Eur J Biochem, 1981 May 15, 116(2), 355 - 8 Exoenzymatic activity of transglycosylase isolated from Escherichia coli; Beachey EH et al.; The possibility that murein transglycosylase of Escherichia coli may function as an exoenzyme to cleave the murein sacculus in a systematic fashion was investigated . Two molecular species of this hydrolytic enzyme have been isolated and characterized: one is associated with the soluble fraction and the other with the envelope fraction of ruptured E . coli cells . The soluble enzyme was employed to digest murein sacculi that had been uniformly labeled with {3H}diaminopimelic acid . The analysis of the reaction product indicated that the enzyme did not cleave the glycan chains randomly . To determine whether transglycosylase released muropeptide first from the N-acetylglucosaminyl or the 1,6-anhydromuramyl ends of the glycan chains, the {3H}diaminopimelate-labeled sacculi were further radiolabeled at their N-acetylglucosaminyl ends with {14C}galactose by a galactosyl transferase reaction . The transglycosylase released galactose-labeled X + X' muropeptides early during the course of digestion, suggesting exoenzymatic cleavage of the glycan chains preferentially from the N-acetylglucosaminyl ends . (X = N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-L-alanyl-D-gamma-glutamyl-meso-diaminopimelic acid; X' = X-D-alanine.) The kinetics of the activity of the membrane-bound enzyme were found to be identical to that of the soluble enzyme, indicating that both molecular species of transglycosylase function as exoenzymes in vitro. Eur J Biochem, 1981 May 15, 116(2), 331 - 5 Outer-membrane vesicles released by normally growing Escherichia coli contain very little lipoprotein; Wensink J et al.; The lipoprotein content of the outer-membrane medium vesicles, which are released from Escherichia coli during normal growth, was compared to the lipoprotein content of the corresponding cellular outer membranes . It was found that the medium vesicles contained only 35% free lipoprotein and almost none of the bound lipoprotein when compared with cellular outer membranes . Medium vesicles also had reduced amounts of protein II and a protein V (Mr = 16 000), while they contained large amounts of pore-forming proteins I and lamB . A mechanism is proposed in which outer membrane vesicles are formed when the outer membrane expands faster than the underlying peptidoglycan layer . The lack or enrichment of individual proteins in medium vesicles may be determined by their interactions with the peptidoglycan-bound lipoprotein complex. Eur J Biochem, 1981 May 15, 116(2), 269 - 76 A proton NMR study of ribosomal protein L25 from Escherichia coli; Kime MJ et al.; A highly folded form of the ribosomal protein L25 from Escherichia coli can be obtained from urea-denatured preparations . Proton NMR data show that this form of the molecule must have a compact, globular tertiary structure . Spectroscopically it is indistinguishable from L25 prepared by methods which avoid denaturing solvents . Thus L25 is a protein which can be reversibly denatured . The stability and solubility of the folded form of the protein are discussed and primary assignments made for a number of resonances in its NMR spectrum . The paper shows that this folded form of the protein can be characterised using NMR spectroscopy . High-resolution NMR spectroscopy provides a sensitive and general way for the characterisation of protein folds. Eur J Biochem, 1981 May 15, 116(2), 227 - 33 Energy is required for maturation of exported proteins in Escherichia coli; Enequist HG et al.; It has been established in numerous cases that proteins which are exported from Escherichia coli are synthesized on membrane-bound polysomes in precursor forms which are proteolytically cleaved to generate the mature species . Here we present evidence that at least one step in the export of proteins requires energy . Energy requirements for processing of the precursors of both the M13 coat protein {Date, T., Zwizinski, C., Ludmerer, S., and Wickner, W . (1980) Proc . Natl Acad . Sci . USA, 77, 827-831; Date, T., Goodman, J . M., and Wickner, W . T . (1980) Proc . Natl Acad . Sci . USA, 77, 4669-4673} and the B subunit of heat-labile enterotoxin {Palva, T., Hirst, T . R., Hardy, S . J . S., Holmgren, J., and Randall, L . L . (1981) J . Bacteriol . in the press} have been demonstrated previously . An energy requirement for the proteolytic processing of an additional five exported proteins is reported here . Studies utilizing an uncA mutant suggest that the form of energy required is proton-motive force . Thus an energized membrane is probably essential for export of most periplasmic and outer membrane proteins. Tijdschr Diergeneeskd, 1981 May 15, 106(10), 501 - 7 {The pathogenesis of coliform mastitis (author's transl)}; Verheijden JH et al.; The present paper is a report on a comparative study of the effects of intravenous or intramammary injection of various doses of E . coli endotoxin in normal and endotoxin-tolerant animals on a number of clinical and clinicochemical parameters . The absence of marked effects on rumen motility following intramammary administration of endotoxins raised serious doubts as to the validity of the common theory of absorption of endotoxins from the udder in these animals . Experimental animals were made tolerant by daily intravenous injections of E . coli endotoxins . Animals tolerant for a single intravenous injection also were tolerant for intravenous infusion of E . coli endotoxins . Intramammary administration of one fifth of the dose of endotoxins for which the animals had been made tolerant, produced a maximum effect on the body temperature and plasma zinc concentrations of cows . In view of these findings, it can be postulated that the systemic symptoms in E . coli endotoxin induced mastitis are not due to the absorption of endotoxins from the udder. Eur J Biochem, 1981 May 15, 116(2), 317 - 22 ADP-ribosylation of proteins in non-infected Escherichia coli cells; Skorko R et al.; Partially purified enzymatic fractions from extracts of Escherichia coli B/r catalyse transfer of the isotope label from {adenine-2,8-(3)H}NAD+ to some bacterial proteins, as well as to hen egg-white lysozyme . The radioactive group in the modified lysozyme was identified as mono(ADP-ribose) . Several bacterial proteins were labelled in vivo with 32P; the presence of the label in the form of an ADP-ribosyl group was shown in one of them. Nature, 1981 May 14, 291(5811), 122 - 6 Genetic analysis of K88-mediated adhesion of enterotoxigenic Escherichia coli; Kehoe M et al.; Four cistrons (adh) involved in the expression of the K88 adhesion system have been identified and mapped . Three of these (adh A, adh B and adh C) are located in a single operon (I) whereas the fourth (adh D) is expressed from a separate promoter (operon II) . The polypeptides encoded by these cistrons have been identified and their role in the formation and regulation of K88 fimbriae (pili) is discussed. Biochim Biophys Acta, 1981 May 14, 659(1), 86 - 98 Affinity chromatography on immobilised triazine dyes . Studies on the interaction with multinucleotide-dependent enzymes; Clonis YD et al.; A systematic investigation into the interaction of several triazinyl dyes with two enzymes from purine metabolism, IMP dehydrogenase (IMP: NAD+ oxidoreductase, EC 1.2.1.14( and adenylosuccinate synthetase (IMP: L-aspartate ligase (GDP-forming), EC 6.3.4.4) has been conducted . Evidence from kinetic inhibition studies, enzyme inactivation with specific affinity labels and specific elution techniques from agarose-immobilised dyes indicate that triazine dyes such as Procion Blue H-B (Cibacron Blue F3G-A), Red HE-3B and Red H-3B are able to differentiate between the nucleotide-binding sites of these enzymes . This information has been exploited to design specific elution techniques for the purification of these enzymes by affinity chromatography. Biochemistry, 1981 May 12, 20(10), 2843 - 52 Identification of proteins at the subunit interface of the Escherichia coli ribosome by cross-linking with dimethyl 3,3'-dithiobis(propionimidate); Cover JA et al.; The 70S ribosomes of Escherichia coli were treated with dimethyl 3,3'-dithiobis(propionimidate) . Under conditions where 40% of the lysine epsilon-amino groups became modified, about 50% of the ribosomes became resistant to dissociation into 30S and 50S subunits when analyzed in the absence of reducing agents on sucrose gradients containing low magnesium concentrations . Dissociation took place in the presence of reducing agents, indicating that the bifunctional reagent had reacted with proteins from both subunits . Proteins were extracted from purified cross-linked 70S ribosomes by using conditions to preclude disulfide interchange . Disulfide-linked protein complexes and non-cross-linked proteins were first fractionated by electrophoresis in polyacrylamide/urea gels at pH 5.5 . The proteins from sequential slices of the urea gel were analyzed by two-dimensional diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis . Monomeric proteins derived from cross-linked dimers appeared below the diagonal of non-cross-linked proteins since the second electrophoresis but not the first is run under reducing conditions to cleave the cross-linked species . Final identification of the constituent proteins in each dimer was made by radioiodination of the cross-linked proteins, followed by two-dimensional polyacrylamide/urea gel electrophoresis in the presence of nonradioactive marker 70S protein . The identification of 11 cross-linked protein dimers which contained one protein from each of the two ribosomal subunits is described . We conclude that the proteins in these cross-linked pairs are located in the regions of contact between the two subunits, i.e., at the "subunit interface". Biochemistry, 1981 May 12, 20(10), 2743 - 8 Medium effects in enzyme-catalyzed decarboxylations; O'Leary MH et al.; Carbon isotope effects and steady-state kinetic parameters have been measured for the decarboxylation of arginine and homoarginine by the pyridoxal 5'-phosphate dependent arginine decarboxylase from Escherichia coli . In water at pH 5.25, 5 degrees C, homoarginine shows an isotope effect k12/k13 = 1.601, indicating that the decarboxylation step is entirely rate determining . In the presence of 16 mol % ethylene glycol under otherwise identical conditions, the decarboxylation rate is increased 3-fold, and the carbon isotope effect is 1.044, indicating that the rate of the decarboxylation step is increased by the presence of the less polar solvent . The decarboxylation or arginine under the same conditions shows a similar trend: in water, the isotope effect is 1.027, decreasing to 1.003 in 16% ethylene glycol, with little change in the steady-state rate . Again, the rate of the decarboxylation step is substantially increased by the presence of the nonpolar solvent . Thus, pyridoxal phosphate dependent enzymatic decarboxylations show a medium effect similar to that observed in a number of nonenzymatic decarboxylations . This suggests that these enzymes may accelerate the decarboxylation step by providing a nonpolar environment . Evidence is also presented that desolvation of the substrate carboxyl group may contribute to catalysis. Biochemistry, 1981 May 12, 20(10), 2707 - 13 Kinetics of pH-dependent interconversion of tryptophanase spectral forms studied by scanning stopped-flow spectrophotometry; June DS et al.; Morino and Snell {Morino, Y., & Snell, E . E . (1967) J . Biol . Chem . 242, 5591-5601} previously showed that the relative amplitudes of the 337- and 420-nm absorption bands of tryptophanase depended on both pH and the nature of a required monovalent cation activator . An investigation of the kinetics of interconversion of the 337- and 420-nm forms following a rapid incremental increase (jump) or decrease (drop) in pH over the range of enzyme stability in 0.2 M KCl at 24 +/- 0.3 degrees C by scanning stopped-flow spectrophotometry showed three distinct time-dependent phases . They were (1) an abrupt phase which is complete in less than 6.5 ms, (2) a fast first-order interconversion of the 420- and 337-nm absorbances, and (3) a slow first-order process involving growth at 355 nm coupled to two decays centered at 325 and 430 nm in the incremental pH jumps and decay at 355 nm with concomitant growth at 430 and 290 nm in the incremental pH-drop experiments . The results of these experiments were analyzed in terms of a scheme involving enzyme forms E alpha, E beta, E beta H+, E gamma, E gamma H+, and E delta . The E alpha form predominates in the absence of activating monovalent cations and absorbs at 420 nm . Those in the beta manifold, E beta and E beta H+, also absorb at 420 nm while those in the gamma manifold, E gamma and E gamma H+, absorb at 337 nm . The form E delta absorbs at 335 nm . E beta H+ and E gamma H+ represent the protonated form of the enzyme in each manifold . Analysis of the abrupt phase showed no significant systematic changes in absorbance above 330 nm for either the pH-jump or pH-drop experiments . The fast second phase involves the first-order interconversion of the beta and gamma manifolds while the slow third phase describes the buildup or decay of the delta manifold . Presumably conformational changes control the rate of these interconversions . The pH dependence of the fast first-order beta to gamma conversion was described and evaluated in terms of five independent equilibrium and rate constants and three independent amplitude terms by simultaneously fitting the amplitude data and first-order rate constants to an equation describing the overall scheme with a nonlinear least-squares program KINFIT4 {Dye, J . L., & Nicely, V . A . (1971) J . Chem . Educ . 48, 443-448} . The pK for protonation of the beta form = 9.70 +/- 0.12, for protonation of the gamma form (337-nm absorber) = 6.77 +/- 0.10, and for the pH-dependent interconversion of the beta and gamma manifolds, pKa = 8.11 +/- 0.04 . The computed equilibrium distribution among the four species of the beta and gamma manifolds showed that E beta H+ and E gamma predominate. C R Seances Acad Sci III, 1981 May 11, 292(17), 987 - 9 {Sequence of the N-terminal end of the periplasmic glutamine binding protein of Escherichia coli K12 . Comparison with other periplasmic proteins}; Marty B et al.; The amino-terminal end of the glutamine binding protein of E . coli K 12 has been sequenced (40 residues) . Homologies with other binding proteins are shown . Likeness is slightly better with the ribose binding protein and with the leucine, isoleucine, valine binding protein. Nucleic Acids Res, 1981 May 11, 9(9), 2121 - 39 Nucleotide sequence of an Escherichia coli tRNA (Leu 1) operon and identification of the transcription promoter signal; Duester G et al.; Fourteen different DNA fragments containing Escherichia coli tRNA genes have been cloned using the vector pBR322 . We report the methods of cloning, the identification of specific tRNA genes, and the presence or absence of rRNA genes on these cloned DNA fragments . In particular, one chimeric plasmid contains a 17.0 kilobase pair EcoRI fragment bearing tRNA(Leu 1) sequences . Using nucleotide sequence analysis we have identified a cluster of three tandem tRNA(Leu 1) genes separated by intergenic spacers of 27 and 34 base pairs, respectively . The nucleotide sequence upstream of the first gene contains a transcription promoter site . A G-C rich sequence (5'-CGCCTCC-3') found between the Pribnow box and initiation site is very similar to the corresponding sequence found in other genes which are under stringent control. Nucleic Acids Res, 1981 May 11, 9(9), 2055 - 73 Requirement for the C-terminal region of middle T-antigen in cellular transformation by polyoma virus; Novak U et al.; Deletions of polyoma virus DNA around the region that codes for the C-terminus of the viral middle T-antigen were created using a transforming fragment (BamH I/EcoR I) of viral DNA cloned in the plasmid vector pAT153 . These species were recloned and assayed for their ability to transform Rat-1 cells in culture . Our results showed that whereas the DNA sequence between the presumed translational termination codon for the viral middle T-antigen and the single viral EcoR I site could be removed with no apparent effect on transformation, the removal of the termination codon itself or any amino acid coding sequences of this protein caused a drastic decrease in the transforming ability of the DNA . Transfection of Rat-1 cells with plasmids that contained viral DNA with deletions which corresponded to the last fourteen or more amino acids of the middle T-antigen never gave rise to cellular transformation. Nucleic Acids Res, 1981 May 11, 9(9), 2207 - 22 Identification of the modified nucleotides produced by covalent photoaddition of hydroxymethyltrimethylpsoralen to RNA; Bachellerie JP et al.; The reaction between RNA and 4'hydroxymethyl-4,5',8-trimethylpsoralen has been studied . Both natural RNA and synthetic RNAs were used . The base specificity of the reaction was found to be the same in natural RNA, homopolymers, and mononucleotides . Uridine was found to be the most reactive base in all cases . The kinetics of formation and reversal of monoadducts and crosslinks has been examined . Paper electrophoretic conditions are described which provide a separation of the monoaddition and crosslinked photoproducts . The relative and absolute amounts of monoadducts and crosslinks can be determined very accurately with this system . Paper electrophoresis provides good separations of the different photoproducts . The mobilities of the products are a simple function of their molecular weights and charges. Nucleic Acids Res, 1981 May 11, 9(9), 2075 - 86 Identification, nucleotide sequence and expression of the regulatory region of the histidine operon of Escherichia coli K-12; Verde P et al.; A restriction fragment has been isolated and its nucleotide sequence determined . This fragment contains sites for RNA polymerase binding, initiation and termination of transcription of the Escherichia coli histidine operon . In vitro transcription of plasmids containing this region generates one single histidine-specific, attenuated, small RNA: the leader RNA . This RNA is more efficiently transcribed when the template DNA is supercoiled . Another promoter was identified on the same fragment of deoxyribonucleic acid by in vitro transcription, DNA sequencing and RNA polymerase binding . Both promoters, transcribing in opposite direction, are very A-T rich and are separated by a G-C rich region containing a palyndromic structure. J Biol Chem, 1981 May 10, 256(9), 4676 - 8 A simple and rapid procedure for the large scale purification of the recA protein of Escherichia coli; Cox MM et al.; A simple and rapid three-step procedure for the large scale purification of the recA protein of Escherichia coli is described . The method depends primarily on a single chromatographic step which is highly specific for recA protein: elution by ATP from single-stranded DNA cellulose . With this procedure, gram quantities of recA protein, greater than 99% pure, can be reproducibility prepared for biochemical and biophysical analysis. J Biol Chem, 1981 May 10, 256(9), 4444 - 9 Incorporation of photosensitive fatty acids into phospholipids of Escherichia coli and irradiation-dependent cross-linking of phospholipids to membrane proteins; Quay SC et al.; In an approach to the study of phospholipid-protein interactions in biological membranes, the photoactivable fatty acids, omega-(m-azidophenoxy)-undecanoic acid (I) and omega-(m-diazirinophenoxy)-hexadecanoic acid (II), were incorporated biosynthetically into the phospholipids of the Escherichia coli fatty acid auxotroph, strain K1060-B5 . The extent of incorporation of the two fatty acids was 43% and 21%, respectively, of the total fatty acid content of the phospholipids . Membrane vesicles prepared from cells grown on the fatty acid supplements and {32P}H3PO4 were irradiated at suitable wavelengths to generate the reactive nitrene or carbene intermediates . Subsequent analysis of solubilized membrane proteins by two-dimensional isoelectric focusing-polyacrylamide gel electrophoresis indicated cross-linking between radioactive phospholipids and an number of proteins . A corresponding experiment with cells grown on oleic acid showed only trace amounts of covalently cross-linked phospholipid-protein adducts . While the extent of cross-linking in vesicles from cells grown on I was only 3 times the background level observed for oleic acid-grown cells, cells grown on II showed 30 times this amount . The present results, together with the previously observed nonreactivity of the nitrene generated from I to undergo C-H insertion, show that the use of carbene precursors such as II is promising for chemical analysis of specific phospholipid-protein interactions in bacterial membranes under biologically meaningful conditions. J Biol Chem, 1981 May 10, 256(9), 4357 - 61 Structural prediction of sugar-binding proteins functional in chemotaxis and transport; Argos P et al.; Comparisons of the D-galactose- and D-ribose-binding protein amino acid sequences and secondary structure predictions with the known primary and three-dimensional structure of L-arabinose-binding protein suggest that the three proteins have similar molecular structures . These studies also indicate an evolutionary relationship among the proteins . One region of striking homology between the galactose- and ribose-binding proteins suggests that this may be th protein-protein contact site for interaction with the membrane-bound chemotaxis receptor . The ligands and the geometry of the galactose binding site are also predicted. Cancer Treat Rep, 1981 May-Jun, 65(5-6), 491 - 4 Radiometric enzyme-inhibition technique for measuring acivicin in plasma; Jayaram HN et al.; A sensitive radiometric enzyme-inhibition assay is described for the determination of acivicin in plasma; it is based on the potent inhibition of carbamyl phosphate synthetase II (CPS) by the drug . Plasma is heated at 95 degrees C for 5 minutes to quantitatively detach bound acivicin . After centrifugation, free drug is quantitated by exposing purified CPS from Escherichia coli to representative aliquots or subdilutions of the resultant supernatants in the presence of L-glutamine, L-aspartic acid, ATP-MgCl2, NaH{14C}O3, and purified L-aspartate transcarbamylase (ATC) from E . coli . Carbamyl phosphate is first synthesized from L-glutamine, ATP-MgCl2, and NaH{14C}O3 by the action of CPS . The unstable carbamyl phosphate thus generated is quickly and quantitatively converted to {14C}carbamyl-L-aspartic acid by the action of ATC utilizing {14C}carbamyl phosphate and L-aspartic acid as substrates . After a 15-minute incubation at 37 degrees c, unreacted NaH{14C}O3 is dissipated at acidic pH and the newly formed {14C}carbamyl-L-aspartic acid is quantitated by scintillation spectrometry . The percent inhibition of the formation of carbamyl-L-aspartic acid through the conjoint actions of CPS and ATC responds in a linear way to the logarithm of the concentration of acivicin between 20 and 200 microM . The unknown concentration of acivicin is determined indirectly by matching the percent inhibition produced by the unknown to the percent inhibition produced by a series of acivicin standards extending over the linear range . This assay is sensitive, adequately reproducible, and easy . It can be used to measure acivicin in the plasma of subjects treated with this new oncolytic agent. Infect Immun, 1981 May, 32(2), 480 - 3 Failure of chlorpromazine to inhibit fluid accumulation caused by Escherichia coli heat-stable enterotoxin in suckling mice; Takeda T et al.; We studied the effect of chlorpromazine on fluid accumulation caused by purified heat-stable enterotoxin (ST) from enterotoxigenic Escherichia coli in suckling mice . We found that chlorpromazine inactivated ST itself in vitro, but did not inhibit the activity of ST in the intestines. J Gen Microbiol, 1981 May, 124(Pt 1), 219 - 23 Some properties of D-mannose isomerase from Escherichia coli K12; Stevens FJ et al.; A second-stage mutant of Escherichia coli K12 designated as strain 806 grew faster on D-lyxose than the mutant strain 805 previously described . Both mutants produced constitutively a novel enzyme, D-mannose isomerase, but strain 806 produced twice as much as strain 805 . The enzyme could fortuitously convert D-lyxose to D-xylulose, which is a normal intermediate in the D-xylose catabolic pathway . The purified enzyme consisted of four subunits each with a molecular weight of about 40 000 . In 0.14 M-Na2SO4, the tetramer dissociated completely into dimers . While the tetramer Km values for D-mannose and D-lyxose were 80 mM and 300 mM, respectively, the dimer Km values for these two sugars were both 300 mM . The amino acid composition of the enzyme was also determined. J Gen Microbiol, 1981 May, 124(Pt 1), 159 - 71 Effect of methyl methanesulphonate on the nucleoid structure of Escherichia coli; Lossius I et al.; Incubation of a strain of Escherichia coli K12 with 25 mM-methyl methanesulphonate (MMS) for 1 h changed the sedimentation coefficient of the nucleoids from 1600S to 850S . When isolated nucleoids were treated with MMS under identical conditions in vitro there was no change in the sedimentation coefficient . Alkaline sucrose-gradient centrifugation of DNA from cells treated with 25 mM-MMS for 1 h indicated that there were approximately 100 breaks plus apurinic sites per chromosome . Titration with ethidium bromide of nucleoids from MMS-treated cells showed that almost all supercoiling had been lost, suggesting that the breaks plus apurinic sites consisted mostly of breaks . Further experiments showed that the apurinic sites were probably created by non-enzymic depurination and that little non-enzymic strand breakage had occurred . The depurinated sites thus created could then serve as substrates for the apurinic-specific endonucleases of the cell, with the result that strand breakage occurred . MMS treatment did not cause any changes in the DNA:RNA ratio of the nucleoids . Removal of MMS followed by a period of incubation resulted in a decrease in the number of breaks plus apurinic sites and an increase in the sedimentation coefficient of the nucleoids . After 2 h incubation in MMS-free medium the sedimentation coefficient of the nucleoids from MMS-treated cells was the same as that of the control; the supercoiling was also partially restored . The effect of MMS on two MMS-sensitive mutants of E . coli, one a polA and the other a recA mutant, was also studied . In both cases MMS caused complete collapse of the nucleoid structure. Mutat Res, 1981 May, 81(3), 265 - 75 Plasmid pKM101-dependent repair and mutagenesis in Escherichia coli cells with mutations lexB30 tif and zab-53 in the recA gene; Blanco M et al.; Bacterial survival after UV irradiation was increased in E . coli K12 lexB30 and tif zab-53 mutants harboring plasmid pKM101 . Mutagenesis in response to UV was observed in these bacteria which, in absence of pKM101, are not UV-mutable . The mutator effect observed in unirradiated wild-type cells containing pKM101 was higher than incubation at 30 degrees C with adenine than at 37 degrees C . This effect was still enhanced by tif mutation, even in the tif zab-53 strain, but it was abolished by lexB30 mutation . In the tif zab-53 (pKM101) strain, repair and mutagenesis of UV-irradiated phage lambda was observed, but not in the lexB30 mutant carrying pKM101 . The pKM101 mutant, pGW1, was unable to protect tif zab-53 bacteria against killing by UV, whereas the protection of lexB30 was intermediate; moreover, it did not promote the mutator effect at 30 degrees C or enhance phage repair and mutagenesis in tif zab-53 cells . All UV-induced bacterial mutations in lexB30 (pKM101) strain were suppressors; in contrast, true revertants were found after UV irradiation of the tif zab-53 (pKM101) cells . We suggest that the constitutive activity of RecA protein is enough for the production of UV-promoted suppressor mutations, whereas true reversions require a more active form of this protein which could exert its effects directly or by acting at a regulatory level on other cellular functions. Rev Infect Dis, 1981 May-Jun, 3(3), 397 - 407 Syndrome of hyperinfection with Strongyloides stercoralis; Igra-Siegman Y et al.; Two patients hyperinfected with Strongyloides stercoralis (an intestinal nematode) are described . Both were both in Puerto Rico and had left the island six to 15 years previously; both were receiving adrenal steroids (one for Hodgkin's disease and the other for Goodpasture's syndrome) . One died shortly after diagnosis, but the other survived the hyperinfection syndrome and complicating bacterial sepsis and meningitis . In addition to our case reports, 103 previously described cases of presumed strongyloides hyperinfection are reviewed . Among 89 patients immunocompromised by therapy or disease, the mortality rate was 86%; bacterial sepsis often contributed to the fatal outcome . In most cases, infection was acquired in an endemic area, sometimes long before the hyperinfection syndrome occurred . The few patients who had never been to an endemic area had a history of prolonged contact with highly soiled material, an observation suggesting cross infection from a contaminated person . When administered in time, thiabendazole, the drug of choice for strongyloidiasis, was effective in 70% of cases . If intestinal infection with S . stercoralis is detected and treated before immunosuppressive therapy is initiated and if a high index of suspicion for the hyperinfection syndrome is maintained while immunosuppressive therapy is given, the mortality from this disease should decrease. Mikrobiologiia, 1981 May-Jun, 50(3), 467 - 70 {Changes in the redox potential in Escherichia coli growth cessation}; Oktiabr'skii ON et al.; Escherichia coli strains K-12 and M-17 were grown in batch and continuous cultures, with pulse addition of glucose or a nitrogen source as limiting substrates; the redox potential decreased when the growth ceased . The maximal deviation of the Eh was 150 mV when the strain K-12 ceased to grow . The Eh decreased rapidly for 25 min in the strain K-12 and for about 10--12 min in the strain M-17; then the Eh returned slowly to the initial level . The second phase took about 150 min in the strain K-12 and 40 to 60 min in the strain M-17 . No changes in the Eh were found when the culture was grown under the anaerobic conditions. Zh Mikrobiol Epidemiol Immunobiol, 1981 May, (5), 65 - 8 {Serologic affinity and specificity of action of pseudotuberculosis and coli-dysentery phages}; Gurleva GG et al.; The study of serological properties, specificity and the range of action has revealed affinity between Y . pseudotuberculosis phages (PST, 3M, Kotlyarova, 2344, 2391), some coliphages (T2, T3, T4) and Sh . dysenteriae phage (dd IV) . The existence of serovar III of Y . pseudotuberculosis phages has been established; to this serovar phage PST belongs . Newly isolated 2344 and 2391 belong to serovar I . The problem of the existence of Y . pseudotuberculosis phages as an independent group is discussed. Eur J Immunol, 1981 May, 11(5), 411 - 7 Sequential expression of B lymphocyte surface antigens in vitro; Abbott J et al.; Serological techniques were used to examine the process of sequential surface antigen expression on differentiating B cells in vitro . A 4-day culture system is described in which bone marrow lymphocytes from neonatal mice acquire in sequence the ability to express Lyb-2, IgM, Ia and IgD in response to a 3-h induction with E . coli lipopoly-saccharide (LPS) . Lyb-2 can be induced on day 1, IgM can be induced after 24 h, Ia after 48 h and IgD only after 96 h in culture . This sequence mimics the order of appearance of B cell surface antigens during ontogeny . When DNA synthesis is blocked from 0.24 h with hydroxyurea (HU), all surface antigens can be induced simultaneously by LPS . Immunoselection of one antigen-bearing population results in the loss of cells bearing other B cell antigens indicating that the surface antigens are induced on the same cells . When both HU and LPS were added to the cultures from the start, IgM appears after 11-14 h, Ia afer 14-15 h and IgD only after 19 h . Induction of antigen was demonstrated by he cytotoxicity assay, quantitative absorption and the protein A sheep red blood cell rosetting assaying . The results obtained show that there is a population of surface IgM-negative precursor B cells in young bone marrow which, when grown in vitro, become sequentially inducible for expression of B surface antigens . Inhibition of DNA synthesis promotes acquisition of the inducible state, and the sequence of antigen expression is correlated with specific time intervals after DNA synthesis has stopped. Can J Biochem, 1981 May, 59(5), 371 - 8 Conformation of cross-linked aspartate transcarbamoylase; Chan WW; We have previously shown that aspartate transcarbamylase loses its substrate cooperativity after modification with a cross-linking reagent . Depending on the presence or absence of substrate analogues during cross-linking, the derivatives resemble the relaxed (R) or taut (T) state, respectively . In the present study, we attempt to characterize the conformation of these derivatives and the effects of ligands . The putative T-state derivative was similar to the native enzyme in its reactivity towards p-hydroxymercuribenzoate and in the increase of reactivity upon addition of succinate . However, unlike the native enzyme it was not activated by succinate at low substrate concentrations . On the other hand, the putative R-state derivative showed greatly enhanced reactivity which was not substantially increased by succinate . In the presence of urea, the native enzyme and the two cross-linked derivatives all resembled the R state . Thus at low substrate concentrations urea activated both the native enzyme and the t-state derivative . In contrast, the effect of urea on the R state derivative is mainly inhibitory . The above results show that the R state has been definitely stabilized whereas the T-state derivative retains some conformational flexibility . Our observations also indicate that the conformational change induced by succinate has two distinct components of which only one is allowed in the T-state derivative. Arch Microbiol, 1981 May, 129(3), 240 - 6 Ultrastructural localization of the maltose-binding protein within the cell envelope of Escherichia coli; Boos W et al.; Logarithmically growing cells of Escherichia coli were fixed with glutaraldehyde and incubated with antimaltose-binding protein Fab coupled to horseradish peroxide (molecular weight of the complex 80,000) . The position of this complex within the cell envelope was determined by reacting with diaminobenzidine-H2O2, staining with osmium tetroxide and processing for thin section electron microscopy . The following observations were made: (i) induction of the maltose-binding protein resulted in swelling and staining of the outer membrane; (ii) the swelling and staining was more prominent in short cells, less prominent or absent in long cells; (iii) rare examples exhibited granular staining in the space between the plasma membrane and the peptidoglycan layer . These stainings were observable mainly in pole caps; (iv) a mutant lacking the receptor for phage lambda showed altered staining pattern . Treatment of glutaraldehyde-fixed cells with EDTA-lysozyme prevented the specific labelling of the maltose-binding protein. Proc Natl Acad Sci U S A, 1981 May, 78(5), 3128 - 32 Origin of replication from Xenopus laevis mitochondrial DNA promotes high-frequency transformation of yeast; Zakian VA; A specific fraction of chromosomal DNA from both yeast and a wide variety of other eukaryotes, but not from Escherichia coli, promotes high-frequency transformation in yeast . The plasmids containing these sequences are maintained as extra-chromosomal molecules in transformed cells . These results suggest that similar or identical sequences are used for the initiation of DNA replication in eukaryotes . To test this hypothesis, several foreign eukaryotic DNAs implicated directly or indirectly in the initiation of DNA replication have been examined for their ability to promote autonomous, extrachromosomal replication in yeast . Simian virus 40 DNA, amplified Xenopus laevis ribosomal DNA, X . laevis 5S ribosomal DNA, X . laevis mtDNA, and five different members of the Alu I family of human middle repetitive DNAs were cloned into the vector YIp5 and used to transform yeast . Of these DNAs, only Xenopus mtDNA promoted high-frequency transformation and extrachromosomal maintenance of YIp5 DNA . A 2.2-kilobase EcoRI fragment from the 17.4-kilobase mtDNA molecule was responsible for these activities . This fragment contains the sequence used for the initiation of replication in Xenopus mitochondria. Proc Natl Acad Sci U S A, 1981 May, 78(5), 2838 - 42 Unwinding of double-stranded DNA helix by dehydration; Lee CH et al.; Conformation changes of the double-stranded DNA helix in response to dehydration were investigated by monitoring, by agarose gel electrophoresis, the linking number of covalently closed circular DNA generated by ligation of linear DNA in the presence of different organic solvents or different temperatures . It was found that: (i) The DNA helix unwinds upon addition of certain organic solvents or elevation of temperature . (ii) The conformational change observed under the experimental conditions is a continuous process in response to the organic solvent concentration . (iii) The delta H of unwinding one linking of the DNA helix is constant at approximately 12.2 +/- 0.4 kcal/mol (1 kcal = 4.184 kJ); the corresponding delta S and d(delta S)/dn are 2nkR and 2kR, in which n is the relative equivalent linking number (referred to the state of delta S = 0 for unwinding) of the DNA, R is the gas constant, and k is equal to 1117/number of base pairs . The delta H, delta S, and d(delta S)/dn for unwinding i linkings are i X 12.2 kcal/mol, 2inkR, and 2ikR, respectively . (iv) d(delta S)/dn, like k, is inversely proportional to the number of base pairs in DNA . (v) Double-stranded DNAs of different chain lengths have average delta S = 35 cal/mol.K for unwinding one linking under the experimental conditions; this corresponds to 127 +/- 14 base pairs per "relative linking." Proc Natl Acad Sci U S A, 1981 May, 78(5), 2707 - 11 Genetic assignment of resonances in the NMR spectrum of a protein: lac repressor; Jarema MA et al.; By using a systematic genetic approach, the resonances in the 19F NMR spectrum of 3-fluorotyrosine-substituted lac repressor protein have been assigned . The NMR data indicate that each monomer of the repressor consists of two distinct and independent domains . One domain, the NH2-terminal sixth of the primary sequence, which has been shown to be very important for DNA binding, is very mobile . The remaining COOH-terminal sequence is more rigid . Ligands of the repressor, which affect its DNA binding capability, lead to conformational changes in the COOH-terminal domain . The approach to the assignment of spectral features taken here can be extended to other systems. Mol Biol (Mosk), 1981 May-Jun, 15(3), 569 - 74 {Binding of Escherichia coli 50S ribosomal subunit proteins with two large 5S RNA fragments}; Maimets TO et al.; Two fragments containing sequences from 1-41 nucleotide (small fragment) and from 42-120 nucleotide (large fragment) were isolated from E . coli 5S RNA T1 RNase partial digest . Affinity chromatography of 50S ribosomal proteins on the immobilized 5S RNA fragments revealed the ability of the large fragment to give a complex only with protein L25 . The small fragment did not bind ribosomal proteins . The intact and reassociated 5S RNA forms a complex consisting of proteins L5, L18, L25. Infect Immun, 1981 May, 32(2), 927 - 36 Plasmids coding for colonization factor antigen I and heat-stable enterotoxin production isolated from enterotoxigenic Escherichia coli: comparison of their properties; McConnell MM et al.; We examined seven enterotoxigenic Escherichia coli strains which produced colonization factor antigen I (CFA/I) . Four of these strains were from South Africa (three serotype O78:H12 and one serotype O63:H-), one was from Ethiopia (O78:H12), and two were from Bangladesh (O78:H11 and O78:H12) . Plasmids coding for CFA/I were mobilized from six of these strains by using resistance or enterotoxin factors . No plasmid was mobilized from the serotype O78:H12 Bangladesh strain . The transconjugants obtained from crosses with the O78 strains also produced heat-stable enterotoxin (ST), and additional investigations showed that CFA/I and ST were coded for by a single non-autotransferring plasmid . These plasmids were fertility inhibition negative, did not restrict any of the coliphages with which they were tested, and were incompatible with each other . Four had molecular weights of approximately 60 X 10(6), and one had a molecular weight of 52 X 10(6) . Like the other CFA/I plasmids, the CFA/I plasmid transferred from the O63:H- strain coded for ST, but this plasmid also coded for heat-labile enterotoxin . In most other respects the properties of this plasmid were similar to those of the CFA/I-ST plasmids previously described . The molecular weight of this plasmid was 65 X 10(6) . The IncT R-factor Rtsl was marked with a transposon for tetracycline resistance and then transferred into the two Bangladesh wild-type strains . Plasmids which coded for tetracycline resistance, CFA/I, and ST were transferred from these strains . These plasmids were incompatible with Rtsl and with the CFA/I-ST plasmids described above and were recombinants between Rtsl and a CFA/I-ST plasmid . Their properties are also described. Infect Immun, 1981 May, 32(2), 748 - 58 Stimulation of nonspecific resistance to infection induced by 6-O-acyl muramyl dipeptide analogs in mice; Matsumoto K et al.; The experimental system utilized in investigating the correlation between the chemical structures of muramyl peptides and their protective activities in the sepsis type of systemic infections caused by Escherichia coli was applied in evaluating the enhancement of resistance to infection induced by 32 synthetic glycopeptide analogs, including 6-O-acyl derivatives and 1-alpha-O-benzyl derivatives of muramyl dipeptide (N-acetyl muramyl-L-alanyl-D-isoglutamine) . In assessing the 6-O-acyl derivatives of muramyl dipeptide, we found that the degree of protective activity was attributable to the kinds of fatty acids introduced . Acylation of the 6-hydroxy group on the muramic acid moiety in muramyl dipeptide with natural mycolic acid or a synthetic fatty acid possessing either an alpha-branched or an alpha-branched, beta-hydroxylated group resulted in a decrease in or a disappearance of the protective activity of muramyl dipeptide . Acylation with a normal fatty acid or an iso fatty acid resulted in a retention or enhancement of muramyl dipeptide activity . The activity of acylated derivatives containing linear fatty acids was stimulated by increasing the chain length up to 18 carbon atoms . The highest degree of protective activity occurred with the derivatives acylated with straight-chain fatty acids, particularly with the derivatives acylated with palmitic acid and arachidic acid . Benzylation of the 1-hydroxy group of muramyl dipeptide resulted in a decrease in or a loss of protective activity. J Med Chem, 1981 May, 24(5), 538 - 44 Quantitative structure-selectivity relationships . Comparison of the inhibition of Escherichia coli and bovine liver dihydrofolate reductase by 5-(substituted-benzyl)-2,4-diaminopyrimidines; Li RL et al.; In our previous publication (Blaney, J . M.; Dietrich, S . W.; Reynolds, M . A.; Hansch, C . J . Med . Chem . 1979, 22, 614), correlation equations were presented for the inhibition of bovine liver and Escherichia coli dihydrofolate reductase (DHFR) by 5-(substituted benzyl)-2,4-diaminopyrimidines . These equations brought out differences in the way these two enzymes interact with substituents, which explain the high selectivity of drugs like trimethoprim . We have tested and further developed these equations in this report . It is of particular interest that our previously published correlation equation for E . coli DHFR accurately predicted the potency of a commercial competitor of trimethoprim (tetroxoprim) now in clinical use . We believe that new and effective competitors for trimethoprim can be designed by means of the two correlation equations. Am Surg, 1981 May, 47(5), 211 - 4 Ultrasonic detection of acute cholecystitis with pericholecystic abscesses; Deitch EA et al.; Perforation of the gallbladder is a life-threatening complication of acute cholecystitis that is often difficult to diagnose at an early stage . Standard radiographic and laboratory tests have not been reliable in identifying patients with this complication . In contrast, biliary sonography correctly diagnosed pericholecystic abscesses preoperatively in three patients with acute cholecystitis . The ultrasonic appearance of acute cholecystitis with a pericholecystic abscess was similar in all three patients . There was an extraluminal fluid collection located contiguous to a thick-walled gallbladder in the fundic region . The fluid collection was constant in location and could be seen in at least two different views . Two of these three patients had acalculous cholecystitis; the initial clinical diagnosis in one was pancreatitis, and in the other alcoholic hepatitis . Biliary sonography, by demonstrating a thickened gallbladder wall in the absence of ascites, strongly suggested that these two patients had acute acalculous cholecystitis, and not hepatitis or pancreatitis . The ultrasonic examination was a critical factor in the decision for prompt surgery instead of continued nonoperative management in these patients . These data suggest that not only can biliary sonography aid in the diagnosis of acute cholecystitis, calculous as well as acalculous, but can also visualize a pericholecystic abscess when it is present. J Clin Invest, 1981 May, 67(5), 1334 - 46 Glomerular dynamics and morphologic changes in the generalized Shwartzman reaction in postpartum rats; Conger JD et al.; The roles of glomerular functional and morphologic changes were examined in the acute renal failure associated with generalized Shwartzman reaction in postpartum Munich Wistar rats . The susceptibility of postpartum rats to acute deterioration in renal function after a 2-h endotoxin infusion was found to be greater than in virgin litter mates: glomerular filtration rate fell by 93% in the former vs . 24% in the latter group (P less than 0.001) . In postpartum rats there were marked changes in platelet count and fibrinogen level (P less than 0.025) compatible with consumption coagulopathy . Renal blood flow and glomerular filtration rate fell from 5.5 +/- 0.9 and 0.74 +/- 0.12 to 2.0 +/- 0.7 and 0.12 +/- 0.01 ml/min, respectively (both P less than 0.001) . Blood pressure did not change . Results of glomerular dynamics studies showed decreases in single nephron filtration rate from 28 +/- 7 to 6 +/- 4 nl/min and in glomerular plasma flow rate from 77 +/- 26 to 23 +/- 12 nl/min (both P less than 0.001) . Afferent net ultrafiltration pressure fell from 20 +/- 3 to 5 +/- 4 mm Hg due to a fall in glomerular capillary hydraulic pressure from 47 +/- 1 to 29 +/- 5 mm Hg (P less than 0.001) . There were four- and twofold increases in afferent and efferent arteriolar resistances, respectively . Less than 20% of glomeruli had evidence of fibrin deposition after 2 h of endotoxin infusion, a time when glomerular filtration rate was reduced by greater than 90% . {1-Sar, 5-Ile, 8-Gly} angiotensin II infusion before endotoxin significantly protected glomerular filtration rate, 62 vs . 7% of control in rats with no preinfusion (P less than 0.01) despite consumption coagulopathy and glomerular fibrin deposition similar to rats without pretreatment . These data suggest that the early deterioration in renal function in the generalized Shwartzman reaction in the postpartum rat is due to major changes in glomerular dynamics induced by neurohumoral agents and that glomerular fibrin deposition plays a lesser pathogenetic role at this time in this disorder . The study does not address the pathogenesis of renal failure in pregnancy nor peripartum renal failure in another species. Clin Chem, 1981 May, 27(5), 673 - 7 A homogeneous fluorescent immunoassay for human immunoglobulin M; Worah D et al.; We describe a homogeneous substrate-labeled fluorescent immunoassay for human IgM . In this competitive-binding method we use a fluorogenic substrate for Escherichia coli beta-galactosidase, N-(6-aminohexyl)-7-beta-galactosyl-coumarin-3-carboxamide, which is covalently coupled to IgM . The fluorescence emission intensity of the labeled IgM at 450 nm (with excitation at 400 nm) is negligible, but when beta-galactosidase is added, the acetal linkage of the galactosyl moiety is hydrolyzed and the product has a greatly enhanced fluorescence . Formation of this fluorescent product is inhibited when antibody specific for IgM is bound to the labeled protein . In competitive-binding reactions, IgM from the serum competes with the labeled IgM for antibody binding sites and consequently the fluorescence produced by the enzymic reaction is related to the IgM concentration . The working range of the assay is between 0.5 and 5.0 g of IgM per liter when a 50-fold predilution of the sera is used . The assay does not cross react significantly with immunoglobulins G or A. J Dent Res, 1981 May, 60(5), 933 - 5 Endotoxin-inactivating potency of hydrogen peroxide: effect on cell growth; DeRenzis FA; An in vitro system was used to study the detoxifying potential of H2O2 on bacterial endotoxin . L929 fibroblasts were exposed to H2O2-treated and -untreated endotoxin . Growth and viability assays were made at 24, 48, 72, and 96 h . The cultures exposed to H2O2-treated endotoxin showed a normal growth rate, whereas the cultures exposed to untreated endotoxin displayed a substantial reduction in growth . Implications are presented as to the potential role of H2O2 in the pathogenesis and treatment of periodontal disease. J Bacteriol, 1981 May, 146(2), 676 - 83 Toxicity of leucine-containing peptides in Escherichia coli caused by circumvention of leucine transport regulation; Tavori H et al.; A variety of leucine-containing peptides (LCP), Phe-Leu, Gly-Leu, Pro-Leu, Ala-Leu, Ala-Leu-Lys, Leu-Phe-Ala, Leu-Leu-Leu, and Leu-Gly-Gly, inhibited the growth of a prototrophic strain of Escherichia coli K-12 at concentrations between 0.05 and 0.28 mM . Toxicity requires normal uptake of peptides . When peptide transport was impaired by mutations, strains became resistant to the respective LCP . Inhibition of growth occurred immediately after the addition of LCP, and was relieved when 0.4 mM isoleucine was added . The presence of Gly-Leu in the medium correlated with the inhibition of growth, and the bacteria began to grow at the normal rate 70 min after Gly-Leu became undetectable . Disappearance of the peptide corresponded with the appearance of free leucine and glycine in the medium . The concentration of leucine inside the LCP-treated bacteria was higher than that in the leucine-treated and the control cultures . We suggest that entry of LCP into the cells via peptide transport systems circumvents the regulation of leucine transport, thereby causing abnormality high concentrations of leucine inside the cells . This accumulation of leucine interferes with the biosynthesis of isoleucine and inhibits the growth of the bacteria. J Bacteriol, 1981 May, 146(2), 564 - 70 Transformation in Escherichia coli: stages in the process; Bergmans HE et al.; Transformation experiments with Escherichia coli recipient cells and linear chromosomal deoxyribonucleic acid (DNA) are reported . E . coli can be rendered competent for DNA uptake by a temperature shock (0 degrees C leads to 42 degrees C leads to 0 degrees C) of the recipient cells in the presence of a high concentration of either Ca2+ or Mg2+ ions . Uptake of DNA into a deoxyribonuclease-resistant form, for which the presence of Ca2+ is essential, was possible during the temperature shock but appeared to occur most readily after the heat shock during incubation at 0 degrees C . When DNA was added to cells that had been heat shocked in the presence of divalent cations only, DNA uptake also occurred . This suggests that competence induction and uptake may be regarded as separate stages . Under conditions used to induce competence, we observed an extensive release of periplasmic enzymes, probably reflecting membrane damage induced during development of competence . After the conversion of donor DNA into a deoxyribonuclease-resistant form, transformants could be selected . It appeared that incubation, before plating, of the transformation mixture in a medium containing high Ca2+ and Mg2+ concentrations and supplemented with all growth requirements increased the transformation frequency . This incubation probably causes recovery of physiologically labile cells. J Bacteriol, 1981 May, 146(2), 453 - 9 Multiple forms of alkaline phosphatase from Escherichia coli cells with repressed and derepressed biosynthesis of the enzyme; Nesmeyanova MA et al.; Isolation of multiple forms of alkaline phosphatase from Escherichia coli cells with repressed and derepressed biosynthesis of the enzyme is reported . Three enzyme forms were isolated from cells with derepressed synthesis, and one form was isolated from cells with repressed enzyme synthesis . The multiple enzyme forms did not differ in pH optimum, thermostability, or the degree of inhibition with orthophosphate; however, they did differ in the relative rate of hydrolysis of different substrates . The addition of substrates to the cells during enzyme derepression resulted in changes of the ratio of the multiple forms. J Bacteriol, 1981 May, 146(2), 435 - 43 Effect of metabolic inhibitors on entry of exogenous deoxyribonucleic acid into Ca2+-treated Escherichia coli cells; Sabelnikov AG et al.; The effect of various metabolic inhibitors (carbonylcyanid-m-chlorophenylhydrazone, nigericin, valinomycin, dicyclocarbodiimide, arsenate, NaF, etc.) and lipid-soluble synthetic ions (tetraphenylphosphonium bromide and tetraphenylboron sodium) on deoxyribonucleic acid (DNA) entry during transformation of Ca2+-treated Escherichia coli cells with plasmid DNA and on cell viability was investigated . In contrast to intact cells, Ca2+-treated E . coli cells were permeable to nigericin, valinomycin, and the other drugs tested . The inhibitors differentially affected {14C}proline active transport, and whereas some drugs inhibited transformation, the effects did not correlate with the effects on transport . The most potent inhibitors of transformation were nigericin, dicyclocarbodiimide, and tetraphenylboron sodium . Carbonylcyanid-m-chlorophenylhydrazone, tetraphenylphosphonium bromide, and valinomycin were relatively inactive . Tetraphenylboron sodium- and nigericin-treated cells bound were plasmid {14C}DNA in the deoxyribonuclease-resistant form than the control and other sample cells . Nevertheless, te penetration of exogenous plasmid DNA into the cell was greatly reduced, at least in case of nigericin . Unlike the other drugs, nigericin and dicyclocarbodiimide drastically affected the cell viability, the former within very short times of interaction . It is concluded that proton motive force does not play any significant role in DNA entry into Ca2+-treated E . coli cells . The results also suggest that adenosine 5'-triphosphate is not required for DNA entry either . The inhibitory effect of certain drugs is discussed in terms of structural perturbations induced by the drugs in cell envelope membranes. Eur J Biochem, 1981 May, 116(1), 93 - 9 The binding of dansylated initiation factor 3 to the 30-S subunit of Escherichia coli: a fluorimetric and biochemical study; Box R et al.; Initiation factor 3 (IF-3) has been labelled using dansyl (1-dimethylaminonaphthalene-5-sulphonyl) chloride under conditions designed to preserve the biological activity of the factor . The sites of modification of the IF-3 have been determined by peptide mapping and sequencing: about six lysines (73, 80, 91, 96, 99, 112) react in various proportions . However, if an IF-3 molecule bears more than one dansyl group on average then its activity is lost . The extent of incorporation is proportional to the amount of dansyl chloride used in the reaction . Spectrofluorimetric analysis of the dansyl-IF-3 leads to the following conclusions . (a) The motion of the dansyl label does not change greatly upon binding to the 30-S subunit . (b) The label is not close enough to any tryptophan group of the ribosome in the 30-S-subunit . IF-3 complex to allow energy transfer . (c) The IF-3 chain is folded so as to bring the tyrosine groups close to the dansyl-binding sites . (d) The stoichiometry of the binding of IF-3 to 30-S ribosomal subunits is close to 1:1 and the binding constant is 2 x 10(7) M-1 . IF-3 also binds non-covalently the fluorescent indicator 8-anilinonaphthalene 1-sulphonate (ANS) with an apparent binding constant of approximately 8000 M-1 . An interaction between ANS and poly(A-U-G), both bound to IF-3, was demonstrated . Combining these results with those for dansyl-IF-3 leads to a model for the interaction between IF-3 and the 30-S subunit involving a combination of 'hydrophobic' and electrostatic attraction between the factor and ribosomal RNA. Eur J Biochem, 1981 May, 116(1), 207 - 13 Effect of threonylcarbamoyl modification (t6A) in yeast tRNA Arg III on codon-anticodon and anticodon-anticodon interactions . A thermodynamic and kinetic evaluation; Weissenbach J et al.; The effect of N-{9-(beta-D-ribofuranosyl) purin-6-ylcarbamoyl}threonine (t6A) adjacent to anticodon U-C-U of yeast tRNA Arg III (where U is a modified U), compared to its unmodified adenosine counterpart, has been evaluated by three independent methods: (a) the polynucleotide-directed binding of tRNA on ribosomes, (b) the ribosome-free trinucleotide binding to the anticodon, (c) the anticodon-anticodon binding test . The results obtained by these three methods indicate a small but significant stabilization effect of t6A on the binding of yeast tRNA Arg III with (a) poly(A,G) in the presence of Escherichia coli ribosomes, (b) free A-G-A triplet, and (c) E . coli tRNA Ser V (anticodon G-G-A) . We therefore conclude that the stabilization effect of t6A occurs on U x A and U x G base pairs adjacent to the 5' side of the modified nucleoside, most probably by stacking. J Bacteriol, 1981 May, 146(2), 718 - 24 Mutations in the ilvY gene of Escherichia coli K-12 that cause constitutive expression of ilvC; Biel AJ et al.; A derivative of Escherichia coli K-12 bearing an ilvC-lac fusion has been studied . beta-Galactosidase formation in this strain is under the control of the ilvC promoter and is therefore induced by the acetohydroxy acids . Derivatives of this fusion strain were isolated that constitutively expressed beta-galactosidase . When an ilvC-containing episome was introduced into these strains, acetohydroxy acid isomeroreductase was also constitutively expressed . The lesions are trans dominant and lie in ilvY, the structural gene specifying a positive control element, v, needed for induction of the isomeroreductase . It was concluded from measurements of beta-galactosidase levels in various diploid strains that, although wild-type v requires inducer to act as a positive control element, it does not act as a repressor in the absence of inducer. J Virol, 1981 May, 38(2), 497 - 503 Specialized transduction with lambda plac5: dependence on recA and on configuration of lac and att lambda; Porter RD et al.; The construction of lambda plac5 transducing phages carrying various lacZ alleles is described . Genetically disabled (N- N- P-) lambda plac transducing the phages were used to study the dependence of specialized transduction on host RecA function and on the location of the lacZ gene in the recipient strain . In the absence of site-specific recombination at att lambda, transduction was completely dependent on host RecA function . Regardless of the configuration of att lambda, lambda plac transducing phages recombined at a 20- to 50-fold higher frequency with F42 lac than with a lac gene located in the cellular chromosome . Deletion mutants of lacZ in the recipient strain were used to show that the probability of lac recombination resulting from lambda plac infection is apparently proportional to the amount of homology between the parental lacZ genes. J Bacteriol, 1981 May, 146(2), 823 - 5 Two classes of region III flagellar genes in Escherichia coli; Kondoh H et al.; We infected various nonflagellated mutants of Escherichia coli with fla-transducing phages and followed the kinetics of the appearance of motility . Our analysis revealed two distinct classes of region III fla genes . Class II fla genes (hag, flaD) functioned 15 min later than class I fla genes (flaN, flaB, flaC, flaO, flaA, flbD, flaQ, flaP) in flagellar morphogenesis . We suggest that the two classes of fla genes are involved in two different stages, initiation (class I) and completion (class II), of flagellar formation. J Bacteriol, 1981 May, 146(2), 804 - 7 Reduction in three iron-regulated outer membrane proteins and protein a by the Escherichia coli K-12 perA mutation; Lundrigan M et al.; We identified four outer membrane proteins (protein a and the iron-regulated proteins 74K, 81K, and 83K) present in reduced amounts of Escherichia coli K-12 perA strains . A comparison of the levels of enterochelin with the levels of 74K, 81K, and 83K suggested that perA acts posttranscriptionally. J Gen Microbiol, 1981 May, 124(Pt 1), 181 - 5 Respiratory biogenesis during the cell cycle of aerobically grown Escherichia coli K12 . The accumulation of iron-sulphur clusters and their orientation in the membrane; Poole RK et al.; The magnitudes of four signals detectable in intact cells by electron paramagnetic resonance spectroscopy, and assigned to membrane-associated protein, increase continuously during the cell cycle of Escherichia coli K12 . Studies on membrane multilayers prepared from cells separated according to their age in the cycle suggest that the orientation within the membrane of the ferredoxin-type signal is invariant throughout the cycle. J Gen Microbiol, 1981 May, 124(Pt 1), 17 - 23 Hybrid plasmids containing the citrate synthase gene (gltA) of Escherichia coli K12; Guest JR; The Clarke-Carbon colony bank containing ColE1-Escherichia coli hybrid plasmids was screened by conjugation for complementation of the citrate synthase lesion of a gltA mutant . Three ColE1-gltA+ plasmids were identified: pLC26-17 (16.3 kilobase pairs), pLC27-18 (16.35 kb) and pLC31-28 (26.0 kb) . The citrate synthase activities of strains containing the hybrid plasmids were amplified 3- to 10-fold . Genetic studies indicated that the smaller plasmids may contain at least part of the succinate dehydrogenase gene (sdh) . A physical map of a 19.4 kb region of the E . coli chromosome containing the citrate synthase gene (gltA) was constructed by restriction analysis with the isolated plasmids . The relative positions of 20 restriction sites were defined and a region (3.1 kb) containing the gltA+ gene was identified in the segment common to all three plasmids. Gene, 1981 May, 13(4), 339 - 46 Recovery of DNA fragments inserted by the "tailing" method: regeneration of PstI restriction sites; Otsuka A; A general method has been developed for the recovery of any DNA fragment inserted into a cloning vehicle containing a single endonuclease PstI site . Endonuclease PstI sites are regenerated by the addition of one or more deoxyguanosine residues to the 3' termini of the PstI-cleaved vehicle by terminal deoxynucleotidyl transferase . Chain elongation by terminal deoxynucleotidyl transferase is then continued with dITP, dATP or dGTP . A plasmid vehicle, pAO1, containing a single PstI site has been constructed . Insertional (foreign) DNA fragments that were "tailed" with dCTP have been annealed to PstI-cleaved pAO1 that was "tailed" with dGTP . When the annealed fragments were used to transform competent Escherichia coli cells, the single-stranded DNA gaps in the recombinant plasmids were repaired . Plasmids recovered from transformed bacteria could be cleaved by PstI into the insertional DNA with dG:dC tracts and linear pAO1 molecules. Can J Biochem, 1981 May, 59(5), 311 - 4 Interaction of selectively spin-labeled 70S ribosomes with tRNAPhe . An electron paramagnetic resonance study; Rodriguez A et al.; 70S ribosomes from Escherichia coli, selectively spin labeled on the SH groups of proteins S18, S12, S21, S17, and L27, were used to study the formation of the tertiary complex ribosome-poly(U)-tRNAPhe . Most of these ribosomal proteins are located in the region of binding of tRNA . The electron paramagnetic resonance observable structural change suggests a loosening of the ribosome structure upon binding of the tRNA molecule. Res Vet Sci, 1981 May, 30(3), 379 - 81 Experimental Escherichia coli and rotavirus infection in lambs; Wray C et al.; Colostrum-deprived lambs were infected with either enteropathogenic Escherichia coli(O9:K30:K99) or rotavirus or a mixture of the E coli and rotavirus . E coli doses of 10(6) and above consistently produced diarrhoea, as did experimental rotavirus infection . When both of the agents were administered, the mortality rate was higher, although the duration of diarrhoea was no greater than that observed when either of the two agents was administered alone. Proc Natl Acad Sci U S A, 1981 May, 78(5), 2937 - 41 Mutations that affect lamB gene expression at a posttranscriptional level; Schwartz M et al.; We previously obtained strains of Escherichia coli in which the beginning of gene lacZ, which codes for beta-galactosidase, is replaced by the beginning of gene lamB, which codes for a maltose-inducible outer membrane protein . In some of these strains the induction (with maltose) of lamB-lacZ hybrid protein synthesis was lethal because of membrane damage resulting from an incomplete export of this protein to the outer membrane . We describe here a class of maltose-resistant mutants obtained from one such strain . Mutants in this class fail to produce the lamB-lacZ hybrid protein but retain the ability to express lacY, which is located distal to the hybrid gene . Some of the mutants carry deletions within the hybrid gene . The others carry point mutations which most probably affect the initiation of translation at the beginning of the hybrid gene . One of these is located in the sequence that codes for the presumed ribosome interaction site on the mRNA . Three others, of which two are located in the coding region (sixth codon), are believed to result in an alteration of mRNA secondary structure such that the accessibility of the ribosome interaction site is reduced. Proc Natl Acad Sci U S A, 1981 May, 78(5), 2913 - 7 Tandem termination sites in the tryptophan operon of Escherichia coli; Wu AM et al.; In vivo, transcription of tryptophan (trp) operon mRNA appears to terminate at a site (trp t) 36 nucleotides after the last structural gene, and efficient function at this site requires the protein factor rho . However, distal nucleotide sequences also seem to play a role in modulating termination at trp t . We report here our in vitro studies of DNA fragments carrying portions of the trp termination region . Transcription of these DNA fragments in a purified system demonstrates that RNA polymerase actually recognizes two different termination sites . Termination at the previously characterized site, trp t, is only 25% efficient, and it is unaffected by the presence of rho factor in vitro . However, addition of rho to the transcription reaction mixture reveals that termination also occurs within a region that we have designated trp t', located about 250 bases past trp t . These two sites behave independently in vitro, whether in the tandem configuration or cloned separately, and their structural features and functional characteristics are quite different . This contrasts with the observation that termination of transcription at the end of the trp operon in vivo appears to require a rho-mediated interaction between trp t and trp t' . The possible involvement of other factors and the significance of multiple termination sites is discussed. Proc Natl Acad Sci U S A, 1981 May, 78(5), 2825 - 9 Molecular cloning and characterization of winter flounder antifreeze cDNA; Lin Y et al.; Double-stranded cDNA was synthesized from partially purified winter flounder antifreeze mRNA and inserted into the endonuclease Pst I site of plasmid pBR322 by the poly(dG).poly(dC) homopolymer extension technique . The recombinant plasmids wee used to transform Escherichia coli . Clones containing antifreeze cDNA inserts were identified by the hybridization-selection technique . One of the inserts, 380 nucleotides in length, was digested with endonucleases Sau3AI and HinfI, which cleaved the insert into three fragments . The nucleotide sequences of these fragments were determined . The cDNA contains the entire coding sequence for a possible antifreeze peptide, including the leader sequence . The predicted amino acid sequence is similar to but not identical to one of the known sequences of antifreeze peptide . Within the cDNA are three segments of repeating sequences . The basic repeating sequence of 11 amino acids is maintained in the amino acid sequence coded by the cDNA and in the antifreeze peptide. Proc Natl Acad Sci U S A, 1981 May, 78(5), 2772 - 6 cDNA clone coding for part of a mouse H-2d major histocompatibility antigen; Kvist S et al.; mRNA coding for mouse major transplantation antigens of the d haplotype was partially purified, copied into double-stranded cDNA, and cloned in Escherichia coli . Clones were selected by their ability to hybridize specifically with mRNA coding for H-2K, D, or L antigens . One of these clones, pH-2d-1, carries a 1200-base-pair insert, comprising the noncoding region, including poly(A) at the 3' end and part of the coding region . A partial sequence of the latter region showed extensive homology with the known amino acid sequences of H-2Kb,Kk, and HLA-B7 antigens . From this comparison, it appears that the coding region extends from amino acid 133 in the second domain, through the third domain, to the cytoplasmic COOH-terminal region . A stretch of 24 hydrophobic or uncharged residues, located 31 amino acids from the COOH-terminal end, could represent the segment that spans the membrane . This is followed on the cytoplasmic side of the membrane by a cluster of basic amino acids and a possible phosphorylation site on a threonine residue. Mol Biol (Mosk), 1981 May-Jun, 15(3), 636 - 52 {Reaction of pyrophosphorolysis catalyzed by Escherichia coli RNA polymerase}; Rozovskaia TA et al.; E . coli RNA polymerase is shown to be capable of catalyzing consecutive DNA-dependent pyrophosphorolysis of RNA in the presence of inorganic pyrophosphate and Mg2+ . Active ternary complex of the enzyme with DNA and nascent RNA is needed for the reaction, the mixure of all the components can not carry out pyrophosphorolysis . The reaction was realized in the absence of added nucleoside triphosphates . Nucleoside triphosphates are low molecular mass products of the reaction . The rate of pyrophosphorolysis of the RNA synthesised for the AI promoter of the DNA of wild type T7 phage and delta D III T7 mutant phage was followed as a function of primary structure of the DNA, temperature, ionic strength and inorganic pyrophosphate concentration . The relative rate pyrophosphorolysis for particular nucleotides in different regions of the RNA can differ by several orders of magnitude depending on the primary structure of the RNA that undergoes pyrophosphorolysis . Ternary complex containing partially pyrophosphorilised RNA is active on the RNA synthesis when pyrophosphate is removed and nucleoside triphosphates are added to the reaction mixture . RNA as short as 70-8 nucleotides long can be produced at the conditions used . It seems that efficient dissociation in this region of RNA limits the pyrophosphorolysis to proceed up to the 5' end of RNA . Ternary complex of RNA polymerase with nascent RNA and DNA is shown to undergo site specific dissociation . The specificity of the dissociation is shown to be a function of the primary structure of RNA and the direction of the reaction . Dissociation occurs at different places along RNA sequence when the RNA is synthesised and when it is pyrophosphorilised. Eur J Biochem, 1981 May, 116(1), 59 - 65 The influence of elongation-factor-Tu . GTP and anticodon-anticodon interactions on the anticodon loop conformation of yeast tRNATyr; Weygand-Durasevic I et al.; The interactions of yeast tRNATyr, spin-labelled at position i6A-37 next to the anticodon, with EF-Tu . GTP and with Escherichia coli tRNAVal (which has a complementary anticodon) have been studied . The immobilization of the spin label upon ternary complex formation shows a conformational change of the anticodon region, although this part of tRNATyr is not in direct contact with the protein, as indicated by RNase T1 digestion . Upon anticodon-anticodon interaction, no conformational change of the anticodon loop of tRNATyr was observed. Eur J Biochem, 1981 May, 116(1), 165 - 70 Nucleotide sequence coding for the respiratory NADH dehydrogenase of Escherichia coli . UUG initiation codon; Young IG et al.; The nucleotide sequence of the structural gene coding for the respiratory NADH dehydrogenase of Escherichia coli has been determined by the chain-termination method . The reading frame for the protein starts with the unusual initiation codon UUG and predicts an amino acid sequence of 434 residues (Mr = 47 304) . The reading frame was confirmed by protein chemical studies including determination of the N-terminal sequence of the protein . The product made in vivo was found to have threonine as its N-terminal residue, indicating that the initiating N-formylmethionine had been removed by post-translational processing. J Virol, 1981 May, 38(2), 761 - 9 Analysis of JC virus DNA purified directly from human progressive multifocal leukoencephalopathy brains; Rentier-Delrue F et al.; Human polyomavirus JC DNA was purified directly from the diseased brain tissue of two patients with progressive multifocal leukoencephalopathy (PML) by a method employing differential salt precipitation (B . Hirt, J . Mol . Biol . 26:365-369, 1967) . Each of the viral genomes (JC-NIH-1 and JC-NIH-2) was molecularly cloned intact in Escherichia coli, using pBR322, at their unique EcoRI (0.00 map unit) and BamHI (0.51 map unit) sites . The JC-NIH-1 genome was approximately 50 base pairs larger and the JC-NIH-2 genome was approximately 50 base pairs smaller than the prototype human polyomavirus JC (Mad-1) DNA . Analysis of the restriction endonuclease cleavage fragments of these two DNAs and the human polyomavirus JC (Mad-1) DNA revealed only slight differences which mapped in a region of the genome extending from 0.67 to 0.74 map unit . From previous homology studies, this region of variance corresponds to the noncoding region to the late side of the origin of DNA replication. J Lab Clin Med, 1981 May, 97(5), 602 - 9 Determination of a specific receptor for formyl-methionyl-leucyl-phenylalanine on th pulmonary alveolar macrophage and its relationship to chemotaxis and superoxide production; Spilberg I et al.; 3H-FMLP, a chemotactic peptide that resembles Escherichia coli chemotactic factor, is chemotactic for PAM, binds specifically to a site on the cell, and induces the generation of superoxide radicals by the cell . Scatchard analysis revealed an equilibrium dissociation constant at 26 degrees C of 1.45 x 10(-8)M and the presence of 1.7 , 10(5) receptors per cell . Binding was not inhibited by a partially purified C5a preparation or by the neutrophil-derived CCF but was inhibited by various N-formylated peptides . The order of potency of each peptide to inhibit 3H-FMLP binding was identical to the order of potency of each peptide to induce generation of superoxide by the PAM . Only small amounts of beta-glucuronidase activity and no lysozyme were detected in the supernatant after incubation of the cells for 30 min with varying concentrations of FMLP. J Bacteriol, 1981 May, 146(2), 660 - 7 Cloning and characterization of the Escherichia coli gene coding for alkaline phosphatase; Berg PE; The Escherichia coli structural gene for alkaline phosphatase, phoA, and a promoter-like mutant of phoA, called pho-1003(Bin) phoA+, were cloned by using plasmid vectors . Initially, these genes were cloned on deoxyribonucleic acid fragments of 28.9 kilobases (kb) . Subsequently, they were subcloned on fragments and 4.8 and then 2.7 kilobases . A restriction map was developed, and phoA was localized to a 1.7-kb region . The promoter end of the gene was inferred by its proximity to another gene cloned on the same deoxyribonucleic acid fragment, proC . The stability of the largest plasmid (33.3 kb) was found to be recA dependent, although the subcloned plasmids were stable in a recA+ strain . Synthesis of alkaline phosphatase directed by the phoA+ and pho-1003(Bin) phoA+ plasmids in a phoA deletion strain was assayed under repressing and derepressing levels of phosphate . These data were compared with the copy numbers of the plasmids . It was found that synthesis of alkaline phosphatase was tightly regulated, even under derepressing conditions: a copy number of 17 enabled cells to synthesize only about twofold more enzyme than did cells with 1 chromosomal copy of phoA+ . Enzyme levels were also compared for cells containing pho-1003(Bin) phoA+ and phoA+. Biokhimiia, 1981 May, 46(5), 771 - 81 {Isolation of ribonucleic acids possessing template activity from Crithidia oncopelti mitochondria}; Zaitseva GN et al.; Three RNA fractions (12SE, 9SE and 8SE) were isolated from highly purified mitochondrial preparations of Crithidia oncopelti . Using inhibitory analysis and competitive hybridization, it was shown that the mitochondrial fractions under study are synthesized in the kinetoplasts and do not belong to ribosomal RNA . It was demonstrated that these fractions possess template activity, since they stimulate protein synthesis in a cell-free system of E . coli . Each fraction is subjected to translation to form one predominant polypeptide with molecular weight of about 20 000, 10 000 and 7000, respectively. Proc Natl Acad Sci U S A, 1981 May, 78(5), 2848 - 52 Target cell specificity of two species of human interferon-alpha produced in Escherichia coli and of hybrid molecules derived from them; Streuli M et al.; Plasmids containing cDNAs for human interferon (IfN) alpha-1, IFN alpha-2, and several hybrids of the two cDNAs, all joined identically to an Escherichia coli lac promoter fragment gave rise, in E . coli, to fused interferons (fIFNs) that had very different target-cell specificities . fIFN alpha-1 had a lower specific activity on human WISH cells than on bovine MDBK cells, while fIFN alpha-2 showed the opposite behavior . fIFN hybrids with the NH2-proximal half of fIFN alpha-2 behaved qualitatively like fIFN alpha-1, and those with the NH2-proximal half of fIFN alpha-2, behaved like fIFN alpha-2 . On mouse L929 cells, fIFN alpha-2 was almost inactive, while fIFN alpha-1 showed relatively high activity . In this case, the fIFN hybrids with the COOH-proximal half of IFN alpha-1 showed activity on mouse cells, while the reciprocal hybrid did not . In many cases, the activity spectrum of the hybrids was very different from that of either parent . We propose that the IFN molecule has either two binding sites or two regions constituting the binding site, one in the COOH- and the other in the NH2-proximal half . The experimental findings can be accounted for if the fits of the two sites to their receptor counterparts on different cell lines are independent of one another. Proc Natl Acad Sci U S A, 1981 May, 78(5), 2767 - 71 In vivo and in vitro detection of the leader RNA of the histidine operon of Escherichia coli K-12; Frunzio R et al.; The DNA of the attenuator region of the histidine operon of Escherichia coli has been transcribed in a purified in vitro system and found to synthesize two major RNA transcripts . The first one, 180 nucleotides long, has been identified as the histidine-specific leader RNA . It contains the coding sequence for the leader peptide {Di Nocera, P . P., Blasi, F., Di Lauro, R., Frunzio, R . & Bruni, C . B . (1978) Proc . Natl . Acad . Sci . USA 75, 4276-4280} and is terminated at the attenuator site . Termination of transcription at this site is extremely efficient in the in vitro system . The leader RNA also has been detected in vivo in a minicell producer strain transformed with plasmids harboring the regulatory region of the histidine operon of E . coli . A second RNA molecule is synthesized in the in vitro system . It has a divergent direction of transcription with respect to the histidine leader RNA, but its role, if any, in the regulation of the histidine operon remains to be ascertained . The existence of the histidine leader RNA lends support to the regulatory mechanism which postulates that regulation of the histidine operon is dependent on the alternative secondary structures that the leader RNA may assume, depending on whether or not the histidine-rich leader peptide is translated. Can J Microbiol, 1981 May, 27(5), 547 - 50 Simple assay and extraction of periplasmic penicillinase in Escherichia coli; Choma CT et al.; Benzylpenicillin was clearly separated from benzylpenicilloic acid by ascending chromatography on a diethylaminoethyl cellulose paper using 0.1 M ammonium acetate as a solvent . Using this chromatographic system, penicillinase was assayed by measuring the formation of {14C}benzylpenicilloic acid from {14C}benzylpenicillin . This assay remedies the lack of specificity of the commonly used iodometric assays . Periplasmic penicillinase was released from Escherichia coli by suspension in a mixture of 1% phenethyl alcohol and 5 mM ethylenediaminetetraacetate (pH 7.0) . This simple extraction method not only facilitates the assay of penicillinase in an E . coli culture, but will also be useful for large-scale purification of periplasmic penicillinase. J Immunol, 1981 May, 126(5), 1883 - 6 Hapten-specific murine colony-forming B cells: in vitro response of colonies to fluoresceinated thymus independent antigens; Pillai PS et al.; The ability to clone hapten-specific B cells in agar and to subsequently trigger their clonal progeny to antibody synthesis was investigated . Fluorescein (FL) specific B cells were purified on FL-gelatin dishes and cultured in semisolid agar for 6 to 7 days; individual colonies were then picked for restimulation in microculture . FL-specific B cells could be cloned as efficiently as unpurified splenic B cells . The number of colonies formed depended on the presence of sheep erythrocytes (SRBC) or E . coli lipopolysaccharide (LPS) in the cultures . An additive number of colonies were observed with SRBC + LPS compared to that of SRBC or LPS alone . The colonies obtained from SRBC-containing cultures were stimulatable at high frequency by various FL-conjugated antigens to yield anti-FL PFC . However, colonies grown with LPS as the only additive were not stimulatable by any of the antigens tested . On the other hand, addition of M phi or SRBC as additional "mitogens" along with LPS in the agar resulted in progeny colonies that could respond in vitro . Although M phi did not increase the number of colonies, their presence enhanced the size and in some cases the frequency of stimulatable colonies . These data complement earlier observations in suggesting that different B cell subpopulations may grow under different cloning conditions . Moreover, the ability to stimulate the clonal progeny of single B cells to antibody synthesis should permit further definition of triggering and tolerance events at the single-cell level. J Bacteriol, 1981 May, 146(2), 552 - 63 Role of ribonucleic acid synthesis in conjugational transfer of chromosomal and plasmid deoxyribonucleic acids; Maturin LJ Sr et al.; A strain of Escherichia coli K-12 containing mutations that allow for the experimental control of RNA and DNA syntheses was constructed to investigate the role that RNA synthesis plays in conjugational DNA transfer when DNA replication is inhibited . The mutations possessed by this strain and its donor derivatives include: (i) thyA, which blocks synthesis of dTMP, causing a requirement for thymine; (ii) deoC, which blocks breakdown of deoxyribose 5-phosphate, permitting growth with low levels of thymine; (iii) pyrF, which blocks synthesis of UMP from OMP, imposing a requirement for uridine; (iv) cdd and pyrG, which block the deamination of cytidine to uridine and the synthesis of CTP from UTP, respectively, causing a requirement for cytidine; (v) codA and codB, which block the deamination of cytosine to uracil and cytosine transport, respectively, preventing the substitution of cytosine for cytidine; and (vi) dnaB, which blocks vegetative but not conjugational DNA replication at 42 degrees C . DNA synthesis can be blocked in the donor strains by the addition of excess uridine when exogenous thymine is not present . We found that RNA synthesis can also be blocked by addition of excess uridine when exogenous cytidine is not present . Blocking RNA synthesis prior to mating, under conditions in which DNA synthesis either is or is not inhibited, depresses DNA transfer . However, under conditions in which DNA synthesis is inhibited, the blocking of RNA synthesis immediately after mating has commenced had no effect on continued conjugational transfer of DNA . Thus, RNA synthesis is needed to initiate but not to continue conjugational DNA transfer. Can J Biochem, 1981 May, 59(5), 379 - 82 Effect of glutathione deficiency on the pool of CoA-glutathione mixed disulfide in Escherichia coli; Loewen PC; The formic acid extracts of several glutathione-deficient strains of Escherichia coli have been assayed for the presence of the mixed disulfide of CoA and glutathione, CoASSG . Strains deficient in gamma-glutamyl-cysteine synthase (EC 6.3.2.2) produced only CoA dimer . Strains deficient in glutathione synthase (EC 6.3.2.3) produced the mixed disulfide of CoA and the gamma-glutamylcysteine dipeptide . The pool size of total CoA in the cell did not change significantly even in the absence of glutathione. J Med Microbiol, 1981 May, 14(2), 223 - 6 Mannose-resistant and eluting haemagglutinins and fimbriae in Escherichia coli; Ip SM et al.; Twenty-three strains of Escherichia coli with mannose-resistant and eluting (MRE) haemagglutinins with eight different patterns of substrate specificity were examined by a variety of electronmicroscope methods . In 12 strains, the presence of MRE haemagglutinins with broad-spectrum patterns 1, 3 and 4 was correlated with that of fimbriae . In 11 strains with MRE haemagglutinins with the less common patterns 6, 7 var., 8, 9 and 10, fimbriae were not found on bacteria in MRE+ cultures, indicating that the latter haemagglutinins are non-fimbrial. J Bacteriol, 1981 May, 146(2), 784 - 9 Escherichia coli 987P pilus: purification and partial characterization; Isaacson RE et al.; The Escherichia coli somatic pilus, 987P, has been purified after removal by homogenization from a 987P+ enterotoxigenic E . coli . Cell-free pili were precipitated by the addition of MgCl2, collected, and dissolved in MgCl2-free buffer . Five cycles of precipitation and dissolving resulted in a preparation of 987P that was judged to be homogeneous based on electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis . In the electron microscope, 987P was rod shaped, having a diameter of 7 nm and an apparent axial hole . Cells and membrane vesicles were not observed in the purified pilus preparation . Electrophoresis of 987P through sodium dodecyl sulfate-polyacrylamide gels resulted in a single band when the sample was denatured in the absence of mercaptoethanol and in two bands when the sample was denatured in the presence of mercaptoethanol . The calculated molecular weight of 987 was variable, depending upon the polyacrylamide concentration and whether mercaptoethanol was included in the denaturing solution . Chemically, 987P is composed primarily of protein but also contains an unidentified amino sugar . The amino terminal amino acid of 987P is alanine and its isoelectric point is pH 3.7 . 987P possesses no detectable hemagglutinating activity. Boll Soc Ital Biol Sper, 1981 Apr 30, 57(8), 887 - 90 {Effect of levamisole on changes in the of hepatic RES induced by chronic ethanol poisoning}; Andreana A et al.; Hepatic RES is depressed in chronic alcholic intoxication and we have previously observed a significant reduction of killing of E.coli by the Kupffer cells of the isolated rat liver . In the present study rats treated with ethanol for 3 weeks, were given levamisole and then the clearance of E . coli, by the isolated liver, was followed . Levamisole 10mg/kg i.p . for 3 days, restored the depression of intracellular killing induced by ethanol . At the dosage of 2,5 mg/kg of levamisole this effect was not present . Changes of humoral factors were not involved in the phenomenon because sera from untreated anaimals were present in the perfusate for all sets of experiments. Nature, 1981 Apr 30, 290(5809), 744 - 9 Structure of catabolite gene activator protein at 2.9 A resolution suggests binding to left-handed B-DNA; McKay DB et al.; The 2.9 A resolution crystal structure of Escherichia coli catabolite gene activator protein (CAP) complexed with cyclic AMP reveals two distinct structural domains separated by a cleft . The smaller carboxy-terminal domain is presumed to bind DNA while the amino-terminal domain is seen to bind cyclic AMP . Model building studies suggest that CAP binds to left-handed B-type DNA, contracting its major groove via two alpha-helices . It is possible that the CAP conversion of right- to left-handed DNA in a closed supercoil, is what activates transcription by RNA polymerase. Biochemistry, 1981 Apr 28, 20(9), 2503 - 12 Role of metal cofactors in enzyme regulation . Differences in the regulatory properties of the Escherichia coli nicotinamide adenine dinucleotide phosphate specific malic enzyme, depending on whether magnesium ion or manganese ion serves as divalent cation; Brown DA et al.; A number of differences in the kinetic and physical properties of the Escherichia coli nicotinamide adenine dinucleotide phosphate (NADP+) dependent malic enzyme have been found, depending upon whether Mg2+ or Mn2+ served to fulfill the divalent cation requirement . The velocity-NADP+ and velocity-cation saturation curves exhibit a simple hyperbolic response in the presence of either metal cofactor, but the affinity for NADP+ (and malate) as well as the Vmax is increased in the presence of Mn2+ . The high affinity of the enzyme for Mn2+ coupled with the increased affinity for substrates indicates that Mn2+ is the preferred cofactor in vitro . With either Mg2+ or Mn2+ as cation, the velocity-malate saturation curves in the absence of effectors are complex at pH 7.45, indicating varying combinations of apparent positive and negative cooperative behavior . Greater initial positive cooperative behavior between malate binding sites is observed with Mg2+ as cation . The enzyme appears to be equally sensitive to inhibition by the allosteric inhibitors reduced nicotinamide adenine dinucleotide (NADH) and oxaloacetic acid (OAA) in the presence of either cation, but the interaction between malate binding sites, in the presence of effectors, varies significantly with the choice of metal cofactor . The inhibitor NADH increases the interaction between malate binding sites in the presence of Mn2+ but has little effect on subunit interaction in the presence of Mg2+ . The inhibitor OAA increases the interaction between malate binding sites in the presence of both cations, with increased positive cooperativity observed with Mn2+ but increased negative cooperativity with Mg2+ . The kinetic data can be explained by a model involving sequential ligand-induced conformational changes of the enzyme, resulting in a mixture of apparent positive and negative cooperative behavior . Alternative explanations involving different classes of noninteracting binding sites or different enzyme forms are also considered . The metal cofactors, Mg2+ and Mn2+, appear to stabilize two distinct conformational states of the enzyme which differ in response to varying substrate and effector concentrations . Altered conformational states of the enzyme in the presence of the two cations are further substantiated by proteolytic digestion studies with the homogeneous enzyme . The results are strikingly similar to previous results reported on the nicotinamide adenine dinucleotide (NAD+) dependent malic enzyme and the NAD+-dependent isocitrate dehydrogenase, supporting the suggestion that metal cofactors function as regulatory entities. Biochemistry, 1981 Apr 28, 20(9), 2497 - 502 Steady-state kinetics and inhibition studies of the aldol condensation reaction catalyzed by bovine liver and Escherichia coli 2-keto-4-hydroxyglutarate aldolase; Grady SR et al.; Two sensitive assays, one which fluorometrically measures only the L isomer of 2-keto-4-hydroxyglutarate after decarboxylation to L-malate and the other which spectrophotometrically determines both enantiomers by reductive amination with glutamate dehydrogenase, are described . By use of these assays, the steady-state kinetics of the aldol condensation of pyruvate with glyoxylate, as catalyzed by 2-keto-4-hydroxyglutarate aldolase from either bovine liver or Escherichia coli, were studied as was the inhibition of this reaction by glyoxylate and other anions . For the E . coli aldolase, double-reciprocal plots are linear except at high (above 5 mM) glyoxylate concentrations; apparent Km values increase with increasing concentrations of the fixed substrate . The data are consistent with an ordered reaction sequence . Inhibition by halides follows the lyotropic or Hofmeister series . Esters are not good inhibitors; mono-, di-, and tricarboxylic acids are increasingly inhibitory . Of the substrate analogues tested, hydroxypyruvate is the most potent inhibitor . Inhibition studies with citrate, acetaldehyde, and glyoxylate (all competitive inhibitors) suggest there are two domains at the active site-the Schiff base forming lysyl residue which interacts with carbonyl analogues (like acetaldehyde) and a center of positive charge which binds anions (like citrate) . In contrast to the bacterial enzyme, liver 2-keto-4-hydroxyglutarate aldolase is inhibited in a competitive manner by much lower concentrations (0.1 mM or even lower) of glyoxylate . Many salts and some carboxylic acids activate the liver enzyme . Similarly, substrate analogues like 2-ketobutyrate and fluoropyruvate are mild activators; no effect is seen with acetaldehyde . Besides glyoxylate, only glyoxal, 2-ketoglutarate, and hydroxypyruvate inhibit the aldol condensation reaction . A uniform value of 1 is found for the number of inhibitor molecules bound per active site of either liver or E . coli 2-keto-4-hydroxyglutarate aldolase. Biochim Biophys Acta, 1981 Apr 27, 653(2), 276 - 87 Isolation and characterization of polysomes from thylakoid membranes of Chlamydomonas reinhardii; Bolli R et al.; Chloroplast polysomes that were originally bound to thylakoid membranes were isolated from the cell wall mutant CW-15 from Chlamydomonas reinhardii . Polysomes were isolated from synchronously grown cells harvested in the middle of the third light period, when the ratio of chloroplast to cytoplasmic polysomes was maximal . Thylakoid membranes were isolated from a chloroplast fraction and polysomes were released by Triton X-100 . Analyses of subunits on sucrose gradients showed that the polysomes consisted predominantly of the 70 S-type ribosomes . The detached polysomes as well as polysomes still bound to the thylakoid membrane were active in in vitro protein synthesis when supplemented with Escherichia coli-soluble factors . The in vitro activity was inhibited by chloramphenicol and aurintricarboxylic acid, but not by cycloheximide. Biochim Biophys Acta, 1981 Apr 27, 653(2), 299 - 302 tRNA methylation . A rapid and simple method for determination of total radioactivity and methylated base distribution in the same sample; Nau F et al.; A simple and rapid method is described for the analysis of in vitro methylated tRNA . The total radioactivity is first determined after trichloroacetic acid-precipitation and filtration . Then the tRNA is hydrolyzed while still on the filter, and the bases are separated by thin-layer chromatography . This method eliminates the need for duplicate sample preparation, and has proved especially useful when the amount of tRNA and/or tRNA methylase is limited. Biochim Biophys Acta, 1981 Apr 27, 653(2), 294 - 8 Effects of rifampicin on the relationship of R6K replicating forms to the folded chromosomal complex of Escherichia coli; Archibold ER et al.; An examination of the relationship of R6K plasmic DNA to the folded chromosome has shown that replicating forms of this plasmid, when compared to the non-replicating forms, were preferentially associated to the apparently mediated by RNA molecules . In this report we show that inhibition of RNA synthesis with rifampicin prior to pulse-labeling cells harboring R6K plasmid DNA resulted in the release of the replicating forms . Analyses of the single-stranded length of rifampicin-released nascent molecules indicate that continuous replication of R6K plasmid DNA to unit length was not affected by the presence of rifampicin . Thus, it appears that complexing was not required for continued synthesis of this plasmid . Further, the inhibition of protein synthesis does not appear to alter the complexing frequency of R6K plasmid DNA to the folded structure . These results suggest that active RNA synthesis is required for maintaining the association of the replicating forms to their replicational site(s). Biochim Biophys Acta, 1981 Apr 27, 653(2), 236 - 47 Determination of the ligand-binding characteristics of several tight-binding mutants of the lactose repressor protein; O'Gorman RB et al.; Several tight-binding mutants of the lactose repressor protein have been characterized with respect to their fluorescence properties and their inducer, operator and nonspecific DNA-binding constants . The tryptophan fluorescence emission spectra for the mutants and the wild-type repressor are quite similar . However, alterations in the Stern-Volmer constants for iodide quenching of the tryptophans in the mutant proteins compared to wild-type suggest differences in the local environment or solvent accessibility for these amino acids in the tight-binding repressors . The inducer-binding affinities and association rate constants of the mutant proteins and protein-operator DNA fragment complexes are also altered compared to wild-type . The extents of these changes vary among the different mutant repressors . The nonspecific DNA-binding affinities of the mutant proteins are 2--3-fold greater than the wild-type repressor, and the affinities of the tight-binding proteins for a 29 base-pair operator DNA fragment are also increased, though to a varying extent depending upon the mutant . The phenotypic behavior of these proteins in vivo can be partially explained by these results obtained in vitro; however, it is likely that there are additional factors responsible for the tight-binding behavior of the proteins that were not detectable in these experiments. Biochim Biophys Acta, 1981 Apr 27, 653(2), 169 - 84 Studies on Escherichia coli chromosome proteins . I . Analysis of the proteins by two-dimensional gel electrophoresis; Moriya T et al.; As an approach for studying the function of chromosome proteins in DNA replication and gene expression, proteins remaining attached to Escherichia coli nucleoids were analyzed by two-dimensional polyacrylamide slab gel electrophoresis . Nucleoids were isolated by gentle lysis of the cells in the presence of a DNA counter-ion such as 1 M NaCl or 5 mM spermidine . In exponentially growing cells, about 100 proteins have been found to exist in the nucleoids . Kinetic studies indicated that the number of chromosome proteins remaining attached varied with time after synchronization . Based on the pattern of the variation, appearance, increase or disappearance of the 29 major proteins, nucleoid proteins were shown to be classified in six different groups (groups A--F) . A strong correlation was observed between the variation of proteins belonging to group D and initiation of DNA synthesis or cell division. J Biol Chem, 1981 Apr 25, 256(8), 3978 - 87 On the fidelity of DNA replication . Nucleoside monophosphate generation during polymerization; Loeb LA et al.; During catalysis by homogeneous procaryotic DNA polymerases, nucleoside monophosphates are generated by a 3' leads to 5'-exonucleolytic activity . Using Escherichia coli DNA polymerase I and poly{d(A-T)} as a template, the contribution of this activity to the fidelity of DNA synthesis has been evaluated by three different criteria . 1) The ratio between the rates of monophosphate generation and incorporation of the noncomplementary nucleotide with Mg2+ as an activating cation was 0.6 +/- 0.6, which is insufficient to account for the high fidelity of polymerization . 2) Inhibition of polymerization by pyrophosphate fails to diminish fidelity, although some kinetic models suggest that optimal error correction via monophosphate release requires the polymerization reaction to be strongly driven by pyrophosphate release . 3) The addition of deoxynucleoside monophosphates in concentrations as great as 10 mM to the reaction mixture does not alter the fidelity of DNA synthesis . These observations argue against the kinetic proofreading mode to account for the fidelity of E . coli DNA polymerase I when copying poly{d(A-T)} in a Mg2+-activated reaction . Furthermore, they suggest that the polymerase may enhance specificity at the base-selection step . However, the 3' leads to 5' exonuclease plays a larger role when the polymerase is activated with Mn2+ and may also be important in copying natural DNA where lower error rates are observed in vitro. J Biol Chem, 1981 Apr 25, 256(8), 3945 - 50 Evidence for histidyl residues at the Zn2+ binding sites of monomeric and dimeric forms of alkaline phosphatase; McCracken S et al.; Chemical modification of Escherichia coli alkaline phosphatase using the group-specific reagent, ethoxyformic anhydride, has demonstrated that 3 histidyl residues/subunit are modified with a concomitant loss of enzyme activity . Reaction with {14C}ethoxyformic anhydride indicates that only three ethoxyformyl groups are incorporated per subunit, confirming that no other amino acid residues are modified under these conditions . Zinc ions protect alkaline phosphatase from inactivation as well as from histidine modification, thus implicating all 3 histidyl residues in Zn2+ binding . The ethoxyformylation reaction was also used to characterize Zn2+ binding sites in immobilized dimeric and monomeric alkaline phosphatase derivatives . The immobilized dimeric alkaline phosphatase was inactivated with ethoxyformic anhydride at a rate similar to that of the soluble enzyme, demonstrating that immobilization did not significantly alter the chemical environment of the Zn2+ binding site . The catalytically inactive, immobilized monomer of alkaline phosphatase was modified more rapidly with ethoxyformic anhydride, demonstrated by the loss of its ability to form functionally active enzyme upon titration with nascent soluble subunits . Moreover, Zn2+ protects the immobilized subunit alkaline phosphatase against this modification, indicating that the isolated subunits of alkaline phosphatase bind Zn2+ . These results are consistent with a model for renaturation of the dimeric enzyme in which individual subunits refold and bind Zn2+ before which individual subunits refold and bind Zn2+ before establishing subunit interactions to regain catalytic activity. J Biol Chem, 1981 Apr 25, 256(8), 3735 - 42 Transport of long and medium chain fatty acids by Escherichia coli K12; Maloy SR et al.; Kinetic, metabolic, and physical parameters of long and medium chain fatty acid transport by Escherichia coli K12 were determined . Uptake of long chain fatty acids (C11-C18:1) mediated by the fadL gene involves concentrative transport . Evidence for this is as follows: (i) characteristic Ki and Vmax values were obtained for long chain fatty acids, (ii) long chain fatty acid transport was inhibited by energy inhibitors, (iii) long chain fatty acids were concentrated 10-fold inside the cell against a concentration gradient, (iv) efflux of transported long chain fatty acids did not occur, and (v) an energy of activation of 11.72 kcal mol-1 and Q10 of 2.3 were obtained for long chain fatty acid transport . The fadL gene product shows some activity with medium chain fatty acids (C7-C10) as well . Medium chain fatty acids also appear to enter the cell by simple diffusion since: (i) medium chain fatty acid transport by fadL strains is not saturable under our assay conditions, (ii) fadL strains do not concentrate medium chain fatty acids against a concentration gradient, and (iii) medium chain fatty acids are available for efflux in fadL strains . Physical parameters of long and medium chain fatty acid transport are also reported . These results present evidence for separate mechanisms of long and medium chain fatty acid transport in E . coli. J Biol Chem, 1981 Apr 25, 256(8), 3831 - 2 Stereospecificity of the ferric enterobactin receptor of Escherichia coli K-12; Neilands JB et al.; Synthetic enterobactin and enantioenterobactin (D-seryl enterobactin) have been examined for the ability to transport iron in Escherichia coli . Failure of the unnatural, D-serine-derived material to support growth of E . coli mutants indicates outer membrane receptor specificity for the naturally occurring complex having an L-seryl backbone and the delta-cis configuration of the Fe(III).catecholate center . Enantioenterobactin was markedly less effective in protecting cells against colicin B compared to synthetic or natural enterobactin. J Biol Chem, 1981 Apr 25, 256(8), 4024 - 32 Characterization of the DNA methylase activity of the restriction enzyme from Escherichia coli K; Burckhardt J et al.; The restriction endonuclease from Escherichia coli K is a multifunctional protein which efficiently methylates heteroduplex DNA (one strand modified and one strand unmodified) in the presence of S-adenosylmethionine (AdoMet), ATP, and Mg2+ . The methylase activity is catalytic, and seems to modify different heteroduplex host specificity sites for E . coli K with equal efficiency . In the methylase reaction, both AdoMet and ATP (or its imido analog) act as allosteric effectors, but AdoMet also serves as a methyl donor . Preincubation of the enzyme with AdoMet eliminates the lag period observed in DNA methylation . The rate of enzyme activation was determined using the AdoMet analog Sinefungin . The result are consistent with the hypothesis that the early steps of AdoMet binding and enzyme activation are common to both restriction and modification reactions. J Biol Chem, 1981 Apr 25, 256(8), 4007 - 16 Human prolactin . cDNA structural analysis and evolutionary comparisons; Cooke NE et al.; Prolactin (Prl), growth hormone, and chorionic sommatomammotropin form a set (the "Prl set") of hormones which is thought to have evolved from a common ancestral gene . This assumption is based on several lines of evidence: overlap in their biological and immunological properties, similarities in their amino acid sequences, and homologies in the nucleic acid sequences of their structural genes . In the current study we report the cloning, amplification in bacteria, and sequence analysis of DNA complementary to Prl mRNA isolated from human pituitary Prl-secreting adenomas . The cloned DNA contains 914 bases, which includes the entire coding sequence of human prePrl as well as portions of the 5- and 3'-untranslated regions of the mRNA . The amino acid sequence predicted by our data differs from a previously reported amino acid sequence in 8 positions . With the results of this study we can now compare in one species the nucleotide sequences of the structural gene coding for each of the hormones of the Prl set . The sequence divergence at replacement sites is used to establish an evolutionary clock for the Prl set of genes . Using this clock, we postulate that the chromosomal segregation of human Prl and human growth hormone occurred about 392 million years ago and that growth hormone and chorionic sommatomammotropin underwent an intrachromosomal recombination within the last 10 million years. Nucleic Acids Res, 1981 Apr 24, 9(8), 1991 - 2002 The A* protein of phi X174 is an inhibitor of DNA replication; Eisenberg S et al.; Extracts prepared from phi X174 infected E . coli cells inhibited in vitro RF replication The inhibition was dependent upon the presence of A* protein in the reaction and served as an assay to highly purify the A* protein . Purified A* protein bound tightly to duplex DNA as well as single-stranded DNA . The binding of the A* protein to duplex DNA inhibited (I) its single-stranded DNA specific endonucleolytic activity; (II) in vitro synthesis of viral (+) single stranded DNA on an A-RFII DNA complex template; (III) ATP hydrolysis by rep protein and unwinding of the strands of RF DNA . We propose that this inhibitory activity is responsible in vivo for the shut off of E . coli chromosome replication during phi X174 infection, and has a role in the transition from semiconservative RF DNA replication to single-stranded DNA synthesis in the life cycle of phi X174. Nucleic Acids Res, 1981 Apr 24, 9(8), 1973 - 89 The construction of new vehicles for the cloning of transcription termination signals; Enger-Valk BE et al.; We have constructed new plasmids that can be used to clone transcription terminator containing DNA fragments between the first gene of the tryptophan (trp) operon and the tetracycline resistance (tet) gene . Both genes are under control of the trp promotor . Therefore the presence of a transcription termination signal on cloned fragments can be monitored by a decrease in expression of the tet gene . The plasmids contain cloning sites at different distances from the translation start signal . Consequently a cloned DNA fragment can be translated in the three possible reading frames, offering the opportunity to distinguish terminators from translation polarity (pseudo-terminators) . The usefulness of the plasmids was shown by the cloning of the trp terminator and of a pseudo-terminator located in the trpB gene. Nucleic Acids Res, 1981 Apr 24, 9(8), 1825 - 39 Detection of the catalytic activities of DNA polymerases and their associated exonucleases following SDS-polyacrylamide gel electrophoresis; Spanos A et al.; A method is described to detect DNA polymerases and nucleases in homogeneous or crude enzyme preparations after electrophoresis in SDS-polyacrylamide gels(2) containing the appropriate template or substrate . DNA polymerases are electrophoresed in a gel containing gapped calf thymus DNA and after a renatur