 52.5 (Cd2+) . Only the Rp isomer of ATP beta S is a substrate in the presence of Mg2+, and the Sp isomer becomes a better substrate in the presence of Mn2+, Co2+, and Zn2+; both isomers are equally good substrates in the presence of Cd2+ . The change in specificity upon replacing Mg2+ by Cd2+ is greater than 1800 at beta-phosphorus and 10 at alpha phosphorus . These results provide a basis for proposing that the lambda screw sense configuration of the beta, gamma-bidentate MgATP complex is the substrate for acetate kinase . In the reverse reaction, both Sp and Rp isomers of ADP alpha S are substrates in the presence of all metal ions tested, the Sp isomer preferred by a factor between 12.3 (Mg2+) and 45.5 (Cd2+) . In the presence of Mg2+, Mn2+, and Co2+, only the Rp isomer of ATP beta S is synthesized from prochiral ADP beta S, while a mixture of Rp and Sp isomers is synthesized in the presence of Zn2+ and Cd2+ . These results are analogous to those for the forward reaction and suggest that the Mg.ADP complex which binds as a substrate in the reverse reaction, and is released as a product in the forward reaction, is the beta-monodentate . The classification of acetate kinase as an enzyme having a type I mechanism (Dunaway-Mariano, D . and Cleland, W . W . (1980) Biochemistry 19, 1506-1515) for kinases, is discussed. Nucleic Acids Res, 1981 Jul 24, 9(14), 3465 - 81 The proteins of the messenger RNA binding site of Escherichia coli ribosomes; Gimautdinova OI et al.; Oligo(U) derivatives with {14C}-4-(N-2-chloroethyl-N-methylamino)benzaldehyde attached to 3'-end cis-diol group via acetal bond, p(Up)n-1UCHRCl as well as with {14C}-4-(N-2-chloroethyl-N-methylamino)benzylamine attached to 5'-phosphate via amide bond, ClRCH2NHpU(pU)6 were used to modify 70S E . coli ribosomes near mRNA binding centre . Within ternary complex with ribosome and tRNAPhe all reagents covalently bind to ribosome the extent of modification being 0.1-0.4 mole/mole 70S . p(Up)n-1UCHRCl alkylates either 30S (n=5,7) or both subunits (n=6,8) . rRNA is preferentially modified within 30S subunit . ClRCH2NHpU(pU)6 alkylates both subunits the proteins being mainly modified . The distribution of the label among proteins differ for various reagents . S4, S5, S7, S9, S11, S13, S15, S18 and S21 are found to be alkylated within 30S subunit, proteins L1, L2, L6, L7/L12, L19, L31 and L32 are modified in the 50S subunit . Most proteins modified within 30S subunit are located at the "head" of this subunit and proteins modified within 50S subunit are located at the surface of the contact between this subunit and the "head" of 30S subunit at the model of Stoffler. Nucleic Acids Res, 1981 Jul 24, 9(14), 3287 - 306 Secondary structure of the large subunit ribosomal RNA from Escherichia coli, Zea mays chloroplast, and human and mouse mitochondrial ribosomes; Glotz C et al.; Short base-paired RNA fragments, and fragments containing intra-RNA cross-links, were isolated from E . coli 23S rRNA or 50S ribosomal subunits by two-dimensional gel electrophoresis . The interactions thus found were used as a first basis for constructing a secondary structure model of the 23S rRNA . Sequence comparison with the 23S rDNA from Z . mays chloroplasts, as well as with the 16S (large subunit) rDNA from human and mouse mitochondria, enabled the experimental model to be improved and extrapolated to give complete secondary structures of all four species . The structures are organized in well-defined domains, with over 450 compensating base changes between the two 23S species . Some ribosomal structural "'switches" were found, one involving 5S rRNA. Biochim Biophys Acta, 1981 Jul 24, 660(1), 16 - 22 NADP-specific isocitrate dehydrogenase from Escherichia coli . V . Multiple forms of the enzyme; Vasquez B et al.; Two forms of NADP-specific isocitrate dehydrogenase (threo-DS-isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) in Escherichia coli have been resolved by polyacrylamide gel isoelectric focusing and electrophoresis . Incubation of the enzyme with Mn2+ plus isocitrate prior to focusing resulted in the formation of an additional form of the enzyme, presumably the enzyme-manganese-isocitrate complex . Glycerol, a cryoprotectant used to stabilize the enzyme during purification and storage, also stabilized in during focusing, but was not necessary during electrophoresis . Thin-layer gel filtration did not reveal any differences in molecular weight between the different species of isocitrate dehydrogenase. Nucleic Acids Res, 1981 Jul 24, 9(14), 3483 - 90 Transient cleavage kinetics of the Eco RI restriction endonuclease measured in a pulsed quench-flow apparatus: enzyme concentration-dependent activity change; Langowski J et al.; We report measurements of the cleavage rate of pBR 322 plasmid DNA by the restriction endonuclease Eco RI as a function of enzyme and DNA concentration . The reaction, which at high excess of enzyme over DNA occurs between 0.2 and 5 seconds, was studied by the means of a microprocessor controlled pulsed quench-flow apparatus . Enzyme concentrations were between 1 and 100 nM with DNA concentrations being 3 to 6 nM (specific Eco RI sites) . The catalytic constants for cleavage of the first and second phosphodiester bonds as measured at high enzyme concentration both have the same value of 0.35 sec-1 and 21 degrees C . At enzyme concentrations comparable to or less than DNA concentration, the rate of the first cleavage is proportional to enzyme concentration, while the second step is independent of concentration . At approx . 10 nM Eco RI endonuclease concentration, a rate increase shows up in both the first and the second cleavage . We suggest that this increase is due to the tetramerization reported by Modrich & Zabel1, which occurs in this concentration range. Nucleic Acids Res, 1981 Jul 24, 9(14), 3219 - 33 Organization of ribosomal RNA gene repeats of the mouse; Kominami R et al.; The organization of the ribosomal RNA (rRNA) genes of the mouse was determined by Southern blot hybridization using cloned rDNA fragments as probes, which could encompass the entire spacer region between two rRNA gene regions . The rRNA genes are organized into tandem repeats of nearly uniform length of about 44 kb . The heterogeneity detected in the nontranscribed spacer appears to be caused by its sequence rather than its length difference . At least three kinds of repetitive sequences are present in the non-transcribed spacer region; two of them are located 13 kb upstream from the 5'-end of 18S RNA gene and the other located 1 to 4 kb downstream from the 3'-end of 28S RNA gene. Biochim Biophys Acta, 1981 Jul 24, 660(1), 142 - 7 Evidence for an essential arginine residue at the active site of Escherichia coli acetate kinase; Wong SS et al.; Escherichia coli acetate kinase (ATP: acetate phosphotransferase, EC 2.7.2.1.) was inactivated in the presence of either 2,3-butanedione in borate buffer or phenylglyoxal in triethanolamine buffer . When incubated with 9.4 mM phenylglyoxal or 5.1 mM butanedione, the enzyme lost its activity with an apparent rate constant of inactivation of 0.079 min-1, respectively . The loss of enzymatic activity was concomitant with the loss of an arginine residue per active site . Phosphorylated substrates of acetate kinase, ATP, ADP and acetylphosphate as well as AMP markedly decreased the rate of inactivation by both phenylglyoxal and butanedione . Acetate neither provided any protection nor affected the protection rendered by the adenine nucleotides . However, it interfered with the protection afforded by acetylphosphate . These data suggest that an arginine residue is located at the active site of acetate kinase and is essential for its catalytic activity, probably as a binding site for the negatively charged phosphate group of the substrates. Nucleic Acids Res, 1981 Jul 24, 9(14), 3403 - 17 Structural complexity of defective-interfering RNAs of Semliki Forest virus as revealed by analysis of complementary DNA; Soderlund H et al.; The 18S defective interfering RNA of Semliki Forest virus has been reverse transcribed to cDNA, which was shown to be heterogeneous by restriction enzyme analysis . After transformation to E.coli, using pBR322 as a vector, two clones, pKTH301 and pKTH309 with inserts of 1.7 kb and 2 kb, were characterized, respectively . The restriction maps of the two clones were different but suggested that both contained repeating units . At the 3' terminus, pKTH301 had preserved 106 nucleotides and pKTH309 102 nucleotides from the 3' end of the viral 42S genome . The conserved 3' terminal sequence was joined to a different sequence in the two clones, and these sequences were not derived from the region coding for the viral structural proteins . The DI RNAs represented by the two clones are generated from the viral 42S RNA by several noncontinuous internal deletions, since the largest colinear regions with 42S RNA are 320 nucleotides in pKTH301, and 430 and 340 nucleotides in pKTH309 . All these fragments had unique RNase T1 oligonucleotide fingerprints, suggesting that they were derived from different regions of 42S RNA. Biochemistry, 1981 Jul 21, 20(15), 4243 - 6 Sulfhydrylcellulose: a new medium for chromatography of mercurated polynucleotides; Feist PL et al.; We have synthesized a new medium, sulfhydrylcellulose, for affinity chromatography of mercurated polynucleotides . It is the product of reaction between aminoethylcellulose and N-acetylhomocysteine thiolactone . Sulfhydrylcellulose carries up to 90 mumol of SH groups/g and is inexpensive, easy to prepare, and stable . Because it binds mercurated RNA specifically and reversibly and exhibits no size discrimination, sulfhydrylcellulose should have wide applications. Boll Soc Ital Biol Sper, 1981 Jul 15, 57(13), 1437 - 42 The characterization of the area of rat mitochondrial DNA containing the large ribosomal RNA gene; Saccone C et al.; The base sequence of the Eco-RI D fragment from rat liver mtDNA, cloned in recombinant plasmid, has been analyzed, This fragment contains the genes for the 16SRNA of the large ribosomal subunit and the tRNA(Leu) . Comparisons between these genes and corresponding regions either in other mitochondrial genomes or in E . coli DNA are presented, that allow some interesting evolutionary and phylogenetic considerations. Nucleic Acids Res, 1981 Jul 10, 9(13), 3139 - 50 Radiation induced DNA double strand breaks are rejoined by ligation and recombination processes; Weibezahn KF et al.; Using the method of filter elution of double stranded DNA under neutral conditions we have shown that most of gamma-ray induced double strand breaks (DSB) are rejoined in both mammalian and bacterial cells . Rejoining also occurs in the G1 phase in V79 Chinese hamster cells and under different growth conditions . Within 8 minutes at 37 C, half the breaks are rejoined . The rejoining in E . coli is equally fast and depends on the presence of DNA ligase . Some of the breaks in E . coli rejoin slowly, and these require rec+ . The non-rejoined DSB are distributed over the DNA without any preference for the nucleosomal or the linker structure in the chromosome . Two kinds of DSB rejoining are discriminated, a fast process of DNA ligation and a slower process involving rec functions. Nucleic Acids Res, 1981 Jul 10, 9(13), 3061 - 75 Cloning of a new mouse foetal beta-globin mRNA sequence; Affara NA et al.; A novel globin cDNA recombinant (pFG5) has been isolated from a 14-15 day Porton mouse foetal liver cDNA library . It codes for a beta-like globin mRNA expressed in foetal liver-derived erythroblasts and erythrocytes but not in adult reticulocytes nor in yolk sac derived nucleated erythrocytes . It is also found in Friend cells induced to differentiate by DMSO . The nucleotide sequence of pFG5 confirms that it does not code for the beta major or beta minor globin chains nor the embryonic epsilon Y2 globin chain; but it is identical to the published partial sequence of the epsilon Y3 globin gene over the region of overlap (78 nucleotides). J Biol Chem, 1981 Jul 10, 256(13), 6796 - 803 A conformational study of thioredoxin and its tryptic fragments; Reutimann H et al.; The absorption, fluorescence, and circular dichroism properties of Escherichia coli thioredoxin, and of its tryptic fragments thioredoxin-T-(1-73) and thioredoxin-T-(74-108), in water and in trifluoroethanol, have been investigated as a function of pH and temperature in order to gain information about their conformational behavior . Both reduced and oxidized thioredoxin have a remarkable conformational stability as judged from CD spectra at various pH values and temperatures . The percentage of secondary structure in solution was calculated using the procedures suggested in the literature, but no satisfactory agreement with the x-ray data could be obtained . The reasons for this discrepancy are discussed . The fluorescence spectrum shows an intense tyrosine contribution, whose intensity is strongly pH-dependent in the acidic region . An interaction with a carboxylate group (tentatively Tyr-49 with Asp-104) is suggested . The pH dependence of the fluorescence intensity of tryptophan in reduced thioredoxin is also surprisingly marked, and a group with pK = 6.4, having a high quenching efficiency in the deprotonated form, is deemed responsible for this behavior . This is ascribed to one of the two reactive cysteine residues, whose negative charge is probably stabilized by Lys-36 . Various spectroscopic/conformational studies have been carried out on the two fragments (1-73) and (74-108) . The salient result is that the (1-73) fragment, which assumes a largely aperiodic structure at neutral and alkaline pH, is able to refold in a globular and stable structure when the pH is below approximately 3.0 . This process is attended by marked changes in the fluorescence and CD spectra . The refolding mechanism and its implications are discussed. J Biol Chem, 1981 Jul 10, 256(13), 6751 - 5 Differences among subfractions of H1 histone in their interactions with linear and superhelical DNA . Circular dichroism; Liao LW et al.; Interactions between subfractions of ox thymus H1 histone and either linear T7 DNA or superhelical PM2 DNA were studied by measuring the circular dichroism of H1:DNA complexes . H1 subfractions differed from one to the next in their effectiveness at distorting the circular dichroic spectrum of DNA by as much as 3- to 4-fold for both forms of DNA . The order of effectiveness of the subfractions was the same at all ionic strengths between 0.05 M and 0.25 M, but the degree of spectral distortion caused by any of the subfractions was sensitive to the salt concentration . At 0 M NaCl and above 3 M NaCl, there was little or no distortion of the spectrum of DNA by any subfraction; the maximum effectiveness for all of the subfractions was at 0.15 M to 0.2 M NaCl whether the DNA was linear or superhelical . Between 0 M and 0.15 M NaCl, the H1 subfractions in free solution underwent a conformational change from a substantially unfolded state to one that is presumably the native state . This was revealed by circular dichroism . In part, this folding of the protein molecules must account for the effect of salt on the ability of H1 to distort the circular dichroism of DNA when the two macromolecules are brought together in complex formation . The distortion of the circular dichroism of DNA by H1:DNA complex formation is thought to be due to side-by-side aggregation of fibers in an asymmetrically ordered array . Apparently, the different H1 subfractions induce formation of H1:DNA complexes that differ in degree of orderliness or in a more complicated geometric parameter of the array, and this is true for superhelical as well as linear DNA. J Biol Chem, 1981 Jul 10, 256(13), 6729 - 35 The role of spermine in preventing misacylation by phenylalanyl-tRNA synthetase; Loftfield RB et al.; Physiological concentrations of polyamines contribute significantly to both the speed and precision of aminoacylation of tRNA . Unphysiologically high concentrations of magnesium ion are required to obtain high rates of synthesis of phenylalanyl-tRNAPheyeast catalyzed by yeast phenylalanine:tRNA ligase in vitro . Under such conditions, rates of misacylation (e.g . the synthesis of Phe-tRNAValE.coli) may be one-fifth of the rate of correct acylation . High rates of correct aminoacylation are achieved in the presence of physiological concentrations of magnesium (1.0 mM) plus spermine (0.2 mM) . Under these conditions, there is almost no misacylation . A kinetic study of the magnesium dependence of aminoacylation shows that the rate-determining transition state for Phe-tRNAPhe synthesis contains, in addition to tightly bound Mg2+, either two spermines (Kd = approximately 50 microM) or 2 Mg2+ ions (Kd = approximately 1.0 mM) . We postulate that the tRNAPhe binds two spermines to form a very compact, very precisely defined structure that easily forms an activated E.S conformer, E.S (Jencks' Circe Effect) . In the absence of spermine, 2 Mg2+ ions bind somewhat more poorly to the tRNA to form a similar but not identical tRNA conformer which, in turn, is less able to form E.S and thus more slowly aminoacylated . Misacylation of noncognate tRNA requires several additional, more loosely bound Mg2+ ions that serve to relax the tRNA structure . Such magnesium-driven relaxation has a minor effect on Km (tRNA), but the resulting floppy structure is able to avoid the barriers against reaction and is amino-acylated thousands of times more rapidly than the compact defined structure. J Biol Chem, 1981 Jul 10, 256(13), 6646 - 50 Isolation and characterization of a protease-nicked thioredoxin; McEvoy M et al.; An altered form of thioredoxin, composed of two peptides with molecular weights of 7 and 5 X 10(3) has been isolated from Escherichia coli B after chromatography on Ultrogel AcA54 . The position of the clip site, determined by amino acid sequencing to lie between Pro(64) and Gly(65), is consistent with a cleavage within the hinge region connecting the two prominent folding domains of thioredoxin . The NH2-terminal domain containing the active site, and the carboxyl-terminal domain correspond to the 7 and 5 X 10(3) fragments, respectively . The two peptides combine to form a tight complex, T'12 . At concentrations below 10(-6) M, the clipped species was not efficiently reduced by thioredoxin reductase, showing only half the activity of native thioredoxin at a protein concentration of 9 X 10(-8) M . The Km of the clipped species for thioredoxin reductase (2.9 X 10(-6) M) was nearly equivalent to that of native thioredoxin (2.2 X 10(-6) M), and the Vmax for both native and clipped thioredoxins were nearly identical . These data are taken to imply a reversible association of the fragments to produce a thioredoxin equivalent to the intact species in its interaction with thioredoxin reductase . Some evidence exists for an unfolding of the 7 X 10(3) fragment upon dissociation of the complex . A functional role for the two-domain structure of thioredoxin is proposed in which the carboxyl domain serves to stabilize the active site within the amino domain and confer the species specificity in the binding of thioredoxin to thioredoxin reductase. J Biol Chem, 1981 Jul 10, 256(13), 6600 - 7 The effect of initiation factor IF-3 on Escherichia coli ribosomal subunit association kinetics; Chaires JB et al.; IF-3 strikingly inhibits the rate of association of 30 S and 50 S subunits to form 70 S ribosome, at all Mg2+ concentrations between 1 and 18 mM, in 60 mM KCl, pH 7.8, at 26 degrees C . The rate of formation of 70 S is determined from the increase in light scattering intensity in stopped flow experiments in which, typically, largely dissociated 30 S-50S mixtures in 2 mM Mg2+ buffer are jumped against high {Mg2+} buffers containing either no or 1-2 eq of IF-3 . The curves can be analyzed using two reactions (the anti-association model): 30 S + 50 S (formula see text) Both forward rates are large: in the physiologically important range, 3-6 mM Mg2+, k1 increases from 0.4 to 15 X 10(6) M-1 s-1, while k2 ranges from 3 to 18 X 10(6) M-1 s-1 . The kinetic curves have a characteristic shape: an early spurt of 70 S particle formation while IF-3 and 50 S particles are competing for the large initial pool of free 30 S subunits, and a slower phase in which 70 S formation is controlled by the release of 30 S subunits from the 30 S.IF-3 complexes (k-2 runs between 0.05 and 0.17 s-1) . The addition of a third reaction, 30 S.IF-3 + 50 S (formula see text) 70 S + IF-3, is not required for an adequate fit, changes the values of k2 slightly, and of k-2 not too much, and yields values of k-3 (0-20 X 10(3) M-1 s-1) probably too small to play a significant physiological role in initiation of protein synthesis unaided by other interactions . The "anti-association" effect of IF-3 on ribosomal subunits is clear-cut . The "pro-dissociation" effect on 70 S ribosomes remains to be demonstrated. J Biol Chem, 1981 Jul 10, 256(13), 6544 - 7 Genetic insertion of nuclear spin labels in the lac repressor; Jarema MA et al.; A general strategy for the insertion of nuclear spin labels throughout the sequence of a protein is illustrated with the Escherichia coli lac repressor . Examples are shown where the 19F nucleus is incorporated using 3-fluorotyrosine, as well as the selective insertion of additional protons . These selectively inserted nuclei give additional resonances in the respective NMR spectra that can be used to probe the structure and function of the proteins. J Biol Chem, 1981 Jul 10, 256(13), 6804 - 10 beta-Galactosidase alpha-complementation . Overlapping sequences; Welply JK et al.; Enzyme activity is restored to two defective beta-galactosidase molecules (M15 protein lacking amino acid residues 11-41 and M112 protein lacking residues 23-31) by incubation with CNBr2 (residues 3-92 of beta-galactosidase) . M15 and M112 proteins (alpha-acceptors) are dimers . Complemented enzyme, like wild type, has a tetrameric structure . Cleavage of CNBr2 with glutamic acid-specific protease yielded a much smaller alp ha-donor (3-41 peptide) which was also effective in complementation, indicating that the M15 protein can supply all of the residues from 42-92 for the structure of complemented enzyme . Treatment of M112 protein/3-41 peptide complemented enzyme with trypsin under very mild conditions followed by examination of the products demonstrated that the alpha-donor pep}tide supplies the NH2-terminal segment of complemented enzyme . Similar trypsin treatment of M15 protein/CNBr2 indicated that in this complemented enzyme the polypeptide region beyond those residues missing in the alpha-acceptor can be provided by either the alpha-donor or the alpha-acceptor . Both M15 protein and M112 protein are more susceptible to mild tryptic proteolysis than complemented enzyme, indicating a more open structure . Several antipeptide antibodies that react with these two proteins do not react with beta-galactosidase . M112 protein, like M15 protein, can be activated by anti-beta-galactosidase but to a much higher level. J Biol Chem, 1981 Jul 10, 256(13), 7064 - 7 Mitochondrial ribosome assembly in Neurospora crassa . Purification of the mitochondrially synthesized ribosomal protein, S-5; LaPolla RJ et al.; In Neurospora, one mitochondrial ribosomal protein (S-5, Mr = 52,000) is synthesized intramitochondrially and is presumably encoded by mitochondrial DNA . We have developed a rapid method for the purification of S-5 which takes advantage of its high affinity for carboxymethyl-Sepharose in the presence of 6 M urea . Using this method, S-5, at purity greater than 95%, can be prepared by column chromatography in a single batch elution step . The amino acid composition of S-5 was determined . Judged by the contents of hydrophilic and basic amino acids, S-5 is more similar to Escherichia coli and yeast ribosomal proteins than to other mitochondrial translation products which are hydrophobic membrane proteins. Nucleic Acids Res, 1981 Jul 10, 9(13), 3159 - 74 Reaction kinetics of some important site-specific endonucleases; Hinsch B et al.; Reaction kinetics of the site-specific endonucleases BamHI, BgIII, C1aI, EcoRI, HpaII, PstI, SaII, SmaI, and XorII were investigated employing some frequently used substrates . Six of these enzymes could be analyzed under steady-state conditions . Kinetic data were obtained from progress curves applying an integrated Michaelis-Menten equation . KM ranged from 4 x 10(-9) M to 4 x 10(-11) M . Activities also spanned two orders of magnitude . In the case of C1aI the analysis of the pre-steady-state kinetics ("burst reaction") allowed the assessment of several rate constants . The rate-limiting step is the very slow dissociation of the enzyme-product complex (0.22 min(-1)) . This complex is formed from the enzyme-bound nicked intermediate at a rate of 1.7 min(-1) . The introduction of the first cut is again faster by a factor of about 6 . SmaI and XorII resembled C1aI in their kinetics . The burst reaction can be used for the easy and unambiguous determination of molar concentrations of site-specific endonucleases in any preparation, which is free of non-specific DNases. Nucleic Acids Res, 1981 Jul 10, 9(13), 3047 - 60 A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system; Garner MM et al.; The use of gel electrophoresis for quantitative studies of DNA-protein interactions is described . This rapid and simple technique involves separation of free DNA from DNA-protein complexes based on differences in their electrophoretic mobilities in polyacrylamide gels . Under favorable conditions both unbound DNA and DNA associated with protein can be quantified . This gel method is applied to the study of the E . coli lactose operon regulatory system . At ionic strengths in the physiological range, the catabolite activator protein (CAP) is shown to form a long-lived complex with the wild type lac promotor, but not with a CAP-insensitive mutant . Formation of a stable "open" or "melted-in" complex of RNA polymerase with the wild type promoter requires the participation of CAP and cyclic AMP . Further, it is demonstrated that even when pre-formed in the presence of CAP-cAMP, the polymerase-promoter open complex becomes unstable if CAP is then selectively removed. Nucleic Acids Res, 1981 Jul 10, 9(13), 2999 - 3014 Molecular cloning of the complete Epstein-Barr virus genome as a set of overlapping restriction endonuclease fragments; Arrand JR et al.; A complete collection of fragments of Epstein-Barr virus DNA, obtained by cleavage with restriction endonuclease Eco RI, has been cloned . Fourteen different internal fragments of the virus genome, derived from linear virion DNA of the B95-8 strain, and sequences corresponding to the terminal regions of virion DNA, derived from intracellular circular EBV DNA isolated from 895-8 cells, were cloned . Sizes of fragments were determined by agarose gel electrophoresis and their sum leads to an estimated molecular weight of 110 x 10(6) for virion DNA . Large Eco RI DNA fragments of special interest were also cloned in cosmids using another source of EBV DNA, that is, to circular viral DNA derived from Raji cells . In order to provide a set of overlapping sequences, all the 29 internal Bam HI fragments of B95-8 virion DNA were cloned in pBR322 . The map location within the viral genome of each cloned DNA fragment was identified by hybridizing to blots of virion DNA cleaved with several different restriction endonucleases. Nucleic Acids Res, 1981 Jul 10, 9(13), 3187 - 98 Fluorescence modification of Escherichia coli 5S RNA; Digweed M et al.; Reaction of 5S RNA with chlorocetaldehyde leads to the conversion of unpaired adenines to the fluorescent 1,N6-etheno-adenine derivatives . Up to 16 of the 23 adenines in free 5S RNA can be modified, the fastest reacting are A29, A34, A57-59 . Partial modification of adenines in this area results in a 20% reduction in the efficiency of 5S RNA incorporation into 50S subunits during reconstitution and a 15% reduction in the activity of these subunits in peptide synthesis . Fluorescence from 1,N6-etheno-adenine is quenched in free 5S RNA and is not detectably further influenced by the binding of proteins E-L5, E-L18 and E-L25, nor by the first stage of the two step E . coli 50S subunit reconstitution procedure . However, the fluorescence is further reduced to near zero after the second step of the reconstitution . Thus, 5S RNS free in solution contains 16 unpaired adenines, those in the region between A29 and A59 particularly accessible to modification by chlorocetaldehyde . This portion of the 5S RNA molecule appears to undergo either a conformational change or interacts with other ribosomal components in the last stage of subunit reassembly. Biochemistry, 1981 Jul 7, 20(14), 4178 - 85 In vitro synthesis of the respiratory NADH dehydrogenase of Escherichia coli . Role of UUG as initiation codon; Poulis MI et al.; The respiratory NADH dehydrogenase of Escherichia coli has been synthesized in vitro in a coupled transcription--translation system with cloned deoxyribonucleic acid (DNA) as template . The identity of the protein produced was confirmed by paper chromatography and electrophoresis of tryptic peptides . {35S}Methionine-labeled tryptic peptides from the in vitro product were shown to comigrate with authentic methionine-containing tryptic peptides from the purified enzyme . Using a transcription-translation system derived from an ndh mutant, it was shown that the enzyme produced in vitro was incorporated into membrane vesicles of the mutant to give functional, cyanide-sensitive NADH oxidase activity . Radiochemical N-terminal sequencing of the synthesized NADH dehydrogenase showed that the product was a mixture of three different species, with N-formylmethionine, methionine, or threonine at the N terminus . The results indicated that only partial N-terminal processing was occurring in vitro and that the first residue of the unprocessed NADH dehydrogenase is N-formylmethionine . Since DNA sequencing has shown that this residue is encoded by UUG {Young, I . G., Rogers, B . L., Campbell, H . D., Jaworowski, A., & Shaw, D . C . (1981) Eur . J . Biochem . (in press)}, this work verifies the role of UUG as a normal initiation codon. Biochemistry, 1981 Jul 7, 20(14), 3996 - 4006 Study of transfer ribonucleic acid unfolding by dynamic nuclear magnetic resonance; Johnston PD et al.; Nuclear magnetic resonance (NMR) measurements of proton exchange were performed on yeast tRNAPhe, and in much less detail on Escherichia coli tRNAfMet, over a range of Mg2+ concentrations and temperatures, at neutral pH and 0.1 M NaCl . The resonances studied were those of ring nitrogen protons, resonating between 10 and 15 ppm downfield from sodium 3-(trimethylsilyl)-1-propanesulfonate, which partake in hydrogen bonding between bases of secondary and tertiary pairs . Methods include saturation--recovery, line width, and real-time observation after a change to deuterated solvent . The relevant theory is briefly reviewed . We believe that most of the higher temperature rates reflect major unfolding of the molecule . For E . coli tRNAfMet, the temperature dependence of the rate for the U8--A14 resonance maps well onto previous optical T-jump studies for a transition assigned to tertiary melting . For yeast tRNAPhe, exchange rates of several resolved protons could be studied from 30 to 45 degrees C in zero Mg2+ concentration and had activation energies on the order of 40 kcal/mol . Initially, the tertiary structure melts, followed shortly by the acceptor stem . At high Mg2+ concentration, relatively few exchange rates are measurable below the general cooperative melt at about 60 degrees C; these are attributed to tertiary changes . Real-time observations suggest a change in the exchange mechanism at room temperature with a lower activation energy . The results are compared with those obtained by other methods directed toward assaying ribonucleic acid dynamics. Biochemistry, 1981 Jul 7, 20(14), 4042 - 9 Changes in the substrate specificities of an enzyme during directed evolution of new functions; Hall BG; Wild-type ebg enzyme, the second beta-galactosidase of Escherichia coli K12, does not permit growth on lactose . As part of a study of the evolution of new enzymatic functions, I have selected, from a lacZ deletion strain, a variety of spontaneous mutants that grow on lactose and other beta-galactoside sugars . Single point mutations in the structural gene ebgA alter the enzyme so that it hydrolyzes lactose or lactulose effectively; two mutations in ebgA permit galactosylarabinose hydrolysis, while three mutations are required for lactobionic acid hydrolysis . Wild-type ebg enzyme and 16 functional mutant ebg enzymes were purified and analyzed kinetically to determine how the substrate specificities had changed during the directed evolution of these new functions . The specificities for the biologically selected substrates generally increased by at least an order of magnitude via increased Vmax and decreased Km for the substrate . These changes were very specific for the selected substrate, often being accompanied by decreased specificities for other related substrates . The single, double, or triple substitutions in the enzymes did not detectably alter the thermal stability of ebg enzyme. Biochim Biophys Acta, 1981 Jul 6, 645(1), 137 - 42 Mode of action of colicin ib: formation of ion-permeable membrane channels; Weaver CA et al.; Addition of purified colicin Ib to whole Escherichia coli cells or cytoplasmic membrane vesicles inhibits their subsequent ability to generate a membrane potential . In addition, this colicin is shown to bring about a voltage-dependent increase in the conductance of an artificial planar bilayer membrane prepared from soybean phospholipids . This results from the formation of ion-permeable channels . These data provide strong evidence that the depolarization of Escherichia coli cells by this colicin results from an Ib-induced increase in membrane permeability to ions. J Submicrosc Cytol, 1981 Jul, 13(3), 291 - 308 Structural transitions of chromatin in isolated Xenopus erythrocyte nuclei . I . The effects of ions; Chegini N et al.; Nuclei from Xenopus erythrocytes have been isolated under varying ionic conditions and their morphology studied by light and electron microscopy . The 200-A beaded chromatin fibre observed in vivo is maintained in media containing MgCl2 or CaCl2, but not in those containing Mn2+ as the sole divalent cation . In the absence of these alkaline-earth metal ions, 100-A filaments predominate . This suggests that the native 200-A fibre in isolated nuclei is also stabilized by specific divalent cations . Gross chromatin condensation within the nucleus occurs independently of whether the chromatin is in the form of 100-A or 200-A fibres, but requires the presence of divalent cations . Lower concentrations of MgCl2 or CaCl2 are necessary as compared with MnCl2 . In the presence of sufficient divalent cations, decreasing KCl concentration causes peripheral condensation, the condensation of chromatin towards the nuclear membrane . Monovalent cation alone does not appear capable of inducing peripheral condensation . The transcriptional capacities of the different morphological types identified have been studied using E . coli RNA polymerase as a probe . Neither the degree of chromatin condensation nor the state of higher-order coiling have a significant effect on the rate of transcription of the DNA . These results are discussed with regard to further in vitro studies on eukaryotic gene activity. Scand J Immunol, 1981 Jul, 14(1), 95 - 8 Dimeric IgA in the rat is transferred from serum into bile but not into milk; Dahlgren U et al.; Intravenous administration of ductus thoracicus lymph with dimeric IgA antibodies against Escherichia coli 06 to lactating rat dams did not result in transfer of IgA antibodies into the milk, although the antibodies were detectable in serum 1 min after the administration and in bile 60 min later . After intravenous injection of serum from bile-duct-occluded (BDO) rats immunized in the Peyer's patches into lactating rat dams . IgA antibodies appeared in the serum and remained there up to 230 min . At this time no IgA antibodies were seen in the milk while they were present in bile . IgG and IgM 06 antibodies did not appear in bile or milk after intravenous administration of lymph or serum from BDO rats. Farmaco {Sci}, 1981 Jul, 36(7), 536 - 50 A new water soluble derivative of 4,5'-dimethylangelicin as a potential agent for photochemotherapy; Guiotto A et al.; A water soluble derivative of 4,5'-dimethylangelicin (I) having a long chain linking an amino group to the planar furocoumarinic moiety, that is 4'-N,N-dimethylaminoe