Biochemistry, 1982 Jun 22, 21(13), 3128 - 36 Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: role of divalent metals in the dimerization and phosphorylation of enzyme I; Hoving H et al.; The function of divalent metal ions (Mg2+ and Mn2+) in the dimerization and phosphorylation of enzyme I has been studied . Only a dimeric form of the enzyme can be phosphorylated {Misset, O., Brouwer, M., & Robillard, G . T . (1980) Biochemistry 19, 883--890; Hoving, H., Lolkema, J . S., & Robillard, G . T . (1981) Biochemistry 20, 87--93} . Kinetic studies of phosphoryl-group exchange between phosphoenolpyruvate and pyruvate and measurements of initial enzyme I phosphorylation rates revealed that a divalent metal ion must be bound to the enzyme to render the dimer active . Mn2+ binding experiments by means of electron paramagnetic resonance showed binding of at least one Mn2+ per unphosphorylated dimer with a binding constant comparable to the activation constant found in the kinetic studies and a 10-fold tighter binding of only one Mn2+ per phosphorylated dimer . Gel filtration experiments provided evidence that divalent metals produce about a 10-fold stabilization of the dimers, in addition to their effect on the specific dimer activity . The stability of the dimer was also strongly dependent on salts such as LiCl, NaCl, KCl, and a series of tetraalkylammonium chlorides . The relative effects of these salts suggest that hydrophobic interactions possibly play a significant role in enzyme I dimerization. Biochemistry, 1982 Jun 22, 21(13), 3077 - 82 Fluorescence studies on the location of L7/L12 relative to L10 in the 50S ribosome of Escherichia coli; Zantema A et al.; The localization of the protein L7/L12 relative to protein L10 in the Escherichia coli ribosome was studied by fluorescence energy transfer . N-{7-(Dimethylamino)-4-methylcoumarinyl}maleimide, coupled to Cys-70 of L10, served as a donor for fluorescein which was attached to Lys-51 or to the N terminus of L7/L12 . The binding of the fluorescein-L7/L12 dimers to a strong and a weak binding site in 50S ribosomes could be distinguished . Therefore, it was possible to measure the distances between Cys-70 of L10 and Lys-51 and the N terminus of each L7/L12 dimer . For L7/L12 in the strong binding site, these two distances are both about 43 A, and for L7/L12 in the weak binding site, both are about 56 A. Biochemistry, 1982 Jun 22, 21(13), 3064 - 9 Cloning, purification, and characterization of beta-cystathionase from Escherichia coli; Dwivedi CM et al.; The Clarke-Carbon clone bank of hybrid plasmid Escherichia coli DNA has been screened for plasmids able to complement an E . coli strain deficient for the production of beta-cystathionase . Clone 4-14 had the ability to complement a deletion mutation at this locus and expressed higher levels of beta-cystathionase than the wild-type strain . The transfer of the plasmid carried by this clone to a strain that constitutively expresses all the enzymes of the methionine biosynthetic pathway results in 100-fold overproduction of beta-cystathionase as compared to wild-type levels . With use of this strain, an efficient three-step purification scheme is described that gives 90% pure enzyme in 54% yield with a specific activity of 215 IU/mg . This enzyme is characterized as to molecular weight (280 000), number of subunits (six), pyridoxal phosphate binding (5.7 mol of pyridoxal phosphate bound/mol of protein, Km of 0.005 mM), amino acid composition, substrate specificity, and kinetic properties. Biochemistry, 1982 Jun 22, 21(13), 3069 - 76 Preparation and characterization of fluorescent 50S ribosomes . Specific labeling of ribosomal proteins L7/L12 and L10 of Escherichia coli; Zantema A et al.; So that the topographic and dynamic properties of the L7/L12--L10 complex in the 50S ribosome of Escherichia coli could be studied, methods and reagents were developed in order to introduce fluorescent groups at specific positions of these proteins . In the case of L7/L12, this was done by attaching an aldehyde group to Lys-51 of the protein by using 4-(4-formylphenoxy)butyrimidate or by converting the amino terminus of L12 into an aldehyde group by periodate oxidation . Subsequent reaction of the aldehyde groups with newly developed hydrazine derivatives of fluorescein and coumarin resulted in specifically labeled L7/L12 derivatives . L10 was modified at the single cysteine residue with N-{7-(dimethylamino)-4-methylcoumarinyl}maleimide . The fluorescent proteins L10 and L7/L12 could be reconstituted into 50S ribosomes . The resulting specifically labeled 50S ribosomes show 25--100% activity in elongation factor G dependent GTPase as well as in polyphenylalanine synthesis . The fluorescent properties of the labeled 50S ribosomes show that these fluorescent derivatives are suitable for energy transfer studies. Biochim Biophys Acta, 1982 Jun 16, 716(3), 316 - 23 Biosynthesis of glycosaminoglycans in peritoneal macrophages from the guinea pig; Takasu Y et al.; The majority of glycosaminoglycans synthesized in peritoneal macrophages from the guinea pig in vitro were secreted into culture medium . The secreted glycosaminoglycans were reduced in size with alkali treatment indicating that the glycosaminoglycans existed in the form of proteoglycans . After the glycosaminoglycans were digested with chondroitinase AC and ABC, the high voltage paper electrophoretic analysis and the descending paper chromatographic analysis indicated the presence of a considerable amount of unsaturated disulfated disaccharides . Based on the enzymatic assay with chondro-4- and 6-sulfatase, the positions of sulfation in the disulfated disaccharide have been identified as the 4- and 6-position of N-acetylgalactosamine . Moreover, the results of the ion-exchange chromatography and the chondroitinase AC and ABC digestion indicated that delta Di-diSE derived from dermatan sulfate . This suggests that peritoneal macrophages are capable of synthesizing oversulfated proteodermatan sulfate as main component . The proportion of synthesized oversulfated dermatan sulfate to the total glycosaminoglycans was independent of the incubation time, and the distribution of oversulfated dermatan sulfate in cell and incubation medium also did not change . After exposure of macrophages to Escherichia coli for 15 min, the incorporation of {35S}sulfate and {3H}glucosamine into the glycosaminoglycans was increased by about 40% with no significant change in the proportion of synthesized oversulfated dermatan sulfate, but the release of glycosaminoglycans into the culture medium remains essentially unchanged . The difference of the existence of oversulfated dermatan sulfate is not yet understood. Biochim Biophys Acta, 1982 Jun 16, 716(3), 420 - 3 Transition temperatures in the sensitivity of Escherichia coli B/r to lysozyme; Scheie P; Lysozyme attacked Escherichia coli B/r in the absence of EDTA or imposed osmotic shocks when the cells were rapidly cooled below specific temperatures . Cells subjected to lysozyme while being cooled to below 20 degrees C began to lose ability to subsequently form colonies . This sensitivity increased with decreasing temperatures and almost all cells cooled to 0 degrees C were affected . Slightly hypertonic solutions did not improve survival . Cells cooled first to as low as 5 degrees C and then subjected to lysozyme while cool did not lose their ability to form colonies subsequent to rewarming . However, 70% of the cells cooled first to 0 degrees C and subjected to lysozyme lost their colony-forming ability . Cell lysis also began when treated near 5 degrees C, but even when treated at 0 degrees C about 50% of the cells maintained their rod shape in the presence of lysozyme . These results are discussed in terms of a possible phase transition in a portion of the cell envelope and/or a transient osmotic swelling as a results of metabolic pumps failing at the low temperatures. Eur J Biochem, 1982 Jun 15, 125(1), 151 - 6 Methods for the detection of single-strand breaks in DNA under neutral conditions and their application in a study on the mechanism of repair of N-methylated purines in mouse cells; Wintersberger E; Considering enzymatic activities found in bacteria and in animal cells, there are two possible mechanisms for repair of N-methylated purines produced by methylating agents such as the mutagen and carcinogen N-methyl-N'-nitro-N-nitrosoguanidine . Both mechanisms involve first an enzymatic removal of the methylated bases by glycosylases . The resulting apurinic sites could then be repaired by (a) direct insertion of the correct bases purine insertases or (b) opening of the polynucleotide chain by apurinic endonuclease followed by repair synthesis . As the methods commonly used to detect lesions induced by methylating agents involve alkali, it was thus far not possible to decide between the above possibilities because apurinic sites are by themselves alkali labile . In this paper I describe two methods which avoid alkali and therefore allow the clarification of some aspects of the repair reaction . One of these methods makes use of 95% formamide at 40 degrees C in place of alkali to denature DNA with pre-existing single-strand breaks, the other measures the capacity of DNA scissions with free 3'-OH groups to act as primer for Escherichia coli DNA polymerase I . Results obtained with both methods make it unlikely that purine insertases play a major role in the repair of apurinic sites . Kinetics of production and repair of single-strand breaks, produced in 3T6 mouse fibroblasts by incubation with N-methyl-N'-nitro-N-nitrosoguanidine, were also examined using the methods of alkaline elution and alkaline sucrose gradient centrifugation. Biochem J, 1982 Jun 15, 204(3), 681 - 8 Alternative-substrate inhibition and the kinetic mechanism of the beta-galactoside/proton symport of Escherichia coli; Page MG et al.; The effects of competing alternative substrates on the rate of uptake by galactoside/proton symport were investigated . These experiments produced a decrease in apparent maximum velocity with increased alternative-substrate concentration that cannot be accounted for by a simple ordered mechanism . This, together with non-linearities in the variation of the apparent kinetic constants with alternative-substrate concentration, can be accounted for by a random mechanism for galactoside and proton binding. Eur J Biochem, 1982 Jun 15, 125(1), 49 - 55 Identification by affinity labeling of potential sites in 23-S rRNA interacting with the 3' end of tRNA; Leitner M et al.; A derivative of tRNAPhe carrying at its 3' end a photolyzable group was chemically synthesized by coupling p-azidobenzoylglycylhydrazide to periodate-oxidized tRNA . The reaction converts the 3'-terminal ribofuranoside residue into a six-membered ring . The binding of p-azidobenzoylglycylhydrazide-tRNA to 70-S ribosomes resembles the binding of Phe-tRNA in its requirement for poly(U) and Mg2+ dependence . Irradiation at wavelengths greater than 300 nm of complexes of p-azidobenzoylglycylhydrazide-tRNA and 70-S ribosomes results in the covalent binding of approximately 20% of the reversibly bound tRNA to the ribosomes . The labeling occurs predominantly at a single site in 23-S rRNA within the sequence included in the 18-S fragment . To identify the modified sequence, an RNase A digest of 23-S rRNA was fractionated on a hydrophobic matrix (Porapak Q) and adsorbed and non-adsorbed fractions were compared by fingerprint analysis . A relatively intense spot was observed in the expected region in the fingerprint of the adsorbed fraction . The presence of an unusual nucleotide in the sequence of this oligonucleotide was demonstrated by a mobility-shift analysis of a partial nuclease P1 digest . The sequence derived is G*-A-A-G-C (the asterisk denoting the site of modification) . Sequences at six positions in the 23-S rRNA are compatible with the sequence of the oligonucleotide determined . Of the six positions five are located within nucleotides 1467-1887 . This region of approximately 400 nucleotides is likely to include the sequence interacting with the 3' end of tRNA and possibly forming part of the peptidyl-transferase center. Nucleic Acids Res, 1982 Jun 11, 10(11), 3531 - 9 Nucleotide sequence of a region of the mitochondrial genome of Aspergillus nidulans including the gene for ATPase subunit 6; Grisi E et al.; A 1500 bp fragment of the Aspergillus nidulans mitochondrial genome contains genes for arginine and asparagine tRNAs, an unassigned reading frame, and the structural gene for ATPase subunit 6 . The tRNA genes possess 66% nucleotide homology and possibly originated by a relatively recent duplication event . The unassigned reading frame displays a low level of homology with the human URF A6L . The predicted amino acid sequence of the A-nidulans ATPase subunit 6 gene is 40% homologous to the yeast polypeptide and includes the short, highly conserved regions also present in the equivalent subunits from other mitochondrial systems and from Escherichia coli. J Biol Chem, 1982 Jun 10, 257(11), 6481 - 7 The receptor for colicin E3 . Isolation and some properties; Imajoh S et al.; A simple method for the isolation of the colicin E3 receptor is described . The receptor was extracted with lithium diiodesalicylate/urea/Triton X-100/EDTA from the cell envelope of Escherichia coli . The combination of affinity chromatography on immobilized protein A of colicin E3 with the efficient extraction led to the preparation of the receptor in a pure form, with a good yield (70%) and high activity . The purified receptor was a pure protein with a molecular weight of 60,000 . The receptor protein was prepared in micelles of Triton X-100, and formed an equimolar complex with each E colicin (E1, E2, and E3), respectively . However, in the absence of the micelles, the receptor protein was easily inactivated. J Biol Chem, 1982 Jun 10, 257(11), 6446 - 51 Assignment of the functional loci in the colicin E1 molecule by characterization of its proteolytic fragments; Ohno-Iwashita Y et al.; We obtained two fragments of colicin E1 by limited proteolysis with thermolysin . Both of the fragments have an ionophore-like activity . One of the fragments, Th1 (Mr = 18,000), induces an increase in membrane ion permeability comparable with that with whole colicin E1 when assayed with liposomes prepared from soybean phospholipids . This suggests that Th1 is an active fragment of colicin E1 . The size of this fragment is about one-third of that of colicin E1 and it was found that the fragment is derived from the COOH-terminal part of colicin E1 . A larger fragment, Th2 (Mr = 39,000), was also obtained on thermolysin treatment of colicin E1, which comprises the two-thirds of colicin E1 from the COOH terminus and has a receptor-binding activity in addition to the same ionophore-like activity of Th1 . Th2 has the ability to kill osmotic shocked cells but not intact cells, suggesting that the NH2-terminal portion of colicin E1, which was lost in Th2, is responsible for the transport of the colicin molecule through the outer membrane . Thus, the regions which possess or correlate to the ionophore-like, receptor-binding, and transmembrane activities of colicin E1 were assigned to the COOH-terminal, central, and NH2-terminal parts of the molecule, respectively. J Biol Chem, 1982 Jun 10, 257(11), 6023 - 7 Identification of an active site cysteine residue in Escherichia coli pyruvate oxidase; Koland JG et al.; The cysteine-directed reagent N-ethylmaleimide rapidly and completely inactivates pyruvate oxidase . This inactivation is correlated with the reaction of one cysteine residue per enzyme monomer . In the presence of the cofactor, thiamin pyrophosphate, the enzyme is not inhibited by N-ethylmaleimide . Furthermore, the N-ethylmaleimide-inactivated enzyme exhibits a very low affinity for the cofactor as determined by a fluorescence quenching technique . The presence of a reactive cysteine residue at the thiamin pyrophosphate binding site is therefore indicated . Although N-ethylmaleimide completely inactivates the enzyme, a second sulfhydryl reagent methylmethanethiosulfonate is only partially inhibitory . It is shown that methylmethanethiosulfonate and N-ethylmaleimide react with the same cysteine residue . Thus, the N-ethylmaleimide-sensitive residue is probably not directly involved in catalysis. J Biol Chem, 1982 Jun 10, 257(11), 6544 - 50 Isolation and characterization of an Escherichia coli mutant lacking tRNA-guanine transglycosylase . Function and biosynthesis of queuosine in tRNA; Noguchi S et al.; An E . coli mutant that lacks tRNA-guanine transglycosylase was isolated by random screening from a collection of Escherichia coli mutants obtained with N-methyl-N'-nitro-N-nitrosoguanidine . The defective gene, named tgt, was mapped at about 9 min on the E . coli chromosome, and the gene order was shown to be phoB-tgt-tsx . tgt was transferred to an E . coli strain with a defined genetic background by P1 transduction to investigate its function . The mutant thus obtained lacked queuosine (2-amino-5-{3S, 4R, 5S)-4,5-dihydroxycyclopent-1-en-3-ylaminomethyl}-7-(beta-D-ribofuranosyl)-pyrro lo-{2,3-D}-pyrimidin-4-one) in tRNA, indicating that the enzyme is actually involved in the biosynthesis of queuosine in tRNA . No clear biological defect was observed in the mutant, and, in fact, it grew slightly faster than the control isogenic strain . tRNATyr lacking queuosine, isolated from the mutant, showed no significant biological difference from normal queuosine-containing tRNA in amino acid acceptor activity or amino acid transfer in a cell-free protein synthesizing system directed by synthetic polynucleotide . The only phenotypic change observed in the mutant thus far is marked reduction of viability when the cells are kept under unsuitable conditions for growth, suggesting that the presence of queuosine in tRNA is important to E . coli for survival in the natural environment. J Biol Chem, 1982 Jun 10, 257(11), 6595 - 9 Effect of deoxynucleoside phosphorothioates incorporated in DNA on cleavage by restriction enzymes; Vosberg HP et al.; DNA synthesized in vitro using deoxynucleoside phosphorothioates as substrates is quite similar to normal DNA in its biochemical properties (Vosberg, H.P., and Eckstein, F . (1977) Biochemistry 16, 3633-3640) . In order to investigate the effect of phosphorothioate groups in DNA on the cleavage pattern of restriction endonucleases phosphorothioate double-stranded, circular, replicative form of fd DNA was synthesized in vitro with Escherichia coli DNA polymerase I using native single-stranded DNA as template and mixtures of three normal nucleotides and one nucleoside phosphorothioate analogue as substrates . The double-stranded products were hybrids with respect to their phosphorothioate content . Restriction analysis of normal and phosphorothioate DNA with the restriction endonucleases Hae III, Bam HI, Hpa II, HindII, Alu I, and Taq I showed that the enzymes were inhibited to different degrees depending on which of the nucleotides was replaced by the phosphorothioate . Most significant, inhibition was seen throughout with those DNAs which contained a phosphorothioate exactly at the cleavage site . Phosphorothioate substitutions at other positions, but still within the recognition sequences, were, except for Alu I, not or weakly inhibitory . Phosphorothioate nucleotides not present in the recognition sequences did not affect at all the fragment patterns . The results show that recognition sequences of restriction endonucleases can be selectively protected against cleavage by base-specific introduction of phosphorothioate groups into DNA. J Biol Chem, 1982 Jun 10, 257(11), 6343 - 50 Escherichia coli sulfite reductase hemoprotein subunit . Prosthetic groups, catalytic parameters, and ligand complexes; Siegel LM et al.; Escherichia coli NADPH-sulfite reductase can be dissociated into an oligomeric flavoprotein and a monomeric hemoprotein (HP) subunit in 4 M urea . HP catalyzes stoichiometric 6-electron reductions of SO32- (to S2-) and of NO2-, as well as 2-electron reduction of NH2OH, with reduced methyl viologen (MV+) as reductant . While Vmax values are highest with the nitrogenous substrates, Km for SO32- is 2 to 3 orders of magnitude less than the Km for NO2- or NH2OH . EPR spectroscopic and chemical analyses show that HP contains one siroheme and one Fe4S4 center per polypeptide . The heme is in the high spin Fe3+ state in HP as isolated . Near-quantitative reduction of the Fe4S4 center to a state yielding a g = 1.94 type of EPR spectrum by S2O42- and/or MV+ could be achieved if HP was converted to either the CN- or CO complex or treated with 80% dimethyl sulfoxide . HP binds one SO32- or CN- per peptide . Binding of these ligands, as well as CO, appears to be mutually exclusive and to involve the heme . The heme Fe3+/Fe2+ potential is shifted from -340 mV in the free HP to -155 mV in the HP-CN- complex . The potential of the Fe4S4 center is approximately 70 mV more negative in the CN- as opposed to the CO-ligated HP (-420 mV), a result which indicates the presence of heme-Fe4S4-ligand interaction in the HP complexes. J Biol Chem, 1982 Jun 10, 257(11), 6292 - 5 Relationship between superhelical density and cruciform formation in plasmid pVH51; Singleton CK et al.; The relative stability of the cruciform state at the large inverted repeat of plasmid pVH51 is measured . At physiological superhelical densities, the cruciform state is present in a high percentage of the plasmid molecules . Investigation of the relationship between negative superhelical density and cruciform prevalence reveals a sharp transition from an undetectable level to a relatively stable state . This transition occurs over the negative superhelical density range of 0.046 to 0.066 . Estimates of the free energy contribution to cruciform formation resulting from loss of negative superhelical turns suggest that about 22 kcal/mol are required to generate the cruciform structure at this site in pVH51. J Biol Chem, 1982 Jun 10, 257(11), 6242 - 50 Protein n, a primosomal DNA replication protein of Escherichia coli . Purification and characterization; Low RL et al.; Protein n, essential in forming the primosome for the in vitro conversion of phi X174 single-stranded (SS) DNA to the duplex replicative form (RF), has been purified about 5000-fold to near homogeneity from Escherichia coli . Protein n is heat- and acid-resistant and N-ethyl-maleimide-sensitive . It appears to be a dimer of 12,000 (+/- 2000)-dalton polypeptides . About 80 molecules of protein n are present/cell . Protein n binding to phi X SS DNA depends on the presence of single-strand binding protein (SSB) . This requirement for SSB reflects a direct interaction of protein n and SSB . About 30 protein n monomers can be bound to an SSB-coated circle . However, in forming the primosome on an SSB-coated phi X circle, an input of only 2-3 protein n monomers is required and 1 monomer bound/circle . Retention of this low level of protein n on SSB-coated phi X SS DNA is dependent upon protein n', a DNA-dependent ATPase (dATPase) that guides primosome assembly . This single protein n monomer is retained in the assembled primosome, which is conserved on the completed parental RF and participates in the next stage of the replicative cycle, production of progeny RF. Biochemistry, 1982 Jun 8, 21(12), 2892 - 904 Spinach siroheme enzymes: Isolation and characterization of ferredoxin-sulfite reductase and comparison of properties with ferredoxin-nitrite reductase; Krueger RJ et al.; Sulfite reductase (SiR) has been purified to homogeneity from spinach leaves . Two forms of the enzyme were separated by hydroxylapatite chromatography . One, with subunit Mr 69 000 appears to be proteolytically cleaved to give rise to the other, with subunit Mr 63 000, during the purification procedure . The two species have identical catalytic activities (on a per heme basis) when reduced methylviologen (MV+) or ferredoxin (Fdr) is used as electron donor for sulfite reduction, and they exhibit nearly identical optical and EPR spectra . Both enzyme forms exist in 50 mM phosphate buffer (pH 7.7) primarily as dimers at 20 degrees C . Spinach SiR contains 1 mol of siroheme and one Fe4S4 center per subunit . The heme iron is the high spin Fe3+ state in the enzyme as isolated . Near quantitative reduction of the Fe4S4 center by dithionite could be achieved if SiR was either converted to the CO complex or treated with 80% dimethyl sulfoxide . Spinach SiR and nitrite reductase (NiR) both catalyze Fdr-or MV+-de-pendent six-electron reductions of SO3(2)- and NO2-, as well as the two electron reduction of NH2OH . Vmax values are highest with the nitrogenous substrates . However, the Km of SiR for So3(2-), and of NiR for NO2-, is at least 2 orders of magnitude less than with either of the other substrates . Rates of reduction with Fdr as electron donor are greater than with MV+ as donor, No immunological cross-reaction could be detected between spinach SiR and Escherichia coli SiR or between spinach SiR and NiR. Biochim Biophys Acta, 1982 Jun 4, 704(2), 345 - 52 Chemical modifications of Escherichia coli L-asparaginase and their effect on plasma clearance rate and other properties; Nickle EC et al.; Escherichia coli asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) has been modified by succinylation, acetylation and the attachment of N,N-dimethyl-1,3-propanediamine and glucuronic acid . The effect of these modifications on plasma clearance rates in mice and on other properties is compared to the effects of modification with lactose and N-acetylneuraminyl lactose studied previously . The t 1/2 values for the acylated enzyme samples (lower pI) were reduced, succinylated asparaginase sharply and the acetylated enzyme less so . The N,N-dimethyl-1,3-propanediamine-modified samples (increased pI) also had lower t 1/2 values, but samples modified with glucuronic acid (reduced pI) showed little change in clearance time . The main conclusion is that the increased t 1/2 value found in the previous work for N-acetylneuraminyl-lactosylated enzyme is not due to the decreased pI value of the modified enzyme, but must be attributed to interference by N-acetylneuraminyl-lactose residues, directly or indirectly, with the mechanism normally used by the mouse to clear itself of injected E . coli asparaginase. Eur J Pharmacol, 1982 Jun 4, 80(4), 369 - 76 Chlorpromazine inhibition of enterotoxin-induced fluid secretion and cAMP production in rat ileum; Olsvik O et al.; A heat-labile enterotoxin prepared from E . coli (EcLT) increased fluid secretion and cAMP production by segments of rat ileum in vivo and in vitro . The effect of this toxin was compared to that of cholera toxin (VcLT) . The increase of cAMP occurred more rapidly after EcLT than after VcLT indicating a difference in the kinetics of uptake or action of the two toxins . Chlorpromazine (CPZ) 5 mg/kg given by intramuscular injection 1 h before application of the toxins inhibited the increase in cAMP levels and the increase in fluid secretion in vivo . CPZ 10(-4) M given together with the toxins to intestinal loops in vitro inhibited the increase in cAMP levels and fluid secretion by this preparation . Scanning electron microscopy revealed that CPZ caused extensive shedding of the fluid-producing mucosal cells. Science, 1982 Jun 4, 216(4550), 1136 - 8 Isolation of human oncogene sequences (v-fes homolog) from a cosmid library; Groffen J et al.; To define the human homolog (or homologs) of transforming sequences (v-fes gene) common to Gardner (GA) and Snyder Theilen (ST) isolates of feline sarcoma virus (FeSV), a representative library of human lung carcinoma DNA in a cosmid vector system was constructed . Three cosmid clones were isolated containing GA/ST FeSV v-fes homologous cellular sequences, within 32- to 42-kilobase cellular inserts representing 56 kilobases of contiguous human cellular DNA . Sequences both homologous to, and colinear with, GA or ST FeSV v-fes are distributed discontinuously over a region of up to 9.5 kilobases and contain a minimum of three regions of nonhomology representing probable introns . A thymidine kinase selection system was used to show that, upon transfection to RAT-2 cells, the human c-fes sequence lacked detectable transforming activity. Clin Chim Acta, 1982 Jun 3, 121(3), 277 - 89 Development of a highly sensitive sandwich enzyme immunoassay for human ferritin using affinity-purified anti-ferritin labelled with beta-D-galactosidase from Escherichia coli; Imagawa M et al.; A highly sensitive sandwich enzyme immunoassay of human ferritin was developed . Polystyrene balls were coated with rabbit anti-human ferritin IgG by physical adsorption, and rabbit anti-human ferritin Fab' was purified by affinity chromatography and labelled with beta-D-galactosidase from Escherichia coli . Using the anti-ferritin-coated polystyrene balls and labelled anti-ferritin, the sensitivity obtained was 23 fg (0.05 amol) of ferritin per tube . The range of serum ferritin levels that could be determined using 0.1 microliter of serum was 0.23-4500 ng/ml, and even 2.3 pg/ml was measurable by using 10 microliter of serum . The coefficients of within-assay (n = 25) and between-assay (n = 10) variations were 5.9-8.8% . The regression equation and coefficient of correlation to a radioimmunoassay were Y(RIA) = 0.92 X(EIA) + 3.0 and 0.99 (n = 78), respectively . The corresponding sandwich radioimmunoassay was less sensitive, partly because the specific radioactivity of 125I-labelled anti-ferritin IgG used was not sufficiently high. J Pharmacobiodyn, 1982 Jun, 5(6), 403 - 9 The immunosuppressive effects of trichothecenes and cyclochlorotine on the antibody responses in guinea pigs; Ito H et al.; Immunosuppressive effects of trichothecenes of Fusarium solani and Fusarium nivale, T-2 toxin and fusarenon-X, and also of a mycotoxin of Penicillium islandicum, cyclochlorotine, were studied by measuring the anti-2,4-dinitrophenyl (DNP) antibody responses in guinea pigs immunized with DNP-bovine serum albumin . Among these mycotoxins, T-2 toxin alone suppressed strongly the anti-DNP antibody responses at a certain sublethal dose . With other mycotoxins, no effect was observed at any sublethal doses tested . All of the mycotoxins, on the other hand, inhibited the in vitro blast transformation of guinea pig splenic cells stimulated with lipopolysaccharide of Escherichia coli (B cell mitogen) or concanavalin A (T cell mitogen), when measured by incorporation of 3H-thymidine into DNA . Their inhibitory activities were independent of the sort of mitogen used . As in the case of the antibody responses, T-2 toxin was most potent in reducing the DNA synthesis, and exhibited about 10 and 1000 times as potent inhibitory activity as fusarenon-X and cyclochlorotine, respectively. Biokhimiia, 1982 Jun, 47(6), 915 - 20 {Early changes in template DNA structure following glucocorticoid injection}; Adler VV et al.; The structure of chromatin is changed at early stages of glucocorticoid hormone interaction with rat hepatocytes . These changes consist in: a) increase of actidine orange binding in rate liver nuclei after injection of the hormone; b) decrease of the number of sites in chromatin which are sensitive to nuclease S1; c) inhibition of the nuclei capacity for DNA synthesis in the presence of E . coli DNA polymerase and d) increase of molecular weight of DNA fragments . The data obtained suggest that at early stages of hormonal induction part of template DNA ruptures is reconstituted, which can result in an increase of DNA molecular weights and changes in chromatin superstructure and the template properties of the protein. Acta Anaesthesiol Scand, 1982 Jun, 26(3), 175 - 9 The effects of high dose methylprednisolone or fluid volume expansion on cerebral haemodynamics and oxygen uptake in endotoxic shock . An experimental study in dogs; Ekstrom-Jodal B et al.; E coli endotoxin has been shown to decrease cerebral blood flow (CBF) and increase cerebral metabolic rate of oxygen (CMRO2) in normocapnic dogs . The aim of this study was to investigate the effects of different clinical treatments on the cerebral and central circulation already under the influence of endotoxin . Thus the animals were treated with either methylprednisolone or a lactated Ringer's solution . CBF, CMRO2 and intracranial pressure were followed . Central haemodynamic parameters, i.e . cardiac output, aortic pressure and pulmonary artery pressure, were also measured . Five dogs were given methylprednisolone (Solu-Medrol) 30 mg/kg 90 min after the endotoxin injection . Following this drug there were no significant changes in CBF when compared to controls . The primarily increased CMRO2 did, however, show a transient decrease . Five animals were treated with a lactated Ringer's solution . (Ringerdex), 30 mg/kg, given intravenously over 20 min starting 90 min after the endotoxin injection . In these animals, the cardiac output as well as CMRO2 returned to the values before endotoxin . CBF did not increase significantly. Zh Mikrobiol Epidemiol Immunobiol, 1982 Jun, (6), 88 - 91 {Phagocyte characteristics in acute suppurative surgical infection}; Belotskii SM et al.; The chemotaxis of PMN against E . coli filtrate, as well as the adherence and phagocytosis of bovine RBC, both uncoated and IgM-, IgG- or C'-coated, were studied in 10 healthy donors, 15 patients with local infections, 22 patients with the onset of the generalized infection and 25 patients with sepsis (including 7 patients with the lethal process) . In patients with local infections the above-mentioned factors remained unchanged during treatment . In patients wit the onset of generalization chemotaxis and immune phagocytosis increased after the beginning of treatment . In septic patients showing a good response to treatment immune adherence and nonimmune, phagocytosis were stimulated . In patients with lethal sepsis a high level of immune adherence, but at the same time suppressed immune phagocytosis were observed. Immunopharmacology, 1982 Jun, 4(3), 253 - 66 Effect of nonsteroidal anti-inflammatory agents on immune complex- and chemotactic factor-induced inflammation; Issekutz AC et al.; Previously we reported that indomethacin and acetylsalicylic acid (ASA) inhibited leukocyte infiltration, blood flow, and vascular permeability during E . coli-induced inflammation in rabbit skin . Here we report the kinetics of these responses and the effects of these drugs on them, when a phagocytic stimulus, such as the immune complexes in the reverse Arthus reaction, or a nonphagocytic stimulus, such as the chemotactic factor in zymosan-activated plasma (ZAP, C5a des Arg), initiate inflammation . Both reactions causes infiltration of 51Cr-labeled leukocytes, an increase in blood flow, measured with 86RbC1, and in vascular permeability, measured with 125I-albumin . In both reactions, all three of these parameters were simultaneously inhibited by indomethacin or ASA . The local injection of prostaglandin E2 (0.5 microgram) at doses which, in control (uninflamed) skin increased only the blood flow, reversed all three inhibitory effects of the drugs . These results indicate that the drug-induced inhibition of leukocyte infiltration was secondary to the inhibition of the vascular responses . Furthermore, the vascular responses in both types of lesions were probably mediated, in large part, by vasodilatory prostaglandins . The magnitude and course of the vascular responses and their sensitivity to the prostaglandin synthetase inhibitors was similar in the Arthus (phagocytic) and in the ZAP (nonphagocytic) lesions . These results suggest that phagocytosis may not be a prerequisite for the generation of prostaglandins and the vascular responses during polymorphonuclear leukocyte-mediated inflammation in vivo. Crit Care Med, 1982 Jun, 10(6), 375 - 7 Effects of dopamine of cerebral circulation and oxygen metabolism in endotoxic shock: an experimental study in dogs; Ekstrom-Jodal B et al.; Experimental endotoxic shock in normocapnic dogs decreases cerebral blood flow (CBF) and increases cerebral metabolic rate of oxygen (CMRO2) . In 6 animals, an iv injection of 1.0-1.5 mg/kg of Escherichia coli endotoxin was followed by iv infusion of dopamine HCI at a rate of 3-24 Micrograms/kg . min . Doses of only 5 micrograms/kg . min maximally increased both CBF and CMRO2 . There were no significant changes in cardiac output and whole body oxygen consumption . Mean aortic pressure and mean pulmonary artery pressure increased slightly after doses of 20 micrograms/kg . min . Results indicate that damage to the blood-brain barrier (BBB) after endotoxin administration allowed circulating monoamines to directly influence cerebral metabolisms. Biull Eksp Biol Med, 1982 Jun, 93(6), 92 - 4 {Molecular size of plasmid R18 and its substituted variants in Escherichia coli cells}; Lushnikov AA et al.; R-substituted plasmids (R18Arg) obtained from plasmid R18 and capable to convert Arg- strains E coli to Arg+ strains at the expense of genetic material mobilized from P . aeruginosa chromosome are physically unstable and exist as molecules of different size . It was shown that the physical size of plasmid R18Arg is 3--5 megadaltons greater than those of plasmid R18. Zh Mikrobiol Epidemiol Immunobiol, 1982 Jun, (6), 40 - 4 {Partial composition of the O-antigens of O-group Escherichia}; Ulisko IN et al.; The partial composition of O-antigens in Escherichia known as the causative agents of escherichioses localized in the intestine and other organs has been studied . Escherichia of groups 01, 02 and 03 are characterized by different partial composition of their O-antigens . This composition can be expressed by the formulae: 01a, 1b; 01a, 1b, 1c; 01a, 1b, 1c, 1d; 01a, 1e; 02a, 2b, 2c; 02a, 2b, 2c, 2d, 2e; 02a, 2b, 2d, 2e, 2g; 02a, 2c, 2e; 06a, 6b; 06a, 6c . No pronounced correlation between the partial composition of O-antigens within the O-group and the presence of different K- or H-antigens in the strain has been established . The scheme of adsorption has been developed and anti-Escherichia factor O-sera have been obtained . The use of these sera allows one to differentiate Escherichia strains, isolated from patients, within the O-groups according to the partial composition of their antigens, which is of both diagnostic and epidemiological importance. Mutat Res, 1982 Jun, 94(2), 299 - 313 Influence of single-stranded DNA-binding protein on recA induction in Escherichia coli; Meyer RR et al.; Two ssb mutants of Escherichia coli, which carry a lesion in the single-strand DNA-binding protein (SSB), are sensitive to UV-irradiation . We have investigated the influence of SSB on the "SOS" repair pathway by examining the levels of recA protein synthesis . These strains fail to induce normal levels of recA protein after treatment with nalidixic acid or ultraviolet light . The level of recA protein synthesis in wild-type cells is about three times greater than ssb cells . This deficiency in ssb mutants occurs in all strains and at all temperatures tested (30-41.5 degrees) . In contrast, the ssb-1 mutation has no effect on temperature-induced recA induction in a recA441 (tif-1) strain . Cells carrying ssb+ plasmids and overproducing normal DNA-binding protein surprisingly are moderately UV-sensitive and have reduced levels of recA protein synthesis . Together these results establish that single-strand DNA-binding protein is involved in the induction of recA, and accounts, at least in part, for the UV sensitivity of ssb mutants . Three possible mechanisms to explain the role of SSB are discussed. Mutat Res, 1982 Jun, 94(2), 285 - 97 The effect of mutations in the uvrD cistron of Escherichia coli on repair resynthesis; Kuemmerle NB et al.; The resynthesis step of the excision repair pathway has been examined in Escherichia coli K12 strains isogenic except for mutations in the uvrD cistron . Strains mutant at the uvrD3, uvrD101, uvrE156, and recL152 loci exhibited slight but distinct differences in their response to ultraviolet radiation . The repair capacity of the uvrD101 mutant was given special attention . Repair resynthesis in this mutant was saturated at fluences greater than about 20 J/m2 . Isopycnic analysis of repaired deoxyribonucleic acid from this strain revealed a two-fold increase over its wild-type counterpart in the amount of repair replication performed after a dose of 15 J/m2 . Sedimentation velocity analysis of DNA after selective photolysis of bromouracil-containing repaired regions showed that the uvrD101 mutation exerted its primary effect on the long-patch component of excision repair . The uvrD101 mutant performed long-patch repair at a larger number of sites than the number repaired by this mode in the wild-type strain; these patches were more extensive in length than the long-patch component in wild-type. J Periodontol, 1982 Jun, 53(6), 368 - 78 Endotoxin penetration into root cementum of periodontally healthy and diseased human teeth; Nakib NM et al.; This study was undertaken to determine the extent of in vitro penetration of E . coli endotoxin into the root cementum of periodontally healthy and diseased teeth . Freshly extracted teeth were washed in distilled water, scaled and divided into two groups of 16 teeth each . Nine diseased and five healthy teeth in the first group were immersed in various concentrations of E . coli endotoxin for 2 to 12 weeks . They were then prepared for indirect immunofluorescence examination after treatment with anti-endotoxin antibody and rhodamine conjugated secondary antibody . Teeth in the second group were prepared for autoradiographic examination by immersing nine diseased and five healthy teeth in tritium labelled E . coli endotoxin for 2 to 12 weeks . The latter technique also allowed for semi-quantitative study of the depth of endotoxin penetration by creating facets on the root at various depths after endotoxin exposure . This technique was also used to investigate the strength of endotoxin binding to the tooth surface by brushing for 1 minute and re-examining the tooth for the presence of endotoxin . Controls included periodontally diseased and healthy teeth . Results of the study showed that (1) endotoxin adheres to the tooth surface without penetration into the root cementum of either periodontally healthy or diseased teeth, and (2) the binding of the endotoxin to the root surface appears to be weak. J Clin Microbiol, 1982 Jun, 15(6), 1062 - 4 Frequency of Escherichia coli strains producing heat-labile toxin or heat-stable toxin or both in children with and without diarrhea in São Paulo; Reis MH et al.; Enterotoxigenic Escherichia coli strains were isolated from 32 (13.4%) of 245 children with diarrhea and from 11 (11.4%) of 96 children of the control group . Strains producing heat-labile toxin were found more frequently in normal children than in children with diarrhea . Strains producing heat-stable toxin and both heat-labile and heat-stable toxins were isolated only from children with diarrhea . Association of these strains with diarrhea was highly significant as shown by statistical analysis . The O:H types and the colonization factors of strains producing heat-stable toxin and both heat-labile and heat-stable toxins are presented. Eur J Biochem, 1982 Jun, 124(3), 577 - 83 Rate of translation and kinetics of processing of newly synthesized molecules of two major outer-membrane proteins, the OmpA and OmpF proteins, of Escherichia coli K12; Crowlesmith I et al.; The rate of synthesis of the OmpA and OmpF proteins, two of the major outer membrane proteins of Escherichia coli K12, was determined . At 25 degrees C both proteins were translated at 6.5 amino acids/s, and the OmpF protein was translated at 15 amino acids/s at 37 degrees C . The former rate corresponded to a synthesis time of just over 50 s for both proteins, which is significantly faster than their reported rates of assembly into the outer membrane at 25 degrees C . The kinetics of processing of the pro-OmpF protein were also investigated in detail, and the pro-OmpF half-life estimated to be 3-5 s at 25 degrees C . However a fraction of the precursor was processed more slowly, which may explain the discrepancy between these data and our earlier published estimate of 30 s . Pro-OmpA protein was processed with similar kinetics . These results demonstrate that the rate-limiting step in the assembly of both proteins into the outer membrane is post-translational and follows the processing step. Eur J Biochem, 1982 Jun, 124(3), 545 - 52 Lactose carrier protein of Escherichia coli . Reconstitution of galactoside binding and countertransport; Wright JK et al.; A procedure for the reconstitution of the lactose carrier protein, a galactoside:proton symporter in Escherichia coli, is described . Starting from cytoplasmic membranes derived from carrier-overproducing strains, essentially all proteins including 89% of the carrier are solubilized by a mixture of dodecyl/tetradecyl polyoxyethylene (n = 9.5) ether and dodecyl O-beta-D-maltoside . In the micellar state the carrier binds substrates with reduced affinity . Addition of E . coli phospholipids and removal of detergents by a hydrophobic column yields small vesicles (50-60-nm diameter) . In these vesicles, about 70% of the carrier is recovered and reconstituted carrier is identical to native carrier in terms of substrate binding . After fusion of the small vesicles into larger vesicles (1-5 micrometers), rapid countertransport of galactosides is demonstrated . Attempts to show active galactoside transport by the imposition of artificial electrical potential or pH gradients were unsuccessful, most likely because the reconstituted vesicles are in fact highly permeable to protons. Am J Vet Res, 1982 Jun, 43(6), 999 - 1002 Thromboxane, prostaglandin I2 (epoprostenol), and the hemodynamic changes in equine endotoxin shock; Bottoms GD et al.; This study had 2 objectives: (i) to correlate plasma thromboxane and prostaglandin I2 (epoprostenol) concentrations with hemodynamic changes occurring in equine endotoxin shock, and (ii) to determine the effects of flunixin meglumine on plasma concentrations of these prostaglandins relative to hemodynamic changes . Shock was induced in 2 groups, each of 4 anesthetized ponies, and in a 3rd group of 2 ponies . Group A ponies were given endotoxin only (and were not treated), and group B ponies were given endotoxin and then treated with flunixin meglumine . Group C ponies were treated with flunixin meglumine 5 minutes before they were fiven endotoxin . Arterial, pulmonary arterial, and central venous pressures were measured and blood samples were collected at 0, 0.1, 0.25, 0.5, 1, 1, 3, and 4 hours after ponies were given the endotoxin . The plasma thromboxane and prostaglandin I2 concentrations were increased in equine endotoxic shock . Increased thromboxane concentration was associated with the high pulmonary arterial and central venous pressures and low arterial blood pressure in the minutes immediately after the ponies were given endotoxin . The increased prostaglandin I2 concentration was associated with systemic hypotension at 1 to 2 hours after endotoxin . Treatment of ponies with flunixin meglumine after endotoxin was given (group B) prevented the prostaglandin I2 rise and the associated hypotension . Treatment with fluixin meglumine before endotoxin was given prevented the increase of the plasma thromboxane and prostaglandin I2 values, along with the associated hemodynamic changes. Am J Vet Res, 1982 Jun, 43(6), 1034 - 40 Effect of passive immunization on phagocytosis of blood-borne Escherichia coli in spleen and liver of turkeys; Arp LH; Clearance of Escherichia coli from blood and localization in tissues were studied by culture and microscopy in nonimmunized and passively immunized turkeys after IV inoculation . In nonimmunized poults, high mortality was associated with persistence of large numbers of E coli in the blood . In immunized poults, rapid clearance of E coli from blood was associated with large numbers of E coli in hepatic sinusoids and fewer bacteria in the spleen . An electron-dense, fibrillogranular coat covered extracellular E coli in immunized poults, but not in nonimmunized poults . Rapid clearance of virulent E coli from blood was markedly enhanced by antibody-dependent phagocytosis in liver. Proc Natl Acad Sci U S A, 1982 Jun, 79(11), 3438 - 41 Role of positive charge on the amino-terminal region of the signal peptide in protein secretion across the membrane; Inouye S et al.; The positively charged amino-terminal region of the signal peptide has been proposed to have an important role at an initial step of protein secretion across the membrane (loop model) . To test this hypothesis, the charge on the amino-terminal region of the signal peptide of the prolipoprotein of the Escherichia coli outer membrane was altered by using synthetic oligonucleotides from +2 to +1, 0, and -1 by guided site specific mutagenesis of a plasmid DNA carrying an inducible lipoprotein gene . The wild-type sequence of this sectio, Met-Lys-Ala-Thr-Lys (+2), was thus changed to Met-Lys-Asp-Thr-Lys (I-1; +1), Met-Ala-Thr-Lys (I-2; +1), Met-Asp-Thr-Lys (I-3; 0), and Met-Glu-Asp-Thr-Lys (I-4; -1) . After induction of lipoprotein production, cells were pulse labeled with {35S}methionine for 10 sec . The lipoprotein of I-1, I-2, and I-3 was assembled in the membrane, although the rates of lipoprotein production progressively decreased as the charge on the signal peptide became more negative . Conversely, in the case of I-4, only a small amount of lipoprotein assembled in the membrane while a large amount of glycerol-unmodified prolipoprotein accumulated in the cytoplasm . This soluble prolipoprotein was gradually and posttranslationally secreted across the membrane to be modified and assembled in the membrane . These results indicate that the positively charged amino-terminal region of the signal peptide plays an important role in efficient protein secretion across the membrane. Infect Immun, 1982 Jun, 36(3), 900 - 6 Effect of parenteral vaccination of dams on intestinal Escherichia coli in piglets with diarrhea; Soderlind O et al.; To evaluate the effect of widely used parenteral vaccination of dams against neonatal colibacillosis, the virulence factors of the intestinal Escherichia coli flora, namely, O serogroup, enterotoxin(s) produced (heat labile, porcine heat stable, and murine heat stable) and adhesins (K88, K99, and 987P antigens) of 149 piglets from different herds in Sweden were investigated . Three categories were investigated: healthy piglets, diarrheal piglets born to unvaccinated dams, and diarrheal piglets born to dams vaccinated with a polyvalent Formalin-killed whole-cell vaccine containing K88 antigen (Porcovac; Hoechst Pharmaceuticals, Hounslow, England) . Piglets less than 1 week old and those 1 to 8 weeks old were evaluated separately . Diarrheal piglets less than 1 week old from vaccinated dams yielded a higher incidence of K99 antigen-positive E . coli of the murine heat-stable enterotoxigenicity type compared with piglets of the same age group from unvaccinated dams . The percentage of diarrheal cases from which E . coli lacking recognized virulence attributes were isolated was also higher in the former compared with the latter group . In the 1- to 8-week-old diarrheal piglets of vaccinated dams, the overall incidence, enterotoxigenicity type, and serotype of the E . coli isolates resembled those of diarrheal piglets less than 1 week of age from unvaccinated herds . Enterotoxigenic E . coli bearing 987P antigen detectable in vitro was rare . Most of the enterotoxigenic isolates lacking K88, K99, and 987P antigens produced only ST . The investigation pinpoints some of the inadequacies of vaccines of the type studied under field conditions. Infect Immun, 1982 Jun, 36(3), 851 - 6 Serotypes of enterotoxigenic Escherichia coli in Thailand and the Philippines; Echeverria P et al.; The serotypes of 386 enterotoxigenic Escherichia coli (ETEC) isolated from 82 individuals with and without diarrhea in Thailand and the Philippines were determined . The 136 strains producing both heat-labile toxin (LT) and heat-stable toxin (ST) belonged to 12 different O serogroups; however, 83% (113/136) were of one of four serogroups (O6, O8, O25, and O78), and 76% of (104/136) belonged to one of seven O:K:H serotypes . Only 14% (28/196) of LT-only-producing ETEC belonged to serogroups most common among LT and ST strains, and these 196 strains belonged to 35 different O:K:H serotypes . Three O serogroups (O20, O27, and O78) accounted for 94% (52/54) of strains producing only ST . Although only 4% (2/54) of ST-only ETEC belonged to the seven serotypes most commonly found among strains which produced LT and ST, 85% of ETEC belonged to three other serotypes, O20:K?:H21, O27:K?:H7, and O78:H- . A total of 46% (37/80) of ETEC of serotypes O6:H16, O8:H9, O25:H42, and O78:H12 were resistant to two or more antibiotics in comparison to 68% (208/306) of ETEC of other serotypes (P less than 0.001) . In Thailand and the Philippines, E . coli which produced LT and ST or ST alone, but not those which produced LT alone, were restricted in their O:K:H serotypes. Infect Immun, 1982 Jun, 36(3), 1238 - 41 Expression of Treponema pallidum antigens in Escherichia coli K-12; Stamm LV et al.; A colony bank of recombinant plasmids harboring Treponema pallidum DNA inserts has been established in Escherichia coli K-12 . By using an in situ immunoassay, we identified four E . coli clones that expressed T . pallidum antigens . Thus, recombinant DNA technology may provide powerful new tools for studying the pathogenesis of T . pallidum infection. J Infect Dis, 1982 Jun, 145(6), 863 - 9 Identification of enterotoxigenic Escherichia coli by colony hybridization using three enterotoxin gene probes; Moseley SL et al.; The applicability of examining clinical specimens with a DNA hybridization technique for genes encoding enterotoxins was examined using enterotoxigenic Escherichia coli (ETEC) that produced both heat-labile toxin (LT) and heat-stable toxin (ST) (24 isolates), ETEC that produced LT only (17 isolates), and ETEC that produced ST only (22 isolates) from Thailand . ETEC was identified with Y-l adrenal cell and suckling mouse assays . All were homologous with radiolabeled fragments of DNA encoding LT or ST of porcine origin (ST-P) or of human origin (ST-H) . Strains of ETEC that produced ST only from rural Thailand were homologous with the ST-H probe only, whereas strains isolated in Bangkok were homologous with the ST-H probe, the ST-P probe, or both probes . The hybridization technique detected ETEC in all stool samples of patients with diarrhea from whom ETEC was isolated and in ETEC-inoculated water containing other species of bacteria . The DNA hybridization assay is useful for characterizing and identifying environmental sources of ETEC. J Hyg (Lond), 1982 Jun, 88(3), 513 - 7 Serotypes of Escherichia coli isolated from patients with recurrent pyogenic cholangitis; Wong WT et al.; Strains of Escherichia coli were isolated from laparotomy specimens from Chinese patients with recurrent pyogenic cholangitis in Hong Kong . A large variety of serotypes were found . Several sites were sampled in each patient . While only one serotype was normally isolated from one site, different sites often yielded different serotypes in the same patient . Generally the 'O' types found did not correspond to those found in the faeces of the Hong Kong Chinese population. Chem Biol Interact, 1982 Jun, 40(2), 141 - 8 Effect of caffeine on nucleotide pools in Escherichia coli; Sandlie I et al.; The influence of caffeine on the intracellular concentration of various nucleoside triphosphates in addition do dTMP and dTDP, in Escherichia coli has been investigated . For most of the nucleoside triphosphates the presence of 10 mM caffeine in the medium resulted in a small increase in pool size 5 min after addition, followed by a slow decrease to the initial concentration . In the case of dTTP, however, the pool size reached a maximum, 2-fold higher than the initial value, 30 min after caffeine addition . This increase in dTTP level is probably due to an effect of caffeine on the DNA synthesis process and synthesis of dTDP-sugars. J Bacteriol, 1982 Jun, 150(3), 1482 - 4 Release of heat-labile enterotoxin subunits by Escherichia coli; Yamamoto T et al.; Most of the heat-labile enterotoxin (LT) synthesized by Escherichia coli is cell associated; however, a small portion of LT (approximately 10%) is released by bacterial cells into the culture supernatant . The LT subunit B (LT-B) produced by a cloned LT-B gene (tox B) was released in amounts equal to the parent LT release . In contrast, no release of LT subunit A (LT-A) or its smaller derivatives was observed in strains containing cloned toxA genes . The data suggest that LT-B is necessary for the release of LT-A across the bacterial membrane. J Bacteriol, 1982 Jun, 150(3), 1479 - 81 Direct participation of lexA protein in repression of colicin E1 synthesis; Ebina Y et al.; Spontaneous colicin E1 production by plasmid RSF2124 in a recA lexA(spr) strain of Escherichia coli was about 10-fold greater than that observed in a wild-type strain . The synthesis was repressed nearly to the level of a recA strain in the presence of the plasmid pMCR551, which carries the lexA gene. J Bacteriol, 1982 Jun, 150(3), 1472 - 5 The cloacin receptor of ColV-bearing Escherichia coli is part of the Fe3+-aerobactin transport system; Bindereif A et al.; Escherichia coli strains which contain the Fe3+-aerobactin transport system specified by the ColV plasmid became deficient in aerobactin-dependent iron transport when they were converted to cloacin-resistant derivatives . An outer membrane protein with a molecular mass of 74,000 daltons was overproduced under iron-limiting growth conditions and was absent in cloacin-resistant mutants . Fe3+-aerobactin protected cells against cloacin . These results suggest that the cloacin receptor protein, controlled by the colV plasmid, also participates in Fe3+-aerobactin transport. J Bacteriol, 1982 Jun, 150(3), 1462 - 6 Cloning of the regulatory genes (ompR and envZ) for the matrix proteins of the Escherichia coli outer membrane; Mizuno T et al.; We have cloned the regulatory gene cluster of Escherichia coli which is composed of at least two distinct genes, ompR and envZ . These genes are known to regulate the production of the outer membrane matrix proteins . The newly formed plasmids were found to complement not only ompR mutations but also envZ mutations . The ompR gene product was identified as a protein of an apparent molecular weight of 28,500. Cancer Res, 1982 Jun, 42(6), 2416 - 9 Influence of temperature on platinum binding to DNA, cell killing, and mutation induction in Escherichia coli K-12 cells treated with cis-diamminedichloroplatinum(II); Brouwer J et al.; Cell killing and specificity of mutation induction by treatment of Escherichia coli K-12 cells with cis-Pt(NH3)2Cl2 at various temperatures have been studied . Survival experiments show that the cell killing by cis-Pt(NH3)2Cl2 is enhanced with increasing temperature . This effect is explained by an increase in the amount of platinum bound to the DNA . The binding is the same in repair-proficient and in repair-deficient cells . However, the mutation induction in the lacI gene is much more strongly enhanced than could be expected from the increased platinum binding . This phenomenon is attributed to a different distribution of the induced lesions by cis-Pt(NH3)2Cl2 at various temperatures and not to an aberrant excision repair . Analysis of the induced lacI mutants revealed an increase in the percentage of nonsense mutants at higher temperature . Among the nonsense mutations, base-pair substitutions at GAG and particularly at GCG sequences are enhanced by the increasing temperature . The results are in agreement with our hypothesis that local denaturation of DNA, known to be promoted at higher temperature, is necessary for the formation of intrastrand cross-links at two guanine bases separated by a third base. Can J Microbiol, 1982 Jun, 28(6), 654 - 9 Inhibition of uridine 5'-diphosphate-N-acetylmuramyl-L-alanine synthetase by beta-chloro-L-alanine in Escherichia coli; Ishiguro EE; The synthesis of the nucleotide precursors for peptidoglycan is regulated by the relA gene in Escherichia coli . Thus, nucleotide precursors labeled with {3H}diaminopimelic acid accumulated in a relA strain but not in an isogenic relA+ strain during amino acid deprivation . Furthermore, nucleotide precursor synthesis was relaxed in the amino acid deprived relA+ strain by treatment with chloramphenicol . Uridine diphosphate-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pentapeptide) was the major component accumulated during the relaxed synthesis of nucleotide precursors in both relA+ and relA strains . The effect of beta-chloro-L-alanine (CLA) on the relaxed synthesis of nucleotide precursors for peptidoglycan was determined . At a low concentration (0.0625 mM) CLA inhibited the synthesis of UDP-MurNAc-pentapeptide and caused the accumulation of UDP-MurNAc-tripeptide . Thus, low concentrations of CLA probably inhibited alanine racemase, as reported previously . Higher concentrations of CLA also inhibited an earlier step in nucleotide precursor synthesis . This was shown to be due to the inhibition of UDP-MurNAc-L-alanine synthetase by CLA . CLA inhibited the activity of this enzyme in cell-free extracts as well as in intact cells. Can J Biochem, 1982 Jun, 60(6), 608 - 12 The inhibition of beta-galactosidase (Escherichia coli) by amino sugars and amino alcohols; Huber RE et al.; Kinetically determined competitive inhibitor constants, which were estimates of the binding capacity of inhibitors interacting with the free enzyme form of beta-galactosidase (Escherichia coli), showed that amino sugars and alcohols inhibited the enzyme much more than did their respective parent sugars and alcohols . In most cases the inhibition was 10-30 times greater, but the inhibition by 1-aminogalactopyranose was about 300 times greater than that of galactopyranose . When the amino groups were acetylated the inhibitory advantage was completely eliminated and partial neutralization of the amino group of an inhibitor by the presence of a group with a negative charge on the same inhibitor decrease the inhibitory advantage . Studies of binding to the galactosyl form of the enzyme (rather than to the free form) indicated that the binding at the "glucose" site was increased only slightly by the presence of the amino group . Overall, the findings suggested that beta-galactosidase has a negative charge near the anomeric carbon binding position of the "galactose" site . Since negative charges are unlikely to be of any importance in binding the normally neutral beta-galactosidase substrates, this charge may be important in stabilizing a positively charged reaction analogous to an oxonium-carbonium ion intermediate. Acta Anaesthesiol Scand, 1982 Jun, 26(3), 163 - 70 Cerebral blood flow and oxygen uptake in endotoxic shock . An experimental study in dogs; Ekstrom-Jodal B et al.; Cerebral blood flow (CBF), cerebral oxygen uptake (CMRO2) and central haemodynamics in anaesthetized dogs with controlled ventilation were studied at intervals for 2 h following an intravenous injection of E . coli endotoxin, 1.0-1.5 mg/kg . CBF showed a 30% decrease within 15 min after the endotoxin administration, while the arterial blood pressure was still not markedly depressed . Autoregulation to arterial blood pressure changes was maintained during endotoxinaemia and the cerebrovascular reaction to changes in arterial carbon dioxide tension (PaCO2) depressed . Normocapnic animals (PaCO2) greater than or equal to 4.0 kPa) showed an increase in CMRO2 of over 40%, that was obvious 1 h after the administration of endotoxin . The intracranial pressure was decreased with 5 min of the administration of endotoxin irrespective of the prevailing arterial blood pressure . Thereafter, it was raised above the control level . Two hours after endotoxin increased protein concentrations in cerebrospinal fluid were seen, results compatible with blood-brain barrier damage and penetration of other substances; e.g . monoamines released during endotoxinaemia could thus be expected to have a direct influence on both cerebral blood flow and metabolism. Eur J Biochem, 1982 Jun, 124(3), 513 - 9 Characterization of monofunctional chorismate mutase/prephenate dehydrogenase enzymes obtained via mutagenesis of recombinant plasmids in vitro; Rood JI et al.; Mutagenesis in vitro has been used to obtain mutant forms of the bifunctional enzyme, chorismate mutase/prephenate dehydrogenase . Plasmid DNA containing the genes that code for the enzyme was treated with hydroxylamine and the resulting products were used to transform strains of Escherichia coli . Two types of mutant were isolated . One contained enzyme which was mutase-positive, dehydrogenase-negative while the other did not exhibit either activity . Kinetic and physical analysis of one of the purified monofunctional enzymes showed that the loss of dehydrogenase activity was due to modification of the binding site for NAD . The results open the way for molecular studies of structure-function relationships with this bifunctional enzyme. J Bacteriol, 1982 Jun, 150(3), 1476 - 8 Threonine prevents derepression of pyridoxine synthesis in Escherichia coli B; Dempsey WB; After 40 min of pyridoxal starvation, pyridoxine phosphate oxidase-less mutants of Escherichia coli B derepressed pyridoxine biosynthesis 13-fold to a rate of 1.7 X 10(-9) mol/h per mg of cells . Threonine at 100 mg/liter prevented this derepression but did not affect the continued synthesis of pyridoxine . Neither serine nor branched-chain amino acids altered the threonine effect. J Ultrasound Med, 1982 Jun-Jul, 1(5), 179 - 84 Gas as a contrast agent and diagnostic aid in abdominal sonography; Burt TB et al.; Gas has a characteristic ultrasound pattern, usually recognized as an area of focal dense echogenicity with acoustic shadowing . It often contains reverberation echoes . Sonographers should be familiar with this pattern and should suspect underlying disease when gas is demonstrated in abnormal or unusual locations . The authors have collected 18 cases; four representative cases are presented to illustrate this principle. Steroids, 1982 Jun, 39(6), 657 - 66 An enzyme immunoassay of estrone in swine serum; Tamamura F et al.; An enzyme immunoassay for estrone in swine serum was established . For this, beta-galactosidase from E . coli was conjugated through estrone-17 (O-carboxymethyl)oxime using a mixed anhydride reaction . The percentage of immunoreactive estrone-17 (O-carboxymethyl)oxime-beta-galactosidase conjugate was estimated to be about 70% . The recovery rate of estrone (25-500 pg) added to 0.05 ml of swine serum averaged 91.4% . The sensitivity of the present enzyme immunoassay was 5 pg/tube . The coefficients of variation (CV) were 5.9-8.2% (within assays) and 4.1-5.9% (between assays), respectively . Estrone values determined by the present enzyme immunoassay were highly correlated with those determined by radioimmunoassay (r = 0.99, P less than 0.005) . This method of enzyme immunoassay was determined to be suitable for the routine assay of serum estrone. Eur J Clin Microbiol, 1982 Jun, 1(3), 178 - 85 Correlation between intestinal immune response to colonization factor antigen/I and acquired resistance to enterotoxigenic Escherichia coli diarrhea in an adult rabbit model; Evans DG et al.; Immunoprotection against diarrhea caused by colonization factor antigen/I (CFA/I)-positive, human-associated, enterotoxigenic Escherichia coli was investigated using the adult rabbit intestinal temporary ligation technique . An oral dose of 1 X 10(8) viable cells of enterotoxigenic Escherichia coli strain H-10407 (078:H11:CFA/I) produced diarrhea in all animals challenged . Rabbits allowed to survive this challenge dose were re-challenged approximately six weeks later with the result that four of seven (57%) did not develop diarrhea . Peroral immunization of rabbits with purified CFA/I elicited protection against challenge with strain H-10407; this protection was dose-related and CFA/I-specific . Immunoprotection did not correlate with a systemic antibody response . CFA/I produced a relatively poor immune response in terms of the number of IgM- and IgG-producing cells in the lamina propria of the animals but did elicit a vigorous increase in the number of intestinal IgA- and anti-CFA/I-producing cells . There was a highly significant inverse relationship between the number of IgA- and anti-CFA/I-producing cells in the lamina propria of the rabbits and the diarrhea response to the challenge strain H-10407 (correlation coefficients of -0.616 and -0.678 respectively) . It is concluded that anti-CFA/I antibody, probably of the IgA class, is the major immune response to orally administered CFA/I and that this response is highly immunoprotective. Kidney Int, 1982 Jun, 21(6), 808 - 12 Effect of iron on acute pyelonephritis and later chronic changes; Guze LB et al.; Administration of iron to rats exacerbated early inflammatory changes of pyelonephritis produced by intravenous inoculation of Escherichia coli . This effect was noted with four of eight strains of E . coli tested and was dependent on bacterial inoculum . Despite this increase in severity of acute pyelonephritis as judged by numbers of bacteria in the kidney and careful gross and microscopic evaluation, there was no enhancement of chronic changes seen 6 months later. Gann, 1982 Jun, 73(3), 470 - 4 Increased antitumor activity of Escherichia coli . L-asparaginase by modification with monomethoxypolyethylene glycol; Kamisaki Y et al.; Escherichia coli L-asparaginase modified with monomethoxypolyethylene glycol, which has no ability to bind to the antibody and almost no immunogenicity, had a longer plasma half-life (32.9+/-2.5 hr) in normal mice than the native enzyme (3.35 +/- 0.45 hr) . Preimmunization of mice with the native enzyme greatly shortened the plasma half-life of the native L-asparaginase (less than 0.1 hr) but did not change that of the modified L-asparaginase (32.2 +/- 2.1 hr) . Treatment of mice bearing asparaginase-sensitive Gardner lymphoma (6C3HED) with L-asparaginases at various doses showed that the modified enzyme had a greater therapeutic effect than the native enzyme . In addition, preimmunization with the native enzyme prevented the antitumor activity of the native enzyme but did not affect the therapeutic efficacy of the modified L-asparaginase against the lymphoma. Gene, 1982 Jun, 18(3), 355 - 60 Human cytomegalovirus DNA: BamHI, EcoRI and PstI restriction endonuclease cleavage maps; Greenaway PJ et al.; The cloned HindIII fragments of human cytomegalovirus (HCMV) strain AD169 DNA were mapped with respect to the BamHI, EcoRI and PstI restriction endonuclease cleavage sites . Composite restriction endonuclease cleavage maps for the entire virus genome were constructed using the previously established linkages between the HindIII fragments. Gene, 1982 Jun, 18(3), 343 - 54 Synthesis of the nutL DNA segments and analysis of antitermination and termination functions in coliphage lambda; Drahos D et al.; The nut antiterminator sequence, when present between a promoter and a terminator, permits the N-mediated antitermination of transcription in phage lambda . The efficiency of nutL was determined by assaying the activity of gene galK placed on a plasmid downstream from the promoter, nutL and terminator modules . As a reference and an estimate of the plasmid copy number, we have used an improved and very reproducible assay for bla activity . Sequences consisting of the 17-bp nutL core flanked by two HindIII cohesive sites were synthesized by the phosphite coupling method, and cloned in proper orientation between the Pp promoter of pBR322 and lambda gene N followed by the tL1 terminator on a galK-expression plasmid . The antitermination efficiencies for two synthetic 17-bp nutL sequences, one wild type and one point mutant at the base of the nutL stem, are similar but substantially reduced in comparison with the native 25-bp nutL sequence cloned at the same site in the otherwise identical galK-expression plasmid . Multiple tandem insertions of the synthetic 17-bp nutL segment successively increase antitermination efficiency, but also to levels below those of comparable plasmids carrying multiple copies of the native 25-bp nutL sequence . Thus, several specific base pairs in the flanking sequences appear to be important for the efficient nut function . In an inverted orientation the 17-bp nutL sequence has lost its antitermination function . It also lost the termination activity exhibited by inversion of the longer 25-bp and 74-bp native nutL sequences. Gene, 1982 Jun, 18(3), 335 - 41 Versatile low-copy-number plasmid vectors for cloning in Escherichia coli; Stoker NG et al.; Small low-copy-number plasmid vectors were constructed by in vitro and in vivo recombinant DNA techniques . pLG338 and pLG339 are derived from pSC105, have a copy number of six to eight per chromosome, and carry genes conferring resistance to tetracycline and kanamycin . pLG338 (7.3 kb) has unique restriction endonuclease sites for BamHI, SalI, HincII, SmaI, XhoI, EcoRI and KpnI, the first five lying within a drug resistance gene . pLG339 (6.2 kb) lacks the KpnI site, but has unique SphI and PvuII sites . These versatile vectors should be useful for cloning many genes coding for membrane and regulatory proteins which cannot be cloned into high-copy-number plasmids. Gene, 1982 Jun, 18(3), 309 - 18 Physical map of the nrdA-nrdB-ftsB-glpT region of the chromosomal DNA of Escherichia coli; Yamada M et al.; Seven pLC plasmids (pLC 3-46, 8-12, 8-24, 8-29, 14-12, 19-24 and 42-17) which complemented nrdA, nrdB, ftsB and/or glpT mutations of Escherichia coli were analyzed . A restriction map of each plasmid was constructed and restriction fragments were subcloned into pBR322 . A physical map of approx . a 15 X 10(6) Mr segment of the chromosomal DNA was deduced from the overlapping region of the pLC plasmids . The pLC plasmids and newly constructed plasmids were examined for the ability to rescue the mutations . The complementation tests defined the location of the genes in the 15 X 10(6) Mr segment in the following order: nrdA-nrdB-ftsB-glpT . Functional nrdAB and ftsB genes were located in the 3.1 X 10(6) Mr EcoRI-PstI fragment. Gene, 1982 Jun, 18(3), 277 - 88 Isolation and characterization of yeast ring chromosome III by a method applicable to other circular DNAs; Devenish RJ et al.; A method is described for the isolation and purification of covalently closed circular (ccc) DNA from yeast (Saccharomyces cerevisiae) . Spheroplasts are lysed at pH 12.45 which denatures linear but not ccc DNA . Next, the lysate is taken through a gentle high-salt-phenol extraction to remove single-stranded DNA . The ccc DNA, recovered by ethanol precipitation, can be further studied by agarose gel electrophoresis, can be cut with restriction endonucleases and can be used to transform Escherichia coli . This method efficiently purifies large (approx . 190 kb) and small (approx . 1.5 kb, TRP1-RI Circle) circular DNAs and thus has general applicability for isolation and purification of plasmids from yeast. Gene, 1982 Jun, 18(3), 261 - 6 Physical localisation and direction of transcription of the structural gene for Escherichia coli ribosomal protein S15; Portier C; The coding sequence for the Escherichia coli ribosomal protein S15 (rpsO) has been shown to lie immediately adjacent to the structural gene for polynucleotide phosphorylase (pnp) . Based on DNA sequencing data, it is deduced that rpsO is transcribed counterclockwise with respect to the standard E . coli genetic map. Gene, 1982 Jun, 18(3), 239 - 46 Selective cloning of Co1E1 DNA initiation sequences using the cloning vector M13 delta E101; Nomura N et al.; DNA sequences required to direct single-strand to double-strand conversion by a phi X174-type mechanism have been detected in Co1E1 plasmid DNA . The sites responsible for this DNA strand initiation, named rriA and rriB, have been localized to the L strand of the HaeII-E fragment and to the H strand of the HaeII-C fragment of Co1E1 . To define rriA and rriB more precisely, random fragments of Co1E1 DNA have been cloned into the single-stranded phage cloning vector M13 delta E101 . This phage contains a viable deletion of the M13 complementary strand origin and makes small turbid plaques . Cloning of DNA initiation determinants into the unique EcoRI site of M13 delta E101 rescues the mutant phage and restores the ability to form clear plaques . DNA sequence analysis of the random, 100-400 bp inserts in clear plaque isolates has localized the rriA and rriB determinants within DNA fragments of about 100 bp . These initiation determinants promote assembly of a functional, multiprotein priming complex, "primosome", identical to that utilized in the in vitro conversion of phi X174 single-stranded DNA to the duplex replicative form. J Reticuloendothel Soc, 1982 Jun, 31(6), 501 - 9 Pseudosecondary antibody response to LPS induced by toxins for macrophages; Remington KM et al.; A single dose of bacterial lipopolysaccharide, administered 21 days after mice had been treated with the macrophage toxins carrageenan, or microparticulate crystalline silica, resulted in a secondarytype antibody response . The phenomenon of pseudosecondary responsiveness was investigated to determine the mechanism for its generation . The results showed that generating a pseudosecondary response was T cell dependent; but except for this, it followed the same kinetic and genetic patterns as a true, two dose of antigens, secondary response . It was concluded that pseudosecondary responsiveness resulted from priming the mice by the consequential effects of the macrophage toxins; thereafter, secondary responsiveness to LPS was generated in a normal manner. Cell, 1982 Jun, 29(2), 319 - 28 On the formation of spontaneous deletions: the importance of short sequence homologies in the generation of large deletions; Albertini AM et al.; Using lacl-Z fusion strains of Escherichia coli we have devised systems that detect deletions of varying lengths . We examined deletions 700-1000 base pairs long, and genetically characterized over 250 spontaneous deletions . Of these, we analyzed 24 by direct DNA sequencing and 18 by inspection of restriction fragment patterns . Deletions of this size occur almost exclusively at short repeated sequences in both (recA+ and recA- strain backgrounds, but are detected 25-fold more frequently in a recA+ background . The frequency of deletion formation correlates with the extent of homology between the short repeated sequences, although other factors may be involved . The largest hotspot, which accounts for 60% of the deletions detected, involves the largest homology in the system (14 of 17 base pairs) . Altering a single base pair within this homology reduces deletion incidence by an order of magnitude . We discuss possible mechanisms of deletion formation and consider its relationship to the excision of transposable elements. J Clin Microbiol, 1982 Jun, 15(6), 1074 - 6 Isopycnic separation of Escherichia coli cultures possessing colonization factor antigen I; Giesa FR et al.; A culture of Escherichia coli possessing colonization factor antigen I was subjected to isopycnic separation on Percoll gradients . The results demonstrated successful division of the culture into two populations: (i) bacteria which cause mannose-resistant hemagglutination and (ii) bacteria which lack the ability to hemagglutinate in the presence of mannose. Eur J Biochem, 1982 Jun, 124(3), 483 - 8 Macromolecular complex of aminoacyl-tRNA synthetases from sheep liver . Identification of the methionyl-tRNA synthetase component by affinity labeling; Brevet A et al.; Both the tRNA aminoacylation and amino-acid-dependent ATP-PPi exchange activities of monomeric trypsin-modified methionyl-tRNA synthetase from sheep liver are lost upon incubation with oxidized initiator tRNAMet . The inactivation, which reflects the formation of a Schiff's base between the 5'-terminal adenosine of tRNA and a lysine within the catalytic site of the enzyme, is accompanied by the covalent attachment of one tRNA molecule per enzyme molecule . The affinity labeling method is applied to the sheep liver complex of Mr 10(6) carrying seven aminoacyl-tRNA synthetase activities, from which the monomeric trypsin-modified methionyl-tRNA synthetase (Mr 68 000) was derived . Upon incubation with oxidized initiator tRNAMet, the methionyl-tRNA synthetase activity of the complex is lost . Of the eleven polypeptide chains composing the high-molecular-weight complex, only one polypeptide chain with Mr 103 000 reacts with the modified tRNAMet . The blocking by periodate-treated tRNA of the methionyl-tRNA synthetase activity in the complex has no effect on the other aminoacyl-tRNA synthetase activities . This strongly argues in favor of the independent parallel functioning of the seven aminoacyl-tRNA synthetases associated in a high-molecular-weight complex. Eur J Biochem, 1982 Jun, 124(3), 435 - 40 Base-excision repair in carrot cells . Partial purification and characterization of uracil-DNA glycosylase and apurinic/apyrimidinic endodeoxyribonuclease; Talpaert-Borle M et al.; Uracil-DNA glycosylase and apurinic/apyrimidinic (AP) endodeoxyribonuclease have been purified from cultured carrot cells . The two enzymes, separated by affinity chromatography on Sepharose-poly(rU), were found to have properties similar to those of the homologous bacterial and mammalian enzymes . The action of AP endodeoxyribonuclease on (dA)230 . (dT, dU)230 partially depyrimidinated by uracil-DNA glycosylase suggests that these two enzymes might act successively to initiate the repair of uracil-containing DNA. Proc Natl Acad Sci U S A, 1982 Jun, 79(12), 3724 - 8 Illegitimate recombination mediated in vitro by DNA gyrase of Escherichia coli: structure of recombinant DNA molecules; Ikeda H et al.; We have developed a cell-free system from Escherichia coli for studying illegitimate recombination between nonhomologous DNA molecules . The recombination is stimulated by oxolinic acid, an inhibitor of DNA gyrase . The stimulation is abolished by coumermycin A1 and is not found in extracts of nalidixic acid-resistant (gyrA) mutants . We therefore inferred that DNA gyrase directly participates in illegitimate recombination, at least in the presence of oxolinic acid {Ikeda, H., Moriya, K . & Matsumoto, T . (1981) Cold Spring Harbor Symp . Quant . Biol . 45, 399--408} . The structure of recombinant DNA molecules formed in the presence of oxolinic acid from a cross between phage lambda and plasmid pBR322 DNAs was analyzed by heteroduplex mapping . Among nine isolates tested, two recombinants were formed by the insertion of the plasmid into the lambda genome . The seven other recombinants had more complicated genome structures . Insertion of pBR322 was accompanied by a deletion on one of the genomes . In all cases, the end points of deletions coincided with one end of the pBR322 insertion . Recombination sites seemed to be distributed randomly on the lambda and pBR322 genomes . Analysis of nucleotide sequences of the recombination junctions proved that the crossover took place between nonhomologous DNA sequences . A model for DNA gyrase-mediated illegitimate recombination is discussed. J Bacteriol, 1982 Jun, 150(3), 1266 - 73 Occurrence and properties of composite transposon Tn2672: evolution of multiple drug resistance transposons; Hanni C et al.; We found Tn2671 (the 23-kb long IS1-flanked r-determinant of NR1-Basel) inserted into the ampicillin resistance gene bla of the Tn3-related transposon Tn902 . The resulting 28-kilobase-long composite transposon Tn2672 (= Tn902 bla::Tn2671) is stable, and it translocates as a unit into various loci including IS1 of the resistance transfer factor of R100-1 . These results are discussed with respect to the evolution of R plasmids providing multiple drug resistance. J Bacteriol, 1982 Jun, 150(3), 1164 - 71 Mapping of two ugp genes coding for the pho regulon-dependent sn-glycerol-3-phosphate transport system of Escherichia coli; Schweizer H et al.; Two genes, ugpA and ugpB, coding for a binding protein-dependent sn-glycerol-3-phosphate transport system, were mapped at 75.3 min on the Escherichia coli chromosome . A Tn10 insertion in ugpA resulted in loss of transport activity but still allowed the synthesis of the sn-glycerol-3-phosphate-binding protein . This Tn10 insertion was found to be linked by P1 transduction to pit, aroB, malA, asd, and livH with 2.5, 2.8, 25, 63.5, and 83% cotransduction frequency . An insertion of Mud (Ampr lac) in ugpB resulted in the loss of the binding protein . ugpB is closely linked to ugpA . It is either the structural gene for the binding protein or located proximal to it . The analysis of the crosses allowed the ordering of the markers in the clockwise direction as follows: aroB, malA, asd, ugpA, ugpB, livH, pit. J Bacteriol, 1982 Jun, 150(3), 1115 - 21 Protein H encoded by plasmid CloDF13 is involved in excretion of cloacin DF13; Oudega B et al.; Excretion of cloacin DF13 was studied in Escherichia coli cells harboring different CloDF13 insertion and deletion mutant plasmids . Insertions of a transposon at position 9.8 or 11.5% of the CloDF13 plasmid blocked the expression of gene H and strongly reduced the specific excretion of cloacin DF13 into the culture medium, but had no effect on the production of cloacin DF13 . Insertions in or deletions of regions of the CloDF13 DNA upstream the cloacin operon did not affect the excretion or production of the bacteriocin . Introduction of a CloDF13 plasmid that encodes for the gene H product in cells harboring a CloDF13 plasmid with an insertion in gene H stimulated the excretion of cloacin DF13 significantly in mitomycin C-induced and in noninduced cultures . Cloacin DF13 in cloacinogenic cells that did not produce the gene H protein was found to be about 90% located in the cytoplasm . In cells that did produce the gene H product, about 30% of the cloacin DF13 molecules were found in the cytoplasm, about 18% were found in the periplasm, about 2% were in the membranes, and about 50% were located in the culture supernatant . Cyclic AMP stimulated the production but not the excretion of cloacin DF13 in cells cultivated in the presence of glucose. J Bacteriol, 1982 Jun, 150(3), 1077 - 84 Origin and direction of mini-R1 plasmid DNA replication in cell extracts of Escherichia coli; Diaz R et al.; Replication of the mini-R1 plasmids pKN177 and pKN182 can be carried out efficiently in cell extracts of Escherichia coli and depends on both transcription and translation . Heteroduplex and restriction analyses indicate that both plasmids are derived from the R1 copy mutant pKN104 by Is1-mediated recombination events without involving structural alterations in the replication region . To ascertain whether the in vitro replication of these miniplasmids corresponds to R1 replication in vivo, the origin and direction of replication were analyzed by electron microscopy of replicative intermediates . It was found that replication starts at a unique origin located within the RepA region at R1 coordinate at 82.4 kilobases and proceeds unidirectionally toward the IS/b sequence . The specification of the origin and the direction of in vitro replication are therefore in full agreement with the pattern observed previously for the in vivo replication of the closely related plasmids R100 and R6-5 . This agreement provides additional evidence that R1 DNA synthesis in vitro employs the same replication mechanism as it does in vivo. J Bacteriol, 1982 Jun, 150(3), 1040 - 7 Use of in vitro gene fusions to study the uxuR regulatory gene in Escherichia coli K-12: direction of transcription and regulation of its expression; Ritzenthaler P et al.; The uxuAB operon is composed of two genes coding for enzymes involved in hexuronate degradation . This operon is negatively controlled by the uxuR and exuR regulatory gene products . Fusions that brought lac gene expression under the control of transcriptional and translational signals within the uxuR gene were used to study uxuR regulation . These fusions were formed on plasmid cloning vectors constructed by Casadaban et al . (J . Bacteriol . 143:971-980, 1980) . The transcriptional direction of the uxuR gene was deduced from the restriction pattern and the phenotypic properties of the new plasmids . The gene is transcribed counterclockwise on the standard Escherichia coli map, as is the uxuAB operon . Introduction in trans of a compatible plasmid carrying a wild-type uxuR gene in the lac fusion plasmid containing strain resulted in a decrease of beta-galactosidase synthesis . It was concluded that expression of the uxuR gene itself is repressed by its own product . The two other types of regulation found in the uxuAB operon, i.e., induction by fructuronate and catabolite control, also apply to the uxuR gene, whereas repression by the exuR repressor does not seem to occur for the uxuR gene. J Bacteriol, 1982 Jun, 150(3), 1016 - 23 The tolC locus of Escherichia coli affects the expression of three major outer membrane proteins; Morona R et al.; tolC mutants, which are resistant to colicin E1 and also highly sensitive to detergents and dyes, were shown to lack the OmpF outer membrane protein . There was little effect on transcription as judged by the use of an ompF-lac operon fusion strain, and the tolC effect was probably due to a post-transcriptional effect . The NmpC protein and protein 2 were also tolC dependent. Biochem J, 1982 Jun 1, 203(3), 769 - 73 Characterization of Neurospora crassa catabolic dehydroquinase purified from N . crassa and Escherichia coli; Hawkins AR et al.; 1 . Neurospora crassa catabolic dehydroquinase has been purified from N . crassa and Escherichia coli . 2 . Protein-sequence and gel-electrophoretic data show that apparently pure, homogeneous native dehydroquinase is a mixture of intact and proteinase-cleaved enzyme monomers . 3 . Protein-sequence data and steady-state kinetics show that the catabolic dehydroquinase gene of N . crassa is expressed with fidelity in E . coli. J Bacteriol, 1982 Jun, 150(3), 1485 - 8 Specialized transduction with lambda plac5: dependence on recB; Porter RD et al.; Genetically disabled lambda plac5 transducing phage derivatives were used to study the recB dependence of recombination during specialized transduction . The frequency of transduction was normalized to colony-forming units, and the end product of recombination was monitored by scoring for addition and substitution transductants . When a chromosomal lac gene was the recipient DNA substrate molecule, both the normalized transduction frequency and the proportion of addition and substitution transductants showed essentially no recB dependence . There was a pronounced recB dependence for both normalized transduction frequency and recombination end product formation when F42 lac was the recipient DNA substrate . recB appears to have no significant role in the recombination that occurs between the two lac regions in an addition transductant . UV irradiation of the transducing phages increased the absolute level of both addition and substitution transductants obtained with a chromosomal lac gene but resulted in a considerable change in the relative frequency of addition versus substitution transductants. Acta Pathol Microbiol Immunol Scand {B}, 1982 Jun, 90(3), 181 - 4 Effect of human leukocyte interferon on phagocytic activity of polymorphonuclear leukocytes; Melby K et al.; The effect of homologous leukocyte interferon HuIFN on the phagocytic activity of human polymorphonuclear (PMN) leukocytes was examined . The PMN cells were obtained from healthy donors and they were grown on coverslips . Escherichia coli labelled with 32P-orthophosphate was phagocytized in the presence of fresh homologous serum during a 15 min incubation period . PMN cells pretreated with interferon for at least 3 hours ingested more bacteria as compared to untreated controls . This effect was dose dependent, in maximal effect being obtained with 10(3) units per ml . The effect was species specific and it was neutralized by antiinterferon globulin. Med Hypotheses, 1982 Jun, 8(6), 635 - 42 Some thoughts on the origin of RNA, and the roles of ribosomes, the nucleolus and the nucleus; Drummond R; The deciphering of DNA's structure led to the formulation of Crick's central dogma, implicit in which is that all RNA is chromosomal in origin . The detection of RNA synthesis at nucleolar sites, plus the similarity between r RNA and nucleolar RNA has firmly established the idea that the nucleolus is responsible for r RNA production . But neither of these considerations actually prove that this is so . An analysis of RNA synthesis in E coli suggests that its r RNA is not chromosomal in origin, but arises by semi conservative replication . The likelihood therefore exists that eukaryotic r RNA also arises by self replication and is not a product of the nucleolus . This concept is favoured by the behaviour of the nucleolus in the differentiating cell in which it seems to have an important role. Eur J Biochem, 1982 Jun, 124(3), 553 - 9 A cleavage site of ribonuclease F . A putative processing endoribonuclease from Escherichia coli; Gurevitz M et al.; A new endoribonuclease activity, RNase F, was partially purified from Escherichia coli cells . This activity can specifically cleave a precursor RNA molecule (of species 1) isolated from T4-infected cells {N . Watson & D . Apirion (1981) Biochem . Biophys . Res . Commun . 103, 543-551} . The cleavage results in products which are very similar to RNA molecules found in the cell, generating a 3'-phosphate and a 5'-hydroxyl groups . The cleavage takes place between a cytosine and an adenine moiety, within a possible loop and stem structure; the cut is in the border between the double-stranded and single-stranded regions of this structure . This specificity of this enzyme could be the introduction of a cleavage near the 3' ends of tRNA molecules and other RNAs like species 1 which could resemble tRNA in their three-dimensional structure. Infect Immun, 1982 Jun, 36(3), 990 - 5 Modulation of natural killer cell activity in mice after interferon induction: depression of activity and depression of in vitro enhancement by interferon; Melder RJ et al.; Splenic natural killer (NK) cell activity of BALB/c and C3H mice was assayed after administration of the interferon inducers Escherichia coli endotoxin or Newcastle disease virus (NDV) . As expected, the NK cell activity rose early in response to the interferon inducers . At 1 to 3 days after an injection of endotoxin, NK activity was hyperesponsive to interferon stimulation . At 5 to 9 days after injection of either endotoxin or NDV, splenic NK activity was depressed, and the spleen cells showed a relative refractoriness to in vitro interferon stimulation . It is postulated that this phenomenon may be related to hyporeactivity, the inability to reinduce interferon after an initial period of interferon production. J Bacteriol, 1982 Jun, 150(3), 1489 - 94 RNase III cleavage of Escherichia coli beta-galactosidase and tryptophan operon mRNA; Shen V et al.; Purified RNase III of Escherichia coli cleaved the initial 479-nucleotide sequence of lac operon mRNA at four specific sites and also gave limited cleavage of trp operon mRNA . This action explains the inactivation of mRNA coding capacity by RNase III in vitro. Biochem Pharmacol, 1982 Jun 1, 31(11), 2005 - 9 Inhibition of Escherichia coli heat-stable enterotoxin effects on intestinal guanylate cyclase and fluid secretion by quinacrine; Greenberg RN et al.; Enterotoxigenic Escherichia coli may produce a heat-stable enterotoxin (ST) that causes diarrheal disease in humans and in animals ST activates particulate guanylate cyclase in intestinal mucosal cells and causes intestinal fluid secretion . In this study, we examined the effects of quinacrine on ST activation of guanylate cyclase and ST-mediated intestinal fluid secretion . Quinacrine significantly reduced ST activation of particulate guanylate cyclase in rat intestinal tissue . Additionally, quinacrine reduced ST-mediated fluid secretion in a rat intestinal loop assay (P less than 0.05) . In the suckling mouse model, subcutaneous quinacrine (0.1 mumole/mouse) reduced ST-induced fluid secretion at a submaximally effective dose of the toxin, but it did not reduce ST-mediated fluid secretion at a near maximally effective dose . Quinacrine (0.1 mumole/mouse) did not significantly reduce intestinal fluid secretion induced by the analog of cyclic GMP, 8-bromo cyclic GMP . However, at a higher concentration of quinacrine (1 mumole/mouse), significant inhibition of 8-bromo cyclic GMP-induced secretion was observed . Inhibition by the antimalarial agent quinacrine of ST-induced fluid secretion, by a block prior to guanylate cyclase activation, suggests a possible role for a phospholipase early in the sequence of events of ST activation of guanylate cyclase . The results suggest that ST may activate membrane phospholipases prior to ST activation of guanylate cyclase. Infect Immun, 1982 Jun, 36(3), 1192 - 8 In vitro adhesion of piliated Escherichia coli to small intestinal villous epithelial cells from rabbits and the identification of a soluble 987P pilus receptor-containing fraction; Dean EA et al.; Type 987P-piliated Escherichia coli adhered in vitro to small intestinal villous epithelial cells and brush borders isolated from adult female rabbits, but not from infant rabbits . K99-piliated E . coli adhered to epithelial cells and brush borders from both adult and infant rabbits . 987P-negative and K99-negative E . coli as well as CFA/I-positive and CFA/I-negative E . coli failed to adhere to epithelial cells or brush borders from either adult or infant rabbits . CFA/II-positive and CFA/II-negative E . coli did not adhere to epithelial cells from infant rabbits . Pretreatment of adult rabbit brush borders with purified 987P pili inhibited adherence of piliated strain 987 . Optimal adherence occurred after 15 min when brush borders and piliated strain 987 were incubated together at 25 degrees C or 37 degrees C in buffers at pH 7 to 8 containing 0.11 to 0.21 M total salts . A receptor-containing fraction that caused aggregation of piliated strain 987 was released into solution when brush borders were stored at 4 degrees C . Receptor activity (aggregation) remained in solution when centrifuged at 10,000 X g for 15 min, but was sedimented at 226,000 X g for 1 h . Receptor activity was abolished by periodate oxidation and pronase digestion. Infect Immun, 1982 Jun, 36(3), 1146 - 53 Adhesion in vitro and in vivo associated with an adhesive antigen (F41) produced by a K99 mutant of the reference strain Escherichia coli B41; Morris JA et al.; A K99-negative mutant of the K99 reference strain Escherichia coli B41 (O101:K99) was isolated (strain B41M) . It did not react with OK antiserum to a K12 K99+ recombinant or with OK antiserum to K99-positive organisms from the O8, O20, or O64 serogroups, but it did react with OK antiserum to K99-positive organisms from the O101 and O9 serogroups . The mutant hemagglutinated sheep erythrocytes and attached in vitro to calf enterocytes when cultured at 37 degrees C but not when grown at 18 degrees C . Attachment was mannose resistant but susceptible to heating and formaldehyde . These properties were associated with the presence of fimbriae . The isolated hemagglutinin migrated to the anode in immunoelectrophoresis experiments, competitively inhibited attachment of strain B41M to calf enterocytes, and could be demonstrated adhering to these cells in vitro by indirect immunofluorescent staining . The anionic hemagglutinin is referred to provisionally as F41 . Germfree piglets infected with strain B41M developed diarrhea within 16 h . Scanning electron microscopy showed groups of bacteria adherent to the microvilli of villous enterocytes . Indirect immunofluorescent staining demonstrated the presence of F41 antigen in vivo. J Bacteriol, 1982 Jun, 150(3), 1302 - 13 Fine-structure deletion map and complementation analysis of the glnA-glnL-glnG region in Escherichia coli; MacNeil T et al.; A total of 399 independent mutants of Escherichia coli were obtained which have point and insertion mutations in the glnA region . Mutants isolated included Gln- and Reg- strains (unable to utilize arginine as a nitrogen source) . Mutations were mapped with 73 deletion-containing derivatives of a lambda gln phage . Complementation analysis was performed with lambda gln derivatives containing point mutations which conferred a Gln- or Reg- phenotype . Deletion mapping and complementation analysis assigned 104 mutations in 24 deletion intervals to glnA . Mutations in Reg- strains were assigned to two genes, glnL and glnG . glnL contained 131 mutations in 12 deletion intervals, and glnG contained 164 mutations in 10 deletion intervals . The gene order is glnA-glnL-glnG, transcribed from left to right . Polarity of insertion mutations indicates that glnL and glnG form from left to right . Polarity of insertion mutations indicates that glnL and glnG form an operon . Complementation analysis of glnA insertion mutations with glnL and glnG mutations showed polarity of glnA onto most glnL and glnG alleles, suggesting that transcription of glnA may proceed into the glnL-glnG operon . All mutations analyzed in glnA conferred a Gln- phenotype . However, we also found that over half of the Gln- strains isolated ater chemical mutagenesis contained point mutations in glnG . Mutants which synthesized a high level of glutamine synthetase in the presence of ammonia (GlnC phenotype) were selected as revertants of a strain with a Tn10 insertion in glnD and were mapped with chromosomal deletions . Results indicate that mutations in 12 and 15 examined strains clearly map outside of glnA, probably in glnL. Biochim Biophys Acta, 1982 May 31, 697(2), 174 - 7 Protein synthesis using poly(2'-halogeno-2'-deoxyadenylic acids) as messenger; Fukui T et al.; Poly(2'-fluoro-2'-deoxyadenylic acid), poly(2'-chloro-2'-deoxyadenylic acid) and poly(2'-bromo-2'-deoxyadenylic acid) are used as messenger RNAs in protein synthesizing systems in vitro . All polynucleotides were active as messengers and {14 C}lysine was incorporated into polypeptides . The initial velocity of polylysine formation was greater using poly(2'-fluoro-2'-deoxyadenylic acid) as messenger than in the case of poly(rA), and all synthetic messengers lived longer in the protein synthetic system. Biochim Biophys Acta, 1982 May 31, 697(2), 255 - 8 Restriction enzyme cleavage of ultraviolet-damaged DNA; Cleaver JE et al.; SV40 and pBR322 DNAs damaged by ultraviolet light were cleaved abnormally by several restriction enzymes because of damage to pyrimidines in the recognition sequences . The use of a tandemly duplicated plasmid provided a particularly sensitive target molecule for detecting pyrimidine dimers and other possible photoproducts . The relative efficiency with which cleavage was blocked (HindIII greater than TaqI greater than EcoRI greater than BamI greater than SalI much greater than Hha I, Hae III) corresponds approximately to the relative frequency of pyrimidine dimer formation in the recognition sequences, but at a slightly higher frequency in potential sites for the non-cyclobutane T-C product . The pyrimidine dimers appear to have a range of influence that extends 1 to 3 basepairs along the DNA molecule . These effects provide clues to the way DNA damage from mutagens and carcinogens can interfere with specific enzyme-DNA interactions. Science, 1982 May 28, 216(4549), 999 - 1001 Ribosomal crystalline arrays of large subunits from Escherichia coli; Clark MW et al.; Crystalline sheets of the 50S ribosomal subunits of Escherichia coli have been formed in vitro . Electron micrographs of these arrays diffract to 35-angstrom resolution . The lattice parameters of the crystals are a = 330 +/- 20 angstroms, b = 330 +/- 30 angstroms, and alpha = 123 degrees +/- 5 degrees, and the space group is most likely p21 . These arrays of ribosomal subunits are sufficiently ordered to resolve such known features of the large ribosomal subunit as the L7/L12 stalk and the central protuberance. Biochemistry, 1982 May 25, 21(11), 2702 - 13 Neutron scattering data on reconstituted complexes of fd deoxyribonucleic acid and gene 5 protein show that the deoxyribonucleic acid is near the center; Gray DM et al.; We have performed low-angle neutron scattering studies on reconstituted complexes of fd DNA and the gene 5 protein that is produced during infection of Escherichia coli by filamentous fd phage . Essentially identical helical complexes have been made with normal protonated DNA or DNA in which at least 87% of the nonexchangeable protons are replaced by deuterium . From neutron scattering profiles of both complexes over a range of D2O/H2O solvent mixtures, the DNA deuteration is shown to have a dramatic influence on the measured cross-sectional radius of gyration . Most importantly, data for the complex containing deuterated DNA lead to a more negative slope in a plot of the square of the cross-sectional radius of gyration vs . the inverse of the solute-solvent contrast, compared with the slope of a plot of data for the complex containing protonated DNA . This means that, in a cross-sectional view of the complex, the DNA is near the center of the structure . By our analysis, the DNA has a cross-sectional radius of gyration of 17.6 +/- 3 A, while the protein has a cross-sectional radius of gyration of about 33.5 A . Therefore, the model for the structure of the helical complex that has been proposed from X-ray diffraction studies on gene 5 protein crystallized with oligodeoxynucleotides {McPherson, A., Jurnak, F., Wang, A., Kolpak, F., Rich, A., Molineux, I., & Fitzgerald, P . (1980) Biophys . J . 32, 155-170} is not valid for the complex in solution . From our neutron diffraction data we have also obtained values for the solvent-excluded volume and mass per unit length . The relation of our findings to the solution structure of the complex is discussed. Biochemistry, 1982 May 25, 21(11), 2656 - 600 Role of arginine in the binding of thiamin pyrophosphate to Escherichia coli pyruvate oxidase; Koland JG et al.; The mode of interaction between Escherichia coli pyruvate oxidase and its cofactor, thiamin pyrophosphate (TPP), was studied with the aid of arginine-directed reagents . The enzyme is rapidly inactivated by either phenylglyoxal or 2,3-butanedione, with the cofactor, TPP, offering partial protection against these reagents . The inactivation by phenylglyoxal was found to be reversible . Experiments with {7-14C}phenylglyoxal showed that while several arginine residues react with this reagent, TPP can prevent the labeling of one such residue . Furthermore, inactivation by 2,3-butanedione is attended by at least a 100-fold decrease in affinity of the enzyme for TPP . These results suggest a direct role for arginine in the binding of the cofactor. Biochemistry, 1982 May 25, 21(11), 2586 - 92 Guanidine hydrochloride induced unfolding of the alpha subunit of tryptophan synthase and of the two alpha proteolytic fragments: evidence for stepwise unfolding of the two alpha domains; Miles EW et al.; The relationship between the domain structure of the alpha subunit of Escherichia coli tryptophan synthase and the mechanism of unfolding of the alpha subunit is investigated . Previous studies of the unfolding of the alpha subunit by increasing concentrations of guanidine hydrochloride or urea detected a partially unfolded form of the alpha subunit at intermediate concentrations of either denaturant . The possibility that this partially unfolded form of the alpha subunit results from the preferential unfolding of one of the two domains of the alpha subunit is now investigated . This study utilizes two proteolytic fragments of the alpha subunit, alpha-1 and alpha-2, which have been shown to refold independently and to correspond to two domains of the alpha subunit . The effects of guanidine hydrochloride concentration on the separate alpha-1 and alpha-2 fragments, on the intact alpha subunit, and on the derivative nicked by trypsin (alpha') are compared by measuring ellipticity at 222 nm and by measuring the susceptibility of tyrosyl residues to chemical modification . The results show that guanidine hydrochloride induced unfolding of the alpha subunit results from the stepwise unfolding of the two domains: the alpha-2 fragment and the corresponding domain in the intact alpha subunit are unfolded by low concentrations of guanidine hydrochloride; the alpha-1 fragment and the corresponding domain in the intact alpha subunit are unfolded by higher concentrations of guanidine hydrochloride. J Biol Chem, 1982 May 25, 257(10), 5852 - 60 Localization and processing of outer membrane and periplasmic proteins in Escherichia coli strains harboring export-specific suppressor mutations; Emr SD et al.; Mutations at three genetic loci (termed prlA,B,C) were previously shown to specifically suppress signal sequence mutations in the lamB gene encoding the outer membrane phage lambda receptor protein of Escherichia coli (Emr, S . D., Hanley-Way, S., and Silhavy, T . J . (1981) Cell 23, 79-88) . The majority of these suppressor mutations map at the prlA locus and are thought to result in an altered ribosomal protein . In this study, we demonstrate that prlA mutations also phenotypically suppress signal sequence mutations in the malE gene encoding the periplasmic maltose-binding protein . For both lamB and malE mutations, suppression is achieved by transporting the export-defective protein to its correct extracytoplasmic location, in some instances with near 100% efficiency . With a single exception, the mutant-exported protein is apparently processed to its normal mature form . These results indicate that prlA-mediated protein export occurs via the usual route, and additional data suggest that the prlA product directly interacts with the mutant signal sequence to restore export . The single prlC allele also suppresses malE signal sequence mutations, whereas the single prlB allele only phenotypically suppresses lamB signal sequence mutations . However, with these latter two suppressors, there is some indication that export of the phage lambda receptor to the outer membrane is not accomplished by the usual route. J Biol Chem, 1982 May 25, 257(10), 5692 - 9 Size classes of products synthesized processively by two subassemblies of Escherichia coli DNA polymerase III holoenzyme; Fay PJ et al.; Two forms of Escherichia coli DNA polymerase III, DNA polymerase III', and DNA polymerase III have been shown to synthesize DNA products via a processive mechanism with product sizes distinctive for each enzyme form . These forms of DNA polymerase III are intermediate in complexity between the core DNA polymerase III and the DNA polymerase