Escherichia coli

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 renaturation treatment, the gel is incubated in a reaction mixture in which one deoxyribonucleoside triphosphate is {alpha-32P}-labelled . Incorporation of radioactivity into DNA is detected at the vicinity of the polymerase band by autoradiography . An associated nuclease activity can be measured after electrophoresis in a gel containing 32P-labelled gapped DNA, when nucleolytic digestion is seen as a clear band in the resulting autoradiogram . The gels can subsequently be stained with Coomassie blue to establish identical molecular weights of polymerase, nuclease and protein bands . Applications of this technique are discussed.

Nucleic Acids Res, 1981 Apr 24, 9(8), 1777 - 88
An intron nucleotide sequence variant in a cloned beta +-thalassaemia globin gene; Westaway D et al.; A 7.5 kb Hsu I restriction fragment of genomic DNA containing a beta-globin gene has been isolated from a patient doubly heterozygous for beta + thalassaemia and a delta beta (Lepore globin fusion gene . This fragment must be derived from the chromosome carrying the beta +-thalassaemia determinant . The gross structure of the cloned gene plus flanking sequences is indistinguishable from that of a normal beta-globin gene . Within in 1606 base-pair transcribed region of the gene there is only one nucleotide difference from the normal beta-globin gene sequence . This is a G leads to A replacement 21 nucleotides upstream from the 3' terminus of the small intron . This nucleotide lies within a 10 base-pair sequence repeated in an inverted configuration near the 5' terminus of the small intron . The nucleotide replacement may result in a precursor mRNA less amenable to RNA splicing than its normal counterpart.

Science, 1981 Apr 24, 212(4493), 403 - 11
Secondary structure of 16S ribosomal RNA; Noller HF et al.; A secondary structure model for 16S ribosomal RNA which is based on available chemical, enzymatic, and comparative sequence data shows good agreement between constraints dictated by the model and a wide variety of experimental observations . The four major structural domains created by the base-pairing scheme correspond closely to RNA fragments isolated after nuclease digestion in the presence of bound ribosomal proteins . Functionally important sites appear to be located in unpaired regions and are phylogenetically highly conserved.

Tijdschr Diergeneeskd, 1981 Apr 15, 106(8), suppl 3:57 - 60
Baby pig diarrhea caused by coccidiosis; Coussement W et al.; An outbreak of coccidiosis on two Belgian farms is described . Diarrhea started in piglets at 9 or 10 days of life . Zero to three pigs died per litter . The morbidity rate varied from 70 to 90 per cent . Histological examination of the intestines revealed shortening of villi and different stages of the life cycle of coccidia were seen in the enterocytes . Virological examination was negative for corona-, corona-like, and rotavirus . A haemolytic E . coli strain was isolated in one case . As for treatment, good results were obtained by the adding of 1 kg amproleum pre-mix per ton sow feed . Scouring pigs were treated orally with 2 cc of an amprol solution once a day . The diarrhea stopped one day after treatment.

Biochem J, 1981 Apr 15, 196(1), 269 - 83
Identification of the AraE transport protein of Escherichia coli; MacPherson AJ et al.; 1 . Two arabinose-inducible proteins are detected in membrane preparations from strains of Escherichia coli containing arabinose-H+ (or fucose-H+) transport activity; one protein has an apparent subunit relative molecular mass (Mr) of 36 000-37 000 and the other has Mr 27 000 . 2 . An araE deletion mutant was isolated and characterized; it has lost arabinose-H+ symport activity and the arabinose-inducible protein of Mr 36 000, but not the protein of Mr 27 000 . 3 . An araE+ specialized transducing phage was characterized and used to re-introduce the araE+ gene into the deletion strain, a procedure that restores both arabinose-H+ symport activity and the protein of Mr 36,000 . 4 . N-Ethylmaleimide inhibits arabinose transport and partially inhibits arabinose-H+ symport activity . 5 . N-Ethylmaleimide modifies an arabinose-inducible protein of Mr 36 000-38 000, and arabinose protects the protein against the reagent . 6 . These observations identify an arabinose-transport protein of Escherichia coli as the product of the araE+ gene . 7 . The protein was recognized as a single spot staining with Coomassie Blue after two-dimensional gel electrophoresis.

Biochemistry, 1981 Apr 14, 20(8), 2081 - 5
Abortive initiation and long ribonucleic acid synthesis; Munson LM et al.; In vitro transcription assays have been used to study the rate of ribonucleic acid (RNA) synthesis from the Escherichia coli lactose promotor mutant lacL8UV5 contained on a 203-bp (base pair) restriction fragment . The half-life of long (63-base) RNA production from heparin-resistant RNA polymerase-promotor complexes was found to be related to the amount of oligonucleotides released during the initiation process (abortive initiation) . Studies indicate that once a ternary complex between the promoter, RNA polymerase, and a newly synthesized RNA seven and nine nucleotides long is formed, abortive initiation is reduced and the rate of synthesis of long RNAs is increased . The promoter for the left inverted repeat of the transposable element Tn5 was also examined . It was observed to have a much slower rate of production of long RNAs, and it released oligonucleotides 4 times as often as the lactose promoter . The correlation between the amount of abortive initiation and the half-time of long RNA production is discussed.

J Biol Chem, 1981 Apr 10, 256(7), 3233 - 9
Stereochemistry and kinetic isotope effects in the decarboxylation of S-adenosylmethionine catalyzed by the pyruvyl enzyme, S-adenosylmethionine decarboxylase; Allen RR et al.; S-Adenosyl-5'-3-methylthio{1-3H}propylamine was prepared by decarboxylation of S-adenosylmethionine in tritiated water, catalyzed by S-adenosylmethionine decarboxylase from Escherichia coli . Degradation of this product to 3-methylthio{1-3H}propylamine followed by oxidation in a coupled assay system involving pea seedling amine oxidase, catalase, and aldehyde dehydrogenase indicated less than 4% of total counts in water and greater than 75% in NADH plus NAD+ . These results allow the assignment of tritium in 3-methylthiopropylamine to the 1-R configuration, establishing that S-adenosylmethionine decarboxylase, a pyruvate-containing decarboxylase, operates via a retentive mode . Tabulation of the available stereochemical results for two pyruvate-containing and three pyridoxal phosphate-dependent amino acid decarboxylases indicates that stereochemistry has been conserved both within each class of amino acid decarboxylases and between structurally distinct classes of decarboxylases . Comparison of the specific activity of 3-methylthio{1-3H}propylamine to the tritiated water employed in the decarboxylation reaction leads to a primary tritium isotope effect of 4.5 under conditions of substrate concentration far in excess of Km . Although a portion of the tritium in 3-methylthiopropylamine could have arisen through an enzyme-catalyzed exchange into decarboxylated adenosylmethionine (leading to an underestimate of the kinetic tritium isotope effect), we are unable to detect enzymatic loss of tritium from decarboxylated S-adenosylmethionine . The magnitude of the observed isotope effect is discussed in the context of a kinetically significant exchange of an active site residue with solvent in the present of enzyme-bound S-adenosylmethionine.

J Biol Chem, 1981 Apr 10, 256(7), 3207 - 12
Function of phospholipids in Escherichia coli . Influence of changes in polar head group composition on the lipid phase transition and characterization of a mutant containing only saturated phospholipid acyl chains; Pluschke G et al.; The cls mutation conferring a defect in cardiolipin synthesis (Pluschke, G., Hirota, Y., and Overath, P . (1978) J . Biol . Chem . 253, 5048-5055) has been introduced into an Escherichia coli strain defective in unsaturated fatty acid synthesis in order to study the effect of changes in polar head group composition on the ordered in equilibrium fluid phase transition of the membrane phospholipids . The defect in cardiolipin formation is compensated by an increase in phosphatidylglycerol content, resulting in a decrease of the midpoint of the phase transition by 6 degrees C . Starvation of the cls mutant strain for the unsaturated fatty acid supplement leads to the incorporation of saturated acyl chains of reduced average length into the phospholipids, and growth is inhibited although the membrane remains in a fluid state . A revertant of this strain is described which retains the parental fabB-, fadE-, and cls markers and grows in the absence of an unsaturated fatty acid supplement . A cls+ derivative of the revertant can multiply in a restricted temperature range (35-43 degrees C) . It contains only saturated phospholipid acyl chains of an anomalously short average length of 14 carbon atoms but has the same polar head group composition as wild type E . coli . The results demonstrate that, under defined conditions, saturated acyl chains of reduced length are functionally equivalent to unsaturated chains.

Nucleic Acids Res, 1981 Apr 10, 9(7), 1657 - 73
Heterogeneity of the 5' terminus of hen ovalbumin messenger ribonucleic acid; Malek LT et al.; The 5'-terminal sequence of hen ovalbumin mRNA was investigated using a novel labeling method . Ovalbumin mRNA was purified by hybridization to complementary DNA coupled to cellulose . The mRNA thus purified was shown to be 97.9% pure by hybridization with plasmid DNA containing sequences to the messengers coding for conalbumin and ovomucoid, the next two most abundant messengers of oviduct . After digestion with RNase T1 and alkaline phosphatase, 5'-terminal capped oligonucleotides were selected by binding to anti-m7G-Sepharose . These were then labeled using RNA ligase and {5'-32P}pCp, separated by two-dimensional gel electrophoresis, and sequenced by partial digestion with base-specific ribonucleases . A nested set of three capped oligonucleotides was identified . Their structures and relative abundances were m7GpppAUACAG, 3% m7GpppACAUACAG, 61+; and m7GpppGUACAUACAG, 36%.

Nucleic Acids Res, 1981 Apr 10, 9(7), 1757 - 64
Translational regulation by ribosomal protein S8 in Escherichia coli: structural homology between rRNA binding site and feedback target on mRNA; Olins PO et al.; It has been previously shown that ribosomal protein synthesis in Escherichia coli is regulated at the level of translation by certain key ribosomal proteins . In the spc operon, S8 regulates the expression of L5 and some of the subsequent genes, while the first two genes (L14 and L24) are regulated independently . We therefore determined the DNA sequence at the junction of the L24 and L5 genes, which corresponds to the putative feedback target for S8 . We show that there is a striking homology between the structure of the mRNA for this region and the known binding site for S8 on 16S rRNA . These results support the theory that the regulation of ribosomal protein synthesis is based on competition between rRNA and mRNA for regulatory ribosomal proteins.

J Biol Chem, 1981 Apr 10, 256(7), 3141 - 4
In vitro synthesis of the F0 and F1 components of the proton translocating ATPase of Escherichia coli; Brusilow WS et al.; Specialized lambda transducing phage DNA containing the unc region of the Escherichia coli chromosome was used as template to direct an in vitro transcription/translation system . The results demonstrated synthesis of seven of the eight polypeptides of the proton translocating ATPase of this organism . The three polypeptides a, b, and c, constituting the F0 portion of the complex, were resolved by sodium dodecyl sulfatepolyacrylamide gel analysis and have apparent molecular weights (Mr = 24,000, 18,000, and 8,000-9,000) similar to the corresponding proteins produced in vivo . In addition, the alpha, beta, delta, and epsilon polypeptides of the F1 portion of the ATPase were also detected and their molecular weights correspond to the in vivo peptides . A 4.3-kilobase HindIII-generated lambda unc DNA fragment was cloned onto plasmid vectors and was demonstrated to contain the genes for the three F0 and two of the F1 (alpha, delta) subunits . In addition, the polypeptides synthesized in vitro were precipitable with antibody prepared against purified F1.

Biochim Biophys Acta, 1981 Apr 3, 673(4), 495 - 503
Cyclic AMP and permeability coefficient of albumin of the isolated rat mesentery . Effects of Escherichia coli endotoxin; Brachet E et al.; We have investigated the mechanisms whereby Escherichia coli endotoxin exerts its exudative effects, by using an isolated rat mesentery placed as a separation membrane between the two compartments of a diffusion cell . The permeability coefficient of albumin (PA) can be easily computed from the equilibration rate of 125I-labeled albumin added to one compartment . E . coli endotoxin increased PA in a concentration-related manner . Direct measurements revealed an early and transient increase in cyclic AMP and prostaglandin E-immunoreactive material . These effects of endotoxin could be inhibited by indomethacin . Calcium-depleted tissues have a low PA, even though cyclic AMP levels could still be increased by endotoxin . It incubations were prolonged beyond 90 min, PA remained elevated, but prostaglandin E and cyclic AMP levels fell to control values . Similar results were observed with trypsin-treated tissues . These results suggest that transmesenteric passage of albumin is increased in the presence of endotoxin . During the earlier part of the incubation (up to 90 min), the effects could be related to a local synthesis of prostaglandin E, and are controlled by cyclic AMP and intracellular calcium levels . During longer incubations (90-280 min) mesothelial exfoliation could occur, allowing free diffusion of albumin through the remaining interstitial tissue.

Eur J Biochem, 1981 Apr, 115(2), 423 - 8
Changes in amount of hypo-modified tRNA having guanine in place of queuine during erythroid differentiation of murine erythroleukemia cells; Shindo-Okada N et al.; The amounts of hypo-modified tRNAs having guanine in place of queuine in murine erythroleukemic cells decreased markedly when the cells differentiated into mature erythroid cells . The amounts of these hypo-modified tRNAs can be determined easily by measuring incorporation of labeled guanine into tRNA with Escherichia coli tRNA--guanine transglycosylase . The decrease was detected at on early stage of erythroid differentiation: namely, before any detectable increase in the percentage of cells containing hemoglobin . The amount of guanine-accepting tRNA species was nearly proportional to the percentage of undifferentiated cells in the population, regardless of the type of inducer used . Decrease in the amounts of hypo-modified tRNAs in the cells was effectively blocked by 12-O-tetradecanoylphorbol 13-acetate, which inhibits differentiation of these cells . 8-Azaguanine, which is known to be substrate of tRNA--guanine transglycosylase, was incorporated almost exclusively into the first position of hypo-modified tRNA in murine erythroleukemic cells when they were pulse-labeled in culture with 8-azaguanine, suggesting strongly that tRNA-guanine transglycosylase in the cells is actually involved in incorporation of 8-azaguanine into tRNA in vivo . The amount of 8-azaguanine incorporated into tRNA in differentiated cells was one third of that in undifferentiated cells, the decrease being parallel with that in the amount of guanine-accepting tRNA in these cells . The results suggest that the appearance of hypo-modified tRNAs in the transformed cells was due to lack of substrate for queuosine biosynthesis in tRNA.

Am J Epidemiol, 1981 Apr, 113(4), 445 - 51
Handwashing to prevent diarrhea in day-care centers; Black RE et al.; Diarrhea has been recognized as a frequent health problem among children enrolled in day-care centers . Thus, we evaluated the effect of a handwashing program in two day-care centers (HWC) on the incidence of diarrhea among children when compared to children in two control centers (CC) . After the program was begun, the incidence of diarrhea at the HWC began to fall and after the second month of the study was consistently lower than that at the CC . The incidence of diarrhea in the HWC was approximately half that of the CC for the entire 35-week study period . Adenoviruses, rotavirus, Giardia lamblia, and enteropathogenic Escherichia coli were found in the stools of a small number of ill children, but not pathogen was identified in the stools of most children with diarrhea . These results suggest that a handwashing program will probably prevent at least some of the diarrhea in day-care centers.

Am J Vet Res, 1981 Apr, 42(4), 650 - 7
Interaction of blood-borne Escherichia coli with phagocytes of spleen and liver in turkeys; Arp LH et al.; The response of splenic and hepatic macrophages to blood-borne virulent and avirulent Escherichia coli was studied in 3-week-old turkeys . Bacterial titers in blood, spleen, and liver were determined for 20 minutes after IV injection of E coli . Spleen and liver were examined by light and electron microscopy . Blood titers of avirulent E coli were reduced to 1/3,000 of their original level in 20 minutes, whereas titers of virulent E coli were only slightly reduced . The E coli localized in macrophages of hepatic sinusoids and splenic reticular sheaths (ellipsoids) . In liver, phagocytosis was more efficient for avirulent E coli than for virulent E coli . In splenic macrophages, phagosomal membranes were separated from ingested avirulent E coli by a prominent space, whereas phagosomal membranes surrounding virulent E coli were wavy and closely apposed to the bacterial surface . The appearance of phagosomes may reflect the capacity of splenic macrophages to kill intracellular E coli . Cultural and histopathologic results indicated that virulent E coli resisted trapping and killing by macrophages of spleen and liver.

J Gen Microbiol, 1981 Apr, 123(Pt 2), 351 - 4
Regulation of anthranilate synthase in Escherichia coli growing in glucose-limited chemostats; Shimizu RW et al.; Strains of Escherichia coli isogenic except for the trpR locus were grown in glucose-limited chemostats . Anthranilate synthase was assayed spectrofluorimetrically to measure trp expression . The specific activity was about ten times greater in the trpR- strain than in the trpR+ strain . In glucose-limited chemostats, the specific activity of anthranilate synthase was independent of growth rate . Both strains produced two to three times more anthranilate synthase in chemostats than in batch culture . The addition of 19 amino acids (not including tryptophan) increased trp expression of anthranilate synthase fivefold in the trpR+ strain.

J Gen Microbiol, 1981 Apr, 123(Pt 2), 323 - 33
Characterization of polysaccharide accumulation in a cell division defective mutant of Escherichia coli 15T-; Schoemaker JM et al.; Escherichia coli 15T-R1, a temperature-dependent cell division mutant, grows into filaments of various lengths (200 to 500 microgram) at 24 degrees C, but divides essentially normally at 37 degrees C . When grown to late-exponential phase at the restrictive temperature, the elongated cells showed discrete areas of increased density at polar regions and other sites in the cytoplasm, when viewed by phase contrast microscopy . Electron microscopy of preparations specifically stained for polysaccharide revealed clusters of granules with a similar distribution pattern to that of the dense areas seen by phase contrast microscopy . The granules were susceptible to alpha-amylase digestion, and chemical analysis of the extracted and purified polysaccharide showed that it consisted of polyglucose, including glycogen . At 24 degrees C the R1 cells contained about twice as much polyglucose and four times as much glucogen as at 37 degrees C.

Clin Exp Immunol, 1981 Apr, 44(1), 38 - 48
Ontogeny of the autoimmune reaction in normal mice to antigens in erythrocytes and gut; Cunningham AJ et al.; Normal mice have large numbers of cells (PFC) making antibody to an autoantigen which is exposed when their own erythrocytes are treated with proteolytic enzymes . Antibody against this antigen can be demonstrated in serum by haemolysis tests against the treated cells; this antibody rises to high levels within 2 to 3 days after injection of E . coli lipopolysaccharide . using quantitative absorption tests we have located the 'bromelain mouse' (BrM) autoantigen in the gastrointestinal tract as well as in erythrocytes; this distribution pattern resembles that of classical blood group antigens . We have described the ontogenetic development of PFC, B cells capable of activation by LPS, serum antibody and antigen . Free antigen is found in the gut shortly after birth . B cells rise rapidly to high levels in the peritoneal cavity, but require a short period of culture to release detectable antibody . PFC and B cells increase more slowly in spleen to adult levels by 3 weeks of age . The serum antibody lags behind PFC development . The pattern is consistent with an early stimulation of B cells in the peritoneal cavity by gut-derived antigen . We discuss the possible relationship of this autoimmune response to high natural responses against other autoantigens in mice, man and other species.

Can J Comp Med, 1981 Apr, 45(2), 207 - 10
Dose-response of ponies to parenteral Escherichia coli endotoxin; Burrows GE; The response of the pony to increasing doses of Escherichia coli endotoxin was evaluated using intravenous and intraperitoneal administration models . Marked changes were seen in all parameters measured following endotoxin administration . Leukopenia (neutropenia, lymphopenia) and thrombocytopenia were not dose-dependent . Similarly, elevated plasma fibrinogen and altered glucose concentrations (hyperglycemia and hypoglycemia), pyrexia and increased lactate/pyruvate ratios were apparent at all endotoxin doses but were not dose related . The widely used packed cell volume and capillary refill time, we well as blood lactate and possibly serum beta-glucuronidase, were increased in a dose-related manner.

Can J Comp Med, 1981 Apr, 45(2), 167 - 72
Nicotinic acid inhibits enterotoxin-induced jejunal secretion in the pig; Forsyth GW et al.; The use of nicotinic acid for preventing intestinal secretion caused by cholera toxin and by the heat-stable enterotoxin of Escherichia coli has been investigated in the weanling pig . Secretory effects were measured in ligated jejunal loops of halothane-anesthetized pigs by dilution of a nonabsorbable marker added to the loop fluid . Different routes of administration and different initial pH values for nicotinate solutions were studied to determine optimal conditions for secretory inhibition . The neutral sodium salt of nicotinic acid had no significant antisecretory activity under any conditions used in these trials . Inhibition of secretion was most effective with partly neutralized nicotinic acid at pH 4.5 added directly to loops containing enterotoxin . Net fluid secretion induced by cholera toxin or heat-stable enterotoxin of E . coli was prevented by this treatment . Reversal of secretion was not accompanied by any measurable changes in cyclic nucleotide concentration in intestinal mucosa . Nicotinic acid antagonism of a secretory step common to cholera toxin and heat-stable enterotoxin of E . coli but subsequent to cyclic nucleotide involvement is indicated by these data.

Avian Dis, 1981 Apr-Jun, 25(2), 312 - 25
Long-term exposure of chickens to three levels of social stress; Gross WB et al.; Cockerels were kept in environments characterized by high (HSS), medium (MSS), or low (LSS) levels of social stress for 3 or 4 months . Chickens raised in an environment of low light intensity (LSS) gained more weight than did those raised under natural lighting . Ability of chickens to produce antibody in response to antigen was greatly reduced, 2(15.4) in the LSS group to 2(3.4) in the HSS group, 1 day after chickens were moved from the LSS environment into the HSS environment . Normal responsiveness returned within 1 week . No long-term environments affected antibody production . After 3 months, chickens in the LSS environment had reduced weight gain and resistance to Escherichia coli infection compared with birds in the HSS environment . Chickens in the MSS environment, compared with those on the HSS and LSS environments, had greater weight gains, superior feed efficiencies, medium plasma corticosterone levels, a better negative correlation between antibody responsiveness and RBC antigens, and better resistance to Mycoplasma gallisepticum challenge . All parameters except antibody responsiveness were such that long-term exposure to HSS or LSS environments appears to be detrimental.

Equine Vet J, 1981 Apr, 13(2), 95 - 8
Prevention of endotoxin-induced arterial hypoxaemia and lactic acidosis with flunixin meglumine in the conscious pony; Moore JN et al.; Bacterial endotoxin injected intravenously into conscious ponies produced alterations in cardiopulmonary and gastrointestinal function . Specifically, tachypnoea, dyspnoea, hypoxaemia, colic, lactic acidosis and diarrhoea resulted from administration of 10 micrograms/kg Escherichia coli endotoxin . Pretreatment of the ponies with a potent prostaglandin synthetase inhibitor, flunixin meglumine, prevented these ill effects of endotoxin.

Proc Natl Acad Sci U S A, 1981 Apr, 78(4), 2155 - 8
Enzymatic reduction of protein-bound methionine sulfoxide; Brot N et al.; An enzyme that catalyzes the reduction of methionine sulfoxide residues in ribosomal protein L12 has been partially purified from Escherichia coli extracts . Methionine sulfoxide present in oxidize {Met}enkephalin is also reduced by the purified enzyme . The enzyme is different from a previously reported E . coli enzyme that catalyzes the reduction of methionine sulfoxide to methionine {Ejiri, S . I., Weissbach, H . & Brot, N . (1980) Anal . Biochem . 102, 393--398} . Extracts of rat tissues, Euglena gracilis, Tetrahymena pyriformis, HeLa cells, and spinach also can catalyze the reduction of methionine sulfoxide residues in protein.

Proc Natl Acad Sci U S A, 1981 Apr, 78(4), 2140 - 4
Capacity for alternating sites cooperativity in catalysis by succinyl-coenzyme A synthetase; Wolodko WT et al.; Succinyl-coenzyme A synthetase {succinate:CoA ligase (ADP-forming), EC 6.2.1.5} of Escherichia coli in an alpha 2 beta 2 tetramer . A histidyl residue in the alpha subunit is phosphorylated as a catalytic intermediate . It has been suggested {Bild, G . S., Janson, C . & Boyer, P . D . (1980) J . Biol . Chem . 255, 8109--8115} that the mechanism of action of this enzyme involves intersubunit cooperativity in which attachment of substrates at one of the two active sites promotes catalytic events at the other . This scheme would require that the two active sites, although otherwise equivalent, should act alternately . We have prepared a hybrid enzyme species that contains one 35S-labeled alpha subunit (dephosphorylated), one nonradioactive alpha subunit (phosphorylated), and two beta subunits per tetrameric molecule . With the aid of a selective chromatographic procedure for the isolation of peptides that contain phosphohistidyl residues, we have shown that each of the alpha subunits undergoes phosphorylation when the hybrid enzyme is exposed briefly to substrates . This result demonstrates that the two active sites are capable of alternate activity and lends support to the concept of alternating sites cooperativity . The half-of-the-sites phosphorylation that occurs with this enzyme is not a consequence of permanent asymmetry or other lack of equivalence of the two alpha subunits.

Proc Natl Acad Sci U S A, 1981 Apr, 78(4), 2072 - 6
Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase; Mulligan RC et al.; Cultured monkey (TC7) and mouse (3T6) cells synthesize an Excherichia coli enzyme, xanthine-guanine phosphoribosyltransferase (XGPRT; 5-phospho-alpha-D-ribose-1-diphosphate:xanthine phosphoribosyltransferase, EC 2.4.2.22), after transfection with DNA vectors carrying the corresponding bacterial gene, Ecogpt . In contrast to mammalian cells, which do not efficiently use xanthine for purine nucleotide synthesis, cells that produce E . coli XGPRT can synthesize GMP from xanthine via XMP . After transfection with vector-Ecogpt DNAs, surviving cells producing XGPRT can be selectively grown with xanthine as the sole precursor for guanine nucleotide formation in a medium containing inhibitors (aminopterin and mycophenolic acid) that block de novo purine nucleotide synthesis . Cells transformed for Ecogpt arise with a frequency of 10(-4) to 10(-5); they appear to be genetically stable in as much as there is no discernible decrease in XGPRT formation or loss on their ability to grow in selective medium after propagation in nonselective medium . Although several of the vector-gpt DNAs can replicate in monkey and mouse cells, none of the transformants contain autonomously replicating vector-gpt DNA . Rather, the gpt transformants contain one to five copies of the transfecting DNA associated with, and most probably integrated into, cellular DNA sequences . In several transformants, vector-coded gene products for which there was no selection are also synthesized . This suggests that recombinant DNAs containing Ecogpt as a selective marker can be useful for cotransformation of nonselectable genes.

J Infect Dis, 1981 Apr, 143(4), 598 - 602
Biweekly prophylactic doxycycline for travelers' diarrhea; Santosham M et al.; A double-blind study to determine the efficacy of biweekly oral doxycycline in the prevention of travelers' diarrhea was conducted among 46 Peace Corps volunteers during their first six weeks in Honduras . The volunteers took either 100 mg of doxycycline per dose or a placebo for three weeks and were observed for an additional three weeks . There was no significant difference in the number of persons with travelers' diarrhea in the two groups (eight of 24 in the doxycycline group and 10 of 22 in the placebo group) in the three weeks when the drug was taken . However, significantly fewer episodes (P less than 0.05) of travelers' diarrhea occurred in the doxycycline group than in the placebo group at the end of the second, third, and fourth weeks . Enterotoxigenic Escherichia coli (ETEC) was the most common pathogen identified . ETEC from 13 (62%) of 21 stool samples were resistant to doxycycline . Biweekly doxycycline was only marginally effective in preventing travelers' diarrhea and did not prevent diarrhea secondary to doxycycline-resistant ETEC.

Eur J Biochem, 1981 Apr, 115(3), 627 - 34
Properties of ribosomes and ribosomal RNAs synthesized by Escherichia coli grown in the presence of ethionine . Normal maturation of ribosomal RNA in the absence of methylation; Chelbi-Alix MK et al.; Analysis of 16-S rRNA synthesized in Escherichia coli D10 (met-) incubated in a medium containing ethionine in place of methionine shows that it lacks most and probably all of the methyl groups present in normal 16-SrRNA but possesses the same 3'-OH, and 5'-phosphate terminal sequences as the latter . 23-S rRNA formed in ethionine-treated cells also contains normal terminal sequences . 5-S rRNAs of normal and ethionine-treated E . coli D10 are identical . These results lead to the conclusion that methylation of ribosomal precursor RNAs is not necessary for their maturation to products with normal chain lengths and does not influence the conformation of 16-S rRNA.

Eur J Biochem, 1981 Apr, 115(2), 279 - 85
Photochemical cross-linking of elongation factor G to 70-S ribosomes from Escherichia coli by 4-(6-formyl-3-azidophenoxy)butyrimidate; Maassen JA et al.; Ribosomal proteins situated at or near the binding site of elongation factor G (EF-G) on the Escherichia coli ribosome have been identified by use of the heterobifunctional cross-linker 4-(6-formyl-3-azidophenoxy)butyrimidate . Four different preparations of EF-G, in which the number of cross-linker molecules coupled to EF-G ranged from four to seven, all cross-linked to 50-S subunit proteins L1, L3 and L11 as well as to 30-S subunit proteins S3 and S4 . Cross-linking of EF-G to ribosomal proteins was tested electrophoretically . In the case of L7/L12 and L11 immunological methods were also used . Cross-linking of EF-G to L1, L3, L11, S3 and S4 is specific as judged from the fact that addition of unmodified EF-G and of thiostrepton results in less cross-linking . The cross-linking data suggests that the binding site for EF-G includes several proteins which are located at the interface between the 30-S and 50-S subunits.

Appl Environ Microbiol, 1981 Apr, 41(4), 1046 - 8
Loss of plasmids during enrichment for Escherichia coli; Hill WE et al.; Enrichment with sodium lauryl sulfate and incubation at 44.5 degrees C resulted in a loss of plasmids and decreased efficiency in the recovery of pathogenic . Escherichia coli strains from foods.

Am J Pathol, 1981 Apr, 103(1), 47 - 55
The in vivo quantitation and kinetics of monocyte migration into acute inflammatory tissue; Issekutz TB et al.; Mononuclear leukocytes isolated from the blood of rabbits, were labeled with 51Cr and returned to the animal intravenously . 51Cr-labeled monocytes disappeared from the blood with a half-life of 39.0 +/- 2.51 hours . Numerous acute inflammatory lesions were produced by the intradermal injection of Escherichia coli into the skin of the back of a rabbit . The animal was sacrificed after 1 hour, and the radioactivity in each lesion was determined . Monocyte accumulation was substantial by the time a lesion was 1 hour old . The maximum rate of accumulation occurred at 3--4 hours, and monocytes continued to enter the lesions at 25% of the maximal rate for at least 24 hours . Monocytes initially migrate into bacterial inflammatory sites simultaneously with neutrophils and histologically become the predominant cell type after 12 hours because they continue to migrate into these lesions long after neutrophils have stopped . The kinetics of monocyte migration is related to other aspects of inflammation.

J Bacteriol, 1981 Apr, 146(1), 54 - 63
Structural and functional properties of colicin M; Schaller K et al.; Colicin M of Escherichia coli Cl139 was isolated in pure form . It consisted of a single polypeptide with a molecular weight of 27,000 +/- 2,000 . Colicin M lysed sensitive cells of E . coli but had to act continuously up to the point when lysis commenced (after 20 min) . Colicin M was largely resistant to hydrolysis by trypsin except when adsorbed to cells . Within 4 to 5 min after addition of colicin M, cells could be rescued by trypsin or sodium dodecyl sulfate . Later, colicin M was apparently inaccessible to these inactivating agents . Killing of cells by colicin M required Ca2+ ions . Cells could be rescued with ethylene glycol-bis(beta-aminoethyl ether)-N,N'-tetraacetate (EGTA) immediately before the onset of lysis . Under these conditions, colicin M remained bound to the cells, and it became again sensitive to trypsin . We conclude that under the influence of EGTA colicin M is removed from its site of action and becomes again accessible to trypsin at the cell surface.

J Bacteriol, 1981 Apr, 146(1), 422 - 5
Blue ghosts: a new method for isolating amber mutants defective in essential genes of Escherichia coli; Brown S et al.; We describe a technique which permits an easy screening for amber mutants defective in essential genes of Escherichia coli . Using this approach, we have isolated three amber mutants defective in the rho gene . An extension of the technique allows the detection of ochre mutants and transposon insertions in essential genes.

J Bacteriol, 1981 Apr, 146(1), 418 - 21
Deoxyribonucleic acid initiation mutation dnaB252 is suppressed by elevated dnaC+ gene dosage; Sclafani RA et al.; The Escherichia coli dnaB252 allele is the only dnaB mutation which confers a deoxyribonucleic acid initiation-defective phenotype on the cell . The presence of a multicopy hybrid plasmid containing the dnaC+ gene in a dnaB252 strain completely suppressed the temperature-sensitive phenotype . It is suggested that at high temperature the dnaB252 protein has a lowered affinity for dnaC protein, and that the formation of a dnaB-dnaC complex is mandatory for initiation.

J Bacteriol, 1981 Apr, 146(1), 41 - 8
Mitomycin C-induced synthesis of cloacin DF13 and lethality in cloacinogenic Escherichia coli cells; van Tiel-Menkveld GJ et al.; Treatment of cloacinogenic cultures with increasing concentrations of mitomycin C induced an increasing synthesis of cloacin DF13 accompanied by a decreasing number of colony-forming cells . Cells grown in the presence of glucose required a 10-fold-higher concentration of mitomycin C for optimal induction of cloacin production than did cells grown with lactate . Release of the cloacin was hampered in glucose-grown cells . Experiments with various CloDF13 insertion and deletion mutants revealed that the transcription of CloDF13 deoxyribonucleic acid sequences adjacent to the cloacin structural gene was essential for mitomycin C-induced lethality.

J Bacteriol, 1981 Apr, 146(1), 352 - 9
Synthesis and degradation of nitrate reductase in Escherichia coli; Hackett CS et al.; The biosynthesis, insertion, and in vivo stability of nitrate reductase were examined by following the amount of labeled enzyme present in both membranes and cytoplasm at varying times after a short pulse of radioactive sulfate . Nitrate reductase levels were measured by autoradiography of immunoprecipitated material after fractionation on sodium dodecyl sulfate-polyacrylamide gels . These experiments demonstrated that subunits A and B were synthesized in the cytoplasm and subsequently inserted into membranes . The insertion of these subunits was dependent upon the synthesis of another protein, and the rate of synthesis of this protein determined the rate of insertion of subunits A and B . The nitrate reductase produced by the chlA mutant was inserted into membranes in the normal fashion, whereas the nitrate reductase produced by the chlC and chlE mutants was poorly incorporated . The nitrate reductase in the wild type was completely stable in vivo under inducing or noninducing conditions, whereas in the chlC and chlE mutants nitrate reductase was degraded extensively in both the cytoplasm and membranes, even under inducing conditions . Under similar conditions, nitrate reductase was stable in the chlA mutant.

J Bacteriol, 1981 Apr, 146(1), 345 - 51
Multiple forms of lysyl-transfer ribonucleic acid synthetase in Escherichia coli; Hirshfield IN et al.; Lysyl-transfer ribonucleic acid synthetase (EC 6.1.1.6) was identified as four polypeptide spots after two-dimensional polyacrylamide gel electrophoresis of whole-cell lysates of Escherichia coli . Identification was made by migration with partially purified enzyme preparations, by peptide map patterns, by mutant analysis, and by correlation of spot intensities with changes in enzyme levels under different growth conditions . Wild-type cells growing at 37 degrees C in glucose minimal medium displayed the enzyme predominantly as two spots (spots I and III) . Growth at 46 degrees C, growth in the presence of alanine or glycyl-L-leucine, or growth of a strain with a mutational deficiency in S-adenosylmethionine synthetase (metK) greatly increased the synthesis of two other spots (spots II and IV) . Polypeptides I and III, but not polypeptides II and IV, had altered isoelectric points in a lysyl-transfer ribonucleic acid synthetase mutant . These data suggest that multiple forms of lysyl-transfer ribonucleic acid synthetase exist in vivo and that they may be encoded by more than one gene.

J Bacteriol, 1981 Apr, 146(1), 331 - 6
Mechanism of lysis of Escherichia coli by ethanol and other chaotropic agents; Ingram LO; Ethanol has been shown to inhibit the assembly of cross-linked peptidoglycan and to induce cell lysis in Escherichia coli . These effects of ethanol appear to result from the weakening of hydrophobic interactions by ethanol rather than from the intercalation of ethanol into membranes . Other chaotropic agents also inhibited cross-linking and induced lysis . The potency of chaotropic anions with regard to this effect followed the expected chaotropic series . Antichaotropic agents, which strengthened hydrophobic interactions, antagonized ethanol-induced lysis . The weakening of hydrophobic interactions by ethanol is proposed as a general mechanism by which ethanol and other chaotropic agents could affect membrane-associated enzyme activities.

J Bacteriol, 1981 Apr, 146(1), 275 - 84
Short deoxyribonucleic acid repair patch length in Escherichia coli is determined by the processive mechanism of deoxyribonucleic acid polymerase I; Matson SW et al.; The lengths of ultraviolet irradiation-induced repair resynthesis patches were measured in repair-competent extracts of Escherichia coli . Extracts containing wild-type deoxyribonucleic acid (DNA) polymerase I introduced a patch 15 to 20 nucleotides in length during repair of ColE1 plasmid DNA; extracts containing the polA5 mutant form of DNA polymerase I introduced a patch only about 5 nucleotides in length in a similar reaction . The repair patch length in the presence of either DNA polymerase corresponded to the processivity of that polymerase (the average number of nucleotides added per enzyme-DNA binding event) as determined with purified enzymes and DNA treated with a nonspecific endonuclease . The base composition of the repair patch inserted by the wild-type DNA polymerase was similar to that of the bacterial genome, whereas the patch inserted by the mutant enzyme was skewed toward greater pyrimidine incorporation . This skewing is expected, considering the predominance of pyrimidine incorporation occurring at the ultraviolet lesion and the short patch made by the mutant enzyme . Since the defect in the polA5 DNA polymerase which causes premature dissociation from DNA is reflected exactly in the repair patch length, the processive mechanism of the polymerase must be a central determinant of patch length.

J Bacteriol, 1981 Apr, 146(1), 269 - 74
Gene organization around the phenylalanyl-transfer ribonucleic acid synthetase locus in Escherichia coli; Comer MM; The organization of seven genes located at about 38 min on the genetic map of Escherichia coli was examined; these genes included pheS and pheT, which code for the alpha and beta subunits of phenylalanyl-transfer ribonucleic acid synthetase, and thrS, the structural gene for threonyl-transfer ribonucleic acid synthetase . Deletion mutants were isolated from an F-prime-containing merodiploid strain and were characterized genetically . Seventeen different kinds of deletions extending into pheS of pheT were identified . These deletions unambiguously defined the gene order as aroD pps himA pheT pheS thrS pfkB . Mutants with deletions covering either pheS or pheT, but not both, were analyzed further by assay of phenylalanyl-transfer ribonucleic acid synthetase . The phenotype of the mutants with a deletion from pfkB through pheS was anomalous; although the pheT gene was apparently still present, its product, the beta subunit, was much reduced in activity.

J Bacteriol, 1981 Apr, 146(1), 200 - 8
Properties of a mutant Escherichia coli phosphoenolpyruvate carboxylase deficient in coregulation by intermediary metabolites; McAlister LE et al.; Phosphoenolpyruvate carboxylase of Escherichia coli is activated by three different mechanisms: contiguous by acetyl coenzyme A, precursor by fructose 1,6-bisphosphate, and compensatory feedback by cytidine 5'-diphosphate (CDP) . Even though each activator can interact independently with the enzyme, synergistic effects are observed with some combinations, namely, fructose 1,6-bisphosphate or CDP (coregulators), with acetyl coenzyme A . A mutant was isolated that has a phosphoenolpyruvate carboxylase which is refractory to activation by fructose, 1,6-bisphosphate and CDP . The mutant enzyme was shown to be active primarily as the dimer and to lack cooperativity in substrate binding . The binding of acetyl coenzyme A and substrate, however, was essentially the same as that of the wild-type enzyme . The mutant cells grew extremely slowly on glucose alone as the sole carbon source . The only defect in the mutant appeared to be the inability of this enzyme to be activated by the coregulators . These data are consistent with the thesis that coregulation by fructose 1,6-bisphosphate or CDP is an essential requirement for the activation in vivo of this enzyme.

J Bacteriol, 1981 Apr, 146(1), 10 - 7
Reconstitution of maltose transport in malB mutants of Escherichia coli through calcium-induced disruptions of the outer membrane; Brass JM et al.; The barrier function of the Escherichia coli outer membrane against low concentrations of maltose in strains missing the lambda receptor was partially overcome by treating the cells for 3 h with 25 mM Ca2+ . Kinetic analysis of maltose-transport revealed a Ca2+-induced shift of the apparent Km of the system from about 100 microM in cells pretreated with Tris to about 15 microM in cells pretreated with Tris plus Ca2+ . In contrast to maltose transport in untreated cells, that of Ca2+-treated lamB cells was inhibited by molecules with a high molecular weight, such as amylopectin (molecular weight, 20,000), and anti-maltose-binding protein antibodies . In addition, lysozyme was shown to attack Ca2+-treated cells in contrast to untreated cells . The Ca2+-induced permeability increase of the outer membrane allowed reconstitution of maltose transport in a mutant missing the maltose-binding protein with osmotic shock fluid containing the maltose-binding protein . Even though Ca2+-treatment allowed the entry of large molecules, the release of the periplasmic maltose-binding protein or alkaline phosphatase was negligible.

Surg Gynecol Obstet, 1981 Apr, 152(4), 427 - 32
Lysosomal enzyme in endotoxin shock; Mori K et al.; Lysosomal enzymes are released in the blood stream in various types of shock . The role of the released enzymes in the development of irreversible shock is still controversial . The present study was undertaken to determine the effect of the administration of lysosomal enzymes in normal rats and rats treated with endotoxin . Lysosomal enzymes were prepared from the isologous liver . They were given either intravenously or intraperitoneally to normal rats . No hemodynamic effect was observed . They were also given to the rats that received the lethal dose of endotoxin . No hemodynamic effect was found in this group . The survival time and the time course of the blood pressure to death did not change . The lysosomal enzyme preparation was given to the rats that received various dosages of endotoxin of less than LD190 . The mortality rate of the rats that received endotoxin was not modified by the administration of the lysosomal enzyme preparation . These results indicate that lysosomal enzymes released into the blood in endotoxin shock may not contribute significantly to the irreversibility of shock.

Proc Natl Acad Sci U S A, 1981 Apr, 78(4), 2330 - 4
A cloned calmodulin structural gene probe is complementary to DNA sequences from diverse species; Munjaal RP et al.; Calmodulin mRNA has been partially purified from a total nucleic acid extract of the electroplax of Electrophorus electricus by oligo(dT)-cellulose chromatography and sucrose gradient centrifugation . A 9- to 10S fraction was determined to contain 39% calmodulin mRNA by translation in a reticulocyte lysate followed by immunoprecipitation with antibodies to calmodulin . Double-stranded cDNA was synthesized from the RNA fraction by using reverse transcriptase from avian myeloblastosis virus . The double-stranded cDNA was joined to pBR322 linearized by restriction endonuclease Pst I and used to transform Escherichia coli RRI . DNAs from 60 tetracycline-resistant cloned hybridized to {32P}cDNA synthesized from the partially purified calmodulin mRNA fraction . By direct DNA sequence analysis, one of these clones, pCM109, was shown to contain calmodulin-specific sequences corresponding to amino acid residues 93--148 of calmodulin or approximately 38% of the peptide-coding region of the calmodulin structural gene sequence . pCM109 was hybridized to DNA isolated from three vertebrate and one plant species by the procedure of Southern . Positive hybridization bands were noted regardless of the DNA source . These data suggest thaat calmodulin gene sequences are evolutionarily conserved, as has been shown to be the case for the primary amino acid sequence.

Proc Natl Acad Sci U S A, 1981 Apr, 78(4), 2478 - 82
Antibody-mediated activation of genetically defective Escherichia coli beta-galactosidases by monoclonal antibodies produced by somatic cell hybrids; Accolla RS et al.; Six hybridomas producing monoclonal antibodies against Escherichia coli beta-galactosidase (beta-D-galactoside galoctohydrolase, EC 3.2.1.23) have been derived from two separate somatic cell fusions . Three of these antibodies can activate defective enzymes produced by strains of E . coli carrying Z-gene point mutations . In antigen excess, one monoclonal antibody shows similar enzyme binding and mutant-activating capacity . Characteristically, the former reaction has a 200-fold higher equilibrium constant . These data provide direct evidence that the enzyme-activation reaction is a single-hit event in which one antibody site favors the correct conformation of one active center of the enzyme . Because each "activating" hybridoma is able to activate several but not all point mutant enzymes tested, it appears that the correction of the genetic defect is produced by binding key sites of the protein three-dimensional structure rather than the sites affected by the mutation.

Proc Natl Acad Sci U S A, 1981 Apr, 78(4), 2460 - 4
Yeast genes fused to beta-galactosidase in Escherichia coli can be expressed normally in yeast; Rose M et al.; A plasmid was constructed that allows the selection in vivo of gene fusions between the Escherichia coli beta-galactosidase gene and the yeast (Saccharomyces cerevisiae) URA3 gene . A large yeast DNA fragment containing the URA3 gene was placed upstream of an amino-terminally deleted version of the lacZ gene . The plasmid vehicle contains sequences that allow selection and maintenance of the plasmid in both yeast and E . coli . Selection for Lac+ in E . coli yielded numerous deletions that fused the lacZ gene to the URA3 gene and flanking yeast sequences, to the bacterial tetracycline-resistance gene from the parent plasmid pBR322, and to the yeast 2-micrometer plasmid DNA . Some of these fusion plasmids produced beta-galactosidase activity when introduced into yeast . One of the fusions to the URA3 gene itself has been shown to place the expression of beta-galactosidase activity under uracil regulation in yeasts.

Eur J Biochem, 1981 Apr, 115(3), 479 - 84
Identification of a 16-S RNA fragment crosslinked to protein S1 within Escherichia coli ribosomal 30-S subunits by the use of a crosslinking reagent: ethyl 4-azidobenzoylaminoacetimidate; Golinska B et al.; The bifunctional reagent ethyl 4-azidobenzoylaminoacetimidate was used to crosslink specifically ribosomal protein S1 to 16-S RNA within 30-S subunits . The reagent was attached to isolated protein S1 . The modified protein was reassociated with protein-S1-depleted 30-S subunits and then crosslinked to the RNA molecule . The covalently bound 16-S RNA-protein S1 complex was isolated and the RNA fragment C-U-A-A-C-G-C-G-U-U-A-A-G-U-C-G-A-C-C-G-C-C-U-G-G-G-G-A-G (positions 861-889) was characterized to be crosslinked to protein S1.

J Gen Microbiol, 1981 Apr, 123(Pt 2), 241 - 7
Escherichia coli 5'-nucleotidase: purification, properties and its release by osmotic shock; Broad DF et al.; Escherichia coli 5'-nucleotidase was purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis . Its molecular weight was estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, by gel exclusion chromatography, and by membrane filtration; values of 66 000, 48 500, and 15 000 to 30 000, respectively, were obtained . The enzyme was completely released from bacteria by osmotic shock treatment . The apparently anomalous behaviour of 5'-nucleotidase in terms of the molecular sieving hypothesis for the release of enzymes by osmotic shock proposed by Smith & Wyatt (1974) and extended by Broad & Smith (1979) is discussed.

Gene, 1981 Apr, 13(3), 251 - 68
A deletion analysis of hybrid phage carrying the US region of Herpes simplex virus type 1 (Patton) . I . Isolation of deletion derivatives and identification of chi-likes sequences; Umene K et al.; The EcoRI-H fragment (15.4 kb) of Herpes simplex virus type 1 (HSV-1) has been cloned in lambda gtWES in both orientations . This fragment contains the entire US region and has about 900 bp of terminal redundant sequences derived from the internal and terminal repeats of the S region . 56 independent plaque-forming deletion derivatives of the lambda gt/WES::EcoRI-H hybrid phage were isolated using either EDTA resistance or ability to grow on Escherichia coli(P2) lysogens as selective methods . The endpoints of these deletions were located using nine restriction enzymes that cleave within the EcoRI-H fragment . All of the deletions have at least one endpoint within the cloned fragment . Several unusual features of the lambda hybrids, including heterogeneity of a particular region in the HSV-1 EcoRI-H fragment and the presence of chi-like sequences in the US region of HSV-1, are discussed.

Gene, 1981 Apr, 13(3), 227 - 37
Isolation of beta-globin-related genes from a human cosmid library; Grosveld FG et al.; A human gene library was constructed using an improved cloning technique for cosmid vectors . Human placental DNA was partially digested with restriction endonuclease MboI; size-fractionated and ligated to BamHI-cut and phosphatase-treated cosmid vector pJB8 . After packaging in lambda phage particles, the recombinant DNA was transduced into Escherichia coli 1400 or HB101 followed by selection on ampicillin for recombinant E . coli . 150 000 recombinant-DNA-containing colonies were screened for the presence of the human beta-globin related genes . Five recombinants were isolated containing the human beta-globin locus and encompassing approx . 70 kb of human DNA.

Proc Natl Acad Sci U S A, 1981 Apr, 78(4), 2302 - 6
Matrix protein in planar membranes: clusters of channels in a native environment and their functional reassembly; Schindler H et al.; Planar bilayers formed from Escherichia coli outer membrane vesicles exhibit conductance properties similar to those previously observed in bilayers reconstituted from aggregates of matrix protein, the major outer membrane protein . Discrete conductance steps are observed, reflecting voltage-dependent transmembrane channels . These exist in clusters which are activated by voltage . After activation, channels close with increasing potentials and reopen reversibly at lower voltage . Depending on the sign of the potential, two distinct closed states of the pores are observed . Cooperative interactions, hysteresis effects, relaxation times, and values of channel conductance depend on cluster size . These properties provide the reference data for the reconstitution of membrane function from individual components . Planar bilayers were formed from vesicles containing either solubilized matrix protein in a homogeneous trimeric state or bacterial glycolipid (lipopolysaccharide), or both . Activation of channel conductance required the presence of glycolipid and the formation of channel clusters, leading to conductance properties of the channels closely resembling those observed in native outer membranes . At very low concentrations of trimers, irreversible association to clusters by lateral diffusion was observed . Nearly quantitative recoveries of channels allowed the assignment of three pores per trimer.

Proc Natl Acad Sci U S A, 1981 Apr, 78(4), 2258 - 62
Deletion mapping of sequences essential for in vivo transcription of the iso-1-cytochrome c gene; Faye G et al.; The 5' termini of yeast CYC1 RNA molecules have been mapped, by nuclease S1 digestion of mRNA . DNA duplexes, to seven locations from 29 to 93 base pairs upstream from the initiating ATG codon . When the CYC1 gene is introduced into yeast in plasmid YEp13, substantially the same transcripts are made . Using this system to study in vivo gene expression, we measured the capacity of enzymatically produced DNA deletions to form the normal set of RNAs . Four regions of 5'-flanking DNA were identified as functional . Sequences within the region -242 to -139 are required for maximal CYC1 transcript formation; their deletion reduces transcription by a factor of 15 but does not change the pattern of 5' ends observed . Deletion of the sequence between -242 and -99 does not further change the overall transcript level but does affect the specificity of CYC1 mRNA starting . A deletion that extends from -242 to -75 causes both an additional shift in the pattern of 5' ends observed and a further large decrease (factor of 10--20) in CYC1 RNA level . Deletions that extend from -242 to -43, and particularly two deletions that extend still closer to the initiating ATG, cause the appearance of an abundant transcript which starts upstream of position -1078 and of minor transcripts starting in the region -325 to -245.

Proc Natl Acad Sci U S A, 1981 Apr, 78(4), 2159 - 63
Overproduction of the free radical of ribonucleotide reductase in hydroxyurea-resistant mouse fibroblast 3T6 cells; Akerblom L et al.; Hydroxyurea inhibits the activity of ribonucleotide reductase (ribonucleoside-diphosphate reductase; 2'-deoxy-ribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) in bacteria and mammalian cells . The reductase from Escherichia coli consists of two nonidentical subunits (B1 and B2) and hydroxyurea acts by specifically destroying a tyrosine free radical of B2 required for enzyme activity . The mammalian enzyme also consists of two nonidentical subunits (M1 and M2), only one of which (M1) has been obtained in pure form . By continuous culture at stepwise increasing drug concentrations, we have now obtained a 3T6 mouse fibroblast cell line with a 100-fold increased resistance to hydroxyurea . Extracts from resistant cells showed a 3- to 15-fold increase in reductase activity . The amount of M1 protein was not increased . The amount of M2 protein could not be measured directly, but the M2 activity in extracts from resistant cells (but not normal cells) showed an EPR spectrum very similar to that of the tyrosine radical of the bacterial B2 subunit . We propose that resistance to hydroxyurea is caused either by overproduction of the complete M2 subunit or by increased generation of the tyrosine radical within the M2 protein . It seems that either alternative mirrors a possible normal regulatory mechanism for the activity of the reductase.

J Virol, 1981 Apr, 38(1), 393 - 7
Ground squirrel hepatitis virus DNA: molecular cloning and comparison with hepatitis B virus DNA; Siddiqui A et al.; Ground squirrel hepatitis virus (GSHV) shares many ultrastructural antigenic, molecular, and biological features with hepatitis B virus (HBV) of humans, indicating that they are members of the same virus group . Both viruses contain small circular DNA molecules which are partially single stranded . Here, we ligated an endonuclease EcoRI digest of GSHV DNA with EcoRI-cleaved plasmid vector pBR322 and cloned recombinant plasmids in Escherichia coli C600 . Two cloned recombinants were characterized . One (pGS2) was found to contain only part of the GSHV genome, and the other (pGS11) was found to contain the entire viral DNA . A restriction endonuclease cleavage map of the GSHV insert in pGS11 and the locations of certain physical features of the virion DNA were determined . The relative positions of the single-stranded region, the unique 5' end of the short DNA strand, and the unique nick in the long DNA strand in GSHV DNA were found to be the same as those previously described for HBV DNA . Hybridization with an HBV {32P}DNA probe containing the apparent coding sequence for the major polypeptide of HBV surface antigen and a probe containing the putative coding sequence for the major polypeptide of the HBV core revealed specific homology with different restriction fragments of GSHV DNA . The two homologous regions had approximately the same locations relative to the single-stranded region, the 5' end of the short strand, and the nick in the long strand in the two viral DNAs . These results suggest that in both viruses the genes for the major HBV surface antigen and core polypeptides have the same locations relative to unique physical features of the viral DNAs.

Eur J Biochem, 1981 Apr, 115(2), 293 - 6
Cation dependence of restriction endonuclease EcoRI activity; Woodhead JL et al.; Restriction endonuclease EcoRI cleaves the DNA sequence 5'd(-G-A-A-T-T-C-) under optimum digestion conditions . A variation in pH and ionic strength can result in EcoRI activity when 5'd(-A-A-T-T-) is cut . A divalent cation, usually Mg2+, is required for enzyme activity, though Mn2+ can also be used . Eight different cations with ionic radius/charge ratios similar to Mg2+ were tested and Co2+ and Zn2+ were also found to act as cofactors for EcoRI . A comprehensive study has been made of the effect of NaCl and pH on the EcoRI/EcoRI transition in the presence of the above four cations . Generally, a decrease in NaCl and/or an increase in pH caused a decrease in enzyme specificity . The changeover depended on the cation . They may be placed in order of their ability to increase EcoRI specificity thus: Co2+ greater than Zn2+ greater than Mg2+ greater than Mn2+ . The Km of EcoRI for ColE1 DNA, in the presence of Co2+, was found to be 0.4 nM, compared to 3 nM with Mg2+, whereas the turnover was only one double-stranded scission/min with Co2+ compared to eight/min with Mg2+ . The implications of all these findings on the enzyme's mechanism are discussed.

Eur J Biochem, 1981 Apr, 115(2), 235 - 9
The major components of the mouse and human genomes . 2 . Reassociation kinetics; Soriano P et al.; The reassociation kinetics of DNA fragments obtained from the major components of the mouse and human genomes (recently isolated in our laboratory) have been investigated . It has been found that the relative amounts of interspersed repeated and unique sequences strikingly differ in the different major components of each genome and in the corresponding major components of the two genomes . Furthermore, within each major component, the interspersed repeated and unique sequences do not differ in dG + dC contents . These findings lead to the general conclusion that the sequence organization of mammalian genomes is not uniform in different chromosomal regions and that it exhibits remarkable variations in different mammals.

Cell, 1981 Apr, 24(1), 235 - 42
A single base-pair alteration is responsible for the DNA overproduction phenotype of a plasmid copy-number mutant; Muesing M et al.; The Cop- plasmid pOP1 delta 6 is a recessive copy-number mutant derived from Col E1.pOP1 delta 6 exists at 200-300 copies per chromosome in E . coli, while Col E1 exists at 10-15 copies per chromosome . We have investigated the molecular basis for DNA overproduction by pOP1 delta 6 by mapping the mutation to a restriction fragment of the plasmid genome, which is about 400 bp from the origin of replication . The mutation is a single base-pair alteration-a GC leads to TA transversion . The alteration changes the nucleotide sequence of two RNA elements known to be synthesized from opposite DNA strands in the same region of the plasmid genome; a small, nontranslated RNA known as RNA1 and the primer RNA required for initiation of DNA replication in vitro . The mutation is located in a GC-rich region of dyad symmetry, which precedes the termination signal for RNA1 transcription . When pOP1 delta 6 DNA is transcribed in vitro, RNA1 is not observed . Rather, several new transcripts of a larger size are observed resulting from readthrough transcription of RNA1 . In conjunction with previous genetic evidence, these results indicate that RNA 1 may be a negative modulator of Col E1 DNA replication and that its secondary structure is critical to its function.

J Bacteriol, 1981 Apr, 146(1), 85 - 92
The region controlling the thermosensitive effect of plasmid Rts1 on host growth is separate from the Rts1 replication region; Yamamoto T et al.; Rts1 is a high-molecular-weight (126 x 10(6)) plasmid encoding resistance to kanamycin . It expresses unusual temperature-sensitive phenotypes, which affect plasmid maintenance and replication, as well as host cell growth . We have cloned the essential replication region of Rts1 from pAK8, a smaller derivative which is phenotypically similar to Rts1 . Restriction endonuclease digests of isolated pAK8 deoxyribonucleic acid were allowed to "self-ligate" (ligation without an additional cloning vector) and subsequently were used to transform Escherichia coli strain 20SO to kanamycin resistance . Screening of these strains for the phenotypes of thermosensitive host growth and temperature-dependent plasmid elimination demonstrated that these two properties were expressed independently . Furthermore, it was shown that the Rts1 replication locus per se is not necessarily responsible for altered host growth at the nonpermissive temperature . The kanamycin resistance fragment of pAK8 was also cloned into pBR322 . Electrophoretic analysis of BamHI restriction enzyme digests of this plasmid and similar digests of an Rts1 miniplasmid has allowed the identification of an 18.6-megadalton fragment carrying the replication locus and a 14.1-megadalton fragment carrying the kanamycin resistance gene.

J Bacteriol, 1981 Apr, 146(1), 409 - 11
Improved mapping of ksgB and integration of transposons near relB and terC in Escherichia coli; Diderichsen B; Tn5 transposons were integrated near relB at 34.2 min . ksgB was mapped at 36.4 min, 2 min from its previously assumed position.

J Bacteriol, 1981 Apr, 146(1), 325 - 30
Synthesis of a precursor to the B subunit of heat-labile enterotoxin in Escherichia coli; Palva ET et al.; Escherichia coli K-12 minicells were employed to investigate the biosynthesis of plasmid-encoded, heat-labile enterotoxin of E . coli . Two polypeptide species related to the B subunit of the toxin were expressed in the minicells . One of these polypeptides (molecular weight, 11,500) was immunoprecipitated by antiserum to cholera toxin . Because the B subunits of heat-labile enterotoxin and cholera toxin have common antigenic sites, we concluded that this species was the mature B subunit . The larger polypeptide (molecular weight, 13,000) is likely to be a precursor of the B subunit because it could be chased into the mature form . This conversion was inhibited by compounds which dissipate proton motive force, suggesting that processing requires energy.

J Bacteriol, 1981 Apr, 146(1), 18 - 23
Effect of gyrB-mediated changes in chromosome structure on killing of Escherichia coli by ultraviolet light: experiments with strains differing in deoxyribonucleic acid repair capacity; von Wright A et al.; Mutations at the gyrB locus were found to decrease the degree of supercoiling of the Escherichia coli chromosome . The effect of a gyrB mutation on the repair of ultraviolet-induced deoxyribonucleic acid damage was studied by following the killing of strains of E . coli K-12 proficient and deficient in deoxyribonucleic acid repair . The effectiveness of both excision and postreplication types of deoxyribonucleic acid repair was found to be altered by this mutation, the former being apparently enhanced and the latter impaired.

J Bacteriol, 1981 Apr, 146(1), 149 - 54
Regulation of galactose operon expression: glucose effects and role of cyclic adenosine 3',5'-monophosphate; Joseph E et al.; We studied the following two aspects of the glucose effect on galactose operon expression in Escherichia coli K-12: catabolite repression and inducer exclusion . Using both inducible and constitutive strains and measuring the rate of promoter-proximal enzyme synthesis, we found that the galactose operon did not seem to exhibit catabolite repression . The only glucose effect on galactose operon expression which we observed was inducer exclusion, as shown by the existence of diauxic growth in the presence of glucose and galactose . This diauxie was not relieved by cyclic adenosine 3',5'-monophosphate . Cyclic adenosine 3',5'-monophosphate did not seem to be an antagonist of any glucose effect on galactose operon expression; its only effect was to stimulate promoter-distal gene expression.

J Bacteriol, 1981 Apr, 146(1), 108 - 16
Pyrimidine metabolism of Bdellovibrio bacteriovorus grown intraperiplasmically and axenically; Rosson RA et al.; Bdellovibrio bacteriovorus grown axenically or intraperiplasmically on Escherichia coli has pathways for the interconversion of pyrimidines and the synthesis of pyrimidine nucleoside 5'-triphosphates similar to those found in the enteric bacteria . Minimal differences in enzyme activities were observed for axenically and intraperiplasmically grown cells . As might be expected for an organism which takes up deoxyribonucleoside 5'-monophosphates per se, high levels of enzymes which catalyze the generation of deoxyribonucleoside triphosphates from monophosphates were found . In addition, all enzymes of the thymine salvage pathway, except for thymidine kinase, were directly demonstrated in wild-type strains . It was possible to demonstrate this activity only indirectly owing to an inhibitor in wild-type extracts . Investigations with inhibitors of pyrimidine interconversion reactions showed that essentially all B . bacteriovorus deoxyribonucleic acid not synthesized from units derived from E . coli deoxyribonucleic acid is made from components of the substrate organism's ribonucleic acid . Evidence for de novo pyrimidine synthesis from the amino acid level was not found for B . bacteriovorus grown on E . coli that had a high protein/deoxyribonucleic acid ratio or on normal E . coli . The potential for de novo pyrimidine synthesis by intraperiplasmically grown B . bacteriovorus, however, cannot be totally ruled out on the basis of these investigations.

Proc Natl Acad Sci U S A, 1981 Apr, 78(4), 2273 - 7
Chemical probing of the tRNA--ribosome complex; Peattie DA et al.; We probed the (Escherichia coli) tRNAPhe--ribosome interaction with the chemical reagents dimethyl sulfate and diethyl pyrocarbonate . This monitored the higher-order structure of the tRNA in this biological complex and identified critical sites in the tRNA molecule involved in binding to the ribosome . The methylation of the N-7 position of guanosine and the N-3 position of cytidine as well as diethyl pyrocarbonate attack on adenosines are sensitive to secondary and tertiary interactions . Here we identify specific bases in E . coli Phe-tRNAPhe affected by the interaction with the ribosome . The 70S ribosome protects the N-3 position of cytidine-74 and 75 in the 3'-terminal C-C-A, suggesting a strong, possibly base pairing, interaction between the ribosome and that universal sequence . The ribosome also induces strong reactivities at the N-7 positions of G-24 and G-46 in the central region of the tRNA molecule near the variable-loop domain as well as less significant reactivities at 11 other guanosines . Two of these, G-10 and G-44, are close to G-24 and G-46 in the center of the molecule; the others (guanosines 1, 5, 6, 18, 19, 63, 65, 69, and 71) are in the coaxial acceptor stem-T stem helix . All of the effects are ribosome induced and occur in the presence or absence of the messenger poly(U) . Prior chemical modification of the anticodon bases as well as the two adjacent 3' purines and, less effectively, four purines in the anticodon stem prevent stable poly(U)-directed ribosome binding . Thus, we identify the 3' terminal C-C-A sequence, near the peptidyl transferase site, and the anticodon stem and loop of tRNAPhe as forming critical contacts with the ribosome . Other regions of the molecule become reactive on ribosome binding, but these do not suggest a significant conformational change being more likely due to a change of environment.

J Bacteriol, 1981 Apr, 146(1), 128 - 32
Inactivation of the ribonucleic acid-processing enzyme ribonuclease E blocks cell division; Goldblum K et al.; The Escherichia coli endoribonuclease ribonuclease E, the enzyme responsible for the processing of precursor 5S ribosomal ribonucleic acid (RNA) from the nascent ribosomal RNA transcript, is thermolabile in rne-3071 mutants . When cells of such a strain were shifted to a nonpermissive temperature, the function of ribonuclease E was almost instantaneously inactivated . However, a threefold linear increase in absorbance took place over a 3-h period, and similar linear increases occurred in all the macromolecules we measured, including deoxyribonucleic acid, RNA, protein, and lipopolysaccharides . Interestingly, during this period, the cells elongated but failed to divide . Thus, these experiments suggest that an early effect of inactivation of the RNA processing enzyme ribonuclease E is a block in cell division.

Chest, 1981 Apr, 79(4 Suppl), 38S - 43S
International conference on byssinosis . Mechanisms of disease induction; Edwards J; From this work and other published data there are at least three distinct compounds that have been shown to be capable of inducing symptoms of byssinosis . There is an aminopolysaccharide-protein complex in cotton plant bracts that acts by causing histamine release in the human lung and also causes necrosis of bronchiolar epithelium . There is endotoxin or endotoxin-like material present in cotton plant bracts that acts by a mechanism other than by causing histamine release . This can induce histologic features of chronic bronchitis over a period of time . There is a polyphenol compound whose action is unlikely to be via complement activation, even though higher titers of antibody to it are present in byssinotic compared with nonbyssinotic subjects . It is postulated that such a compound is held in the lungs by antibody and exerts its effect on the pulmonary vasculature, altering capillary resistance . It also produces pathologic change as might be expected from its ability to precipitate proteins and activate complement.

J Gen Microbiol, 1981 Apr, 123(Pt 2), 343 - 9
Role of membrane potential and ATP in complex formation between Escherichia coli male cells and filamentous phage fd; Yamamoto M et al.; Mutant strains of Escherichia coli male cells defective in Ca2+,Mg2+-dependent ATPase (unc) were constructed and tested for their ability to form a complex between sex pili and the filamentous phage fd under conditions where either the membrane potential or the cellular concentration of ATP was lowered . The uncoupler carbonyl cyanide m-chlorophenylhydrazone and the respiratory inhibitor cyanide, as well as valinomycin-K+ and colicin E1, all markedly diminished complex formation, indicating that the maintenance of a membrane potential, but probably not the pH gradient, is essential for the formation of the complex . Since complex formation with freshly centrifuged cells (which initially lacked sex pili) as well as with preincubated cells (in which pre-existing pili were available for complex formation) was inhibited by exposure to the inhibitors, energy seems to be required for both the reappearance (probably assembly) and the maintenance of sex pili on the cell surface . Brief exposure of freshly centrifuged cells to arsenate resulted in only partial inhibition of complex formation . However, marked inhibition of complex formation was observed following exposure to arsenate of preincubated cells possessing sex pili . This indicates that compounds such as ATP may also be required for maintenance of sex pili on the cell surface.

Pathol Biol (Paris), 1981 Apr, 29(4), 237 - 9
{Enterotoxigenic Escherichia coli and temperate areas childhood diarrheas (author's transl)}; Albouy C et al.; We test for CFA and LT toxin 159 E . coli strains issued from endemic diarrheas in hospitalized children . Three strains belong to the CFA1 group but only one is LT toxin producing . This last one is isolated in feces of a child coming from topical areas.

J Bacteriol, 1981 Apr, 146(1), 260 - 8
General method, using Mu-Mud1 dilysogens, to determine the direction of transcription of and generate deletions in the glnA region of Escherichia coli; MacNeil D; A general, genetic technique for determining the direction of transcription for bacterial genes is presented . By comparing the phenotype of Mu-Mud1 dilysogens with the phenotype of deletion-containing derivatives, the direction of transcription for the gene containing Mud1 can be unambiguously determined . This method can generate a series of strains containing deletions with predetermined endpoints, and strains with duplications of the region containing the Mud1 insertion . In Escherichia coli, the glnA and glnG genes are transcribed in the same direction.

J Bacteriol, 1981 Apr, 146(1), 251 - 9
Location of an F-pilin pool in the inner membrane; Moore D et al.; Polyacrylamide gel analysis of {35S}methionine-labeled membrane preparations from Escherichia coli has revealed the presence of five polypeptides present only in the membranes of cells containing the conjugative plasmid F . In addition to the previously reported product of traT, polypeptides migrating with apparent molecular weights of 100,000, 23,500, 12,000, and 7,000 were resolved . Membrane preparations from F traJ mutants lacked these polypeptides, indicating that all of these proteins are tra gene products . The 7,000-molecular-weight polypeptide comigrated with unlabeled purified F-pilin protein . About 4 to 5% of the total radioactive label in whole membrane preparations was present in this polypeptide, indicating the existence of a substantial pool of membrane-associated F-pilin . The polypeptide could be extracted from whole membrane preparations with Triton X-100 and was found in the inner membrane fraction of membranes separated by sucrose density centrifugation.

Biochemistry, 1981 Mar 31, 20(7), 1907 - 18
Escherichia coli deoxyribonucleic acid dependent ribonucleic acid polymerase transcriptional pause sites on SV40 DNA F1; Reisbig RR et al.; We have studied elongation of SV40 DNA F1 by E . coli RNA polymerase looking specifically at the length of the transcript as a function of time . By running the transcription reactions at 18 degrees C with limited enzyme and adding heparin or rifampicin after elongation has started, we have achieved almost exclusive initiation from the SV40 DNA preferred promotor size {Zain, B . S., Weissmann, S . M., Lebowitz, P., & Lewis, A . M., Jr . (1973) J . Virol . 11, 682-693} . In the region within 1500 nucleotides of the initiation we observe nine prominent sites and a number of minor site where hesitation during elongation occurs . The positions of these hesitation points or pause sites are not effected by changes in the salt concentration, the simultaneous lowering of the concentrations of all the NTPs, or by increases in the RNA polymerase concentration, implying that the pause sites are a consequence of the RNA, DNA, and RNA polymerase ternary complex . The pause sites are not an artifact of the lowered temperature (18 degrees C) used in the experiments since they are also observed at 37 degrees C . The first four of these sites have been sequenced by using the 3'-O-methyl analogues of the ribonucleotide triphosphates . We have found no sequence homology between the pause sites . The kinetics of the pause reactions do not fit a first-order model but do correspond to a scheme were continuation through a pause site and termination at a pause site are both represented . For one of the pause sites, the relaxation time for continuation through the pause site was determined to be approximately 2.5 min and for the termination approximately 50 min at 18 degrees C . If the concentration of one of the NTPs is lowered to 10 muM, the strength of a pause site can be increased if that NTP is contained in the pause . Also, minor pause sites are observed at regions in the RNA sequence which are rich in the NTP that has the lowered concentration . When GTP is replaced by ITP during transcription, a new set of pause site quite different from the normal sites of hesitation are observed . The major new pause sites occur at or near sequences in the RNA which are rich in I-U residues preceded by a region rich in C residues . This indicates, as has been previously noted, that sequences where the DNA.RNA hybrid is quite stable followed by a region that is very unstable may cause termination . When BrUTP replaced UTP, very little effect was observed on the pause sites . The addition of p termination factor causes termintion to increase in all the pause sites with a length greater than 300 nucleotides . In the type of experiments performed here, those pause sites had continuation relaxation times greater than 45 s at 37 degrees C . This implies that regardless of the nature of a pause, p will cause at least some termination at all hesitation sites with a relaxation time greater than 45 s . All the results are discussed in terms of a kinetic model for the termination of elongation.

Biochemistry, 1981 Mar 31, 20(7), 1874 - 80
Biochemical and biological effects of nonionic nucleic acid methylphosphonates; Miller PS et al.; Oligodeoxyribonucleoside methylphosphonates with base sequences complementary to the anticodon loop of tRNALys and to the -ACCA-OH amino acid accepting stem of tRNA were prepared by chemical synthesis . Oligodeoxyadenosine methylphosphonates form stable, triple-stranded complexes with both poly(U) and poly(dT) . These analogues selectively inhibit cell-free aminoacylation of tRNALys (E . coli) but have no effect on aminoacylation of tRNALys (rabbit) . The extent of inhibition is temperature dependent and parallels the ability of the oligomer to bind to poly(U), which suggests that inhibition occurs as a result of oligomer binding to the -UUUU- anticodon loop of tRNALys (E . coli) . The failure of the oligodeoxyadenosine methylphosphonates to inhibit tRNALys (rabbit) amino-acylation suggests that there may be a difference between the structure of tRNALys or its interaction with aminoacyl synthetase in the Escherichia coli and rabbit systems . The oligodeoxyadenosine analogues also effectively inhibit polyphenylalanine synthesis in cell-free translation systems derived from both E . coli and rabbit reticulocytes . The extent of inhibition parallels the Tm values of the oligo(A) phosphonate-poly(U) complexes and suggests that the inhibition is a consequence of complex formation with the poly(U) message . Tritium-labeled oligodeoxyribonucleoside methylphosphonates with a chain length of up to nine nucleotidyl units are taken up intact by mammalian cells in culture . All the oligomer analogues tested inhibited, to various extents, colony formation by bacterial, hamster, and human tumor cells in culture.

Biochemistry, 1981 Mar 31, 20(7), 1710 - 6
Dihydrofolate reductase hysteresis and its effect of inhibitor binding analyses; Baccanari DP et al.; Escherichia coli dihydrofolate reductase was shown to follow slow transient kinetics (hysteresis) . Nonlinear reaction velocities were detected during the enzyme assay and required 10-15 min to reach a steady-state rate . The degree of hysteresis was influenced by the enzyme concentration and the order of substrate addition . Incubation of the enzyme with NADPH before addition of dihydrofolate resulted in slow initial velocities that increased up to 2-fold during the course of the assay . Increasing the enzyme concentration from 0.2 to 1 nM resulted in diminished hysteresis . NADPH-initiated reactions were linear at all enzyme concentrations tested . Certain drugs had profound effects on hysteresis . Pyrimethamine practically eliminated the hysteresis of dihydrofolate-started reactions, whereas trimethoprime augmented the non-linearities in the sense that hysteresis was detected in both enzyme- and NADPH-started reactions . The shape of these reaction tracings makes trimethoprim is not a slow-binding inhibitor when assayed under conditions that eliminate hysteresis . Contrary to this, sulfamethoxazole did not affect hysteresis or augment inhibition of the enzyme by trimethoprim . Sulfamethoxazole alone (at 6 mM) did not inhibit the hysteresis and allow reliable determinations of Ki values of both weak and tight binding inhibitors . For example, Ki values for pyrimethamine, trimethoprim, and methotrexate were found to be 214 nM, 1.3 nM, and 0.021 nM, respectively.

Biochemistry, 1981 Mar 31, 20(7), 2041 - 7
Genetic identification and purification of the respiratory NADH dehydrogenase of Escherichia coli; Jaworowski A et al.; Escherichia coli membrane particles were solubilized with potassium cholate . An NADH:ubiquinone oxidoreductase was resolved by hydroxylapatite chromatography of the solubilized material . This enzyme has been identified as the respiratory NADH dehydrogenase since it is absent in chromatograms of solubilized material from an ndh mutant strain . Such mutants lack membrane-bound NADH oxidase activity and have previously been shown to have an inactive NADH dehydrogenase complex {Young, I . G., & Wallace, B . J . (1976) Biochim . Biophys . Acta 449, 376-385} . The respiratory NADH dehydrogenase was amplified 50- to 100-fold in vivo by using multicopy plasmid vectors carrying the ndh gene and then purified to homogeneity on hydroxylapatite . Hydroxylapatite chromatography of cholate-solubilized material from genetically amplified strains purified the enzyme approximately 800- to 100-fold relatively to the activity in wild-type membranes . By use of a large-scale purification procedure, 50-100 mg of protein with a specific activity of 500-600 mumol of reduced nicotinamide adenine dinucleotide oxidized min-1 mg-1 at pH 7.5, 30 degrees C, was obtained . Sodium dodecyl sulfate gel electrophoresis of the purified enzyme showed that the enzyme consists of a single polypeptide with an apparent Mr of 45 000.

Biochemistry, 1981 Mar 31, 20(7), 1902 - 6
Properties of purified ribonuclease P from Escherichia coli; Kole R et al.; The purified protein moiety of ribonuclease P (EC 3.1.26.5) from Escherichia coli, a single polypeptide of molecular weight approximately 17 500, has not catalytic activity by itself on several RNA substrates . However, when it is marked in vitro with an RNA species called M1 RNA, RNase P activity is reconstituted . The rate at which the purified RNase P cleaves any particular tRNA precursor molecule depends on the identity of that tRNA precursor.

Biochim Biophys Acta, 1981 Mar 26, 653(1), 9 - 17
tRNA chemical methylation . In vitro and in vivo formation of 1,7-dimethylguanosine at high concentrations of methylating agents; Kanduc D; The methylation patterns produced in Escherichia coli B tRNA by a range of concentrations of the weak carcinogen dimethyl sulphate were examined with the following results: 1 . 1,7-Dimethylguanosine was found to be formed in high amounts in the tRNA methylation reaction at high concentrations of methylating agent . 2 . The dialkylated compound was recovered mainly in the form of derivatives, the spectral and chromatographic behaviour of which varied according to the procedures used for their isolation . Similar results were obtained for the in vivo methylation of rat-liver tRNA: after administration of a very high dose of the powerful carcinogen dimethylnitrosamine, 1,7-dimethylguanosine was found in rat-liver tRNA . Moreover, the analysis of the time-course of nucleic acid methylation indicated that this dialkylated product was still present in rat-liver tRNA when the major product of alkylation, 7-methylguanine, had almost completely disappeared.

Biochim Biophys Acta, 1981 Mar 26, 653(1), 1 - 8
Regulation of small RNAs in Escherichia coli . Alteration in the intracellular concentrations of small RNAs during amino acid and energy starvation; Pao CC et al.; The accumulation of low molecular weight RNAs in Escherichia coli cells following amino acid or energy source starvation was examined using two-dimensional polyacrylamide gel electrophoresis . 32P-labeled small RNA prepared from serine- or isoleucine-starved stringent strain (relA+) cells was shown to display gel patterns that were grossly different from that of unstarved cells . It appears that the deprivation of serine or isoleucine has little or no inhibitory effect on the accumulation of transfer RNA cognate to the deprived amino acid . This is demonstrated by a relative increase in the concentrations of small RNAs that can be charged with serine or isoleucine following starvation of these amino acids . However, small RNAs labeled during starvation of phenylalanine or energy source showed gel patterns similar to that of control cells . This suggested a heterogenous response in the accumulation of some low molecular weight RNAs, presumably transfer RNAs, following starvation of different amino acids.

J Biol Chem, 1981 Mar 25, 256(6), 3125 - 9
Characterization of new membrane lipoproteins and their precursors of Escherichia coli; Ichihara S et al.; By labeling cells heavily with {3H}glycerol or {3H}-palmitic acid several new species of lipoproteins, in addition to Braun's lipoprotein and a peptidoglycan-associated lipoprotein called PAL, were found in the envelope of Escherichia coli . The new lipoproteins were immunochemically different from both Braun's lipoprotein and PAL . A strain lacking the structural gene for Braun's lipoprotein contained new lipoproteins and PAL . In addition to Braun's lipoprotein and PAL, four new lipoproteins were found to be localized in the outer membrane, while other two species were found in the cytoplasmic membrane . The localization of one species is unknown . We previously reported that, on treatment of cells with globomycin, a precursor of Braun's lipoprotein accumulated in the cell envelope (Hussain, M., Ichihara, S., and Mizushima, S . (1980) J . Biol . Chem . 255, 3707-3712) . Similarly, the putative precursors of new lipoproteins and that of PAL accumulated in globomycin-treated cells . These precursors contained glycerol and fatty acid(s) as that of Braun's lipoprotein did . It is suggested that the structures of the "signal" region and the mechanisms of processing of all the lipoproteins of E . coli are similar.

J Biol Chem, 1981 Mar 25, 256(6), 3118 - 24
A mutant thioredoxin from Escherichia coli tsnC 7007 that is nonfunctional as subunit of phage T7 DNA polymerase; Holmgren A et al.; Thioredoxin was purified to homogeneity from the Escherichia coli mutant tsnC 7007 that is defective in phage T7 DNA replication and previously shown to contain a missense thioredoxin . Tryptic peptide maps of reduced and carboxymethylated 7007 thioredoxin combined with amino acid sequence analysis revealed one amino acid substitution; Gly-92 in thioredoxin is exchanged to an aspartic acid residue in the 7007 protein . The missense thioredoxin gave no activity with the gene 5 protein of phage T7 in the complementation to active T7 DNA polymerase . It competitively inhibited the complementation of wild type thioredoxin and gene 5 protein and formed a complex with the gene 5 protein that was retained by antithioredoxin Sepharose . The 7007 thioredoxin has reduced catalytic activity with thioredoxin reductase, ribonucleotide reductase, or as a protein disulfide reductase . The apparent Km value of 7007 thioredoxin as a substrate for thioredoxin reductase was increased 3-fold relative to normal thioredoxin, and the Vmax value was decreased 7-fold . The position of GLy-92 in the known three-dimensional structure of thioredoxin-S2 is correlated with the changed functional properties of the substituted mutant protein.

J Biol Chem, 1981 Mar 25, 256(6), 2669 - 74
Molecular properties of acyl carrier protein derivatives; Rock CO et al.; Acyl carrier protein (ACPSH) functions as the acyl carrier in fatty acid biosynthesis . The acyl moieties are bound to the sole sulfhydryl of the protein located on the 4'-phosphopantetheine prosthetic group . Disulfide-linked dimers of ACPSH were formed by the reaction of ACPSH with acyl-ACP or the mixed disulfide of ACPSH and thionitrobenzoate . The formation of ACP dimers was established by electrophoresis, gel filtration, and sedimentation equilibrium . ACP purified from stationary phase Escherichia coli B cells was found to exist primarily as a mixed disulfide with glutathione . This species was identified by gel electrophoresis amino acid analysis and 31P NMR spectroscopy . A non-denaturing gel electrophoresis system was developed that allows the comparison of the effects of various protein and sulfhydryl modifications on the stability of the ACP protein moiety to pH-induced denaturation . In general, attachment of hydrophilic ligands to the sulfhydryl of ACPSH resulted in less stable protein structures whereas the presence of a hydrophobic thioester resulted in stabilization of the protein conformation . The less stable ACP structures were found to have 31P NMR chemical shifts displaced downfield from ACPSH and the more stable acyl-ACP derivatives were found to have chemical shifts displaced upfield from ACPSH.

J Biol Chem, 1981 Mar 25, 256(6), 2635 - 43
Codon reading and translational error . Reading of the glutamine and lysine codons during protein synthesis in vitro; Lustig F et al.; The reading of glutamine and lysine codons during protein synthesis in vitro has been investigated using an MS2-RNA-programed system derived from Escherichia coli . Under conditions when either glutaminyl-tRNA1Gln (s2UUG) or glutaminyl-tRNA2Gln (CUG) was the only source of glutamine for protein synthesis both tRNAs were able to read the glutamine codons CAA and CAG as indicated by the incorporation of labeled glutamine into the pertinent coat protein tryptic peptides . On the other hand, when the two glutamine tRNAs competed for the codon CAA the reading efficiency of the anticodon s2UUG, which reads the codon according to the wobble rules, was almost 40 times higher than that of the competing anticodon CUG, which reads the codon by "two out of three," i.e . it cannot form a regular base pair with the third codon position . In reading the codon CAG the anticodon CUG was approximately eight times more efficient than the anticodon s2UUG . The lysyl-tRNA1Lys (CUU) could not alone sustain any detectable coat protein synthesis in the MS2 system indicating that there was no significant reading of the lysine codon AAA . This conclusion is supported by the outcome of experiments where lysyl-tRNA1Lys (CUU) and lysyl-tRNA2Lys (s2UUU) competed for the codon AAA . The reading efficiency of the anticodon CUU was less than 1% of that of the competing s2UUU which represents the limit of resolution of our experimental system . When the two lysine tRNAs competed for the codon AAG the anticodon CUU was about four times more efficient than s2UUU . These results are discussed in the context of the two out of three hypothesis, which attempts to relate the frequency of such reading to the hydrogen bonding properties of the codon nucleotides.

Nucleic Acids Res, 1981 Mar 25, 9(6), 1463 - 82
Molecular analysis of the protamine multi-gene family in rainbow trout testis; Gedamu L et al.; We have synthesized a family of double-stranded cDNAs (ds cDNAs) using as a template the family of highly purified protamine mRNAs from rainbow trout testis . Individual pure protamine cDNA components were isolated by cloning this family of protamine ds cDNAs in a plasmid vector (pMB9) . Clones containing protamine sequences were characterized by restriction mapping and by a positive hybrid-selected translation assay, which allowed us to correlate particular cDNAs with particular protein components . To allow more detailed comparisons, complete nucleotide sequences were determined for selected protamine clones . We have detected at least 5 distinctly different coding sequences, which nevertheless show at least 82% homology, and which have probably arisen by repeated gene duplication . These very highly conserved coding sequences do however contain a distinctly variable region near the 5'-end of the mRNA (N-terminus of the protein), corresponding to the major sites of serine phosphorylation . Since the amino acid sequences predicted by our DNA sequences were slightly different from those previously published (1), we have independently determined the amino acid sequences of protamine components CI, CII, CIII from our own source of trout testis . These new peptide sequences are completely consistent with those predicted by our nucleotide sequences . The 3'-untranslated regions of the protamine mRNAs are, surprisingly almost as highly conserved as the coding regions . Both coding and 3'-noncoding portions appear to be under a similar degree of selective pressure and evolutionary constraint to remain constant.

Nucleic Acids Res, 1981 Mar 25, 9(6), 1365 - 81
The complete nucleotide sequence of mouse immunoglobin gamma 2a gene and evolution of heavy chain genes: further evidence for intervening sequence-mediated domain transfer; Yamawaki-Kataoka Y et al.; We have determined the complete nucleotide sequence (1990 base pairs) of mouse immunoglobulin gamma 2a gene, and compared it with the sequences of other gamma subclass genes so far sequenced, i.e . gamma 1 and gamma 2b genes . Divergence of the nucleotide sequence between a compared pair of the gamma genes varies extensively among different segments of the gene . For example, comparison of the gamma 2a and gamma 2b genes has revealed a remarkable homology in a long continuous segment (about 900 bases) that covers from the 3' portion of the first intervening sequence to the third intervening sequence . However, there is no particular segment of the gamma gene that is conserved universally among the three gamma genes . These findings suggest that, during their evolution, segments of the gamma genes had been scrambled between different subclass genes through recombinations within intervening sequences, thus providing further evidence for the intervening sequence-mediated domain transfer hypothesis . We have discussed several possible phylogenic trees which can explain the difference of divergence in various segments of the gamma genes.

Nucleic Acids Res, 1981 Mar 25, 9(6), 1271 - 89
Hairpin-loop formation by inverted repeats in supercoiled DNA is a local and transmissible property; Lilley DM; Short inverted repeat sequences adopt hairpin stem-loop type structures in supercoiled closed circular DNA molecules, demonstrated by S1 nuclease cleavage . Fine mapping of cleavage frequencies is in good agreement with expected cleavage patterns based upon the interaction between an unpaired loop and a sterically bulky enzyme molecule . Whilst the topological properties of underwound DNA circles depend ultimately upon reduced linkage, necessarily a global molecular property, hairpin loop formation is an essentially local property . Thus molecular size is unimportant for the S1 hypersensitivity of the Co1E1 inverted repeat . Furthermore, a 440 bp Sau3AI, EcoRI fragment of Co1E1 which contains the inverted repeat has been cloned into pBR322 whereupon it exhibits S1 cleavage similar to Co1E1 in the supercoiled recombinant molecule . The effect is therefore both local and transmissible . Direct competition, between inverted repeats in the recombinant, coupled with close examination of flanking sequences, enables some simple 'rules' for base pairing in hairpin loops to be formulated . Whilst limited G-T and A-C base pairing appears not to be destabilising, A-G, T-C or loop outs are highly destabilising.

J Biol Chem, 1981 Mar 25, 256(6), 2761 - 6
High resolution experimental and theoretical thermal denaturation studies on small overlapping restriction fragments containing the Escherichia coli lactose genetic control region; Hillen W et al.; The distribution of thermal stability in the Escherichia coli lac control region is evaluated from the melting behavior of 5 short (80-219 base pairs (bp)) sequenced DNA restriction fragments containing various parts of this sequence . The thermal denaturation of these fragments was measured at 3 salt concentrations . The previous notion that the melting curves for small fragments are sharp and asymmetric in 0.01 M Na+ and broadened and less asymmetric at 0.105 and 0.505 M Na+ is confirmed and the possible explanations are discussed . The existence of two thermodynamic boundaries in this region is also confirmed . The exact location of the boundary upstream of the cyclic AMP receptor protein (CAP) binding site is accurately determined from melting experiments at 260 and 282 nm . The secondary boundary located between the promoter and operator sequence is apparent at the two higher salt concentrations and begins to disappear at the lower salt concentration . The physical interpretation of the melting experiments is compared to the results of theoretical predictions derived from the known sequence of the fragments.

Clin Chim Acta, 1981 Mar 19, 111(1), 27 - 32
Galactose and galactose-1-phosphate spot test for galactosemia screening; Misuma H et al.; A simple spot test to measure galactose and galactose-1-phosphate in blood-impregnated filter paper was studied as a screening test for galactosemia in the newborn . A 3-mm disc punched from blood-impregnated filter paper card was fixed with acetone-methanol and incubated in a reaction mixture containing galactose dehydrogenase and alkaline phosphatase . This reaction mixture was then spotted on DEAE-cellulose paper, dried, and observed under a UV-lamp . The minimum amount of galactose detected by this procedure was 2 mg/dl . Estimation of galactose and galactose-1-phosphate with this procedure correlated well with estimation by bacterial assay.

Nature, 1981 Mar 19, 290(5803), 264 - 7
Identification of mutations affecting replication control of plasmid Clo DF13; Stuitje AR et al.; The bacteriocinogenic plasmid Clo DF13, originally isolated from Escherichia cloacae, is stably maintained in Escherichia coli to the extent of about 10 copies per cell . Its replication resembles that of many other small, multicopy plasmids; plasmid-encoded protein is not required but plasmid-specific genetic information is involved in regulation of replication as both conditional and nonconditional copy-number mutants of Clo DF13, and transcomplementable copy-number mutants of plasmid Col E1 have been described . The sequences essential for replication of Col E1 (refs 16, 17) and Clo DF13 (refs 18, 19) have been identified within a region surrounding the replication origin . Initiation of Col E1 replication is preceded by transcription of the origin region, providing the RNA primer at the origin . However, transcription in the opposite direction results in a small transcript of approximately 100 nucleotides (RNA-100) for both Col E1 (refs 21, 22) and Clo DF13 (ref . 23) . Data suggest that Col E1 RNA-100 acts as a negative control element for the initiation of replication . We show here that single base transitions in the RNA-100 cistron of Clo DF13 can result in a nonconditional increase in plasmid copy-number . Also, sequence analysis has revealed that a specific base transition in a DNA region, apparently involved in both termination and initiation of transcription towards the replication origin, results in a thermosensitive plasmid copy-number.

Biochemistry, 1981 Mar 17, 20(6), 1476 - 81
Multiple isotope effect probes of glutamate decarboxylase; O'Leary MH et al.; The enzymatic decarboxylation of glutamic acid shows a carbon isotope effect k12/k13 = 1.018 at 37 degree C, pH 4.7 . In D2O under otherwise identical conditions, k12/k13 = 1.009 . Under the same conditions solvent isotope effects are Vmax H2O/Vmax D2O = 5.0 and (Vmax/Km)H2O/(Vmax/Km)D2O = 2.6 . With the assumption that the carbon isotope effect on the decarboxylation step is in the usual range (1.05--1.07), it is possible to derive relative rates and solvent isotope effects for all steps in the enzyme mechanism . Substrate binding in approximately 2-fold weaker in H2O than in D2O, probably because of the desolvation which accompanies binding of the substrate to the enzyme . A proton inventory analysis of the reaction shows that the Schiff base interchange has a large solvent isotope effect composed of relatively small contributions from at least four separate sites . A conformation change probably accompanies this step . The decarboxylation step shows a solvent isotope effect of approximately 2 . Schiff base interchange and decarboxylation are both partially rate determining . The pH dependence of the isotope effects indicates that the initial step in the reaction can occur by way of two different pathways.

Biochemistry, 1981 Mar 17, 20(6), 1640 - 5
Procedure for purification of Escherichia coli ribonucleic acid synthesis termination protein rho; Finger LR et al.; An improved purification procedure is described for the rho transcription termination factor of Escherichia coli . The method involves lysozyme--sodium deoxycholate lysis, Polymin P fractionation, and chromatography on phosphocellulose, poly(uridylic acid)--Sepharose, and AMP--agarose . The method yields up to 9 mg of electrophoretically pure protein from 200 200 g of E . coli MRE 600 . From quantitative amino acid analysis rho is calculated to have an E280nm (1%) of 3.7 +/- 0.3 . The purified rho has an ATPase specific activity of 32 nmol of Pi released min-1 microgram-1 when poly(cytidylic acid) is used as a cofactor, and it functions effectively in termination of T7 DNA transcription . A subunit molecular weight of 48 000 for rho was determined by phosphate-buffered sodium dodecyl sulfate--polyacrylamide gel electrophoresis . The amino acid composition and circular dichroism spectrum in the far-ultraviolet for rho are presented.

Biochemistry, 1981 Mar 17, 20(6), 1646 - 52
Cloning of rat alpha-fetoprotein 3'-terminal complementary deoxyribonucleic acid sequences and preparation of radioactively labeled hybridization probes from cloned deoxyribonucleic acid inserts; Liao WS et al.; Double-stranded complementary deoxyribonucleic acid (cDNA) was synthesized from rat yolk sac alpha-fetoprotein (AFP) mRNA, inserted into the PstI site of plasmid pBR322 by an oligo(deoxyguanylic acid).oligo(deoxycytidylic acid) joining technique, and cloned in Escherichia coli chi 1776 . A plasmid containing an inserted AFP double-stranded cDNA with a contiguous poly(adenylic acid) {poly(A)} segment was identified and subsequently employed in a new method for preparing AFP-specific hybridization probe . Following an initial digestion of the AFP plasmid with HindIII to create an open, recessed 3' end, lambda exonuclease III was employed to remove the DNA strand opposite the coding strand of the cDNA insert . Oligo(thymidylic acid) was then annealed to the poly(A) segment and employed as primer for E . coli DNA polymerase I to synthesize a 32P-labeled cDNA copy of the AFP coding strand . The single-stranded cDNA product was easily isolated by sedimentation through isokinetic alkaline sucrose gradients . Hybridization with this AFP-specific cDNA probe showed that the yolk sac contained a 6-fold greater concentration of AFP mRNA than that of the fetal liver . AFP mRNA was also found in the normal adult liver, but at a much lower level than in the fetal liver . The concentrations of AFP mRNA in Morris hepatomas 7777 and 8994, however, were significantly elevated to a 2- to 3-fold higher concentration that in the fetal liver.

Biochemistry, 1981 Mar 17, 20(6), 1612 - 7
Early steps in the path of nascent ribonucleic acid across the surface of ribonucleic acid polymerase, determined by photoaffinity labeling; DeRiemer LH et al.; The photoaffinity probes beta-(4-azidophenyl) adenosine 5'-diphosphate (N3PhppA) and beta-(4-azidophenyl) adenylyl-(3'--5')-uridine 5'-diphosphate (N3PhppApU) were used to determine the RNA polymerase subunit contacts made by the 5' ends of three nascent RNA chains . Ternary enzyme-poly{d(A-T)}.oligonucleotide complexes were prepared in which the nascent oligonucleotide contained a photoaffinity label at the 5' end and a 32P radiolabel only at the 3' end . The length of the RNA was fixed at two, three, or four nucleotides . Photolysis of the ternary complexes was followed by dissociation, polyacrylamide gel electrophoresis, autoradiography, and scintillation counting . With a dinucleotide probe, the enzyme subunits labeled were beta' (71%) and sigma (21%) . Photolysis of the ternary complex containing trinucleotide RNA also resulted in labeling of the beta' (64%) and sigma (35%) subunits . With a tetranucleotide, the beta' subunit was very heavily labeled (88%), and a small amount of labeling of the beta (7%) and sigma (4%) subunits was observed . The alpha subunit was not labeled with any of the probes . These results imply that a conformational change, possibly involving dissociation of the sigma subunit, occurs in the enzyme as the ribonucleotide is elongated from a tri- to a tetranucleotide.

Biochemistry, 1981 Mar 17, 20(6), 1606 - 12
Synthesis of mono- and dinucleotide photoaffinity probes of ribonucleic acid polymerase; DeRiemer LH et al.; The abortive initiation reaction of RNA polymerase has been used to prepare adenylyl-(3'--5')-uridine 5'-phosphate (pApU) in 74% yield from AMP and UTP . The reactive intermediate p-azidophenyl phosphorimidazolidate has been prepared by starting from p-nitrophenyl phosphate . Reaction of this compound with the terminal phosphates of adenosine 5'-phosphate and adenylyl-(3'--5')-uridine 5'-phosphate gives the corresponding beta-substituted 5'-diphosphates . These products are incorporated into the 5' (leading) end of RNA by RNA polymerase (Escherichia coli) and can be photoactivated at a specific stage of RNA elongation . The dinucleotide photoaffinity label beta-(4-azidophenyl) adenylyl-(3'--5')-uridine 5'-diphosphate stimulates RNA synthesis more strongly than adenylyl-(3'--5')-uridine.

Eur J Biochem, 1981 Mar 16, 115(1), 29 - 38
The catalytic mechanism of glutamyl-tRNA synthetase of Escherichia coli . A steady-state kinetic investigation; Kern D et al.; The sequence of substrate binding and of end-product dissociation at the steady state of the catalytic process of tRNAGlu aminoacylation by glutamyl-tRNA synthetase from Escherichia coli has been investigated using bisubstrate kinetics, dead-end and end-product inhibition studies . The nature of the kinetic patterns indicates that ATP and tRNAGlu bind randomly to the free enzyme, whereas glutamate binds only to the ternary enzyme . tRNAGlu . ATP complex . Binding of ATP to the enzyme hinders that of tRNAGlu and vice versa . After interconversion of the quaternary enzyme . substrates complex the end-products dissociate in the following order: PPi first, AMP second and Glu-tRNA last . In addition to its role as substrate and as effector with ATP for the binding of glutamate, tRNAGlu promotes the catalytically active enzyme state . Whereas at saturating tRNAGlu concentration the catalysis is rate-determining, this conformational change can be rate-determining at low tRNAGlu concentrations . The results are discussed in the light of the two-step aminoacylation pathway catalyzed by this synthetase.

Eur J Biochem, 1981 Mar 16, 115(1), 133 - 41
Studies on T4-head maturation . 2 . Substrate specificity of gene-49-controlled endonuclease; Kemper B et al.; The substrate specificity of 49+-enzyme was investigated in vitro . The enzyme showed a marked preference for rapidly sedimenting T4 DNA (greater than 1000 S) when helix-destabilizing proteins from Escherichia coli or phage T4 were added to the reaction . Regular replicative T4 DNA (200-S DNA) or denatured T4 DNA was not cleaved by the enzyme in the presence of these proteins but if they were omitted from the reaction both DNAs become good substrates for the enzyme . 200-S DNA was cleaved at its natural sites of single strandedness which occur at one-genome intervals . Gaps in T4 DNA which were constructed by treatment of a nicked DNA with exonuclease III were also cleaved by 49+-enzyme in the absence of helix-destabilizing proteins . Single-stranded T4 DNA was extensively degraded and up to 50% of the material was found to be acid-soluble in a limit digest . The degradation products were predominantly oligonucleotides of random size . No preference for a 5'-terminal nucleotide was observed in material from a limit digest with M13 DNA . Double-stranded DNA was nicked upon exposure to 49+-enzyme and double-strand breakage finally occurred by an accumulation of single-strand interruptions . No acid-soluble material was produced from native T4 DNA . The introduction of nicks in native DNA did not improve its properties as a substrate for the enzyme . Double-stranded DNA was about 100-fold less sensitive to the enzyme than single-stranded DNA.

C R Seances Acad Sci III, 1981 Mar 16, 292(11), 701 - 4
{Biosynthesis of colicin A : existence of pauses in the translation of messenger RNA}; Varenne S et al.; Evidence has been obtained for the existence of nascent polypeptide chains, that transiently accumulate as discrete size classes, due to discontinuities in the translation process of the colicin A mRNA . Existence of these discontinuities is independent of the hot cell of colicinogenic factor, and from the induction of synthesis by mitomycin C . The information that leads to the existence of discontinuities in the mRNA translation is contained in the plasmid Col A and, very likely, in the Col A gene itself.

Biochem J, 1981 Mar 15, 194(3), 989 - 98
The construction, identification and partial characterization of plasmids containing guinea-pig milk protein complementary DNA sequences; Craig RK et al.; A complementary DNA (cDNA) plasmid library has been constructed in the plasmid pAT153, using poly(A)-containing RNA isolated from the lactating guinea-pig mammary gland as the starting material . Double stranded cDNA was inserted into the EcoRI site of the plasmid using poly(dA . dT) tails, then transformed into Escherichia coli HB101 . From the resulting colonies we have selected and partially characterized plasmids containing cDNA copies of the mRNAs for casein A, casein B, casein C and alpha-lactalbumin . However, the proportion containing casein C cDNA was exceptionally low, and these contained at best 60% of the mRNA sequence.

Biochem J, 1981 Mar 15, 194(3), 783 - 7
Phospholipid requirements for the reconstitution of complex-III vesicles exhibiting controlled electron transport; Nelson BD et al.; Phospholipid requirements for the reconstitution of Complex-III vesicles exhibiting respiratory control (electron-transport control) were studied . Vesicles prepared from pure phosphatidylethanolamine gave maximal control ratios . Phosphatidylcholine alone did not support respiratory control, although these vesicles were capable of maintaining stable K+-diffusion gradients . Apparently Complex III cannot insert into a bilayer of phosphatidylcholine . Formation of mixed phosphatidylcholine/phosphatidylethanolamine (6:1, w/w) vesicles was sufficient, however, to allow Complex-III insertion and to restore respiratory control . Mixtures of acidic phospholipids with either phosphatidylethanolamine or phosphatidylcholine did not improve respiratory control over that obtained with pure phosphatidylethanolamine . Phosphatidylethanolamine from bovine heart mitochondria, soya beans or Escherichia coli was equally effective in reconstituting respiratory control, suggesting that the specificity is referable to the head group and not to the fatty-acid moiety.

Biochim Biophys Acta, 1981 Mar 13, 658(1), 54 - 63
Localization and partial purification of a neutral-active phospholipase A2 from BCG-induced rabbit alveolar macrophages; Lanni C et al.; The localization and partial purification of a Ca2+-dependent, membrane associated phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) from BCG-induced rabbit alveolar macrophages is described . Phospholipase A activity was determined using autoclaved Escherichia coli, the phospholipids of which were labelled in the 2-acyl position with {1-14C}oleate . Sonicated macrophages or granule preparations exhibited maximal phospholipase A2 activity at pH 7.0, with 5 mM Ca2+ . Activity was quantitatively recovered in the pellet after centrifugation of homogenates at 100 000 x g, indicating that the enzyme is membrane-associated . At least two populations of macrophage granules were separated that contained phospholipase A2 activity . Plasma membranes enriched 15-fold with respect to alkaline phosphodiesterase I were devoid of phospholipase activity . The enzyme was purified 1278-fold in a yield of 34%, was active over a broad pH range, and was extremely sensitive to low concentrations of Ca2+ . Mg2+ and Mn2+ would not substitute for Ca2+, 1 mM EDTA completely inhibited enzymatic activity . Absolute specificity for the 2-position was demonstrated using 1-{1-14C}stearyl-2-acyl 3-sn-glycerophosphorylethanolamine as substrate . Phospholipase A2 activity was inhibited by the nonsteroidal anti-inflammatory agent indomethacin; the amount of drug required for 50% inhibition was 5 . 10(-4) M.

Nature, 1981 Mar 12, 290(5802), 154 - 7
onc sequences (v-fes) of Snyder-Theilen feline sarcoma virus are derived from noncontiguous regions of a cat cellular gene (c-fes); Franchini G et al.; Type C sarcoma viruses are genetic recombinants containing portions of replication-competent helper viruses linked to sarcoma virus-specific sequences (generically designated onc genes) which are thought to be required for acute fibroblast transformation . The onc elements of different avian and mammalian sarcoma viral isolates are each homologous to subsets of cellular DNA sequences which have no well-defined role in normal cells . Because of the lack of significant homology between helper viral genes and cellular onc sequences, the recombinational mechanisms which facilitate the formation of sarcoma viral genomes remain unclear . In Moloney murine sarcoma virus, viral onc (or v-mos) and cellular onc (or c-mos) sequences exhibit complete and uninterrupted homology as determined by heteroduplex and restriction enzyme analyses of molecularly cloned DNA . By contrast, the cellular counterparts of the onc elements of Rous sarcoma virus (G . Cooper and R . Parker, personal communication), avian erythroblastosis virus (B . Vennstrom, personal communication), Abelson leukaemia virus (D . Baltimore, personal communication), Harvey sarcoma virus (E . Scolnick, personal communication) and simian sarcoma virus (R . Gallo, personal communication) are now known to contain intervening sequences which do not appear in the respective viral genomes . Here we report the use of the Southern blot technique to examine cat cellular DNA sequences (c-fes) homologous to the onc gene (v-fes) of Snyder-Theilen feline sarcoma virus (ST-FeSV) . We used cloned DNA 'probes' containing defined portions of the ST-FeSV genome to show that v-fes sequences originate from at least four noncontiguous sequences in cat cellular DNA, separated from each other by intervening sequences.

Nucleic Acids Res, 1981 Mar 11, 9(5), 1111 - 21
The nucleotide sequence at the 3'-end of Neurospora crassa 25S-rRNA and the location of a 5.8S-rRNA binding site; Kelly JM et al.; The sequence of 110 nucleotides adjacent to the 3'-end of Neurospora crassa 25S-rRNA has been derived by chemical sequencing methods . Sequences present between 40 and 85 nucleotides of the 3'-end were found to complement sequences at the 3'- and 5'-ends of 5.8S-rRNA . Interaction was shown to occur between 5.8S-rRNA and a specific 3'-terminal fragment of 85 nucleotides derived from 25S-rRNA . We have also demonstrated that the nucleotide sequence at the 3'-end of N . crassa 5.8S-rRNA (-UCAUUOH) is different from the published sequence (-UUUUOH) which was derived from rDNA.

J Biol Chem, 1981 Mar 10, 256(5), 2504 - 7
Processing in vivo of precursor maltose-binding protein in Escherichia coli occurs post-translationally as well as co-translationally; Josefsson LG et al.; The mechanism of synthesis of maltose-binding protein (Mr = 38,500), an exported periplasmic protein in Escherichia coli, was investigated in vivo . A precursor to maltose-binding protein (Mr - 41,000), which is identical to the precursor polypeptide synthesized in vitro in a cell-free system, can be detected in vivo indicating that it is not processed to mature size until the polypeptide chain is terminated . The population of incomplete, nascent polypeptide chains of maltose-binding protein was found to contain NH2 termini characteristic of both precursor and mature protein demonstrating that processing occurs co-translationally as well as post-translationally . However, the polypeptide containing the signal sequence must reach a critical size of Mr - 33,000 before any processing takes place.

J Biol Chem, 1981 Mar 10, 256(5), 2307 - 14
NADH inhibition and NAD activation of Escherichia coli lipoamide dehydrogenase catalyzing the NADH-lipoamide reaction; Wilkinson KD et al.; A unique form of inhibition by NADH and partial reversal by NAD+ has been demonstrated with Escherichia coli lipoamide dehydrogenase . Substrate inhibition by NADH is consistent with its reduction of the active two-electron reduced enzyme intermediate to the inactive four-electron reduced form . NAD+ partially overcomes this inhibition by mass action reversal of this reduction . NAD+ activation is only partial since the presence of both NAD+ and NADH forces the accumulation of two binary enzyme-pyridine nucleotide complexes . These are intermediates in the two-electron to four-electron reduction of the enzyme and thus are not on the catalytic pathway . NAD+ is also shown to inhibit by binding to the oxidized enzyme to give a dead-end complex . From the steady state rate equations, it is apparent that the degree of inhibition will depend on the oxidation-reduction potential for two- to four-electron reduction of the enzyme . Thus, the wide variation in the severity of NADH inhibition between the E . coli and pig heart enzymes is explained by quantitative differences in the basic lipoamide dehydrogenase mechanism . A possible physiological role for this type of inhibition as a mechanism of control in E . coli is discussed.

J Biol Chem, 1981 Mar 10, 256(5), 2324 - 8
Cloning of the yeast methionyl-tRNA synthetase gene; Fasiolo F et al.; A pool of random wild type yeast DNA fragments obtained by partial Sau IIIA restriction enzyme digestion and inserted in the Bam HI site of the hybrid yeast Escherichia coli plasmid ((pFL1) has been used to transform to prototrophy a methionyl-tRNA synthetase-impaired mutant requiring methionine . In the numerous prototroph strains recovered at least two independent clones have been obtained which show nonchromosomic inheritance character and an approximately 30-fold increase in methionyl-tRNA synthetase activity as compared to the wild type . Measurement of the Km for methionine in the transformed yeast cells indicates that the activity has been restored by decreasing the Km for methionine to the same level as found for the wild type methionyl-tRNA synthetase . Southern blotting experiments show that the yeast DNA's fragments inserted in the two independent plasmids share a common sequence which must correspond at least partly to the structural gene for methionyl-tRNA synthetase . They also suggest that the methionyl-tRNA synthetase gene is differently orientated in the two plasmids

J Biol Chem, 1981 Mar 10, 256(5), 2143 - 53
Sequence analysis of the DNA encoding the Eco RI endonuclease and methylase; Greene PJ et al.; The Eco RI endonuclease and methylase recognize the same hexanucleotide substrate sequence . We have determined the sequence of a fragment of DNA which encodes these enzymes using the chain-termination method of Sanger (Sanger, F., Nicklen, S., and Coulson, A . R . (1977) Proc . Natl . Acad . Sci . U . S . A . 74, 5463-5467) . The amino acid sequences of both enzymes were derived from the DNA sequence . The coding regions selected include the only open translational frames of sufficient length to accommodate the enzymes . They coincide with previously established gene boundaries and orientation . The predicted amino acid sequences correlate well with analyses of the purified protein . Comparison of the nucleotide and protein sequences reveals no homology between the endonuclease and methylase which might provide insight into the origin of the restriction-modification system or the mechanism of common substrate recognition . Based on secondary structure predictions, the two enzymes also have grossly different molecular architecture . The base composition of the sequence is 65% A + T, and the codon usage is significantly different from that observed in several Escherichia coli chromosomal genes . In some cases, frequently selected codons are recognized by minor tRNA species . A spontaneous mutation in the endonuclease gene was isolated . Serine replaces arginine at residue 187 . In crude extracts, Eco RI specific cleavage is approximately 0.3% wild type.

J Biol Chem, 1981 Mar 10, 256(5), 2140 - 2
Partial NH2- and cooh-terminal sequence analyses of Eco RI DNA restriction and modification enzymes; Rubin RA et al.; NH2- and COOH-terminal amino acid sequences of the Eco RI restriction and modification enzymes have been determined . The results allow localization of the coding regions within the DNA segment which controls activity of both enzymes . Processing of the endonuclease is limited to removal of NH2-terminal formylmethionine whereas, in the case of the methylase, formylMet-Ala is removed.

J Biol Chem, 1981 Mar 10, 256(5), 2109 - 12
Isolation of Escherichia coli mutants with elevated levels of membrane enzymes . A trans-acting mutation controlling diglyceride kinase; Raetz CR et al.; We have developed a rapid autoradiographic colony assay for detecting mutants with elevated levels of certain biosynthetic enzymes . Four Escherichia coli strains in which the specific activity of the membrane enzyme diglyceride kinase is increased 5-10-fold have been obtained with this approach . The mutant kinase has the same thermal denaturation profile and subcellular localization as the wild type . Five other membrane enzymes involved in phospholipid bilayer assembly are unaffected . In one of these strains (GK-1) the mutation (dgkR-1) responsible for the elevated kinase has been mapped at a new site near minute 92, while the previously identified structural gene (dgk) lies near minute 90 . When the structural gene for the kinase (dgk) is cloned on a multi-copy vector-like ColE1, the kinase can be overproduced 5-10-fold on the basis of gene dosage (Lightner, V . A., Larson, T . J., Tailleur, P., Kantor, G . D., Raetz, C . R . H., Bell, R . M., and Modrich, P . (1980) J . Biol . Chem . 255, 9413-9420) . Introduction of such hybrid plasmids into a mutant harboring dgkR-1 leads to a multiplicative (rather than additive) effect, resulting in specific activities of diglyceride kinase that are 35-75-fold higher than normal . These results show that dgkR-1 is a trans-acting mutation and suggest the existence of novel regulatory proteins (or metabolites) that direct the expression of certain membrane enzymes.

J Biol Chem, 1981 Mar 10, 256(5), 2098 - 101
Mössbauer spectroscopic studies of Escherichia coli sulfite reductase . Evidence for coupling between the siroheme and Fe4S4 cluster prosthetic groups; Christner JA et al.; Escherichia coli NADPH-sulfite reductase is a complex hemoflavoprotein with an alpha 8 beta 4 subunit structure . The beta-subunits each contain one siroheme and a tetranuclear iron-sulfur center (Fe4S4) . Isolated beta-monomers can catalyze the 6-electron reduction of sulfite to sulfide . We have studied the beta-monomers with Mossbauer and EPR spectroscopy . The data show conclusively that the siroheme and the Fe4S4 cluster are strongly exchange-coupled . This is proven by the observations that (a) the two chromophores share a single electronic spin and (b) the addition of 1 electron to oxidized sulfite reductase changes the environments of 5 iron atoms . Spin-sharing is demonstrated in oxidized and 2-electron-reduced sulfite reductase and strongly implicated in 1-electron-reduced material . Thus, sulfite reductase provides the first example of an active site where a heme and an iron-sulfur cluster are closely linked as a functional unit, probably via a common bridging ligand.

J Biol Chem, 1981 Mar 10, 256(5), 2252 - 7
Stringent control of RNA synthesis in the absence of guanosine 5'-diphosphate-3'-diphosphate; Pao CC et al.; Severe curtailment of RNA synthesis and widespread readjustment of cellular activities, together with an increase of guanosine 5'-diphosphate-3'-diphosphate (ppGpp) have been demonstrated in Escherichia coli cells starved for amino acid or energy . The rates of growth and RNA synthesis are reduced by shifting the growth temperature from 40 degrees C to 20 degrees C . The intracellular pool of ppGpp diminishes under such conditions . Furthermore, the accumulation of ppGpp normally attainable by either amino acid- or energy-limitation can be totally blocked by a downshift of temperature imposed prior to the starvation . However, the synthesis of stable RNA is still stringently restricted under these conditions . Two other nucleotides were also effected . The intracellular level of phantom spot (Gallant, J., Shell, L., and Bittner, R . (1976) Cell 7, 75-84) decreased upon temperature fall . Guanosine 5'-diphosphate-3'-monophosphate, whose concentrations have been linked to stringent response and stable RNA synthesis, did not change by the simple temperature downshift, but increased following amino acid limitation even when a downshift of temperature was imposed before the starvation . These results suggest that ppGpp is not always needed for inhibition of stable RNA synthesis during stringent response, and that a compound such as guanosine 5'-diphosphate-3-monophosphate may be involved in the stringent regulation of stable RNA synthesis, at least under the temperature downshift conditions.

Nature, 1981 Mar 5, 290(5801), 26 - 9
Homology and concerted evolution at the alpha 1 and alpha 2 loci of human alpha-globin; Liebhaber SA et al.; The identical structure of two racially distinct alpha 1-globin alleles and the high degree of homology between the alpha 1- and alpha 2-globin loci indicate that mechanisms exist for suppression of allelic polymorphisms and for exchange of genetic information within the alpha-globin gene complex.

Nature, 1981 Mar 5, 290(5801), 29 - 33
Homologous pairing can occur before DNA strand separation in general genetic recombination; West SC et al.; In the presence of ATP and Mg2+, purified Escherichia coli recA protein promotes the formation of joint molecules between closed circular duplex DNA and homologous circular single-stranded DNA carrying a short annealed fragment . The presence of this fragment is essential for pairing between molecules . In similar conditions recA protein is unable to act as a helicase and does not cause strand separation of the fragment from the single-stranded circle . Thus, homologous pairing between DNA molecules can take place without prior unwinding of a free end.

Nature, 1981 Mar 5, 290(5801), 65 - 7
Dual expression of lambda genes in the MOPC-315 plasmacytoma; Bothwell AL et al.; The expression of two kappa light chain immunoglobulins in the MPC-11 mouse myeloma is well established, the two protein products being apparently from RNA transcripts derived from separate, rearranged kappa alleles in the MPC-11 genome . Recently, the characterization of kappa-related RNAs and protein products in several lambda-producing myelomas has indicated that multiple expression of light chain RNAs is a common event in myelomas and other cells of the B-lymphocyte lineage . These studies suggest that, although many light chain alleles may function to make RNA and protein in a given B-lymphocytic cell, only one complete, functional light chain is generally translated from the RNAs present in a single cell . The myeloma, MOPC-315, synthesizes and secretes an antibody which has an alpha heavy chain and a lambda II light chain . The DNA of MOPC-315 either has no kappa genes or has only a fragment of one, but it certainly has no kappa genes in the embryonic configuration . Rearrangement of its lambda genes has been observed but the exact nature of the rearrangement is not known . Because initial observations suggested that an immunoglobulin-related protein other than alpha and lambda II was present in MOPC-315 cells, we undertook to derive molecular cDNA clones from the MRNA in MOPC-315 tumour cells . Analysis of the clones has now identified two lambda chain mRNA species: a normal lambda II chain mRNA and another which directs the synthesis of a deleted form of a lambda I protein . The nucleotide sequence of the deleted lambda I mRNA shows that it resulted from a joining of the sequence encoding amino acid 31 of the variable region directly to the constant region coding sequence.

Biochemistry, 1981 Mar 3, 20(5), 1245 - 52
Unambiguous determination of the stereochemistry of nucleotidyl transfer catalyzed by DNA polymerase I from Escherichia coli; Brody RS et al.; Nucleotidyl transfer catalyzed by DNA polymerase I from Escherichia coli proceeds with greater than 97% inversion of configuration at P alpha of the alpha-phosphorothioate analogue of dATP . This is shown by experiments in which dAMPS,18O2 is stereospecifically phosphorylated to (Sp)-dATP alpha S, alpha 18O2, which is then copolymerized with dTTP by DNA polymerase . The product of the polymerization is degraded to dAMPS,18O by methods that do not affect the configuration of the phosphorothioate . After the dAMPS,18O is stereospecifically phosphorylated, the resulting (Sp)-dATP alpha S, alpha 18O is copolymerized as before with dTTP . The 18O is found in the displaced pyrophosphate by mass spectral analysis and so must have been in the pyrophosphate bridge of (Sp)-dATP alpha S, alpha 18O . Since this 18O was originally non-bridging in (Sp)-dATP alpha S, alpha 18O2, the phosphorothioate configuration must have been inverted in the polymerization reaction . This confirms the determination of P . M . J . Burgers & F . Eckstein {(1979) J . Biol . Chem . 254, 6889-6893}, who used kinetic correlations based on the stereoselectivity of snake venom phosphodiesterase to deduce the stereochemistry of this reaction.

Biochemistry, 1981 Mar 3, 20(5), 1061 - 4
Physical studies on the ribosomal protein S2 from the Escherichia coli 30S; Georgalis Y et al.; The protein S2 has been isolated from the 30S subunit of Escherichia coli A19 ribosomes {Littlechild, J., & Malcolm, A.L . (1978) Biochemistry 17, 3363-3369} . This salt-extracted protein is soluble and does not aggregate at salt concentrations of 0.3-0.4 M as used under reconstitution conditions . This differs from the S2 protein extracted by the acetic acid and urea method . The molecular weight from sedimentation equilibrium was found to be 29 200, and the protein was found to have a S0(20,w) value of 2.36S . The apparent specific volume at 20 degrees C was 0.726 mL.g(-1), and the D0(20,2) was 7.37 x 10(-7) cm(2)s(-1) . The value for intrinsic viscosity was found to be 6.42 mL.g(-1) . An axial ratio of (5-6):1 for a prolate ellipsoid of revolution was estimated by using these parameters . The circular dichroism and proton magnetic resonance studies show that protein S2 has both substantial amounts of alpha helix and beta-pleated sheet in solution and appears as a "folded" protein and not a random coil structure.

Biochemistry, 1981 Mar 3, 20(5), 1133 - 9
Ribonucleic acid synthesis termination protein rho function: effects of conditions that destabilize ribonucleic acid secondary structure; Richardson JP et al.; The dependence fo rate of adenosine 5'-triphosphate (ATP) hydrolysis catalyzed by ribonucleic acid (RNA) synthesis termination protein rho from Escherichia coli with T7 RNA as cofactor is used to probe the nature of the interaction between rho and RNA . In general, reaction conditions that destabilize the secondary structure of the RNA enhance its cofactor activity . This is indicated by the effects of MgCl2 concentration, spermidine, temperature, dimethyl sulfoxide, and pretreatment of the RNA with formaldehyde . These results suggest that a functional interaction between rho and RNA depends either on the presence of a sufficiently large single-stranded region in the RNA or on the ability of rho to unwind double helices in the RNA . It is also shown that changes in reaction conditions that increase RNA secondary structure and decrease the rho protein adenosine triphosphate phosphohydrolase (rhoATPase) activity with isolated T7 RNA also decrease the stringency of rho action in RNA synthesis termination . On the other hand, monovalent salts decrease rhoATPase activity with isolated T7 RNA and binding of rho to T7 RNA independently of the MgCl2 concentration and thus the relative stability of the RNA secondary structure.

Carbohydr Res, 1981 Mar 2, 89(2), 211 - 20
Synthesis of alpha- and beta-glycosides containing spin labels, as probes for studies of carbohydrate-protein interaction; Plessas NR et al.; Nitroxide spin-labeled alpha-D-glycopyranosides were synthesized in good yield and in a highly stereoselective manner by reaction of per-O-benzyl-alpha-D-glycopyranosyl bromides with 2,2,6,6-tetramethyl-4-piperidinol under the bromide ion-catalyzed conditions devised by Lemieux et al . After hydrogenolysis, the deblocked intermediates were oxidized to give the desired, spin-labeled alpha-D-glycopyranosides . Nitroxide spin-labeled beta-D-glycopyranosides, as well as a beta-maltoside, were synthesized by standard methods . The synthesis is also described of 2-amino-2-deoxy-D-glucose and -D-galactose derivatives having a spin label at C-2, and of the spin-labeled compound 1-{4-(beta-D-galactopyranosyloxy)phenyl}-3-(2,2,6,6-tetramethylpiperidin-1-oxyl -4-yl)-2-thiourea.

Hokkaido Igaku Zasshi, 1981 Mar, 56(2), 217 - 31
{Studies on the mobilities of granulocytes and the chemotactic factor production by human mononuclear cells (author's transl)}; Nakayama M; Various types of granulocyte mobilities and chemotactic factor production mononuclear cells were studied by the modified agarose plate methods . Three types of granulocyte mobilities, namely random mobility, chemotaxis, and chemokinesis, are easily measured by the agarose plate method . Enhanced granulocyte random mobility by chemotactic factor was observed even when no gradient of chemotactic factor existed . This chemokinetic response decreased in cord blood leukocytes which was known to have the decreased chemotactic response compared to normal adults control . It was speculated that not only chemotaxis but also chemokinesis play an important role in inflammatory reactions in vivo . The degree of disturbance of chemotactic response of cord blood leukocytes which was observed in agarose plate method, was not so apparent as that which was reported by using Boyden chamber method . It seemed that the deformability of granulocytes contributed on the results of chemotaxis in Boyden chamber method . When zymosan activated serum was used as chemoattractant, the chemotactic response of cord blood leukocytes was more impaired than when E . coli derived factor was used . The same results were obrained when chemotactic response of granulocytes from pediatric patients with malignancies were examined . It was concluded that various chemotactic factors should be used as the chemoattractant when the chemotaxis of granulocytes in any disease was examined . One of the chemotactic factors by granulocytes is cell derived chemotactic factor for granulocytes . Production of chemotactic factor for granulocytes by human mononuclear cells was studied in agarose plate method . It was designated as the chemotactic factor for granulocytes, CFG . CFG was produced by human mononuclear cells when they stimulated with LPS or anti beta 2 microglobulin . CFG producing cells were glass adherent and carbonyl iron phagocyting cells . So it was suggested that CFG producing cells were monocytes . This CFG could not attract monocytes, though zymosan activated serum could attract both monocytes and granulocytes . Moreover, anti human C5 serum could not abrogate the chemotactic activity of CFG . It seemed that CFG was different from complement derived chemotactic factor, C5a . Furthermore, specific antiserum against human C3 or IgG could not also abrogate the chemotactic activity of CFG.

Cytometry, 1981 Mar, 1(5), 342 - 5
Evaluation of selected aerosol-control measures on flow sorters; Merrill JT; Flow sorters produce microdroplets as part of their normal operation, and if these microdroplets escape into the room, they are potentially hazardous . We have tested several aerosol-control measures on a commercial flow sorter . To accomplish this, T-4 phages were introduced into the sorter's liquid jet through the sample injection tube, and culture plates containing lawns of T-4-sensitive Escherichia coli bacteria were exposed around the sorter for each operational configuration . After the exposure, the plates were incubated and then scored for plaques . A single phage-containing microdroplet landing on a plate was sufficient to cause a plaque of lysed bacteria to form . The number of plaques was thus an indicator of how much aerosol was released for each configuration . Aerosols were controlled most effectively by catching the central, undeflected stream in a vacuum-exhausted tube; this technique, coupled with the manufacturer's vacuum-exhaustion of the air around the sorting location, produced no plaques . Several failure modes were tested, including having the central stream hit the outside of the catch-tube or deflection plates, and loss the manufacturer's vacuum-exhaustion system . Many flow sorters allow the underflected stream to splash into a flask or beaker, an approach that produces the most plaques if instrument-failure modes are excluded . The simple addition of a catch-tube removed this major contributor to the aerosol production.

J Med Chem, 1981 Mar, 24(3), 304 - 8
Puromycin analogues . Effect of aryl-substituted puromycin analogues on the ribosomal peptidyltransferase reaction; Lee H et al.; A series of ortho- and para-substituted L-phenylalanylpuromycin analogues were synthesized and evaluated as substrates for the peptidyltransferase reaction of Escherichia coli ribosomes . Kinetic results reveal that substitution of the p-methoxy group of the puromycin molecule alters the peptidyltransferase activity of the molecule with the following decreasing order of substrate efficiencies: p-NH2 greater than p-NHCOCH3 greater than p-NO2 = p-NHCO(CH2)2CH3 greater than p-NHCOCH2Br . However, the inability of the ribosome to tolerate a nitro group at the ortho position of the phenylalanine ring precluded the use of the photosensitive puromycin analogue, 2-nitro-4-azidophenylalanylpuromycin aminonucleoside (7a), as a photoaffinity label for the peptidyltransferase site.

Mech Ageing Dev, 1981 Mar, 15(3), 279 - 95
Quantitative measures of aging in the nematode Caenorhabditis elegans . I . Population and longitudinal studies of two behavioral parameters; Bolanowski MA et al.; As a first step in the quantitative characterization of senescence in the nematode Caenorhabditis elegans, we have studied movement wave frequency, defecation frequency, and whole-body water efflux as a function of age . Populations of C . elegans, strain N2, were cultured monoxenically on E . coli lawns at 20 degrees C . The median lifespan in such populations was approximately 12 days . Population mean movement wave frequency declined linearly with age (slope = -4.66 waves/minute per day) . The decline in population mean defecation frequency (defecations per minute) was multiphasic, consisting of (1) a rapid decline (slope = -0.233 defecations/minute per day) from day 3 to day 6, (2) no apparent trend from day 6 to day 9, and (3) a gradual decline (slope = -0.089 defecations/minute per day) from 9 to day 14 . Animals alive on or after day 15 were not observed to defecate . In longitudinal studies, individual animals exhibited linear declines in movement wave frequency and multiphasic declines in defecation frequency . For future population studies, the age-dependent declines in movement and defecation frequency appear sufficiently large and reproducible to a multiparametric description of senescence in C . elegans . One physiological parameter, 3H2O efflux, was found to be age-independent and to consist of two first-order rates . The half-times of the slow and fast efflux rates were approximately 15 and approximately 2.1 minutes, respectively . The two half-times and the fractions of 3H2O exhibiting the two half-times were invariant with age.

J Gen Microbiol, 1981 Mar, 123(Pt 1), 193 - 6
Incompatibility of citrate utilization plasmids isolated from Escherichia coli; Ishiguro N et al.; The 57 conjugative Cit plasmids isolated from 72 citrate-utilizing (Cit+) Escherichia coli strains from various sources were classified into four groups on the basis of their genetic properties . Escherichia coli K13 strains carrying these Cit plasmids could utilize cis-aconitate or tricarballylate, in addition to citrate.

Biochem J, 1981 Mar 1, 193(3), 861 - 7
Prosthetic groups of the NADH-dependent nitrite reductase from Escherichia coli K12; Jackson RH et al.; A substantially improved purification of Escherichia coli NADH-dependent nitrite reductase was obtained by purifying it in presence of 1 mM-NO2- and 10 microM-FAD . The enzyme was obtained in 20% yield with a maximum specific activity of 1.04 kat . kg-1: more than 95% of this sample subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis migrated as a single band of protein . This highly active enzyme contained one non-covalently bound FAD molecule, and, probably, 5 Fe atoms and 4 acid-labile S atoms per subunit . No FMN, covalently bound flavin or Mo was detected . The spectrum of the enzyme shows absorption maxima at 386, 455, 530 and about 575 nm with a shoulder at 480--490 nm . The Soret-band/alpha-band absorbance ratio is about 4:1 . These spectral features are characteristic of sirohaem, apart from the maximum at 455nm, which is attributed to flavin . The enzyme also catalyses the NADH-dependent reduction of horse heart cytochrome c, 2,6-dichlorophenol-indophenol and K3Fe(CN)6 . The presence of sirohaem in E . coli nitrite reductase explains the apparent identity of the cysG and nirB gene of E . coli and inability of hemA mutants to reduce nitrite.

J Biochem (Tokyo), 1981 Mar, 89(3), 855 - 9
Studies on oxygen-insensitive nitrofuran reductase in Escherichia coli B/r; Tatsumi K et al.; Oxygen-insensitive nitrofuran reductase in Escherichia coli B/r was clearly resolved by DEAE-cellulose column chromatography into two components, one NADPH-linked, and the other both NADPH- and NADH-linked . It is known that the strain requires resistance to nitrofurazone in two mutational steps . It is known that the the first step mutants had no NADPH-linked component and the second step ones had neither this component nor the NAD(P)-H-linked one . The NADPH- and NADH-linked activities of the latter component were similarly inactivated by heat or urea treatment . In addition, it was found that these activities were significantly inhibited by dicoumarol, an NAD(P)H dehydrogenase inhibitor, to similar extents . These results suggest that the activities of the NAD(P)H-linked component originate from a single enzyme . On the other hand, the NADPH-linked component was less sensitive to heat, urea and dicoumarol.

Mol Biol (Mosk), 1981 Mar-Apr, 15(2), 403 - 7
{Entry of double-and single-stranded linear DNA in Ca2+-treated Escherichia coli cells}; Sabel'nikov AG et al.; Hydroxylapatite chromatography technique was used to investigate the conformational changes undergone by exogenous linear DNA during the uptake by Ca2+-treated EScherichia coli cells . Both single- (heat denatured) and double-stranded DNAs were examined . While native DNA preserved its double-strandedness during the uptake, the heat denatured (single-stranded) DNA was rapidly converted to the double-stranded form . This process was energy-dependent and is supposed to be dependent on gene recA product.

Mol Biol (Mosk), 1981 Mar-Apr, 15(2), 316 - 22
{Non-coordinated transcription of RNA polymerase beta,beta'-polypeptide genes and adjacent ribosomal protein genes in Escherichia coli cells}; Lideman LF et al.; When E . coli protein synthesis was blocked by chloramphenicol (100 micrograms/ml) or by essential amino acid deprivation, the transcription rates of rplKAJL genes (the ones for L11, L4, L10 and L7/L12 ribosomal proteins) and adjacent rpoBC genes (genes for RNA polymerase beta- and beta'-polypeptides) have been non-coordinately changed . The level of the gene transcription rate was obtained from RNA--DNA hybridization assays with E . coli pulse-labelled RNA and pJC703 or pJC720 plasmid DNA . The transcription of ribosomal protein genes has been found to be uncoupled with translation and controlled by the allelic state of relA gene . Conversely, the effective transcription of proBC gene was relA independent and coupled with translation of the mRNA . Chloramphenicol-induced transcription polarity within rplKAJL-rpoBC chromosome region can be suppressed by 10 micrograms/ml rifampicin.

Mol Biol (Mosk), 1981 Mar-Apr, 15(2), 298 - 309
{Membrane proteins in Escherichia coli: effect of orthophosphate and mutation on regulatory genes of secreted alkaline phosphatase}; Tsfasman IM et al.; To elucidate the regulatory function of membranes during the biosynthesis of secreted alkaline phosphatase in E . coli, the protein compositions of membranes and periplasm in the E . coli wild strain and two mutants on regulatory phoS and phoRc genes of alkaline phosphatase have been studied during repressed and derepressed biosyntheses of enzymes . The biosynthesis of one of the membrane proteins is regulated by exogenous orthophosphate in parallel with alkaline phosphatase and a number of other periplasmic proteins (three of them have not been described in literature before) . This regulation is determined by phoS gene common to alkaline phosphatase . The mutants of regulatory genes of alkaline phosphatase have a decreased content of membrane proteins or completely lack them . Mutations in the phoRc gene result in the loss of alkaline phosphatase and two membrane proteins and do not affect the biosynthesis of periplasmic proteins . Mutation in phoS gene diminishes the content of the other two membrane proteins, one of which is assumed to be the product of gene phoS--phosphate binding protein . The possible membrane localization of products of regulatory phoRc and phoS genes of alkaline phosphatase and participation of membrane proteins in the enzyme biosynthesis and its regulation are discussed.

J Clin Microbiol, 1981 Mar, 13(3), 606 - 8
Unique temperature-sensitive nutritional requirements of bacteremic Escherichia coli isolates; Welch WD et al.; Of 50 strains of Escherichia coli isolated from blood cultures of bacteremic patients, 14 (28%) were unable to grow on minimal medium at 42 degrees C, compared to only 2 of 50 nonbacteremic strains . In 7 of the 14 bacteremic strains, growth at 42 degrees C was restored by adding nicotinic acid . These unique temperature-sensitive auxotrophic patterns warrant evaluation as a marker correlating with clinical pathogenicity in E . coli.

Ann Microbiol (Paris), 1981 Mar-Apr, 132(2), 141 - 8
{Adherence of enteropathogenic "Escherichia coli" to mononuclear cells from human blood (author's transl)}; Avril JL et al.; Two toxigenic strains of Escherichia coli possessing a colonization factor antigen (CFA) respectively CFA/I and CFA/II, were studied for adherence to mononuclear cells from human blood of 12 subjects . Significant differences were observed between these two strains in their ability to adhere, using as control strains their laboratory-passed derivatives which no longer possess CFA . Differences were observed in bacterial adhesion ability of mononuclear cells from different subjects but, in the whole, CFA+ bacteria were able to adhere to few cells from each subject only . These results are in opposition to the hypothesis that it is conceivable that histocompatibility antigens expressed on cells play a role in the attachment of bacteria as the first step in an infection.

Proc Natl Acad Sci U S A, 1981 Mar, 78(3), 1786 - 90
Evidence that rnmB is the operator of the Escherichia coli recA gene; Volkert MR et al.; rnmB281 leads to high constitutive levels of recA protein such that no increase after UV-inducing treatment occurs . The mutation maps in or near the portion of recA corresponding to the NH2-terminal end of the protein . Examination of the recA proteins from rnmB+ recA-/rnmB281 recA+ heterozygotes suggests that both rnmB alleles are cis-acting and codominant . This is the behavior expected from alleles of a regulatory gene such as an operator or promoter of recA . The possibility that rnmB mutations occur in the promoter of recA . The possibility that rnmB mutations occur in the promoter of recA, though not ruled out, seems unlikely based on the structure of the regulatory region of recA . This suggests that rnmB mutations are operator constitutive mutations of the recA gene and should be called recAo mutations . The UV-irradiation responses of recAo+ and recAo281 strains, both recA+, are compared and inferences are drawn about the roles of large amounts of recA protein in producing the responses.

Proc Natl Acad Sci U S A, 1981 Mar, 78(3), 1717 - 21
A mutation downstream from the signal peptidase cleavage site affects cleavage but not membrane insertion of phage coat protein; Russel M et al.; Morphogenesis of filamentous phage includes synthesis of the phage major coat protein in precursor form, its insertion into the host cell plasma membrane, its cleavage to the mature form of the protein, and its assembly there into virions . The M13 mutant am8H1R6 encodes a coat protein in which leucine replaces glutamic acid as residue 2 of the mature protein {Boeke, J . D., Russel, M . & Model, P . (1980) J . Mol . Biol . 144, 103-116} . The coat protein precursor produced by this variant is a poor substrate for the Escherichia coli signal peptidase both in vivo and in vitro . This pre-coat protein, which is eventually processed and assembled into viable phage particles, is associated with the membrane fraction of the infected cell . We conclude that the domain recognized by the signal peptidase extends beyond the signal peptide itself . Furthermore, membrane association and signal peptide cleavage can be separated temporally under conditions that permit membrane insertion, cleavage, and phage assembly.

Proc Natl Acad Sci U S A, 1981 Mar, 78(3), 1567 - 71
Tritium exchange on transfer RNA: slowly exchanging protons sensitive to a change in the dihydrouridine stem; Ramstein J et al.; Measurements of tritium exchange on tRNA were made for periods from 0.5 min to 8 hr after separation from labeled solvent . The exchange curve was analysed in terms of three kinetic classes of exchanging protons with half-lives of 5 hr (12 protons), 0.54 hr (37 protons) and about 3.5 min (58 protons) at 0 degrees C in 0.14 M K+/10 mM Mg2+ . The behaviour under varying ionic conditions of protons in the slowest exchange class and of some protons in the intermediate class suggests that they are dependent on the tertiary structure of the molecule . Moreover, in the same range of exchange times characteristic of these latter protons, about 9 more protons were observed in the case of a mutant form of tRNA Trp, the UGA-suppressor species, than in the wild-type tRNATrp . These two species differ only in base 24 in the dihydrouridine stem . This dynamic difference between the wild-type and suppressor species may be related to a functionally important difference in coupling between the conformation of the molecule and interactions at the anticodon.

Proc Natl Acad Sci U S A, 1981 Mar, 78(3), 1391 - 5
Human insulin prepared by recombinant DNA techniques and native human insulin interact identically with insulin receptors; Keefer LM et al.; Human insulin synthesized from A and B chains separately produced in Escherichia coli from cloned synthetic genes (prepared by the Eli Lilly Research Laboratories, Indianapolis, IN) was characterized by examining its interaction with human cultured lymphocytes, human circulating erythrocytes in vitro, and isolated rat fat cells . The binding behavior of the biosynthetic insulin with human cells was indistinguishable from that of native human or porcine insulins, with respect to affinity, association and dissociation kinetics, negative cooperativity, and the down-regulation of lymphocyte receptors . Similarly, the biosynthetic insulin was as potent as the native insulins in stimulating lipogenesis in isolated rat fat cells . We also examined the receptor binding characteristics of 125I-labeled human and porcine insulins monoiodinated solely at Tyr-A14, which were obtained by means of high-performance liquid chromatography of the iodination reaction mixture (this material was prepared by B . Frank, Eli Lilly Research Laboratories) . In all aspects studied, the pure {TyrA14-125I}iodoinsulins were superior as tracers to the monoiodoinsulin purified by the more conventional method of gel filtration.

J Virol, 1981 Mar, 37(3), 1087 - 9
Procoat, the precursor of M13 coat protein, inserts post-translationally into the membrane of cells infected by wild-type virus; Date T et al.; In growing cells infected by wild-type coliphage M13, the synthesis of procoat protein is completed before it inserts into the plasma membrane ane is converted to coat protein.

J Infect Dis, 1981 Mar, 143(3), 440 - 6
Diarrhea due to Escherichia coli strain RDEC-1 in the rabbit: the peyer's patch as the initial site of attachment and colonization; Cantey JR et al.; A strain of Escherichia coli (RDEC-1) has been described that in rabbits colonizes the intestine; adheres to mucosal epithelial cells of the ileum, cecum, and colon; and causes diarrhea by an unknown mechanism . This study attempted to determine the location of the bacteria in the rabbit intestine during the unexplained six- to seven-day interval between bacterial inoculation and onset of diarrhea . Specimens of ileum, cecum, colon, ileal Peyer's patches, sacculus rotundus, and appendix from control rabbits and from rabbits killed at intervals after inoculation with RDEC-1 bacteria were examined by light and direct fluorescence microscopy . Bacteria in large numbers attach to the tips of the Peyer's patch lymphoid follicles by 24 hr, but they did not attached to ileal, cecal, or colonic mucosa until three days after inoculation . The lag time between inoculation and onset of diarrhea was probably due to the need for the bacteria first to attach to and then colonize the Peyer's patch lymphoid follicles . The intestinal mucosa was probably colonized by bacteria shed from the Peyer's patches.

Cell, 1981 Mar, 23(3), 681 - 7
Termination of DNA replication in vitro at a sequence-specific replication terminus; Germino J et al.; The replication terminus of the drug resistance factor R6K has been cloned into the plasmid vectors pBR313 and pBR322 . When the exogenously added DNA is replicated in vitro using cell extracts prepared from Escherichia coli, the plasmid replication terminus temporarily arrests the progression of the unidirectionally moving replication fork at or near the cloned terminator sequence . When the relative location of the terminator sequence is changed with respect to the replication origin, the point of arrest of the replication fork shifts correspondingly to the new location of the terminator . Termination of replication takes place in vitro regardless of whether the cell extracts used in the in vitro reaction are prepared from E . coli with a resident terminus sequence containing plasmid . From these observations we conclude that the termination of replication in vitro is identical or very similar to that observed in vivo, membrane association is not necessary for the activity of the replication terminus and the terminus sequence does not code for a transacting factor necessary for termination of replication . Therefore, any transacting factor which may be needed for the termination of replication must be coded by the host chromosome.

Can J Biochem, 1981 Mar, 59(3), 158 - 64
Cloning and expression of fumarate reductase gene of Escherichia coli; Lohmeier E et al.; Mutants of Escherichia coli deficient in fumarate reductase activity and therefore unable to grow anaerobically with fumarate as an electron acceptor have been isolated . By F+-mediated conjugation and complementation with the mutant host, two E . coli: Col E1 recombinant DNA plasmids have been identified from the Clarke and Carbon Colony Bank which carry the structural genes for fumarate reductase . Bacteria harboring either of these plasmids express about ten times the normal level of fumarate reductase . Enzyme purified from the two sources, plasmid-carrying and plasmidless E . coli, have identical physical and kinetic properties indicating that both the 69 000 and 25 000 dalton polypeptides are amplified . Regulation of plasmid-encoded enzyme, like the chromosomally encoded enzyme, is dependent upon the presence of fumarate and anaerobiosis.

Biochimie, 1981 Mar, 63(3), 235 - 40
Effect of oligonucleotide AGAGGAGGU on protein synthesis in vitro; Trudel M et al.; The oligonucleotide AGAGGAGGU, complementary to the 3' end of 16S RNA has been shown to inhibit 70S initiation complex formation on E . coli ribosomes (Taniguchi, T . and Weissmann, C., 1978 . Nature, 275, 770-772) . We have prepared this nonanucleotide in larger quantities by a combination of DEAE cellulose-urea chromatography and reverse phase (RPC 5) chromatography . The inhibitory effect of AGAGGAGGU on initiation complex formation has been confirmed . Furthermore, when added to a complete system for in vitro protein synthesis, the translation of Q beta RNA was inhibited by the nonanucleotide . No selectivity was observed in the inhibition of the coat protein and replicase protein synthesis . When both Q beta RNA and pAUG were present, some stimulation of pAUG binding to 70S ribosomes was observed on addition of AGAGGAGGU, as previously reported (Taniguchi and Weissmann, ibid) . No effect was observed in the absence of Q beta RNA . This observation is discussed.

Appl Environ Microbiol, 1981 Mar, 41(3), 670 - 4
Solar radiation induces sublethal injury in Escherichia coli in seawater; Kapuscinski RB et al.; Sublethal injury was noted in Escherichia coli after cells were exposed to solar radiation . Injury was detected by differential plate counts between complete and minimal media that were observed with sunlight-exposed cells but not with cells kept in the dark . Since addition of catalase or pyruvate to minimal medium overcame or repaired this injury, the catalase system appeared to be the site of injury.

Diabetes Care, 1981 Mar-Apr, 4(2), 220 - 2
Internalization of 125I-insulin by IM-9 cultured human lymphocytes: a comparison between A14-monoiodo-pork and biosynthetic human insulin; Carpentier JL et al.; Human insulin synthesized from A- and B-chains separately produced in Escherichia coli from cloned genes was characterized by examining its interaction biochemically and morphologically with IM-9 cultured human lymphocytes . Biosynthetic human insulin (BHI) behaves similarly to pork insulin with respect both to its binding properties and to the rate and magnitude at which it is internalized by the cells.

Diabetes Care, 1981 Mar-Apr, 4(2), 193 - 5
Comparison of the biologic activity of biosynthetic human insulin and natural pork insulin in juvenile-onset diabetic subjects assessed by the glucose controlled insulin infusion system; Klier M et al.; To assess the biologic activity of biosynthetic human insulin (BHI) synthesized by Escherichia coli, six insulin-dependent juvenile-onset diabetic subjects were studied with BHI and natural pork insulin, by means of the glucose controlled insulin infusion system (GCIIS) . First, after an overnight normalization of blood glucose levels, the 24-h insulin requirement was determined while the patients were consuming a diet of 30 kcal/kg . Then, the amount of glucose necessary to maintain normal blood glucose levels during a 5-h intravenous infusion of BHI and pork insulin, respectively, was assessed . Both studies demonstrate that in the insulin-dependent diabetic subject, BHI is at least as effective as natural pork insulin and may, therefore, be useful for the treatment of insulin-dependent diabetes mellitus.

Am J Physiol, 1981 Mar, 240(3), H368 - 74
An observed pressor effect of the cerebellum during endotoxin shock in the dog; Janssen HF et al.; The current study investigates the possibility that the cerebellum may be involved in the regulation of mean arterial pressure (MAP) during endotoxin shock in the anesthetized dog . The effect of intravenously injected Escherichia coli endotoxin on MAP in the cerebellectomized dog was compared to that observed in the intact animal . Even though removal of the cerebellum did not significantly affect MAP in a control group, the cerebellectomized animal (unlike the intact animal) was unable to recover from the initial hypotension typically seen immediately following an intravenous endotoxin injection . Previous investigators have demonstrated that stimulation of fastigial nuclei in the cerebellum increases MAP via beta-adrenergic activation of the renin-angiotensin system . Captopril (SQ 14,225, an angiotensin I-converting enzyme inhibitor) was used to determine whether this system could be responsible for the maintenance of MAP during endotoxin shock . When continuously infused into the intact dog given endotoxin, captopril suppressed MAP to a level similar to that of the cerebellectomized group . A similar response pattern to endotoxin was also observed in animals with a spinal transection at C2.

Am J Physiol, 1981 Mar, 240(3), H348 - 53
Pulmonary injury and prostaglandin production during endotoxemia in conscious sheep; Demling RH et al.; Prostaglandins F2 alpha, E2, and I2 (as 6-keto-PGF1 alpha) and TxA2 (as TxB2) were measured by radioimmunoassay in plasma and lymph from 12 conscious sheep with chronic lung lymph fistulas given Escherichia coli endotoxin (2-10 micrograms/kg) and followed for 24 h . Endotoxin produced a two-phase pulmonary injury . Phase 1 was characterized by transient severe pulmonary hypertension and increased lymph flow rate (QL) . Plasma and lymph PGF2 alpha concentrations increased from base-line values of 0.13 +/- 0.08 and 0.30 +/- 0.10 ng/ml to 0.96 +/- 0.37 and 2.8 +/- 0.80 ng/ml, respectively . Values for TxB2 increased from 0.7 +/- 0.1 to 5.5 +/- 1.1 ng/ml in lymph and to 3.2 +/- 0.6 in plasma . Plasma PGI2 increased from 0.48 +/- 0.29 to 4.97 +/- 1.21 ng/ml and lymph PGI2 from 1.80 +/- 0.73 to 14.19 +/- 2.79 ng/ml . Phase 2 was characterized by moderately elevated pulmonary vascular pressures and a maintained high flow rate of protein-rich lymph . Lung lymph and plasma PGF2 alpha concentrations returned to base line . Lymph PGI2 decreased significantly to 5.23 +/- 2.47 ng/ml, whereas plasma PGI2 decreased to 2.70 +/- 1.07 ng/ml . We conclude that prostaglandins, particularly PGF2 alpha and prostacyclin, are released from the lung after endotoxemia and appear in lung lymph as sensitive indicators of pulmonary microvascular injury . Prostanoid production appears to temporally correspond with changes in the pulmonary microcirculation.

J Bacteriol, 1981 Mar, 145(3), 1456 - 8
Repair of thermal damage to the Escherichia coli nucleoid; Pellon JR et al.; The folded chromosome or nucleoid of Escherichia coli was analyzed by low-speed sedimentation in neutral sucrose gradients after heat treatment (30 min at 50 degrees C) and subsequent incubation of cells at 37 degrees C for various times . Heat treatment resulted in in vivo association of the nucleoids with cellular protein and in an increase in sedimentation coefficient . During incubation at 37 degrees C, a fraction of the nucleoids, from heated cells, because dissociated from cellular protein and regained their characteristic sedimentation coefficients . The percentage of nucleoids which returned to their control sedimentation position in the sucrose gradients corresponded to the percentage of cells able to repair thermal damage as assayed by enumeration on agar plates.

J Bacteriol, 1981 Mar, 145(3), 1445 - 7
Regulation of ribosomal protein synthesis in an Escherichia coli mutant missing ribosomal protein L1; Jinks-Robertson S et al.; In an Escherichia coli B strain missing ribosomal protein L1, the synthesis rate of L11 is 50% greater than that of other ribosomal proteins . This finding is in agreement with the previous conclusion that L1 regulates synthesis of itself and L11 and indicates that this regulation is important for maintaining the balanced synthesis of ribosomal proteins under physiological conditions.

J Bacteriol, 1981 Mar, 145(3), 1432 - 5
Complementation tests between alkaline phosphatase-constitutive mutants (phoS and phoT) of Escherichia coli; Levitz R et al.; Complementation tests between phoS and phoT mutations showed that they belong to the same cistron . Homozygosis of a heterozygotic partial diploid resulted from allelic transfer from the chromosome to the F' episome.

J Bacteriol, 1981 Mar, 145(3), 1425 - 7
Two succinic semialdehyde dehydrogenases are induced when Escherichia coli K-12 Is grown on gamma-aminobutyrate; Donnelly MI et al.; When Escherichia coli K-12 was grown on gamma-aminobutyrate, a second succinic semialdehyde dehydrogenase, dependent upon oxidized nicotinamide adenine dinucleotide or oxidized nicotinamide adenine dinucleotide phosphate and distinct from that induced by gamma-aminobutyrate, was gratuitously induced by succinic semialdehyde.

J Bacteriol, 1981 Mar, 145(3), 1413 - 6
Deoxyribonucleic acid replication in permeable and fully viable Escherichia coli cells; Boye E et al.; Escherichia coli cells made permeable with a hypotonic tris(hydroxymethyl)aminomethane buffer utilized exogenous deoxyribonucleoside triphosphates to perform semiconservative replication . The rate of replication was the same as in cells made permeable with toluene or sucrose.

J Bacteriol, 1981 Mar, 145(3), 1397 - 403
Tris(hydroxymethyl)aminomethane buffer modification of Escherichia coli outer membrane permeability; Irvin RT et al.; The effect of tris(hydroxymethyl)aminomethane (Tris) buffer on outer membrane permeability was examined in a smooth strain (D280) and in a heptose-deficient lipopolysaccharide strain (F515) of Escherichia coli O8 . Tris buffer (pH 8.00) was found to increase outer membrane permeability on the basis of an increased Vo of whole-cell alkaline phosphatase activity and on the basis of sensitivity to lysozyme and altered localization pattern of alkaline phosphatase . The Tris buffer-mediated increase in outer membrane permeability was found to be dependent upon the extent of exposure to and concentration of the Tris buffer . The Tris buffer effects were demonstrated not to be due to allosteric activation of cell-associated alkaline phosphatase and were specific for Tris buffer . Exposure of cells to Tris resulted in the release of a limited amount of cell envelope component . Investigators utilizing Tris buffer are cautioned that Tris is not physiologically inert and that it may interact with the system under investigation.

J Bacteriol, 1981 Mar, 145(3), 1386 - 96
Citrate-tris(hydroxymethyl)aminomethane-mediated release of outer membrane sections from the cell envelope of a deep-rough (heptose-deficient lipopolysaccharide) strain of Escherichia coli O8; Irvin RT et al.; A heptose-deficient lipopolysaccharide strain of Escherichia coli O8, strain F515, was found to release portions of its outer membrane when cells were exposed to 10 mM citrate buffer (pH 2.75) for 30 min and subsequently exposed to 100 mM tris(hydroxymethyl)aminomethane buffer (pH 8.00) . The outer membrane component release was found to be composed of protein, lipopolysaccharide, phospholipid (cardiolipin, phosphatidylethanolamine, and phosphatidylglycerol), and alkaline phosphatase . The outer membrane component was released from the cell envelope in the absence of cell lysis, as no glucose-6-phosphate dehydrogenase activity or succinic dehydrogenase activity was detected . Morphologically, the outer membrane component appeared to consist of laminar fragments and vesicles which had an associated alkaline phosphatase activity.

J Bacteriol, 1981 Mar, 145(3), 1351 - 8
Genetic and biochemical characterization of periplasmic-leaky mutants of Escherichia coli K-12; Lazzaroni JC et al.; Periplasmic-leaky mutants of Escherichia coli K-12 were isolated after nitrosoguanidine-induced mutagenesis . They released periplasmic enzymes into the extracellular medium . Excretion of alkaline phosphatase, which started immediately in the early exponential phase of growth, could reach up to 90% of the total enzyme production in the stationary phase . Leaky mutants were sensitive to ethylenediaminetetraacetic acid, cholic acid, and the antibiotics rifampin, chloramphenicol, mitomycin C, and ampicillin . Furthermore, they were resistant to colicin E1 and partially resistant to phage TuLa . Their genetic characterization showed that the lky mutations mapped between the suc and gal markers, near or in the tolPAB locus . A biochemical analysis of cell envelope components showed that periplasmic-leaky mutants contained reduced amounts of major outer membrane protein OmpF and increased amounts of a 16,000-dalton outer membrane protein.

J Bacteriol, 1981 Mar, 145(3), 1334 - 41
Rifampin resistance mutations that alter the efficiency of transcription termination at the tryptophan operon attenuator; Yanofsky C et al.; Rifampin-resistant mutants of Escherichia coli were isolated which had altered patterns of resistance or sensitivity to the inhibitory compounds 5-methyltryptophan and 5-methylanthranilate . The levels of tryptophan (trp) operon polypeptides in different rifampin-resistant mutants were elevated or reduced, in a manner consistent with their sensitivity to the two analogs . Complementation tests established that the mutations were in rpoB, the structural gene for the beta subunit of ribonucleic acid polymerase . Introduction of these rpoB mutations into mutant strains which terminate transcription abnormally at the trp operon attenuator established that the rpoB mutations alter trp operon expression by increasing or decreasing transcription termination at the attenuator . The rpoB mutations affected transcription termination at the attenuator only in strains which were able to form what is thought to be a ribonucleic acid termination structure . These findings suggest that alteration of the beta subunit of ribonucleic acid polymerase directly or indirectly affects ribonucleic acid polymerase's recognition of the transcription termination signal at the trp operon attenuator.

J Bacteriol, 1981 Mar, 145(3), 1317 - 24
Resolution of distinct selenium-containing formate dehydrogenases from Escherichia coli; Cox JC et al.; Formate dehydrogenase, a component activity of two alternative electron transport pathways in anaerobic Escherichia coli, has been resolved as two distinguishable enzymes . One, which was induced with nitrate reductase as a component of the formate-nitrate reductase pathway, utilized phenazine methosulfate (PMS) in preference to benzyl viologen (BV) as an artificial electron acceptor and appeared to be exclusively membrane-bound . A second formate dehydrogenase, which was induced as a component of the formate hydrogenlyase pathway, appeared to exist both as a membrane-bound form and as a cytoplasmic enzyme; the cytoplasmic activity was resolved completely from the PMS-linked activity on a sucrose gradient . When E . coli was grown in the presence of 75Se-selenite, a 110,000-dalton selenopeptide, previously shown to be a component of the PMS-linked enzyme, was induced and repressed with this activity . In contrast, an 80,000-dalton selenopeptide was induced and repressed with the BV-linked activity and exhibited a distribution similar to the BV-linked formate dehydrogenase in cell fractions and in sucrose gradients . The results indicate that the two formate dehydrogenases are distinguishable on the basis of their artificial electron acceptor specificity, their cellular localization, and the size of their respective selenoprotein components.

J Bacteriol, 1981 Mar, 145(3), 1239 - 48
Genetic and physiological characterization of a spontaneous mutant of Escherichia coli B/r with aberrant control of deoxyribonucleic acid replication; Choung KK et al.; Strain TJK16, a low-thymine-requiring thyA deoB derivative of Escherichia coli B/r A, was found to have an increased initiation mass due to a mutation in a gene affecting the control of initiation of deoxyribonucleic acid replication . In contrast to temperature-sensitive initiation mutants, initiation in TJK16 was not temperature sensitive . By phage P1 transduction, it was found that the mutation lies within a small region of the chromosome between dnaA and gyrB; this region includes dnaN and recF . Coumermycin-resistant derivatives of B/r and TJK16 had the same initiation mass as their coumermycin-sensitive parents, and TJK16 had the same sensitivity to coumermycin as the B/r parent, suggesting that the initiation mutation is not in gyrB.

J Bacteriol, 1981 Mar, 145(3), 1232 - 8
Growth rate-dependent control of chromosome replication initiation in Escherichia coli; Churchward G et al.; The initiation mass, defined as cell mass per origin of deoxyribonucleic acid replication (optical density units at 460 nm of culture/origins per milliliter of culture), reflects the intracellular concentration or activity of a hypothetical factor that controls initiation of chromosome replication in bacteria . In Escherichia coli B/r, the initiation mass was found to increase about twofold with increasing growth rate between 0.6 and 1.6 doublings per h; at higher growth rates it remained essentially constant (measured up to 2.4 doublings per h) . A low-thymine-requiring (thyA deoB) derivative of E . coli B/r, strain TJK16, was found to have a 60 to 80% greater initiation mass than B/r which was independent of the replication velocity and not related to the thyA and deoB mutations . It is suggested that TJK16 had acquired, during its isolation, a mutation in a gene affecting the initiation of deoxyribonucleic acid replication . The initiation age was not altered by this mutation, but other parameters, including deoxyribonucleic acid concentration and cell size, were changed in comparison with the B/r parent, as expected from theoretical considerations.

J Bacteriol, 1981 Mar, 145(3), 1196 - 208
Change in intracellular pH of Escherichia coli mediates the chemotactic response to certain attractants and repellents; Repaske DR et al.; Changes in the membrane potential, pH gradient, proton motive force, and intracellular pH of Escherichia coli were followed during the chemotactic responses to a variety of potentially membrane-active compounds . Lipophilic weak acids, decreases in extracellular pH, and nigericin each caused a repellent response . Lipophilic weak bases, increases in extracellular pH, and valinomycin in the presence of K+ each caused an attractant response . Changes in membrane potential, pH gradient, and proton motive force did not correlate with the behavioral responses to these treatments, but changes in intracellular pH did correlate . Furthermore, the strength of the response to a weak acid was correlated with the magnitude of the change of the intracellular pH, and many compounds which could alter the intracellular pH were found to be chemotactically active . Apparently these attractants and repellents are not detected by specific chemoreceptors but rather are detected via the ability of cells to sense and respond to changes in intracellular pH . The pathway of sensory transduction which proceeds through methyl-accepting chemotaxis protein I was found to be involved in the response to a change in intracellular pH.

Am J Obstet Gynecol, 1981 Mar 1, 139(5), 535 - 9
The role of prostaglandins in endotoxemia and comparisons in response in the nonpregnant, maternal, and fetal models . II . Alterations in prostaglandin physiology in the nonpregnant, pregnant, and fetal experimental animal; O'Brien WF et al.; The present study evaluates the response of the nonpregnant ewe, the pregnant ewe, and the fetal lamb with regard to prostaglandin physiology after E . coli endotoxin administration . Baseline levels are reported and response to a stimulator (E . coli endotoxin) and an inhibitor (indomethacin) of prostaglandin synthesis is evaluated . The results reveal that the prostaglandin response to stimulation or inhibition relies upon the baseline levels and that acute alterations of prostaglandin ratios may occur with inhibition of prostaglandin synthesis . The fetus reacts differently than the pregnant and nonpregnant animal and appears to be resistant to the effects of endotoxin, which may be secondary to the elevated baseline fetal prostaglandin levels.

Arch Pathol Lab Med, 1981 Mar, 105(3), 160 - 3
The alternate complement pathway . A possible role in a patient with focal glomerular sclerosis; Stapleton FB et al.; A boy had focal segmental glomerular sclerosis after the resolution of an unusual transient functional defect in activation of the alternate complement pathway . Prior to 1 year of age, the patient suffered repeated serious bacterial infections that were associated with an inability to opsonize Escherichia coli ON 2 in vitro . Serum levels of complement components were normal . Shortly after resolution of the complement defect, nephrotic syndrome developed . Properdin and C3 were identified in sclerotic glomeruli, which suggests that the ability to activate the alternate complement pathway played a role in the pathogenesis of glomerular sclerosis.

J Neurochem, 1981 Mar, 36(3), 793 - 7
Highly sensitive immunoassays for three forms of rat brain enolase; Kato K et al.; Highly sensitive enzyme immunoassay systems for three forms (alpha alpha, alpha gamma, and gamma gamma) of rat brain enolase were prepared by use of specific antisera to two distinct subunits (alpha and gamma) of the isozymes and beta-D-galactosidase from Escherichia coli as label . Less than fmol-levels of the homologous dimer forms (alpha alpha and gamma gamma) could be determined with the corresponding antibody F(ab')2-bound solid-phase and the antibody Fab'-beta-D-galactosidase complex . The hybrid form (alpha gamma) could also be assayed specifically by use of the antibody to one subunit as solid-phase, and the antibody to another subunit as labelled complex with a minimum detectable sensitivity of 1 fmol.

J Bacteriol, 1981 Mar, 145(3), 1150 - 3
Threonine as a carbon source for Escherichia coli; Chan TT et al.; Threonine can be used aerobically as the sole source of carbon and energy by mutants of Escherichia coli K-12 . The pathway used involves the conversion of threonine via threonine dehydrogenase to aminoketobutyric acid, which is further metabolized by aminoketobutyric acid ligase, forming acetyl coenzyme A and glycine . A strain devoid of serine transhydroxymethylase uses this pathway and excretes glycine as a waste product . Aminoketobutyric acid ligase activity was demonstrated after passage of crude extracts through Sephadex G100.

Gene, 1981 Mar, 13(2), 153 - 61
Construction of plasmids that produce phage P22 repressor; Poteete AR et al.; In a series of plasmid constructions, the c2 (repressor) gene of phage P22 was cloned in a multicopy plasmid and expressed at increasing level . The final result of these constructions is a plasmid that maintains a level of approx . 200 times as much repressor as is found in a lysogen . A series of increasingly virulent phage mutants was isolated by plating sequentially on host cells with increasing levels of repressor . The methods used in the constructions should be applicable to obtaining elevated expression of cloned genes in other systems.

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