Escherichia coli

J Chromatogr, 1986 Oct 10, 368(1), 113 - 24
Hydrophobic interaction chromatography of incompletely methylated transfer RNA from Escherichia coli on octyl-sepharose; Sindhuphak T et al.; Phenylalanine-specific transfer RNA from methionine-starved relaxed Escherichia coli K12 separates into two components when chromatographed on Octyl-Sepharose . The difference in elution between the two tRNAs has been shown to depend on the methyl group in the highly modified 2-methylthio-N-6-isopentenyladenosine . The first eluted tRNAPhe lacks this methyl group, while the last eluted tRNAPhe is fully methylated . Other differences in the modification patterns have no effect on the elution from Octyl-Sepharose . The elution pattern of tyrosine- and serine-specific tRNAs, also normally containing ms2i6A, is similar.

Biochim Biophys Acta, 1986 Oct 9, 861(2), 361 - 7
Fluorescence polarization studies on Escherichia coli membrane stability and its relation to the resistance of the cell to freeze-thawing . II . Stabilization of the membranes by polyamines; Souzu H; The effects of polyamines, spermine, spermidine and putrescine on the stabilization of the membrane organization of Escherichia coli cells were studied using measurements of fluorescence polarization change of extrinsic fluorescence probes in membrane specimens as a function of temperature . The effects of the polyamines on the restoration of the cell viability after freeze-thawing were also investigated . In logarithmic-phase membrane specimens, polyamines depressed the polarization ratio increase below the transition temperatures in a dose-dependent manner . The physiologically relevant concentration of polyamines repressed the ratios to the same levels as are obtained with the stationary-phase specimens . In the stationary-phase specimens, no effect of polyamines on repression of the polarization increase was observed . A preliminary exposure of logarithmic-phase cells to polyamines protected the cells from the reduction of viability in freeze-thawing . However, a considerably high concentration and a certain length of preincubation time were required in order to an effect to be exerted . These results indicate that the intracellular polyamines could stabilize the membrane organization of logarithmic-phase cells to the same extent as in the stationary-phase cell membranes . It is conjectured that the membrane stability which is mediated by the polyamines results in cellular resistance to freeze-thawing, as it is attained by increasing the growth phase of the cells.

Biochim Biophys Acta, 1986 Oct 9, 861(2), 353 - 60
Fluorescence polarization studies on Escherichia coli membrane stability and its relation to the resistance of the cell to freeze-thawing . I . Membrane stability in cells of differing growth phase; Souzu H; Physical properties of Escherichia coli membrane lipids in logarithmic- and stationary-phase cells were studied by measuring the fluorescence polarization change of cis- and trans-parinaric acid as a function of temperature . In aqueous dispersions of phospholipids extracted from cytoplasmic and outer membranes of cells of differing growth phase, a similar polarization increase was observed over the range from physiological temperature to below 0 degrees C, and nearly the same transition ratios were obtained in all samples . The cytoplasmic membrane of both of the growth-phase cells showed a higher polarization ratio above the transition temperatures, compared to that in the aqueous dispersion of phospholipids . The polarization ratios below the transition temperatures of these specimens were lower than the value obtained with the lipids, especially in the stationary-phase specimens . The outer membrane specimens showed a similar polarization change but the transition temperature ranges were considerably higher both in the logarithmic- and the stationary-phase specimens, compared to those in the cytoplasmic membrane specimens . Freeze-thawing of logarithmic-phase cells showed the emergence of activity of certain enzymes which are known to be located in the membranes . The stationary-phase cells did not suffer from any such deleterious effect and maintained a high level of cell viability in a similar treatment . These results indicate that in the stationary-phase cell membranes lipids are in a highly ordered state, and the lipid state causes a membrane stability which results in the high resistance of the cell to freeze-thawing.

Biochim Biophys Acta, 1986 Oct 8, 851(3), 457 - 68
Rapid redox equilibrium between the mitochondrial Q pool and cytochrome b during triphasic reduction of cytochrome b by succinate; Chen M et al.; The reliability of monitoring the redox reactions of cytochrome b using the different wavelengths employed by different authors has been reexamined . It was found that 562-575 nm is suitable in succinate: cytochrome c reductase but not in mitochondria, in which case 562-540 nm is a better pair . Direct optical measurements of the redox reaction kinetics of the mitochondrial Q pool using a commercial dual-wavelength spectrophotometer are possible when succinate is used as the electron donor . Using the correct wavelength pair, and with malonate to slow down the electron input, the reduction course of cytochrome b was still triphasic but a plateau or a turn replaced the oxidation phase previously reported by several authors . At the same time, the reduction course of the Q pool was also triphasic, and in perfect match with that of cytochrome b . Destruction of the Rieske iron-sulfur cluster by British anti-Lewisite (BAL) + O2 treatment or prereduction of the high-potential components made the reduction of both Q and b monophasic . The plot of log (Q/QH2) against log (b3+/b2+) gave a straight line with an n value of 1.7 for cytochrome b at pH 7.4 . This n value rose to 2.0 at pH 6.5 and dropped to 1.4 at pH 8.5 . On the other hand, the mid-point potential of cytochrome b relative to that of the Q pool remained essentially unchanged between pH 6.5 and 8.4 . BAL treatment had a small effect on the midpoint potential of cytochrome b relative to that of the Q pool and had no effect on the n value . Addition of quinone homologues and analogues extended the plateau phase in the reduction of cytochrome b, but exogenous quinones did not equilibrate rapidly with cytochrome b . It was concluded that the appearance of the plateau between the two reduction phases of Q and b is caused by the rapid delivery of electrons to the high-potential components of the respiratory chain as envisaged in the Q cycle; the unexpected n value for cytochrome b suggests a concerted reduction by QH2 of two species of cytochromes b-562.

Biochim Biophys Acta, 1986 Oct 8, 851(3), 385 - 94
Effect of disulfide cross-linking between alpha and delta subunits on the properties of the F1 adenosine triphosphatase of Escherichia coli; Bragg PD et al.; Under very mild oxidizing conditions the delta subunit of the F1-ATPase of Escherichia coli can be crosslinked by a disulfide linkage to one of the alpha subunits of the enzyme . The cross-linked ATPase resembles the native enzyme in the following properties: specific activity; activation by lauryldimethylamine N-oxide (LDAO); binding of aurovertin D and ADP; cross-linking products with 3,3'-dithiobis(succinimidyl propionate); binding to ATPase-stripped everted membrane vesicles and the N,N'-dicyclohexylcarbodiimide sensitivity of the rebound enzyme . However, the rebound crosslinked ATPase differed from the native enzyme in lacking the ability to restore NADH oxidation - and ATP hydrolysis-dependent quenching of the fluorescence of quinacrine to ATPase-stripped membrane vesicles . It is proposed that the delta subunit is involved in the proton pathway of the ATPase, and that this pathway is affected in the alpha delta-cross-linked enzyme . The mechanism for activation of the ATPase by LDAO was examined . Evidence against the proposal of Lotscher, H.-R., De Jong, C . and Capaldi, R.A . (Biochemistry (1984) 23, 4140-4143) that activation involves displacement of the epsilon subunit from an active site on a beta subunit was obtained.

Biochemistry, 1986 Oct 7, 25(20), 6127 - 32
Evidence for a protonmotive force related regulatory system in Escherichia coli and its effects on lactose transport; Plate CA et al.; Strains of Escherichia coli with mutations in the eup (energy-uncoupled phenotype) locus do not grow on nonfermentable carbon sources, have reduced growth yields on limiting glucose, are insensitive to colicins A and K, exhibit resistance to aminoglycoside antibiotics, and are defective in protonmotive force coupled active transport . eup mutations do not result in lowered protonmotive force . Here we show that deenergization of a eup+ strain results in the appearance of a new low KT, low Vmax form of the lactose carrier; in a strain deleted of the eup locus, deenergization does not evoke the low KT, low Vmax form of the lactose carrier . Cells bearing a eup point mutation and exhibiting the Eup- phenotype possess the low KT, low Vmax form of the lactose carrier even when energized . In addition to affecting the kinetic parameters of the lactose carrier, the eup point mutation also reduces the KT and Vmax of the proline carrier . On the basis of these findings, we suggest that the normal eup gene product mediates a novel regulation of lactose carrier function following deenergization . The defect in proline and lactose transport caused by eup point mutations may stem from an altered eup product aberrantly mediating the regulation under energized conditions . Finally, the pleiotropy associated with eup point mutations may be indicative of those protonmotive force driven functions that are subject to eup regulation.

Biochemistry, 1986 Oct 7, 25(20), 5992 - 8
An engineered intersubunit disulfide enhances the stability and DNA binding of the N-terminal domain of lambda repressor; Sauer RT et al.; Site-directed mutagenesis has been used to replace Tyr-88 at the dimer interface of the N-terminal domain of lambda repressor with cysteine . Computer model building had suggested that this substitution would allow formation of an intersubunit disulfide without disruption of the dimer structure {Pabo, C . O., & Suchanek, E . G . (1986) Biochemistry (preceding paper in this issue)} . We find that the Cys-88 protein forms a disulfide-bonded dimer that is very stable to reduction by dithiothreitol and has increased operator DNA binding activity . The covalent Cys88-Cys88' dimer is also considerably more stable than the wild-type protein to thermal denaturation or urea denaturation . As a control, Tyr-85 was replaced with cysteine . A Cys85-Cys85' disulfide cannot form without disrupting the wild-type structure, and we find that this disulfide bond reduces the DNA binding activity and stability of the N-terminal domain.

Biochemistry, 1986 Oct 7, 25(20), 5882 - 9
Direct ATP photolabeling of Escherichia coli recA proteins: identification of regions required for ATP binding; Banks GR et al.; When the Escherichia coli RecA protein is UV irradiated in the presence of {alpha-32P}ATP, a labeled protein--ATP adduct is formed . All the experimental evidence indicates that, in forming such an adduct, the ATP becomes specifically immobilized in the catalytically relevant ATP binding site . The adduct can also be identified after irradiation of E . coli cell lysates in a similar manner . This direct ATP photolabeling of RecA proteins has been used to identify regions of the polypeptide chain involved in the binding of ATP . The photolabeling of a RecA protein that lacks wild-type carboxy-terminal amino acids is not detectable . A RecA protein in which the amino-terminal sequence NH2-Ala-Ile-Asp-Glu-Asn- is replaced by NH2-Thr-Met-Ile-Thr-Asn-Ser-Ser-Ser- is only about 5% as efficiently photolabeled as the wild-type protein . Both of these RecA protein constructions, however, contain all the elements previously implicated, directly or indirectly, in the binding of ATP . ATP-photolabeled RecA protein has also been chemically cleaved at specific amino acids in order to identify regions of the polypeptide chain to which the nucleotide becomes covalently photolinked . The evidence is consistent with a region comprising amino acids 116-170 . Thus, this work and that of others suggest that several disparate regions of the unfolded polypeptide chain may combine to form the ATP binding site upon protein folding or may influence binding through long-range effects.

Biochemistry, 1986 Oct 7, 25(20), 5872 - 81
Interaction of recA protein with a photoaffinity analogue of ATP, 8-azido-ATP: determination of nucleotide cofactor binding parameters and of the relationship between ATP binding and ATP hydrolysis; Kowalczykowski SC; The binding and cross-linking of the ATP photoaffinity analogue 8-azidoadenosine 5'-triphosphate (azido-ATP) with recA protein have been investigated, and through cross-linking inhibition studies, the binding of other nucleotide cofactors to recA protein has also been studied . The azido-ATP molecule was shown to be a good ATP analogue with regard to recA protein binding and enzymatic function by three criteria: first, the cross-linking follows a simple hyperbolic binding curve with a Kd of 4 microM and a cross-linking efficiency ranging from 10% to 70% depending on conditions; second, ATP, dATP, and adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S) specifically inhibit the cross-linking of azido-ATP to recA protein; third, azido-ATP is a substrate for recA protein ATPase activity . Quantitative analysis of the cross-linking inhibition studies using a variety of nucleotide cofactors as competitors has shown that the binding affinity of adenine-containing nucleotides for recA protein decreases in the following order: ATP-gamma-S greater than dATP greater than ATP greater than adenylyl beta,gamma-imidodiphosphate (AMP-PNP) much greater than adenylyl beta,gamma-methylenediphosphate (AMP-PCP) approximately adenine . Similar competition studies also showed that nearly all of the other nucleotide triphosphates also bind to recA protein, with the affinity decreasing in the following order: UTP greater than GTP approximately equal to dCTP greater than dGTP greater than CTP . In addition, studies performed in the presence of single-stranded DNA demonstrated that the affinity of ATP, dATP, ATP-gamma-S, and AMP-PNP for recA protein is significantly increased . These results are discussed in terms of the reciprocal effects that nucleotide cofactors have on the modulation of recA protein--single-stranded DNA binding affinity and vice versa . In addition, it is demonstrated that nucleotide and DNA binding are necessary though not sufficient conditions for ATPase activity . The significance of this result in terms of the possible requirement of critically sized clusters of 15 or more recA protein molecules contiguously bound to DNA for ATPase activity is discussed.

Biochemistry, 1986 Oct 7, 25(20), 5920 - 8
An RNA polymerase mutant with reduced accuracy of chain elongation; Blank A et al.; A new Escherichia coli RNA polymerase mutant was isolated which exhibited reduced accuracy of chain elongation in vivo and in vitro . The novel isolation procedure consisted of simultaneous selection for rifampicin resistance and screening for increased leakiness of an early, strongly polar nonsense mutation of lacZ, one of a special class of mutations whose leakiness reflects mainly transcriptional rather than translational errors . The spontaneous mutant thus isolated displayed a 3-4-fold increase in the leakiness of two different lacZ mutations of this class . Transduction analysis indicated that a single mutation, mapping in or very near the rpoB gene for the beta subunit of RNA polymerase, conferred both rifampicin resistance and increased nonsense leakiness . In an in vitro fidelity assay, homogeneous RNA polymerases from the mutant and parent strains exhibited error rates of 1/0.90 X 10(5) and 1/2.0 X 10(5), respectively, for the poly{d(A-T)} X poly{d(A-T)}-directed misincorporation of noncomplementary GMP . These error rates were verified by product analyses which further revealed that GMP was misincorporated in place of AMP in the synthesis of poly{r(A-U)} . The error rate of wild-type K12 RNA polymerase from a different source was 1/2.0 X 10(5), while that of a hybrid RNA polymerase, containing mutant core enzyme and wild-type sigma subunit, was 1/0.64 X 10(5) . These error rates confirmed the selection of a transcriptional accuracy mutant . The error frequencies observed are much lower than those reported in other in vitro assays . The safeguards used to avoid artifactually enhanced misincorporation, and to thereby quantitate lower error rates, are discussed.

Biochemistry, 1986 Oct 7, 25(20), 5914 - 9
The sigma subunit of RNA polymerase contacts the leading ends of transcripts 9-13 bases long on the lambda PR promoter but not on T7 A1; Bernhard SL et al.; The sigma subunit of RNA polymerase is responsible for specific initiation of RNA synthesis at promoter sites on DNA . sigma dissociates shortly after initiation . Photoaffinity-labeling experiments performed on transcription complexes with two different DNA promoters, which have highly homologous control sequences upstream from the transcribed regions, have revealed that the sigma subunit of RNA polymerase is contacted by the 5' ends of quite different lengths of nascent RNA in each transcription complex . On the other hand, the labeling of subunits beta beta' is quite similar for both promoters, and the alpha subunit is not labeled in either case . The results of transcription experiments on the phage lambda PR promoter show that sigma can be photoaffinity labeled by RNA chains that are 9-13 nucleotides long and thus remains associated with the core enzyme at least to that point . But on the A1 promoter of phage T7 DNA, photoaffinity labeling of sigma ceases with the trinucleotide . Thus release of sigma from the vicinity of nascent RNA depends not merely on the length but on the sequence of the transcript . For the T7 A1 promoter, sigma labeling ceases while the leading end of the RNA is still base paired to the DNA template; thus, it appears that there is at least one site on the enzyme that interacts with the growing transcript/template hybrid, in a sequence-dependent way, to effect sigma release.(ABSTRACT TRUNCATED AT 250 WORDS)

FEBS Lett, 1986 Oct 6, 206(2), 193 - 8
Chain folding in the dihydrolipoyl acyltransferase components of the 2-oxo-acid dehydrogenase complexes from Escherichia coli . Identification of a segment involved in binding the E3 subunit; Packman LC et al.; The state of assembly of the pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes was examined after the dihydrolipoyl acyltransferase (E2) component of each enzyme system had been subjected to varying degrees of limited proteolysis . Dissociation of the dihydrolipoyl dehydrogenase (E3) component accompanied specifically the excision of a homologous segment of each E2 chain that connects the N-terminal lipoyl domain(s) with a C-terminal catalytic domain . The latter remains aggregated as a 24-mer and retains its capacity to bind the 2-oxo-acid decarboxylase (E1) component . The relevant segment of the E2o chain from the 2-oxoglutarate dehydrogenase complex was isolated and shown to be a folded protein which still binds to E3.

J Biol Chem, 1986 Oct 5, 261(28), 13000 - 5
Steady-state kinetic studies of superoxide dismutases . Saturative behavior of the copper- and zinc-containing protein; Fee JA et al.; The mechanism of the Cu-Zn-containing superoxide dismutase (SD) was studied using a stopped-flow spectrophotometric system capable of forming aqueous solutions of O2- having initial concentrations up to approximately 5 mM . By lowering the temperature to 5.5 degrees C, we were able to observe saturation of the enzyme . At 5.5 degrees C and pH 9.3, the Michaelis-Menten parameters extracted from the kinetic traces were turnover number (TN) approximately 1 X 10(6) s-1, Km approximately 3.5 X 10(-3) M . Under our conditions, the average rate at which O-2 binds to the active site, TN/Km is 0.26 X 10(9) M-1 s-1 . TN was decreased in the presence of D2O, and a solvent isotope effect of TNH/TND approximately 3.6 was measured while TN/Km was essentially unaffected by D2O . TN was increased by the presence of the general acid, ND4+ . These observations, by analogy to earlier work with Fe X SD from Escherichia coli (Bull, C., and Fee, J . A . (1985) J . Am . Chem . Soc . 107, 3295-3304), suggest that H2O serves to donate the protons required to form product H2O2 . Values of Km and TN for the zinc-deficient enzyme were found to be approximately a factor of 2 less than those obtained for the holoenzyme under identical experimental conditions, whereas TN/Km was largely unchanged . The imidazolate bridge is thus not essential for catalytically competent extraction of a proton from the solvent.

J Mol Biol, 1986 Oct 5, 191(3), 355 - 65
Regulated yeast promoters produced by DNA rearrangements selected in vivo; Scherer S; DNA rearrangements that activated a promoterless his3 gene were selected in vivo . DNA segments that promote the expression of his3 were identified in Ty1 DNA sequences and a variety of sites in the vector DNA . These elements appear to function when placed in either orientation relative to his3 but not when placed at the 3' end of the his3 gene . Promoter elements regulated by carbon source, nitrogen source, pyrimidines or galactose were characterized . The assembly of more complex regulatory elements and transposons from these units is discussed.

J Biol Chem, 1986 Oct 5, 261(28), 12988 - 93
ATP-independent renaturation of complementary DNA strands by the mutant recA1 protein from Escherichia coli; Bryant FR et al.; In an effort to clarify the requirement for ATP in the recA protein-promoted renaturation of complementary DNA strands, we have analyzed the mutant recA1 protein which lacks single-stranded DNA-dependent ATPase activity at pH 7.5 . Like the wild type, the recA1 protein binds to single-stranded DNA with a stoichiometry of one monomer per approximately four nucleotides . However, unlike the wild type, the mutant protein is dissociated from single-stranded DNA in the presence of ATP or ADP . The ATP analogue adenosine 5'-O-3' (thiotriphosphate) appears to stabilize the binding of recA1 protein to single-stranded DNA but does not elicit the stoichiometry of 1 monomer/8 nucleotides or the formation of highly condensed protein-DNA networks that are characteristic of the wild type recA protein in the presence of this analogue . The recA1 protein does not catalyze DNA renaturation in the presence of ATP, consistent with the dissociation of recA1 protein from single-stranded DNA under these conditions . However, it does promote a pattern of Mg2+-dependent renaturation identical to that found for wild type recA protein.

J Mol Biol, 1986 Oct 5, 191(3), 313 - 20
malM, a new gene of the maltose regulon in Escherichia coli K12 . II . Mutations affecting the signal peptide of the MalM protein; Rousset JP et al.; malM is the last gene of the malK-lamB-malM operon of Escherichia coli K12 . It encodes a periplasmic protein . Mutations affecting the hydrophobic core of the N-terminal extension of the MalM protein have been isolated . They result in an increase in amount and specific activity of a MalM-LacZ hybrid protein . This result confirms that the signal peptide of the MalM protein is functional.

J Mol Biol, 1986 Oct 5, 191(3), 333 - 40
Probing the structural domains and function in vivo of Escherichia coli DNA topoisomerase I by mutagenesis; Zumstein L et al.; Insertion and deletion mutagenesis within the gene topA of Escherichia coli encoding DNA topoisomerase I was carried out to test the existence of subdomains in the enzyme and the relationship between the slow-growth topA- phenotype and the known DNA relaxation activity of the enzyme . All mutants that show no detectable DNA relaxation activity in cell extracts fail to complement the temperature-sensitive growth defect of strain AS17 topAam harboring a plasmid-borne temperature-sensitive suppressor tRNA . All mutants that show partial or full levels of DNA relaxation activity in cell extracts (relative to activity in extracts of wild-type cells) can complement this defect . The carboxyl-proximal 25% of the enzyme appears to be in a domain that is dispensable both in terms of the catalytic function of the enzyme and its biological role . Analysis of the mutant enzyme also indicates that the formation of the covalent topoisomerase-DNA complex is correlated with the DNA relaxation activity, which supports the notion that the covalent complex is an obligatory intermediate in the catalysis of DNA topoisomerization.

J Mol Biol, 1986 Oct 5, 191(3), 321 - 31
Complete nucleotide sequence of the topA gene encoding Escherichia coli DNA topoisomerase I; Tse-Dinh YC et al.; The nucleotide sequence of a 4071 base-pair long segment containing the gene topA encoding Escherichia coli DNA topoisomerase I and its flanking regions has been determined . The gene encodes a total of 864 amino acids from the ATG start to a TAA termination codon, of which the first f-Met appears to be removed after translation; the calculated molecular weight of the translated protein is 97,413 . Mapping of promoters by deletion of sequences upstream from the ATG initiation codon indicates the existence of at least two promoters that direct transcription into topA.

J Mol Biol, 1986 Oct 5, 191(3), 483 - 93
Interconversion of active and inactive 30 S ribosomal subunits is accompanied by a conformational change in the decoding region of 16 S rRNA; Moazed D et al.; Zamir, Elson and their co-workers have shown that 30 S ribosomal subunits are reversibly inactivated by depletion of monovalent or divalent cations . We have re-investigated the conformation of 16 S rRNA in the active and inactive forms of the 30 S subunit, using a strategy that is designed to eliminate reversible ion-dependent conformational effects that are unrelated to the heat-dependent Zamir-Elson transition . A combination of structure-specific chemical probes enables us to monitor the accessibility of pyrimidines at N-3 and purines at N-1 and N-7 . Chemically modified bases are identified by end-labeling followed by analine-induced strand scission (in some cases preceded by hybrid selection), or by primer extension using synthetic DNA oligomers . These studies show the following: The transition from the active to the inactive state cannot be described as a simple loosening or unfolding of native structure, such as that which is observed under conditions of more severe ion depletion . Instead, it has the appearance of a reciprocal interconversion between two differently structured states; some bases become more reactive toward the probes, whilst others become less reactive as a result of inactivation . Changes in reactivity are almost exclusively confined to the "decoding site" centered at positions 1400 and 1500, but significant differences are also detected at U723 and G791 in the central domain . This may reflect possible structural and functional interactions between the central and 3' regions of 16 S rRNA . The inactive form also shows significantly decreased reactivity at positions 1533 to 1538 (the Shine-Dalgarno region), in agreement with earlier findings . The principal changes in reactivity involve the universally conserved nucleotides G926, C1395, A1398 and G1401 . The three purines show reciprocal behavior at their N-1 versus N-7 positions . G926 loses its reactivity at N-1, but becomes highly reactive at N-7 as a result of the transition of the inactive state . In contrast, A1398 and G1401 become reactive at N-1, but lose their hyper-reactivity at N-7 . The possible structural and functional implications of these findings are discussed.

J Mol Biol, 1986 Oct 5, 191(3), 303 - 11
malM, a new gene of the maltose regulon in Escherichia coli K12 . I . malM is the last gene of the malK-lamB operon and encodes a periplasmic protein; Gilson E et al.; The structure and expression of the distal part of the malK-lamB operon in Escherichia coli was studied . DNA sequencing was performed as far as a HinfI restriction site located 1313 base-pairs downstream from gene lamB . The open reading frame, formerly called molA, which begins 245 base-pairs downstream from gene lamB, is longer than was initially thought, and was renamed malM . It could encode a protein of 306 amino acid residues . The complete malM open reading frame was cloned under control of the tac 12 promoter . In maxicells, the resulting plasmid permitted tac12-promoted synthesis of two polypeptides, encoded by gene malM, with apparent molecular weights of 37 X 10(3) and 34.5 X 10(3) . We provide strong evidence that the 34.5 X 10(3) Mr protein is derived from the 37 X 10(3) Mr protein by processing at the amino-terminal end, and that this processed form is located in the periplasmic space . We show that the chromosomal malM gene is expressed as part of the malK-lamB operon, and that its product is periplasmic . Finally, we demonstrate with nuclease S1 mapping experiments that the mRNA terminates at a typical rho-independent terminator located about 45 base-pairs beyond the end of gene malM, which is thus the last gene of the malK-lamB operon.

Avian Dis, 1986 Oct-Dec, 30(4), 766 - 71
Induction, collection, and partial characterization of induced respiratory macrophages of the turkey; Ficken MD et al.; Respiratory macrophages (RM) of the turkey were elicited with a 1:4 (v/v) suspension of incomplete Freund's adjuvant in sterile phosphate-buffered saline injected directly into the abdominal air sacs . RM were purified by passage through a Ficoll-Hypaque gradient resulting in 95.7 +/- 5.9% purity and 94.8 +/- 12.3% viability . On days 7 and 9 postinjection, adequate numbers (7.15 +/- 5.47 X 10(6) macrophages per turkey) of RM for in vitro experiments were obtained . RM of the turkey demonstrated the ability to adhere to glass, phagocytize Zymosan A, and kill Escherichia coli in vitro.

J Antimicrob Chemother, 1986 Oct, 18 Suppl C, 67 - 76
New observations regarding evolution of trimethoprim resistance; Tennhammar-Ekman B et al.; A clinically isolated strain of Escherichia coli, resistant to more than 1000 mg/l of trimethoprim, expressed chromosomal dihydrofolate reductase to a level 200-fold higher than that of drug sensitive E . coli K-12 strains, and this high cellular enzyme activity was found to increase further when the cells were cultured in the presence of trimethoprim . The induced increase in enzyme activity was dependent on the drug concentration . The increase was six-fold at 100 mg/l of trimethoprim . The aberrantly regulated dihydrofolate reductase gene mediating trimethoprim resistance could be transduced into E . coli K-12 or moved by recombination into an F' factor and then transferred into trans position in relation to the corresponding chromosomal gene . In either of these positions, the synthesis of dihydrofolate reductase could be induced to increase by adding trimethoprim to the culture medium . The observed induction was dependent on protein synthesis, since it could be abolished by chloramphenicol . No other folic acid analogue was found to induce increased expression of the dihydrofolate reductase gene . Also thymine starvation had no effect . Two further clinical isolates of E . coli, highly resistant to trimethoprim, were shown to produce drug resistant, plasmid-mediated dihydrofolate reductases, which were distinct from the earlier known enzyme types I and II.

Am J Vet Res, 1986 Oct, 47(10), 2193 - 6
Effects of ketanserin on pulmonary hemodynamics, lung mechanics, and gas exchange in endotoxemic pigs; Olson NC et al.; The effects of ketanserin on pulmonary hemodynamics, lung mechanics, and gas exchange were determined in anesthetized 10- to 14-week-old pigs after they were endotoxemic for 1 or 4.5 hours . Saline solution was given to controls (group 1) . Escherichia coli endotoxin (055-B5) was infused IV at a dosage of 5 micrograms/kg for 1 hour (group 2) . In group 3, endotoxin was infused at 5 micrograms/kg the first hour plus a continuous infusion of endotoxin at 2 micrograms/kg/hr . Ketanserin, a specific serotonin receptor antagonist, was infused IV (300 micrograms/kg) after pigs were endotoxemic for 1 or 4.5 hours (groups 2 and 3, respectively) . At 1 hour of endotoxemia, mean pulmonary artery pressure and pulmonary vascular resistance were increased, and cardiac index was decreased . Ketanserin caused a small attenuation of the increases in mean pulmonary artery pressure and pulmonary vascular resistance, indicating that serotonin may have a small role in the endotoxin response at 1 hour . At 4.5 hours of endotoxemia, mean pulmonary artery pressure, pulmonary vascular resistance, alveolar dead space ventilation, and alveolar-arterial oxygen gradient were increased, and cardiac index and lung dynamic compliance were decreased; ketanserin significantly attenuated the endotoxin-induced changes in cardiac index, mean pulmonary artery pressure, pulmonary vascular resistance, and lung dynamic compliance . Ketanserin also decreased the blood temperature after pigs were endotoxemic for 4.5 hours . However, the endotoxin-induced increases (at 4.5 hours) in alveolar-arterial oxygen gradient and alveolar dead space ventilation were not acutely reversed by ketanserin.(ABSTRACT TRUNCATED AT 250 WORDS)

Am J Vet Res, 1986 Oct, 47(10), 2187 - 92
Dexamethasone-induced attenuation of cardiopulmonary dysfunction in endotoxemic calves; Olson NC et al.; Effects of dexamethasone on pulmonary hemodynamics, pulmonary mechanics, and gas exchange were determined in anesthetized (pentobarbital sodium) and paralyzed (pancuronium bromide) calves (9.4 +/- 0.4 weeks old) during 5 hours of endotoxemia . Escherichia coli endotoxin (055-B5) was infused IV at 20 micrograms/kg the 1st hour, followed by a continuous infusion at 10 micrograms/kg/hour for the following 4 hours . Dexamethasone (5 mg/kg) was given IV 18 hours and 1 hour before endotoxin administration, and was also administered IV (1 mg/kg/hr) during endotoxemia . Endotoxin induced large increases in pulmonary artery pressure, pulmonary vascular resistance, alveolar-arterial O2 gradient, alveolar dead-space ventilation, postmortem gravimetric lung weight of bloodless lung, albumin and total protein concentrations in bronchoalveolar lavage fluid, and the number of neutrophils recovered from bronchoalveolar lavage fluid . Endotoxin induced decreases in the cardiac index, dynamic lung compliance, and PaO2 . Dexamethasone attenuated most of the cardiopulmonary responses induced by endotoxin, especially during the first 3 hours of endotoxemia . Dexamethasone blocked endotoxin-induced increases in bronchoalveolar lavage albumin, total protein, and neutrophil content . Therefore, glucocorticoids modify endotoxin-induced pulmonary injury in calves, possibly by limiting mobilization of endogenous arachidonic acid.

Eur J Biochem, 1986 Oct 1, 160(1), 101 - 8
tRNA (adenine-N1)-methyltransferase from Dictyostelium discoideum . Purification, characterization and developmental changes in activity; Mutzel R et al.; An enzyme activity transferring methyl groups from S-adenosylmethionine to endogenous tRNA was detected in the cytosol of aggregative Dictyostelium discoideum amoebae . This enzyme was purified more than 1000-fold and was characterized as a tRNA (adenine-N1-)-methyltransferase . Kinetic analysis yielded a K0.5 for S-adenosylmethionine of 0.27 microM and competitive inhibition by S-adenosylhomocysteine showed an I0.5 of 0.26 microM . The tRNA methyltransferase activity was stimulated by monovalent cations and the pH optimum was 7.3 . tRNAs isolated from D . discoideum as well as from other eucaryotic sources could be methylated only to a minor extent . In contrast, Escherichia coli tRNA accepted up to 0.6 mol methyl group/mol tRNA, suggesting that the target nucleotide is unmethylated in procaryotic tRNA, but is commonly methylated in tRNAs from eucaryotic organisms . The activity of the methyltransferase increased 4-6-fold during cell differentiation from the vegetative to the aggregative stage.

J Bacteriol, 1986 Oct, 168(1), 40 - 8
Organization and expression of genetic determinants for synthesis and assembly of type 51 R bodies; Kanabrocki JA et al.; Type 51 R bodies are produced by all bacterial endosymbionts (Caedibacter taeniospiralis) of Paramecium tetraurelia that confer the hump-killer trait upon their hosts . Type 51 R-body synthesis by C . taeniospiralis is required for expression of the hump-killer trait . The genetic determinants for type 51 R-body synthesis by C . taeniospiralis 47 have been cloned and expressed in Escherichia coli . In this communication we describe three species of polypeptides required for R-body synthesis and the organization of their genetic determinants . Each polypeptide species is controlled by a separate gene that is expressed as an independent transcriptional unit possessing regulatory signals that are recognized by E . coli . Two polypeptide species of 10 and 18 kilodaltons are required for R-body synthesis but apparently are not structural subunits . The third polypeptide species (13 kilodaltons) is the major structural subunit . R-body assembly involves polymerization reactions that result in high-molecular-mass polypeptide complexes, primarily composed of the 13-kilodalton polypeptide species, that appear to be the result of covalent cross-linking between structural subunits . The results presented here have been suggested to apply to the assembly and structure of all type 51 R bodies, but not necessarily to other R-body types.

Photodermatol, 1986 Oct, 3(5), 261 - 70
Photobiological activity of certain pyranocoumarin derivatives: potential agents for the photochemotherapy of psoriasis; Baccichetti F et al.; The photobiological properties of a series of pyranocoumarin derivatives having linear (xanthyletines) and angular structure (seselins) have been studied; while the linear derivative carrying the methyl geminal group, typical of the parent natural compound, appeared to be entirely inactive in all tests performed, very probably because of the steric hindrance in the dark interaction with DNA, 4 substances lacking in this group showed a marked capacity for inhibiting DNA synthesis in Ehrlich cells . In particular, 4,6-dimethyl-8-desmethylseselin proved to be about 7 times more active than 8-MOP . Practically all compounds were capable of inducing cross-links in DNA, but this feature, marked in the linear compounds, is very much reduced in the angular ones; this property appears to be clearly related to the mutagenicity in the light and with the skin phototoxicity, which are both marked in the former and low or absent in the latter . In the dark, while all compounds are non-mutagenic in the absence of metabolic activation, in the presence of microsomial enzymes pyranocoumarins become mutagens; only the xanthyletine derivative carrying a geminal methyl group at the 8 position was not activated, suggesting that the enzymes metabolize the pyranic ring.

Pediatrie, 1986 Oct-Nov, 41(7), 549 - 52
{Hemolytic-uremic syndrome and Escherichia coli enterocolitis . Apropos of 2 cases}; Palcoux JB et al.; The authors report two cases of HUS that presented as colitis due to E . Coli . They emphasize the interest--from an epidemiological point of view--of looking for E . Coli in stools of children with HUS and serotyping them.

Arch Microbiol, 1986 Oct, 146(1), 80 - 6
Effects of iron-limitation of Escherichia coli on growth, the respiratory chains and gallium uptake; Hubbard JA et al.; The effects of iron limitation on growth, the composition and function of the respiratory chains, and gallium uptake in Escherichia coli have been studied . Decreasing the iron concentration in a defined medium using Chelex resin gave lower growth yields in both continuous culture and prolonged batch culture . In the former, iron-limited (entering {Fe} less than or equal to 2.0 microM) cells exhibited diminished respiration rates, respiration-driven proton translocation quotients, and levels of non-haem iron and cytochromes . The cellular concentration of haemoprotein b-590 (a cytochrome alpha 1-like hydroperoxidase) decreased 20-fold on iron limitation, whilst a CO-binding pigment with an absorption maximum in the dithionite-treated form near 500 nm appeared . Gallium(III) (9 microM) added to iron-limited, but not iron-sufficient, cultures diminished growth yields further; cells grown with low entering concentrations of iron took up less gallium than iron-sufficient cells . These results are attributed to the interference by gallium(III) with siderophore-mediated metal uptake . Gallium also stimulated iron uptake and was itself accumulated by iron-sufficient cells, suggesting that gallium(III) also affects the iron transport system(s) of low affinity.

Acta Chir Scand, 1986 Oct, 152, 569 - 75
Treatment with prostaglandin E1 in a porcine model of early adult respiratory distress syndrome; Modig J et al.; The effects of treatment with PGE1 were evaluated in a porcine model of early adult respiratory distress syndrome induced by endotoxaemia . Spontaneously breathing pigs under ketamine anaesthesia were infused i.v . with E . coli endotoxin (10 micrograms X kg-1 X h-1) for 6 h . Thirteen pigs were given endotoxin, and 11 pigs were treated with a continuous infusion of PGE1, 0.25 microgram X kg-1 X min-1 for 4 h, beginning 2 h after start of endotoxin and established lung injury . Four pigs served as controls and receiving only PGE1 (0.25 microgram X kg-1 X min-1) during the whole observation period of 6 h . PGE1 treatment did not influence the decline in platelet and polymorphonuclear cell counts, whereas it markedly decreased the pulmonary hypertension induced by endotoxaemia . The increased extravascular lung water returned towards baseline after institution of PGE1 . The increased venous admixture was not significantly influenced by PGE1 . Treatment with PGE1 induced an exacerbated hypotensive state in the endotoxaemic animals primarily due to vasodilation . The decline in cardiac output and oxygen delivery noted in endotoxaemic pigs were not influenced by PGE1 and survival was not improved . Although one should be extremely careful in extrapolating these data to the clinical situation the results from the present study suggest the need for optimum volume replacement when starting PGE1 infusion in endotoxin-induced ARDS.

Acta Chir Scand, 1986 Oct, 152, 561 - 8
Lung mechanics with relation to pulmonary haemodynamics, gas exchange and extravascular lung water in mechanically ventilated endotoxaemic pigs; Forsgren P et al.; In a porcine model employing a continuous i.v . infusion of E . coli endotoxin the pathophysiology of early adult respiratory distress syndrome was studied with main emphasis on the early changes in lung mechanics and their relation to changes in pulmonary haemodynamics, gas exchange and extravascular lung water in intermittent positive pressure ventilated (IPPV) pigs under ketamine anaesthesia . Six animals served as controls and revealed no major physiological changes . Nine animals received endotoxin and developed significant changes in lung mechanics with increases in end-inspiratory pressure (32%), expiratory resistance (29%) and decrease in total dynamic lung compliance (27%) . Changes in dynamic compliance and pulmonary haemodynamics displayed a 2-phase reaction . Venous admixture showed a rapid increase at with the increase in mean pulmonary arterial pressure (r = -0.8) and with the increase in venous admixture (r = -0.7) . Extravascular lung water did not increase significantly . The decrease in dynamic compliance is most likely explained by peripheral airway constriction . A contributory factor might be pulmonary microvascular constriction with vascular stasis and mechanical compression of small airways . The increased venous admixture is best explained by a bronchiolar and microvascular constriction, i.e . a "dry" ventilation/perfusion inequality and not consequent to oedema . IPPV seems to counteract the increase in extravascular lung water.

Sex Transm Dis, 1986 Oct-Dec, 13(4), 237 - 44
Immunochemical characterization and purification of Treponema pallidum antigen TpD expressed by Escherichia coli K12; Hindersson P et al.; The immunochemical properties of the Treponema pallidum antigen TpD, as expressed by Escherichia coli K12, was investigated by crossed immunoelectrophoresis in which an affinity-purified antibody to this antigen was used . Two immunologically cross-reacting components of TpD with different mobility were demonstrated . Affinity-purified antibodies were used in obtaining purified TpD and in determining the cellular localization of TpD in T . pallidum by immunoelectron microscopy . TpD was localized on the surface of methanol-fixed T . pallidum . Twenty sera from patients with secondary syphilis and 20 sera from nonsyphilitics were examined in crossed immunoelectrophoresis . All sera from patients with secondary syphilis and none from nonsyphilitics contained antibodies to the TpD components . Because TpD seems to be surface associated and a major immunogen during infection with T . pallidum, this antigen might be useful for development of a vaccine against syphilis and for development of improved methods for serodiagnosis of syphilis.

J Interferon Res, 1986 Oct, 6(5), 519 - 26
Purification and characterization of recombinant mouse interferon-beta; Matsuda S et al.; Recombinant mouse interferon-beta (rMuIFN-beta) produced in Escherichia coli was purified to homogeneity and characterized . The purified protein exhibited a single band of Mr 19,900 on SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions, and also exhibited a single band on native polyacrylamide gel electrophoresis at pH 4.3 . The observed molecular weight corresponded to that of the polypeptide moiety of natural MuIFN-beta of Mr 19,700 . The amino acid composition and the amino-terminal sequence of the purified rMuIFN-beta were identical to those predicted from cDNA sequence . These results indicate that the purified protein is a nonglycosylated MuIFN-beta, which forms no disulfide-linked dimer and probably exists as a monomeric form.

Aust Vet J, 1986 Oct, 63(10), 327 - 31
Hyperacute Escherichia coli mastitis of cattle in the immediate post-partum period; Frost AJ et al.; The pathology of hyperacute coliform mastitis was studied in 5 post-parturient cows . In all infected quarters infiltration of neutrophils was negligible . In all except one case there was severe damage to the ductular and secretory system, involving most areas of the gland . Bacteria were dense in infected alveoli, and there was evidence of substantial phagocytosis of bacteria by the secretory epithelium . The exception showed a large lesion in the middle of the gland from which the spread was ductular; other infections were consistent with spread via the teat canal . The organisms were largely confined to the ductular/secretary lumen and there was little invasion of the parenchyma . The severity of the disease was considered due to the absence of the inflammatory response seen in mid lactation.

Anal Biochem, 1986 Oct, 158(1), 12 - 9
Automated thermometric enzyme immunoassay of human proinsulin produced by Escherichia coli; Birnbaum S et al.; We have determined and monitored the production and release of human proinsulin by genetically engineered Escherichia coli cells . Several M9 media samples were analyzed sequentially after centrifugation with the aid of a rapid automated flow-through thermometric enzyme-linked immunosorbent assay (TELISA) system . The response time was 7 min after sample injection and a single assay was complete after 13 min . Insulin concentrations in the range of 0.1-50 micrograms/ml could be determined . The TELISA method correlated well with conventional radioimmunoassay determinations . Standard curves were reproducible over a period of several days even when the immobilized antibody column was stored at 25 degrees C in the enzyme thermistor unit . Thus, immediate assay start up was possible.

Virus Res, 1986 Oct, 6(1), 85 - 99
Variation in the temporal expression of overlapping baculovirus transcripts; Mainprize TH et al.; To investigate gene expression from the Autographa californica nuclear polyhydrolysis virus genome (AcNPV), complementary DNA (cDNA) was synthesized from polyadenylated RNA transcribed at 2 h and 10 h postinfection (p.i.) and then cloned into Escherichia coli using plasmid pUC-9 . Eighteen 2 h cDNA plasmids were homologous to five distinct regions of the viral genome, while forty-nine 10 h cDNA plasmids were homologous to 15 regions including the five regions transcribed at 2 h . Temporal expression of polyadenylated RNA transcribed from diverse regions of the genome was examined using Northern blot hybridization with the above 2 and 10 h cDNA probes . All regions displayed overlapping sets of RNAs . In addition to HindIII-I/EcoRI-F (IF) and HindIII-B2/EcoRI-H (B2H), several, but not all, regions showed a sequential appearance of higher molecular weight RNAs as the infection progressed . Each overlapping set of RNAs exhibited unique characteristics including variations in the number of overlapping transcripts, their temporal regulation, and their relative abundance during the course of infection.

Vet Q, 1986 Oct, 8(4), 266 - 73
Anorexia during febrile conditions in dwarf goats . The effect of diazepam, flurbiprofen and naloxone; van Miert AS et al.; The most common sign of febrile diseases is anorexia, which develops at a time when adequate caloric and micronutrient availability may be critical . In order to study the relationship of fever and anorexia, feed intake in dwarf goats was studied under conditions of fever and antipyresis . Furthermore, experiments were done to establish whether a feed intake stimulant would override the anorexia during febrile conditions . Infection with Ehrlichia phagocytophila and i.v . injection of Escherichia coli endotoxin (0(111) B4, 0.1 microgram/kg body weight) both resulted in increased rectal temperatures and significant reductions in feed intake . Administration of the antipyretic drug flurbiprofen (1 mg/kg) to febrile animals inhibited the temperature responses, but food intake was still suppressed . Diazepam (0.06 mg/kg), a feed intake stimulant, did not override the anorexia associated with fever . Blocking the febrile response of E . coli LPS-injected goats with flurbiprofen plus diazepam or with flurbiprofen plus naloxone (0.1 mg/kg) did not antagonise their reduced feed intake either . The effects of these drugs and of endotoxin on rumen motility adds an interesting aspect to their activities in the CNS, since the CNS has been shown to regulate various aspects of forestomach motility, which in turn could alter feeding behaviour . Moreover, our findings are consistent with the hypothesis that the suppression of feed intake might depend on the release of interleukin-1.

Mol Cell Biol, 1986 Oct, 6(10), 3349 - 56
UV light-induced cyclobutane pyrimidine dimers are mutagenic in mammalian cells; Protic-Sabljic M et al.; We used a simian virus 40-based shuttle vector plasmid, pZ189, to determine the role of pyrimidine cyclobutane dimers in UV light-induced mutagenesis in monkey cells . The vector DNA was UV irradiated and then introduced into monkey cells by transfection . After replication, vector DNA was recovered from the cells and tested for mutations in its supF suppressor tRNA marker gene by transformation of Escherichia coli carrying a nonsense mutation in the beta-galactosidase gene . When the irradiated vector was treated with E . coli photolyase prior to transfection, pyrimidine cyclobutane dimers were removed selectively . Removal of approximately 90% of the pyrimidine cyclobutane dimers increased the biological activity of the vector by 75% and reduced its mutation frequency by 80% . Sequence analysis of 72 mutants recovered indicated that there were significantly fewer tandem double-base changes and G X C----A X T transitions (particularly at CC sites) after photoreactivation of the DNA . UV-induced photoproducts remained (although at greatly reduced levels) at all pyr-pyr sites after photoreactivation, but there was a relative increase in photoproducts at CC and TC sites and a relative decrease at TT and CT sites, presumably due to a persistence of (6-4) photoproducts at some CC and TC sites . These observations are consistent with the fact that mutations were found after photoreactivation at many sites at which only cyclobutane dimers would be expected to occur . From these results we conclude that UV-induced pyrimidine cyclobutane dimers are mutagenic in DNA replicated in monkey cells.

Mol Gen Genet, 1986 Oct, 205(1), 9 - 13
Structure and function of dnaQ and mutD mutators of Escherichia coli; Takano K et al.; The nucleotide sequences of the recessive dnaQ49 and the dominant mutD5 mutator were determined . The dnaQ49 mutator has a single base substitution in the dnaQ gene, thus causing one amino acid change, 96Val (GTG)----Gly (GGG), in the DnaQ protein (epsilon subunit of DNA polymerase III holoenzyme) . The mutD5 mutator possesses two base substitutions in the same gene, resulting in two amino acid changes, 73Leu (TTG)----Trp (TGG) and 164Ala (GCA)----Val (GTA), which were designated the mutD52 and mutD51 mutations, respectively . Construction of chimaeric genes carrying one or two of these mutations revealed: either mutD51 or mutD52 alone causes the dominant mutator phenotype when present in a multi-copy plasmid; mutator phenotype when present in a low-copy plasmid; the dominant mutD51 mutator activity is suppressed by the dnaQ49 mutation when both mutations are present in the same gene . Based on these findings, we devised a model for the action of these mutators.

Can J Microbiol, 1986 Oct, 32(10), 763 - 4
Effect of heat on endotoxin in plasma and in pyrogen-free water, as measured in the Limulus amoebocyte lysate assay; Piotrowicz BI et al.; Four modes of heating endotoxin in plasma and two different times of heating endotoxin in pyrogen-free water were compared . There were no significant differences in standard curves after heating endotoxin in plasma at 100 degrees C for 1 and for 10 min . However, the standard curve after heating for 10 min at 75 degrees C had a significantly less steep slope, and after heating for 10 min at 56 degrees C, it was completely flat . Heating of endotoxin in pyrogen-free water for 1 min also resulted in the flattening of the standard curve, which was even more pronounced after 10 min of heating.

Kidney Int, 1986 Oct, 30(4), 474 - 80
Roles for thromboxane A2 and leukotrienes in endotoxin-induced acute renal failure; Badr KF et al.; Bolus i.v . administration of 100 micrograms/kg of E . Coli lipopolysaccharide endotoxin (LPS) to adult male Munich-Wistar rats (N = 18) resulted in a progressive fall in RBF and GFR from 6.9 +/- 0.2 SE and 1.1 +/- 0.05 ml/min to minimal values at 50 minutes of 3.8 +/- 0.4 and 0.32 +/- 0.08 (P less than 0.05), respectively, without a fall in mean arterial pressure . At 50 minutes, renal cortical generation rates of PGE2 (1075 +/- 108 pg/mg tissue), 6 keto PGF1 alpha (221 +/- 41 pg/mg), and TxB2 (106 +/- 12 pg/mg) were significantly higher than those of vehicle-treated control rats (N = 10, PGE2 = 466 +/- 107, 6 keto PGF1 alpha = 94 +/- 3, and TxB2 = 35 +/- 3 pg/mg), and morphologic examination revealed normal histology with notable absence of leukocytes and platelets . Pretreatment of a third group of nine rats with TxA2 synthetase inhibitor UK-37.248 (dazoxiben, 10 mg/kg) selectively abolished the LPS-induced rise in TxB2 (29 +/- 3 pg/mg), but not PGE2 (837 +/- 62 pg/mg) or 6 keto PGF1 alpha (179 +/- 5 pg/mg), prevented the fall in RBF at 50 minutes (6.3 +/- 0.4 ml/min), and allowed for significant preservation of GFR (0.67 +/- 0.08 ml/min).(ABSTRACT TRUNCATED AT 250 WORDS)

J Hyg (Lond), 1986 Oct, 97(2), 273 - 80
A rapid and simple method for the detection and enumeration of Escherichia coli in cleansed shellfish; Humphrey TJ et al.; A multiple-tube technique based on peptone water incubated at 44 degrees C for 24 h followed by detection of indole was found to be sensitive and specific for the detection of Escherichia coli in oysters and mussels . The method has the advantage of providing rapid results and is both less expensive and less time-consuming than other MPN techniques.

J Hyg (Lond), 1986 Oct, 97(2), 229 - 36
An outbreak of gastroenteritis on a passenger cruise ship; O'Mahony M et al.; In an outbreak of gastroenteritis on board a cruise ship 251 passengers and 51 crew were affected and consulted the ship's surgeon during a 14-day period . There was a significant association between consumption of cabin tap water and reported illness in passengers . Enterotoxigenic Escherichia coli were isolated from passengers and crew and coliforms were found in the main water storage tank . Contamination of inadequately chlorinated water by sewage was the most likely source of infection . A low level of reported illness and late recognition of the outbreak delayed investigation of what was probably the latest in a series of outbreaks of gastrointestinal illness on board this ship . There is a need for a national surveillance programme which would monitor the extent of illness on board passenger cruise ships as well as a standard approach to the action taken when levels of reported illness rise above a defined level.

Clin Physiol, 1986 Oct, 6(5), 415 - 22
Effects of a thromboxane antagonist (BM 13.177) during endotoxin-induced pulmonary vasoconstriction in sheep; Huttemeier P et al.; We investigated the effect of a thromboxane antagonist, BM 13.177, during endotoxin-induced pulmonary vasoconstriction in sheep . In control animals intravenous E-coli endotoxin (1 microgram/kg) caused a transient increase of pulmonary artery and airway pressure paralleled by large concentration increases of TXB2: in comparison peak plasma concentrations of 6-keto-PGF1 alpha (a prostacyclin metabolite) were small and delayed in time . Pre-treatment with BM 13.177 (bolus 5 mg/kg), followed by 0.75 mg/kg/min intravenously) abolished the rise of pulmonary artery and airway pressure . Plasma concentrations of TXB2 and 6-keto-PGF1 alpha were similar to controls . These and previous investigations imply that BM 13.177 specifically antagonizes TXA2 on the putative receptor in pulmonary vascular and airway smooth muscle.

J Clin Microbiol, 1986 Oct, 24(4), 615 - 9
Hemadsorption and enzyme-linked immunosorbent assay nitrocellulose replica methods for identification of colonization factor antigen (CFA)-positive Escherichia coli colonies and for isolation of CFA-negative mutants; Lopez-Vidal Y et al.; Methods were developed that allow demonstration of individual colonies carrying colonization factor antigen (CFA) I or CFA/II or E8775-type antigen in mixed bacterial cultures on solid media . These methods are based on mannose-resistant hemadsorption or CFA enzyme-linked immunosorbent assay (ELISA) on nitrocellulose replicas of the cultures allowing simultaneous analysis of up to 200 colonies per plate . The sensitivity and specificity of the CFA ELISA nitrocellulose replica method were 97 and 99%, respectively, for CFA/I-carrying colonies and 99 and 100% for CFA/II-positive colonies; corresponding figures for the quicker and simpler hemadsorption modification were somewhat lower . Both methods seem to be useful for studying excretion of CFA-carrying bacteria in feces, as indicated by studies in rabbits infected with enterotoxin-producing Escherichia coli in a nonligated-intestine model . By initially absorbing CFA-carrying bacteria on erythrocytes and then performing nitrocellulose replicas of agar colonies of the nonabsorbed bacteria, CFA-deficient mutants could be identified by the hemadsorption method, as well as by the CFA ELISA . Treatment of CFA-carrying bacteria with antiserum against CFA and complement also resulted in enrichment of spontaneous CFA-deficient mutants that could be identified by the replica methods . Several stable CFA-deficient mutants from enterotoxin-producing E . coli carrying CFA/I, CFA/II, or E8775 were isolated by these approaches.

Genetics, 1986 Oct, 114(2), 375 - 92
Induction of intrachromosomal recombination in yeast by inhibition of thymidylate biosynthesis; Kunz BA et al.; The biosynthesis of thymidylate in the yeast Saccharomyces cerevisiae can be inhibited by antifolate drugs . We have found that antifolate treatment enhances the formation of leucine prototrophs in a haploid strain of yeast carrying, on the same chromosome, two different mutant leu2 alleles separated by Escherichia coli plasmid sequences . That this effect is a consequence of thymine nucleotide depletion was verified by the finding that provision of exogenous thymidylate eliminates the increased production of Leu+ colonies . DNA hybridization analysis revealed that recombination, including reciprocal exchange, gene conversion and unequal sister-chromatid crossing over, between the duplicated genes gave rise to the induced Leu+ segregants . Although gene conversion unaccompanied by crossing over was responsible for the major fraction of leucine prototrophs, events involving reciprocal exchange exhibited the largest increase in frequency . These data show that recombination is induced between directly repeated DNA sequences under conditions of thymine nucleotide depletion . In addition, the results of this and previous studies are consistent with the possibility that inhibition of thymidylate biosynthesis in yeast may create a metabolic condition that provokes all forms of mitotic recombination.

Eur J Biochem, 1986 Oct 1, 160(1), 83 - 91
3-Azi-1-methoxybutyl D-maltooligosaccharides specifically bind to the maltose/maltooligosaccharide-binding protein of Escherichia coli and can be used as photoaffinity labels; Thieme R et al.; Maltooligosaccharides with two to six (alpha 1-4)-linked glucose residues, carrying at their reducing end a 3-azi-1-methoxybutyl group in either alpha or in beta glycosidic linkage, were synthesized . These maltooligosaccharide analogues inhibit maltose uptake via the maltose-binding-protein-dependent transport system in Escherichia coli . The concentration of half-maximal inhibition of maltose transport, at 15 nM concentration, decreases with increasing chain length of the analogue, levelling off at 40 microM after a chain length of four glucose residues in the alpha series and at 350 microM after a chain length of three glucose residues in the beta series . The inhibition of maltose transport occurs at the level of the periplasmic maltose-binding protein . 3-Azi-1-methoxybutyl alpha-D-{3H}maltotrioside was bound by the maltose-binding protein with a Kd of 0.18 mM . Irradiation at 350 nm of purified maltose-binding protein in the presence of 4 microM of this substrate labeled the protein covalently; labeling was prevented by 1 mM maltose . Using a crude preparation of periplasmic proteins two proteins were labeled, the maltose-binding protein and alpha-amylase . Thus, 3-azi-1-methoxybutyl alpha-D-maltooligosaccharides are potent photoaffinity labels for proteins with maltooligosaccharides-binding sites.

Eur J Biochem, 1986 Oct 1, 160(1), 61 - 7
Molecular cloning and expression of cDNA for human antileukoprotease from cervix uterus; Heinzel R et al.; We have isolated cDNA clones for the human antileukoprotease HUSI-I, an elastase inhibitor, from a library, containing cDNA inserts made from human cervix uterus . A library of 10 000 recombinants was screened using a mixture of 16 different oligodeoxyribonucleotides which correspond to amino acids 79-84 and one 20mer oligodeoxyribonucleotide corresponding to amino acids 19-26 . Two overlapping cDNA clones, containing the entire coding sequence and part of the 5'- and 3'-untranslated region, were isolated . DNA sequence data showed that our clone corresponds with the available protein sequence data . For expression, the cDNA fragment was inserted in a derivative of plasmid pPLc236 and expressed under the control of lambda PL promoter . Expression of antileukoprotease was proven by Western blot analysis and inhibition of chymotrypsin.

Arch Surg, 1986 Oct, 121(10), 1173 - 6
Effect of heparin and heparin fractions on experimental abscess formation; Prinz RA et al.; To evaluate the effectiveness of heparin and heparin fractions in decreasing abscess formation, rats were divided into six groups . A fibrin clot containing 10(9) live Escherichia coli was placed in the peritoneal cavity of each rat . Group 1 (controls) received daily subcutaneous (SQ) injections of 0.1 mL of saline solution . Group 2 received daily intramuscular injections of gentamicin, 12.5 mg/kg . Group 3 received a daily SQ dose of 30 U of porcine heparin . In addition to gentamicin, group 4 received heparin, group 5 received heparin fraction PK10169, and group 6 received heparin fraction CY216, all in daily SQ doses of 30 U . Survivors were killed at ten days and examined for intra-abdominal abscesses . All group 1 animals developed abscesses . Abscess formation was significantly decreased in all groups receiving gentamicin . When used with gentamicin, neither heparin nor heparin fractions decreased the number of abscesses formed when compared with gentamicin alone . Heparin or heparin fractions in combination with gentamicin did decrease abscess size significantly when compared with controls.

Arch Biochem Biophys, 1986 Oct, 250(1), 54 - 62
Reductive repression in Escherichia coli K-12 is mediated by oxygen radicals; Hertz R et al.; Cyclic AMP (cAMP) content and the expression of cAMP-dependent phenotypes were positively correlated with respiration capacity in respiration-deficient mutants of Escherichia coli K-12 ("reductive repression," R . Hertz, and J . Bar-Tana, (1982) Arch . Biochem . Biophys . 213, 193-199) . Reductive repression in respiration-deficient mutants could not be accounted for by respective changes in either the energy charge of adenine nucleotides or the redox state of pyridine nucleotides but could be ascribed to an increased formation of oxygen radicals under conditions of limited respiration . Scavengers of superoxide radicals eliminated reductive repression in respiration-deficient mutants with a concomitant increase in cAMP content . Such scavengers also effected a partial escape from permanent glucose catabolite repression, thus indicating a possible role played by oxygen radicals in both repression modes.

Am Surg, 1986 Oct, 52(10), 564 - 7
Increased lethality of endotoxemia in murine frostbite; Spillert CR et al.; Because frostbite (FB) is associated with increased intravascular coagulability, it is reasonable to assume that endotoxin, by enhancing platelet aggregation, will adversely affect FB . Swiss mice (25 +/- 2 g) were anesthetized, and the tails of the animals totally immersed in a freezing solution of equal volumes of ethylene glycol and water (-18 C) for 8 min . The tails were then thawed at room temperature (24 C) . Half an hour after removal from the freezing solution, the animals were given either (Group A) 0.1 cc saline I.P . or (Group B) 0.1 mg E . coli endotoxin (055:B5; 1/3 LD50 dose) in 0.1 cc saline IP . A third group (Group C), was given the same dose of endotoxin but was not subjected to frostbite . Survivals in each group at 2 weeks were as follows: (A) 14/14 (100%), (B) 4/20 (20%), (C) 13/14 (93%) . Using Fisher's exact test, A versus B P less than .001; B versus C P less than .001; A versus C NS . The data presented here emphasize the increased lethality of endotoxemia in murine FB.

Am J Physiol, 1986 Oct, 251(4 Pt 1), E470 - 6
Lipoprotein lipase-suppressing mediator in serum of endotoxin-treated rats; Bagby GJ et al.; The conditions under which lipoprotein lipase-suppressing mediator is present in serum of endotoxin-treated rats was determined in this study . The suppression of lipoprotein lipase activity in 3T3-L1 cells was used as a bioassay for mediator in serum . Endotoxin (0.1-10 micrograms/ml) and serum from control rats did not suppress lipoprotein lipase activity . Maximum suppression of cell lipoprotein lipase activity (70%) by serum from endotoxic rats required a cell exposure time of 5 h . At the highest dose of endotoxin used (1 mg/100 g), significant suppression was achieved when cells were incubated with 0.5% serum from endotoxic rats (P less than 0.05) . Serum obtained 2-3 h after endotoxin injection possessed the maximal ability to suppress lipase activity, but suppressing activity was not present in serum collected 8 h after endotoxin . Rats rendered tolerant to endotoxin by 5 daily injections (0.1 mg/100 g) did not contain detectable levels of mediator in serum after endotoxin injection . The results demonstrate that the presence of lipoprotein lipase activity-suppressing mediator is transitory after in vivo exposure of naive rats to endotoxin, but does not appear in serum of endotoxin tolerant rats.

Proc Natl Acad Sci U S A, 1986 Oct, 83(19), 7405 - 9
Promoters selected from random DNA sequences; Horwitz MS et al.; We have selected a group of Escherichia coli promoters from random DNA sequences by replacing 19 base pairs at the -35 promoter region of the tetracycline resistance gene tetr of the plasmid pBR322 . Substitution of 19 base pairs with chemically synthesized random sequences results in a maximum of 4(19) (about 3 X 10(11)) possible replacement sequences . From a population of about 1000 bacteria harboring plasmids with these random substitutions, tetracycline selection has revealed several functional -35 promoter sequences . These promoters have retained only partial homology to the -35 promoter consensus sequence . In three of these promoters, the consensus alignment shifts 10 nucleotides downstream, allowing the RNA polymerase to recognize another Pribnow box from within the original pBR322 sequence . Two of the sequences promote transcription more strongly than the native promoter . This technique may have application for the selection of additional DNA sequences with varied biological activity.

Proc Natl Acad Sci U S A, 1986 Oct, 83(19), 7182 - 6
Isolation and characterization of a cDNA encoding a human liver/bone/kidney-type alkaline phosphatase; Weiss MJ et al.; Alkaline phosphatases (ALPs) {orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1} isolated from human liver, bone, and kidney (L/B/K) exhibit very similar biochemical and immunologic properties that differentiate them from other human ALPs, such as those characteristically found in placenta and intestine . Despite their similarities, the L/B/K ALPs produced in different tissues show slight physical differences . To examine structural and evolutionary relationships between the various ALPs, a cDNA corresponding to L/B/K ALP mRNA has been isolated . A lambda 11 cDNA expression library was constructed using poly(A) RNA from the osteosarcoma cell line Saos-2 and screened with anti-liver ALP antiserum . The 2553-base-pair cDNA contains an open reading frame that encodes a 524 amino acid polypeptide with a predicted molecular mass of 57.2 kDa . This ALP precursor protein contains a presumed signal peptide of 17 amino acids followed by 37 amino acids that are identical to the amino-terminal sequence determined from purified liver ALP . In addition, amino acid sequences of several CNBr peptides obtained from liver ALP are found within the cDNA-encoded protein . The deduced L/B/K ALP precursor polypeptide shows 52% homology to human placental ALP and 25% homology to Escherichia coli ALP precursor polypeptides . Sixty percent nucleotide homology exists between the human L/B/K and placental cDNAs over the protein coding regions . The 5' and 3' untranslated regions of the L/B/K ALP cDNA, 176 and 805 base pairs, respectively, show no homology to the corresponding regions of placental ALP cDNA.

Proc Natl Acad Sci U S A, 1986 Oct, 83(19), 7177 - 81
Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli: a method for site-specific mutagenesis; Mandecki W; A DNA double-strand break can be efficiently repaired in Escherichia coli if an oligodeoxyribonucleotide is provided to direct the repair . The oligonucleotide must be at least 20 residues long and have a sequence identical to sequences flanking the break . The phenomenon can be used to introduce defined mutations into DNA in the area of a double-strand break . To obtain mutants, the oligonucleotide that carries a mutation and the denatured linearized plasmid DNA are introduced into E . coli by transformation . No enzymatic manipulation in vitro is required . The mutants can constitute up to 98% of the total number of transformants obtained . The efficiency of mutagenesis decreases as the distance between the mutation and the plasmid cleavage site increases . The universality of the method was tested by introducing mutations into four genes, using four plasmids and three E . coli strains, as well as eight restriction enzymes to linearize DNA . Several models of the oligonucleotide-directed DNA double-strand break repair are discussed.

Mutat Res, 1986 Oct, 175(2), 41 - 5
Differential enhancement of spontaneous transition mutations in the lacI gene of an Ung- strain of Escherichia coli; Fix DF et al.; In this communication, the contribution of cytosine deamination to spontaneous mutagenesis in the lacI gene of E . coli was examined . In a wild-type strain, 75% of the amber mutations recovered were G:C----A:T transitions and 60% of these were at the 5-methylcytosine spontaneous hotspots Am6, Am15 and Am34 . In a strain deficient for uracil-DNA glycosylase (Ung-), 96% of the amber mutations were G:C----A:T transitions while only 15% of these occurred at the hotspot sites . This shift in the mutational distribution demonstrates that cytosine deamination is a potent mutagenic process, which is enhanced in the absence of glycosylase . Moreover, some amber sites were greatly enhanced in the Ung- strain while others were only slightly enhanced . This result suggests that the rate of cytosine deamination at individual sites may be influenced by surrounding base composition . Therefore, we examined the neighboring sequences and found a strong correlation between the fold-increase in mutation and the A/T richness of the surrounding sequence . It is suggested that A/T-rich regions denature more often, forming transient single strands in which cytosine residues would be expected to deaminate more readily.

J Urol, 1986 Oct, 136(4), 960 - 3
Immunology of pyelonephritis . VII . Effect of allopurinol; Roberts JA et al.; The inflammatory response and the respiratory burst of bacterial phagocytosis have been shown to be at least partially responsible for the renal damage from infection . In addition, we have shown that renal blood flow decreases following infection . Hypoxanthine is produced in ischemic tissue during the anaerobic metabolism of adenosine monophosphate (AMP) . During reperfusion hypoxanthine is metabolized to uric acid and superoxide in the presence of xanthine oxidase . The toxicity of this oxygen radical was prevented by preventing its formation with pretreatment with allopurinol, an xanthine oxidase inhibitor . The data suggest that xanthine oxidase may be the enzyme responsible for the respiratory burst of phagocytosis, as well as preventing reperfusion damage which occurs after ischemia.

J Bacteriol, 1986 Oct, 168(1), 86 - 95
Characterization by deletion and localized mutagenesis in vitro of the promoter region of the Escherichia coli ompC gene and importance of the upstream DNA domain in positive regulation by the OmpR protein; Mizuno T et al.; The ompC gene codes for a major outer membrane protein whose expression is regulated by the ompR and envZ genes . Two sets of promoter deletion mutants, with upstream and downstream deletions, were constructed on a plasmid in vitro, and their promoter activity was studied by connecting them with the lacZ gene . The DNA sequence for the ompC promoter, including the -35 and -10 regions and the mRNA start site, was defined at the region about 100 base pairs upstream from the ATG initiation codon for the pro-OmpC protein . An additional 61-base-pair sequence extending upstream from the -35 region was required for the ompC promoter to function fully . After targeting the upstream region of the ompC promoter fused to the lacZ gene on a plasmid, in vitro-localized mutagenesis was performed to isolate cis-dominant mutations that affect ompC transcription . Four mutant groups, each of which had common phenotypes for expression and regulation of the gene, were identified . The individual groups also had common base substitutions . In two of the groups, the common base substitutions were localized in the upstream region of the ompC promoter, whereas in the other two they were localized in the -35 region . From these results, the upstream region of the ompC promoter was considered to be the domain responsible for activation by the ompR gene product.

J Bacteriol, 1986 Oct, 168(1), 55 - 64
Isolation and characterization of Escherichia coli mutants defective for phenylpropionate degradation; Burlingame RP et al.; Mutants of Escherichia coli defective in catabolism of 3-phenylpropionate, 3-(3-hydroxyphenyl)propionate, or both were isolated after mutagenesis with ethylmethane sulfonate . Nine phenotypically distinct classes of mutants were identified, including strains lacking each of the first five enzyme activities for the degradation of these compounds and mutants pleiotropically negative for some of these activities . Characterization of these mutants was greatly facilitated by the use of indicator media in which accumulation of 3-(2,3-dihydroxyphenyl)propionate or 2-hydroxy-6-ketononadienedioic acid led to the formation of dark red or bright yellow colors, respectively, in the medium . Assays with wild-type and mutant strains indicated that 3-phenylpropionate (or its dihydrodiol), but none of the hydroxylated derivatives tested, induced the synthesis of enzymes for its conversion to 3-(2,3-dihydroxyphenyl)propionate . The remaining enzymes were induced by the 2- or 3-hydroxy or 2,3-dihydroxy derivatives of 3-phenylpropionate, with the 2-hydroxy compound acting as an apparent gratuitous inducer . Metabolism to nonaromatic intermediates appeared to be unnecessary for full induction of any pathway enzyme . One unusual class of mutants, in which 2-keto-4-pentenoate hydratase appeared to be uninducible, indicated a level of control not previously shown in meta-fission catabolic pathways.

J Bacteriol, 1986 Oct, 168(1), 464 - 6
SOS-associated division inhibition gene sfiC is part of excisable element e14 in Escherichia coli; Maguin E et al.; The cell division inhibition gene sfiC and the excisable element e14, both associated with the SOS response in Escherichia coli, are located at 25 min on the E . coli map . Blotting with a fragment of e14 DNA showed a strict correlation between the presence of e14 and the sfiC+ genotype . Introduction of only e14 into a recA- sfiC- strain made the strain sfiC+ . These results show that the sfiC gene is part of e14.

J Bacteriol, 1986 Oct, 168(1), 434 - 6
Localization of the structural gene for threonine dehydrogenase in Escherichia coli; Ravnikar PD et al.; The threonine dehydrogenase (tdh) gene of Escherichia coli, cloned within the plasmid pDR121, was inactivated in vitro by inserting a segment of DNA carrying the chloramphenicol acetyltransferase (cat) gene . The insertionally inactivated tdh gene was then transferred by homologous recombination into the E . coli chromosome by the procedure of Winans et al . (J . Bacteriol . 161:1219-1221, 1985) . Mating experiments, followed by P1-mediated two- and three-point crosses, enabled us to localize tdh near min 81.2 . The order with respect to known markers is mtl-cysE-tdh-pyrE.

J Bacteriol, 1986 Oct, 168(1), 412 - 6
Analysis of spontaneous base substitutions generated in mismatch-repair-deficient strains of Escherichia coli; Leong PM et al.; We used the lacI system of Escherichia coli to examine the distribution of base substitution mutations occurring spontaneously in different mismatch-repair-deficient strains . The examination of almost 1,200 nonsense mutations generated in strains carrying the mutS, mutH, and mutU alleles confirmed that transitions are highly favored over transversions . The detailed analysis of relative mutation rates at different sites revealed that the pattern of hot spots and cold spots is strikingly similar in each of the three strain backgrounds, strongly supporting the notions that the products of the three genes are part of the same system and that in the absence of any of the components the entire system fails to function . The distribution of mutations occurring in the absence of mismatch repair defined a pronounced topography of the lacI gene . There was no obvious correlation of the hot spots or cold spots with either nearest-neighbor sequences or A X T richness of the immediate surrounding sequence.

J Bacteriol, 1986 Oct, 168(1), 294 - 302
Nucleotide sequence of the phoM region of Escherichia coli: four open reading frames may constitute an operon; Amemura M et al.; The phoM gene is one of the positive regulatory genes for the phosphate regulon of Escherichia coli . We analyzed the nucleotide sequence of a 4.7-kilobase chromosomal DNA segment that encompasses the phoM gene and its flanking regions . Four open reading frames (ORFs) were identified in the order ORF1-ORF2-ORF3 (phoM)-ORF4-dye clockwise on the standard E . coli genetic map . Since these ORFs are preceded by a putative promotor sequence upstream of ORF1 and followed by a putative terminator distal to ORF4, they seem to constitute an operon . The 157-amino-acid ORF1 protein contains highly hydrophobic amino acids in the amino-terminal portion, which is a characteristic of a signal peptide . The 229-amino-acid ORF2 protein is highly homologous to the PhoB protein, a positive regulatory protein for the phosphate regulon . The ORF3 (phoM gene) protein contains two stretches of highly hydrophobic residues in the amino-terminal and central regions and, therefore, may be a membrane protein . The 450-amino-acid ORF4 protein contains long hydrophobic regions and is likely to be a membrane protein.

J Bacteriol, 1986 Oct, 168(1), 251 - 6
Molecular analysis of the UV protection and mutation genes carried by the I incompatibility group plasmid TP110; Glazebrook JA et al.; The imp genes, responsible for the UV protection and mutation effects of the I incompatibility group plasmid TP110, have been cloned into vector plasmids, and their products have been analyzed . The genetic information required for expression of these properties was carried in a continuous DNA sequence of approximately 1.7 kilobases, encoding the production of two proteins with molecular weights of 11,000 and 51,000 . The genetic arrangement of this system therefore appears similar but not identical to the functionally related umuDC and mucAB operons . A third protein with a molecular weight of 40,000 was produced from sequences downstream from imp and could be overproduced by high-level transcription through the imp genes . This protein was not required for the protection and mutation properties.

J Bacteriol, 1986 Oct, 168(1), 228 - 36
DNA and amino acid sequence analysis of structural and immunity genes of colicins Ia and Ib; Mankovich JA et al.; The nucleotide sequences for colicin Ia and colicin Ib structural and immunity genes were determined . The two colicins each consist of 626 amino acid residues . Comparison of the two sequences along their lengths revealed that the two colicins are nearly identical in the N-terminal 426 amino acid residues . The C-terminal 220 amino acid residues of the colicins are only 60% identical, suggesting that this is the region most likely recognized by their cognate immunity proteins . The predicted proteins for the colicin immunity proteins would contain 111 amino acids for the colicin Ia immunity protein and 115 amino acids for the colicin Ib immunity protein . The colicin immunity proteins have no detectable DNA or amino acid homology but do exhibit a conservation of overall hydrophobicity . The colicin immunity genes lie distal to and in opposite orientation to the colicin structural genes . The colicin Ia immunity protein was purified to apparent homogeneity by a combination of isoelectric focusing and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The N-terminal amino acid sequence of the purified Ia immunity protein was determined and was found to be in perfect agreement with that predicted from the DNA sequence of its structural gene . The Ia immunity protein is not a processed membrane protein.

J Bacteriol, 1986 Oct, 168(1), 213 - 20
Participation of the dnaK and dnaJ gene products in phosphorylation of glutaminyl-tRNA synthetase and threonyl-tRNA synthetase of Escherichia coli K-12; Wada M et al.; The heat shock proteins DnaK and DnaJ of Escherichia coli participate in phosphorylation of both glutaminyl-tRNA synthetase and threonyl-tRNA synthetase . When cellular proteins extracted from the dnaK7(Ts) and dnaJ259(Ts) mutant cells labeled with 32Pi at 42 degrees C were analyzed by two-dimensional gel electrophoresis, no phosphorylation of these proteins was observed when they were compared with those from wild-type cells.

J Bacteriol, 1986 Oct, 168(1), 152 - 9
Molecular cloning of the genes for lipid A disaccharide synthase and UDP-N-acetylglucosamine acyltransferase in Escherichia coli; Crowell DN et al.; Several enzymes have been discovered recently in crude extracts of Escherichia coli that appear to be involved in the biosynthesis of the lipid A component of lipopolysaccharide . Two of these are lipid A disaccharide synthase and UDP-N-acetylglucosamine acyltransferase . Lipid A disaccharide synthase activity is barely detectable in cells harboring a lesion in the lpxB (pgsB) gene . We subcloned the lpxB gene from plasmid pLC26-43 of the Clarke and Carbon collection (L . Clarke and J . Carbon, Cell 9:91-99, 1976) and localized it to a 1.7-kilobase-pair fragment of DNA counterclockwise of dnaE on the E . coli chromosome . Furthermore, we discovered a new gene (lpxA) located adjacent to and counterclockwise of lpxB that encodes or controls UDP-N-acetylglucosamine acyltransferase . Our data prove that lpxB and lpxA are transcribed in the clockwise direction and suggest that they may be cotranscribed.

J Bacteriol, 1986 Oct, 168(1), 140 - 51
Induction kinetics and cell surface distribution of Escherichia coli lipoprotein under lac promoter control; Hiemstra H et al.; The induction kinetics and surface accessibility of the outer membrane lipoprotein were studied in an Escherichia coli strain with the lpp gene under control of the lac promoter . Free lipoprotein appeared rapidly after induction with isopropyl-beta-D-thiogalactopyranoside and reached a steady-state level after 30 min . The newly induced lipoprotein was slowly bound to the peptidoglycan layer . Immunological methods were developed to detect lipoprotein accessible at the cell surface after various pretreatments as well as peptidoglycan-bound lipoprotein at the surface of isolated peptidoglycan sacculi with specific antibodies in combination with 125I-protein A . With these methods an increase in lipoprotein molecules at the cell surface and bound to the peptidoglycan sacculus could be detected following induction . The topology of newly synthesized lipoprotein was examined in thin sections as well as at the cell surface and the surface of the peptidoglycan sacculus with immunoelectron microscopy . Ultrathin cell sections, whole cells, and isolated peptidoglycan sacculi showed lipoprotein distributed homogeneously over the entire surface.

J Bacteriol, 1986 Oct, 168(1), 132 - 9
Origin of transfer of IncF plasmids and nucleotide sequences of the type II oriT, traM, and traY alleles from ColB4-K98 and the type IV traY allele from R100-1; Finlay BB et al.; The complete nucleotide sequences of the ColB4-K98 (ColB4) plasmid transfer genes oriT, traM, and traY as well as the traY gene of R100-1 are presented and compared with the corresponding regions from the conjugative plasmids F, R1, and R100 . The sequence encoding the oriT nick sites and surrounding inverted repeats identified in F was conserved in ColB4 . The adenine-thymine-rich sequence following these nick sites was conserved in R1 and ColB4 but differed in F and R100, indicating that this region may serve as the recognition site for the traY protein . A series of direct repeats unique to the ColB4 plasmid was found in the region of dyad symmetry following this AT-rich region . This area also encodes 21-base-pair direct repeats which are homologous to those in F and R100 . The traM gene product may bind in this region . Overlapping and following these repeats is the promoter(s) for the traM protein . The traM protein from ColB4 is similar to the equivalent products from F, R1, and R100 . The traY protein from ColB4 is highly homologous to the R1 traY gene product, while the predicted R100-1 traY product differs at several positions . These differences presumably define the different alleles of traM and traY previously identified for IncF plasmids by genetic criteria . The translational start codons of the ColB4 and R100-1 traY genes are GUG and UUG, respectively, two examples of rare initiator codon usage.

J Virol, 1986 Oct, 60(1), 317 - 9
Temperate coliphage HK253: attachment site and restricted transduction of proAB mutants of Escherichia coli K-12; Poon AP et al.; Temperate coliphage HK253 integrates near the proAB locus on the Escherichia coli K-12 chromosome . It can bring about specialized transduction of proAB and phoE mutants of E . coli, but it is incapable of general transduction . One of the proline-transducing particles was found to be nondefective.

Protein Eng, 1986 Oct-Nov, 1(1), 29 - 35
Altered specificities of genetically engineered alpha 1 antitrypsin variants; Jallat S et al.; Seven active site variants of human alpha 1-antitrypsin (alpha 1AT) were produced in Escherichia coli following site-specific mutagenesis of the alpha 1AT complementary DNA . alpha 1AT (Ala358), alpha 1AT (Ile358) and alpha 1AT (Val358) were efficient inhibitors of both neutrophil and pancreatic elastases, but not of cathepsin G . alpha 1AT (Ala356, Val358) and alpha 1AT (Phe358) specifically inhibited pancreatic elastase and cathepsin G respectively . The most potent inhibitor of neutrophil elastase was alpha 1AT (Leu358), which also proved to be effective against cathepsin G . The alpha 1AT (Arg358) variant inactivated thrombin with kinetics similar to antithrombin III in the presence of heparin . Electrophoretic analysis showed that SDS-stable high mol . wt complexes were formed between the mutant inhibitors and the cognate proteases in each case . These data indicate that effective inhibition occurs when the alpha 1AT P1 residue (position 358) corresponds to the primary specificity of the target protease . Moreover, alteration of the P3 residue (position 356) can further modify the reactivity of the inhibitor . Two of the variants have therapeutic potential: alpha 1AT (Leu358) may be more useful than plasma alpha 1AT in the treatment of destructive lung disorders and alpha 1AT (Arg358) could be effective in the control of thrombosis.

Proc Natl Acad Sci U S A, 1986 Oct, 83(19), 7292 - 6
Nucleotide sequence of rat preputial gland beta-glucuronidase cDNA and in vitro insertion of its encoded polypeptide into microsomal membranes; Nishimura Y et al.; We have selected the rat preputial gland beta-glucuronidase as a model protein to study the sorting of newly synthesized lysosomal hydrolases to the lysosome . The complete coding sequence of beta-glucuronidase messenger RNA was determined from the sequences of a group of overlapping cDNA clones isolated from preputial gland cDNA libraries . The beta-glucuronidase mRNA primary translation product contains 648 amino acids, including an amino-terminal signal sequence of 22 residues . The polypeptide has four potential sites for the addition of asparagine-linked core oligosaccharides . A 376-residue segment of beta-glucuronidase shows extensive homology (23% sequence identity) to a portion of Escherichia coli beta-galactosidase . This homology most likely reflects an evolutionary relationship between the bacterial and eukaryotic enzymes and the conservation of structural features necessary for the glycosidase activity of both proteins . Translation of mRNA synthesized in vitro by transcription of a cDNA containing the entire beta-glucuronidase coding region yielded a polypeptide that was immunoprecipitated with anti-beta-glucuronidase antiserum and had the same electrophoretic mobility as the primary translation product of natural beta-glucuronidase mRNA . In the presence of microsomal membranes, the in vitro-synthesized beta-glucuronidase underwent cotranslational incorporation into the microsomes, as indicated by removal of the signal sequence and the addition of several oligosaccharide chains . The beta-glucuronidase cDNA will provide a useful tool to study the mechanism of mannose phosphorylation and other aspects of the sorting of lysosomal enzymes to lysosomes.

Protein Eng, 1986 Oct-Nov, 1(1), 17 - 21
Expression of the amino terminal part of synthetic human growth hormone gene and somatomedin-like activity of expressed protein; Doi T et al.; We have constructed three different plasmids containing parts of the human growth hormone gene using chemically synthesized oligomers and cloned them for the purpose of expressing them in Escherichia coli . AB, B and BC gene segments corresponding to ABhGH (residue 1-138), BhGH (residue 44-138) and BChGH (residue 44-192) were placed under the control of a tryptophan promoter in the expression vector . Upon induction with 3-indolylacrylic acid, ABhGH accumulated in cells but the BhGH and BChGH segments were not detected appreciably . Northern blotting analysis showed that the amount of mRNA transcribed from the AB gene segment was about ten-fold higher than that from the B or BC gene segment . ABhGH was found to have insulin-like growth factor I (IGF-I) activity, which could be explained by the hydrophilicity curves of these proteins.

J Ultrastruct Mol Struct Res, 1986 Oct-Dec, 97(1-3), 187 - 96
Transverse sectioning of plastic-embedded immunolabeled cryosections: morphology and permeability to protein A-colloidal gold complexes; Stierhof YD et al.; In order to provide data for meaningful interpretation and quantitation of immunogold labeling on cryosections their morphology and permeability to protein A-gold were evaluated: We studied plastic sections of immunogold-labeled ultrathin and semithick cryosections cut perpendicular to the original cryosection plane . Various soluble and insoluble antigens in different specimens (hemoglobin and histone H5 in chicken erythrocytes, tubulin in Leishmania cells, and outer membrane protein OmpA in Escherichia coli) were fixed with glutaraldehyde-formaldehyde, formaldehyde, or periodate-lysine-paraformaldehyde and incubated with specific antibodies and protein A-gold of different sizes . The cryosection surface may be rough or smooth depending both on the sectioned material and on dehydration and drying artifacts or possibly on the cutting process itself . Well-preserved sections are capable of withstanding considerable deformation without showing clefts or cracks . If the sectioned specimen is sufficiently fixed, protein A-gold is not able to enter the IgG-labeled sections significantly but follows surface irregularities . However, gold particles can be detected within visibly damaged sections.

Proteins, 1986 Oct, 1(2), 125 - 33
Cloning, expression, and nucleotide sequence of livR, the repressor for high-affinity branched-chain amino acid transport in Escherichia coli; Antonucci TK et al.; The livR gene encoding the repressor for high-affinity branched-chain amino acid transport in Escherichia coli has been cloned from a library prepared from the episome F106 . The inserted DNA fragment from the initial cloned plasmid, pANT1, complemented two independent, spontaneously derived, regulatory mutations . Subcloning as well as the creation of deletions with Bal31 exonuclease revealed that the entire regulatory region is contained within a 1.1-kb RsaI-SalI fragment . Expression of the pANT plasmids in E . coli minicells showed that the regulatory region encodes one detectable protein with an apparent molecular weight of 21,000 . DNA sequencing revealed one open reading frame of 501 bp encoding a protein with a calculated MW of 19,155 . The potential secondary structure of the regulatory protein has been predicted and it suggests that the carboxy terminus may fold into three consecutive alpha helices . These results suggest that the livR gene encodes a repressor which plays a role in the regulation of expression of the livJ and the livK transport genes.

J Gen Microbiol, 1986 Oct, 132 ( Pt 10), 2677 - 84
Effect of alkylating agents on the expression of inducible genes of Escherichia coli; Vericat JA et al.; Increasing doses of alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine, diethyl sulphate and ethylmethane sulphonate cause an inhibition of the expression of the recA and sfiA genes of wild-type Escherichia coli . This behaviour was not observed in a lexA56 mutant which has a defective LexA repressor that is unable to bind to the SOS operator . Furthermore, an ada-1 mutant showed the same behaviour as the wild-type strain indicating that the adaptive proteins are not responsible for the inhibition of recA and sfiA at high doses of alkylating agents . These results suggest that the inhibitory effect of these alkylating agents may be found in the interaction between the LexA repressor and the control regions of sfiA and recA . On the other hand, high doses of either UV light or mitomycin C produced only a slight decrease in the induction of recA and sfiA, whereas bleomycin had no effect . The fact that a repressor structurally related to LexA repressor, such as LacI protein, showed the same behaviour as the LexA repressor when a Lac+ strain was treated with alkylating agents, suggests that these compounds can modify the binding abilities of repressors to DNA, producing a limited or even abolished release of repressors, and so decreasing the expression of inducible genes.

Mol Gen Genet, 1986 Oct, 205(1), 28 - 33
The effect of spo0 mutations on the expression of spo0A- and spo0F-lacZ fusions; Yamashita S et al.; We have constructed spo0A-lacZ and spo0F-lacZ fusions with a temperate phage vector and have investigated how spo0 gene products are involved in the expression of each of these genes . The expression of spo0A-lacZ and spo0F-lacZ was stimulated at about the time of cessation of vegetative growth in Spo+ cells . This stimulation of spo0A-lacZ was impaired by mutations in the spo0B, D, E, F or H genes but was not affected by mutations in the spo0J or K genes . Similar results were obtained with the spo0F-lacZ fusion . The effect of the spo0A mutation on spo0A-lacZ expression was characteristic: the spo0A-directed beta-galactosidase activity found during vegetative growth was significantly enhanced in the spo0A mutant . This result suggests that spo0A gene expression is auto-regulated being repressed by its own gene product . Another remarkable observation was the effect of the sof-1 mutation, which is known to be a spo0A allele; it suppressed the sporulation deficiency of spo0B, spo0D and spo0F mutants . The spo0A-lacZ stimulation, which is impaired by any one of these spo0 mutations, was restored by the additional sof-1 mutation.

Biochimie, 1986 Oct-Nov, 68(10-11), 1211 - 5
The shikimate pathway . V . Fluorine-containing analogues of 3-deoxy-D-arabino hept-2-ulosonate-7-phosphate (DAHP); Le Marechal P et al.; (3R) and (3S) 3-deoxy-3-fluoro-7-phospho-D-arabino hept-2-ulosonic acids (3R and 3S-3F-DAHP) the 3-fluoro analogues of DAHP were synthesized from the corresponding 2-deoxy-2-fluoro hexose-6-phosphates . 3R- and 3S-3F-DAHP were tested as substrates for 3-dehydroquinate synthetase from E . coli . Determination of kinetic parameters showed that their apparent Km and Vm were in the same order of magnitude for these two compounds . Further conversion of 3R- and 3S-3F-DAHP into (6R) and (6S) 6-fluoro dehydroshikimate and (6R) and (6S) 6-fluoro shikimate, respectively, was investigated and results are discussed.

J Bacteriol, 1986 Oct, 168(1), 341 - 7
Control of developmental gene expression by cell-to-cell interactions in Myxococcus xanthus; Gill RE et al.; The ssbA mutants of Myxococcus xanthus behave as if they are unable to produce a cell-to-cell signal required for normal development . They are unable to form fruiting bodies or spores on developmental medium . They do sporulate, however, if allowed to develop in mixtures with wild-type cells . Fusions of developmentally induced promoters of M . xanthus to the Escherichia coli lacZ gene were used to characterize the effect of the ssbA mutations on developmental gene expression . Each of the five independent fusions tested was found to be dependent upon the ssbA+ allele for full expression . The ssbA mutants were able to express each of these fusions if the mutants were allowed to develop in mixtures with wild-type (Lac-) cells . These results cannot be explained on the basis of genetic exchange . The data are consistent with regulation of gene expression mediated by cell-to-cell interactions.

J Antimicrob Chemother, 1986 Oct, 18 Suppl C, 35 - 41
Role of transposition and homologous recombination in the rearrangement of plasmid DNA; Nies BA et al.; The multi-resistance plasmid pBP16 was used to analyse the variability of R-factors from clinical isolates and the molecular structures and processes involved . The observed rearrangement in pBP16 and its derivatives included inversion, deletion, replicon fusion and dissociation, and transposition . All these events could be traced to the presence and activity of multiple copies of the IS-element IS160 within pBP16 . Since IS-elements are common in R-factors, it is likely that they are one of the main reasons for plasmid instability and that they are involved in a major way in plasmid evolution.

Mol Gen Mikrobiol Virusol, 1986 Oct, (10), 39 - 44
{Cloning of DNA fragments of the genetic transfer factor pAP39}; Krivskaia KS et al.; Large HindIII digested fragments of the plasmid pAP39 have been cloned on the cosmid vector pHC79 . The study of the structure of HindIII fragments of plasmid pAP39 in the recombinant plasmids has shown that these fragments are represented by f1 + f2 fragments from the plasmid pAP1055, by f1 + f6 fragments from the plasmid pAP1056, by f2 + f3 fragments from the plasmid pAP1057 and by two f3 fragment from the plasmid pAP1058 . Physical maps of the recombinant plasmids have been constructed . The plasmid pAP39 is shown to contain two functionally active tra regions.

Mol Cell Biol, 1986 Oct, 6(10), 3433 - 42
Isolation and characterization of expressible cDNA clones encoding the M1 and M2 subunits of mouse ribonucleotide reductase; Thelander L et al.; Mammalian ribonucleotide reductase consists of two nonidentical subunits, proteins M1 and M2, which are differentially regulated during the cell cycle . We have isolated expressible cDNA clones of both subunits from an Okayama-Berg cDNA library made with mRNA from hydroxyurea-resistant, M2 protein-overproducing mouse TA3 cells . Expression of M2 protein could be demonstrated by electron paramagnetic resonance spectroscopy after transfection of COS-7 monkey cells with the plasmid . Electrophoresis and blot analyses of the parent and hydroxyurea-resistant TA3 mRNA revealed two M2 transcripts, a major one of 2.1 kilobases and a minor one of about 1.6 kilobases . Restriction endonuclease mapping of the corresponding cDNAs indicated that the two mRNAs differed only in the length of the 3' untranslated ends . By contrast, there was only one mRNA corresponding to the M1 protein, and its mobility corresponded to about 3.1 kilobases . The hydroxyurea-resistant TA3 cells contained a 50- to 100-fold excess of the M2 mRNAs over that of the parent cells and a 10-fold excess of the M1 mRNA . However, a Southern blot analysis of the corresponding genomic DNA sequences showed that the M2 gene was amplified fivefold but the M1 gene was still single copy . The complete nucleotide sequence of the 2,111-base-pair-long M2 cDNA revealed an open reading frame coding for 390 amino acids, which corresponds to a molecular weight of 45,100 . The mouse M2 protein sequence was quite homologous to the equivalent protein in the clam Spisula solidissima, while the homology to the smaller subunits of Epstein-Barr virus, herpes simplex virus type 2, and Escherichia coli ribonucleotide reductases were less pronounced.

Mol Gen Genet, 1986 Oct, 205(1), 56 - 65
Characterization of the gene products produced in minicells by pSM1, a derivative of R100; Armstrong KA et al.; At least ten polypeptides larger than 6 kilodaltons (K) are produced in minicells from the miniplasmid pSM1 in vivo . pSM1 (5804 bp) is a small derivative of the drug resistance plasmid R100 (ca . 90 kb) and carries the R100 essential replication region as well as some non-essential functions . Cloned restriction fragments of pSM1 and plasmids with deletions within pSM1 sequences were used to assign eight of the ten observed polypeptides to specific coding regions of pSM1 . Two of these polypeptides were identified as RepA1 and RepA2, proteins encoded by the essential replication region of pSM1/R100 . The nucleotide sequence consisting of 885 bp outside the essential replication region is presented here . This sequence contains an open reading frame, orf4, for a protein 22.9 K in size, and one of the pSM1-encoded polypeptides was identified as the orf4 gene product . Five additional polypeptides were shown to be the products of other open reading frames mapping outside the essential replication region . Specific functions have been assigned to four of these polypeptides and tentatively to the fifth.

Mol Gen Genet, 1986 Oct, 205(1), 160 - 3
T-even type phages can change their host range by recombination with gene 34 (tail fibre) or gene 23 (head); Riede I; T-even type phages recognize their cellular receptors with the tip of their long tail fibres . The gene products involved in receptor recognition are proteins 37 and 38 . While screening libraries of phage K3 with a probe of gene 38 from phage T2, a class of weakly hybridizing clones was found in addition to the expected clones of gene 38 of K3 . One of these clones was identified as being from gene 23 of the phage which codes for the major head subunit; another clone originated from gene 34, which codes for the proximal half of the long tail fibres . Neither gene product 23 nor 34 is involved in receptor recognition . Phages can recombine with the DNA of the gene 23 and gene 34 clones and change the host range.

Mol Gen Genet, 1986 Oct, 205(1), 134 - 45
A new cell division operon in Escherichia coli; Gill DR et al.; At 76 min on the E . coli genetic map there is a cluster of genes affecting essential cellular functions, including the heat shock response and cell division . A combination of in-vivo and in-vitro genetic analysis of cell division mutants suggests that the cell division gene fts E is the second gene in a 3 gene operon . A cold-sensitive mutant, defective in the third gene, is also unable to divide at the restrictive temperature, and we designate this new cell division gene fts X . Another cell division gene, fts S, is very close to, but distinct from, the 3 genes of the operon . The fts E product is a 24.5 Kd polypeptide which shows strong homology with a small group of proteins involved in transport . Both the fts E product and the protein coded by the first gene (fts Y) in the operon have a sequence motif found in a wide range of heterogeneous proteins, including the Ras proteins of yeast . This common domain is indicative of a nucleotide-binding site.

Mol Gen Genet, 1986 Oct, 205(1), 127 - 33
The carboxy-terminal region of haemolysin 2001 is required for secretion of the toxin from Escherichia coli; Gray L et al.; As a first step in the detailed analysis of the mechanism of secretion of haemolysin, we sought to identify sequences or domains within haemolysin A (HlyA) that are essential for its secretion . For this purpose we examined the properties of a deletion and Tn5 insertions into the region of the HlyA gene encoding the C-terminal part of the protein, since both of these are relatively simple to generate . We showed that removal of 27 amino acids from the C-terminus of HlyA is sufficient to inhibit secretion drastically, although the residual polypeptide is still haemolytically active . Cellular fractionation studies showed that haemolytic activity does not accumulate in large amounts within the periplasmic space during normal secretion . More significantly, activity does not appear to accumulate within this compartment when the export functions hlyB and hlyD are removed . These results are consistent with a mechanism in which interaction of the C-terminus of HlyA with the secretion machinery, located in the inner membrane, is followed by direct transfer of haemolysin to the medium.

Mol Gen Genet, 1986 Oct, 205(1), 115 - 21
Regulation of transcription of the chromosomal dnaA gene of Escherichia coli; Kucherer C et al.; By comparative S1 analysis we investigated the in vivo regulation of transcription of the chromosomal dnaA gene coding for a protein essential for the initiation of replication at the chromosomal origin . Inactivation of the protein in dnaA mutants results in derepression, whereas excess DnaA protein (presence of a DnaA overproducing plasmid) leads to repression of dnaA transcription . Both dnaA promoters are subject to autoregulation allowing modulation of transcriptional efficiency by at least 20-fold . Increasing the number of oriC sequences (number of DnaA binding sites) in the cell by introducing oriC plasmids leads to a derepression of transcription . Autoregulation and binding to oriC suggest that the DnaA protein exerts a major role in the regulation of the frequency of initiation at oriC . The efficiency of transcription of the dnaA2 promoter is reduced in the absence of dam methylation, which is involved in the regulation of oriC replication.

Genetika, 1986 Oct, 22(10), 2398 - 407
{Mutations predetermined by the primary structure of DNA}; Salganik RI et al.; This study is concerned with an experimental verification of hypotheses postulating the involvement of self-complementary nucleotide sequences in the formation of deletions and insertions . It was suggested that deletions can arise in the regions of self-complementary nucleotide sequences, which allows the formation of the hairpin structures in a single-stranded DNA, arising during excision repair . These hairpin structures can be eliminated by nucleases or during DNA replication . Insertions can arise as a result of homologous recombination, when a migrating DNA strand contains a self-complementary sequence which forms hairpin structure . Model experiments were carried out with the pBR322 plasmid . A plasmid DNA with premutational damage in the palindrome-containing region was constructed by in vitro dimethylsulfate modification of one strand of EcoRI-BamHI restriction fragment . The plasmid was used for transformation of Escherichia coli . Restriction mapping and nucleotide analysis of the mutant DNAs demonstrated that they all contained deletions . The end points of the deletions coincide with the palindrome . To model homologous recombination, a plasmid with D-loop was constructed . A single-stranded DNA fragment containing palindrome forming a hairpin structure was introduced into the plasmid DNA and covalently fixed in the complex . When E . coli cells were transfected with this DNA, plasmid mutants containing insertions predetermined by palindromic structure arose . The evolutionary role of mutations predetermined by primary DNA structure is discussed.

EMBO J, 1986 Oct, 5(10), 2569 - 76
A domain of SV40 capsid polypeptide VP1 that specifies migration into the cell nucleus; Wychowski C et al.; In order to identify the determinants responsible for the nuclear migration of simian virus 40 (SV40) polypeptide VP1, the 5'-terminal portion of the SV40 VP1 gene was fused with the complete cDNA sequence of poliovirus capsid polypeptide VP1 and the hybrid gene was inserted into an SV40 vector in place of the normal SV40 VP1 gene . Deletions of various length were generated in the SV40 VP1 portion of the hybrid gene, resulting in a set of truncated genes encoding 2-40 NH2-terminal amino acids from SV40 VP1, followed by poliovirus VP1 . Monkey kidney cells were infected by the deleted hybrid viruses in the presence of an early SV40 amber mutant as helper, and the subcellular localization of the fusion proteins was determined by indirect immunofluorescence using an anti-poliovirus VP1 immune serum . The presence of the first 11 NH2-terminal amino acids from SV40 VP1 was found to be sufficient to target the fusion protein to the cell nucleus . Deletions extending from the NH2- towards the COOH-terminal end of the protein were next generated . Transport of the SV40 VP1-poliovirus VP1 fusion polypeptide to the nucleus was abolished when the first eight amino acids from SV40 VP1 were deleted . Thus the sequence of the first eight NH2-terminal amino acids of SV40 VP1 appears to contain a nuclear migration signal which is sufficient to target the protein to the cell nucleus.

DNA, 1986 Oct, 5(5), 345 - 56
Molecular cloning and expression in Escherichia coli of equine type I interferons; Himmler A et al.; Using human interferon-alpha 2 (IFN-alpha 2) and IFN-beta DNA to probe an equine genomic library we isolated recombinant phages containing genes for equine interferon-alpha (EqIFN-alpha), interferon-beta (EqIFN-beta), and interferon-omega (EqIFN-omega) . Sequence and hybridization analyses of these genes reveal that the equine genome contains gene families of each of these three type I interferon classes . The mature proteins of EqIFN-alpha are 71-77% homologous to human IFN-alpha polypeptides, and, when expressed in E . coli, possess antiviral activity on both equine and human cells . By contrast, EqIFN-beta is only 59% homologous to its human counterpart and shows activity only on equine cells.

J Anim Sci, 1986 Oct, 63(4), 1307 - 13
Diarrhea: the nemesis of the artificially reared, early weaned piglet and a strategy for defense; Lecce JG; Rearing early weaned piglets artificially for the purpose of increasing the efficiency of the sow is an attractive management concept . However, high death losses resulting from diarrhea in artificially reared piglets have dampered enthusiasm for early weaning . Enterotoxigenic Escherichia coli, transmissible gastroenteritis virus and rotavirus are the three main enteropathogens responsible for causing the diarrhea . The enteropathogens infect the small intestine, which produces a secretory or malabsorptive diarrhea . In nature, the nursing piglet is protected from the enteropathogens by antibody bathing his gut . The source of the antibody is the dam's colostrum and milk . It should be possible to protect artificially reared, early weaned piglets from enteropathogens by feeding them diets that contain antibodies to putative enteropathogens.

Biull Eksp Biol Med, 1986 Oct, 102(10), 459 - 61
{Effect of transposons on the system regulating derepressed plasmid tra-genes}; Shchipkov VP et al.; The ability of standard plasmids of six Fin-groups to inhibit the functions of genes transferring derepressed F-like plasmids has been studied . It is shown that transposons incorporation into the structure of individual plasmids alters the regulatory system of plasmid tra-genes.

Proc Natl Acad Sci U S A, 1986 Oct, 83(19), 7547 - 51
Cloning and expression of cDNA for human diazepam binding inhibitor, a natural ligand of an allosteric regulatory site of the gamma-aminobutyric acid type A receptor; Gray PW et al.; Diazepam binding inhibitor (DBI) is a protein that displaces ligands bound to the beta-carboline/benzodiazepine recognition site, an allosteric modulatory site of the type A gamma-aminobutyric acid receptor complex . An incomplete rat cDNA clone coding for DBI was isolated . This rat sequence was utilized to identify a cDNA clone that encoded the entire 104 residues of human DBI . This sequence was engineered for expression in E . coli, and recombinant DBI exhibits identical biochemical and antigenic characteristics of natural human DBI . DBI is encoded by a multigene family of at least five members, but a single gene appears to account for the majority of DBI expression . DBI is expressed in a tissue-specific manner . Expression is found in central nervous system tissues and appears to extend to peripheral tissues rich in the peripheral type of high-affinity benzodiazepine recognition sites . The role of these sites and DBI in adrenal gland, testis, and kidney remains to be determined.

Proc Natl Acad Sci U S A, 1986 Oct, 83(19), 7192 - 6
Expression of the complete human T-cell leukemia virus type I pX coding sequence as a functional protein in Escherichia coli; Giam CZ et al.; Human T-cell leukemia virus type I (HTLV-I), a virus associated with adult T-cell leukemia, contains a long open reading frame (LOR) in the 3' end of its genome between the env region and the 3' long terminal repeat (LTR) . This open reading frame encodes a 40-kDa protein (designated p40x) that has been implicated as a positive control element for transcription from the HTLV-I LTR in a phenomenon known as trans-activation . We now report the expression of the complete p40x coding sequence as a 40-kDa protein in Escherichia coli . The p40x protein produced in bacteria is shown, using the protoplast fusion technique, to possess biological activity by its ability to trans-activate a HTLV-I LTR-chloramphenicol acetyltransferase plasmid that is stably integrated into the genome of mouse L cells . This stimulatory activity could be detected within 2 hr after fusion, suggesting the possibility of a direct role for p40x in trans-activation of the HTLV-I LTR . The production of p40x in large quantities in E . coli, together with the rapid protoplast fusion assay for its biological activity, should facilitate the analysis of p40x mutants and the elucidation of the molecular mechanism of trans-activation.

J Bacteriol, 1986 Oct, 168(1), 72 - 80
Cloning and expression of Mycobacterium bovis BCG DNA in "Streptomyces lividans"; Kieser T et al.; The ability of "Streptomyces lividans" to use the expression signals of genes from Mycobacterium bovis BCG was tested in vivo by using gene fusions . Random DNA fragments from M . bovis BCG were inserted into promoter-probe plasmids in Escherichia coli and in "S . lividans." Comparison with promoter activity detected with random DNA fragments from the respective hosts suggested that "S . lividans" efficiently utilizes a high proportion of mycobacterial promoters, whereas a smaller fraction are expressed, and expressed more weakly, in E . coli . M . bovis BCG DNA fragments were also inserted into the specially constructed translational fusion vector (pIJ688) in "S . lividans." pIJ688 contains the kanamycin phosphotransferase gene (neo) from transposon Tn5, truncated at its amino terminus, as the indicator . The results suggested that "S . lividans" uses M . bovis BCG translational signals almost as efficiently as its own signals . Moreover, several hybrid proteins with an M . bovis BCG-derived amino terminus seemed to be reasonably stable in "S . lividans." These experiments indicate that "S . lividans" may be a suitable host for the expression of Mycobacterium leprae and Mycobacterium tuberculosis genes from their own signals . This is a precondition for the expression of entire biosynthetic pathways, which could be valuable in the production of diagnostic and therapeutic agents . The vectors may also have wider applications for the analysis of gene expression in Streptomyces.

J Bacteriol, 1986 Oct, 168(1), 440 - 3
Requirement of the Escherichia coli dnaA gene product for plasmid F maintenance; Kline BC et al.; There are DnaA protein-binding sites in at least one F origin of replication, and only potentially leaky dnaA(Ts) mutations had ever been used in previous studies indicating that F replication was independent of the dnaA gene product . Here we show that an Escherichia coli dnaA::Tn10 host which does not make a dnaA gene product cannot sustain autonomous or integrated F plasmid maintenance.

J Bacteriol, 1986 Oct, 168(1), 276 - 82
Effects of DNA gyrase inhibitors in Escherichia coli topoisomerase I mutants; Pruss GJ et al.; Relaxation of titratable supercoils in bacterial nucleoids was measured following treatment of topA mutants with coumermycin or oxolinic acid, inhibitors of DNA gyrase . Relaxation occurred after treatment of the mutants with either inhibitor . We detected no significant difference in relaxation between topA- and topA+ strains treated with coumermycin . This finding, together with previous observations, supports the idea that relaxation caused by coumermycin probably arises from the relaxing activity of gyrase itself . The source of DNA relaxation caused by oxolinic acid was not identified . Nucleoid supercoiling can be increased by adding oxolinic acid to a strain that carries three topoisomerase mutations: delta topA, gyrB225, and gyrA (Nalr) (S . H . Manes, G . J . Pruss, and K . Drlica, J . Bacteriol . 155:420-423, 1983) . We found that this increase in supercoiling requires partial sensitivity to the drug and at the delta topA and gyrA mutations . Full resistance to oxolinic acid in the presence of the delta topA, gyrB225, and gyrA mutations was conferred by an additional mutation that maps at or near gyrB.

J Bacteriol, 1986 Oct, 168(1), 179 - 85
Metastable regulation of type 1 piliation in Escherichia coli and isolation and characterization of a phenotypically stable mutant; Spears PA et al.; Type 1 piliation in Escherichia coli exhibits phase variation due to the inversion of a small, ca . 300-base-pair, element that regulates pilA (fimA), the gene that encodes the structural subunit of pili (Abraham et al., Proc . Natl . Acad . Sci . USA 82:5724-5727, 1985) . We have used the inversion as an assay to characterize a stably piliated mutant . The mutant strain did not exhibit the pilA ON and pilA OFF colonial variants characteristic of the wild type; rather, every clone produced a level of pilA expression intermediate between ON and OFF wild-type populations . The mutant phenotype was conferred by a lesion at a previously undescribed locus between hemA and trpA, which we have termed pilG . Examination of the pilA promoter region in four pilG mutant populations indicated that the phenotypic stability conferred by the pilG mutation was not due to an inability to carry out the inversion . Rather, all pilG mutant populations consisted of approximately equal mixtures of ON and OFF individuals . We suggest that pilG mutants may undergo such rapid switching of the pilA promoter that populations exhibit an intermediate level of pilA expression and phenotypic stability.

J Bacteriol, 1986 Oct, 168(1), 13 - 21
Control of sensitivity to inactivation by H2O2 and broad-spectrum near-UV radiation by the Escherichia coli katF locus; Sammartano LJ et al.; Mutations in the Escherichia coli katF gene (hydroperoxidase II) result in sensitivity to inactivation by H2O2 and broad-spectrum near-UV (NUV; 300 to 400 nm) radiation . Another mutation, nur, originally described as conferring sensitivity to inactivation by broad-spectrum and monochromatic NUV, also confers sensitivity to inactivation by H2O2 . Genetic analysis via transduction suggests that the nur mutation allele of the katF locus . As previously reported for broad-spectrum and monochromatic NUV wavelengths, the sensitivity of a particular strain to H2O2 inactivation is also independent of the recA and uvrA alleles . Extracts of nur and katF strains lack catalase (hydroperoxidase II) as revealed by polyacrylamide gels stained for such activity, which is consistent with the genetic results.

Mutat Res, 1986 Oct, 163(1), 3 - 13
Introduction, rescue and expression of plasmid genes in mammalian cells and Escherichia coli; Zakour RA et al.; A shuttle-vector system is described for the study of mutational specificity in mammalian cells . Using a plasmid (pGKTK) carrying the E . coli galactokinase gene (gk) and the herpes simplex virus thymidine kinase gene (tk), we demonstrate the introduction of a foreign gene into the chromosome of a mammalian cell (TK- mouse fibroblasts) and its efficient rescue back into E . coli . This system makes use of two genes, each of which can expressed in both E . coli and mammalian cells, thereby permitting one marker to be the mutational target and the other to maintain stable integration in the host . In addition, expression of both genes in bacteria makes it possible to deletion map mutants to facilitate their sequencing . In the case of a putative single-copy transformant (T8), about half of the rescued plasmids are identical in size and restriction pattern to the original plasmid . Each of these expressed the tk gene, indicating the fidelity of the rescue system.

Cancer Res, 1986 Oct, 46(10), 4984 - 90
Differential effects of recombinant human leukocyte interferons on cell surface antigen expression; Greiner JW et al.; Human leukocyte (alpha) interferon (IFN-alpha) is composed of a multigene family within which at least eight different species have been expressed in Escherichia coli, isolated, and shown to exert a wide range of biological activities on different human target cells . In this study we utilized eight species of IFN-alpha (A, B, C, D, F, I, J, and K) and investigated their respective capabilities to alter the proliferation of a human breast carcinoma cell line (MCF-7) . The antigens studied were all constitutively expressed on the MCF-7 cell surface: the Mr 180,000 carcinoembryonic antigen; a high molecular weight (greater than 10(6} glycoprotein, termed tumor-associated glycoprotein 72; and a major HLA histocompatibility antigen . The level of expression of each antigen was measured by the binding of monoclonal antibodies B1.1, B72.3, and W6/32, respectively . A high degree of diversity was found among the various IFN-alpha species with respect to their ability to enhance antigen expression and inhibit MCF-7 cell growth . The two most potent species, IFN-alpha A and IFN-alpha B, were found to increase the expression of tumor antigens as well as the HLA determinant by 2-5-fold . In contrast, IFN-alpha D and IFN-alpha J were virtually inactive in altering antigen expression but did inhibit the growth of MCF-7 cells . The remaining IFN-alpha species, -alpha C, -alpha F, -alpha I, and -alpha K, exerted an intermediate range of activities for both antigen enhancement and inhibition of MCF-7 cell growth . The relative ability of each species of IFN-alpha to inhibit MCF-7 cell growth appeared to be independent of their effectiveness in augmenting antigen expression . IFN-alpha D and IFN-alpha J, the two species that failed to alter tumor antigen expression, did, however, seem to interact with the interferon receptor since they inhibited MCF-7 cell growth and competed with other IFN-alpha species for the increase in carcinoembryonic antigen, tumor-associated glycoprotein 72, or HLA expression . A comparison of the concentrations of each IFN-alpha necessary to enhance antigen expression revealed that the surface HLA determinant was approximately 10-fold more sensitive to enhancement than was the tumor antigen, carcinoembryonic antigen . The individual members of the IFN-alpha family thus differ extensively in their ability to alter the level of antigen expression on the surface of MCF-7 breast carcinoma cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Avian Dis, 1986 Oct-Dec, 30(4), 781 - 7
Biological and immunological characterization of pili of Escherichia coli serotypes O1, O2, and O78 pathogenic to poultry; Suwanichkul A et al.; Pili of Escherichia coli serotypes O1, O2, and O78 pathogenic to poultry were isolated and purified by sucrose-density-gradient centrifugation . Each serotype expressed only one type of pilus . The pili of the three serotypes had similar densities and were morphologically similar by electron microscopy . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, however, showed that they were slightly different in subunit molecular mass . Slide agglutination, immunodiffusion, and immunoblot tests were used to test for antigenic relationships between these pili and reference pili . Pili of serotype O78 were type 1, but pili of serotypes O1 and O2 were not, as once believed . However, pili of serotype O2 reacted positively with anti-type 1 serum in immunoblot assay, suggesting the presence of some common antigenic epitopes among these pili.

Avian Dis, 1986 Oct-Dec, 30(4), 687 - 9
Immunogenicity of an Escherichia coli multivalent pilus vaccine in chickens; Gyimah JE et al.; Immunogenicity of an oil-emulsified Escherichia coli multivalent pilus vaccine was evaluated in 4-week-old chickens . The vaccine contained 180 micrograms of pilus protein from each of serotypes O1 and O78 and 170 micrograms of pilus protein from serotype O2 . Chickens were vaccinated twice subcutaneously at 4 and 6 weeks old and challenged via the posterior thoracic air sac with E . coli serotype O1, O2, or O78 2 weeks after the last vaccination . Unvaccinated challenged chickens suffered 8% to 26% mortality; no vaccinated chickens died . Vaccinated chickens had very mild gross lesions in the air sacs, livers, and pericardial sacs and eliminated E . coli more efficiently than the unvaccinated challenged chickens . The results showed that a multivalent pilus vaccine protects chickens against active respiratory infection.

Biochimie, 1986 Oct-Nov, 68(10-11), 1181 - 7
Nucleotide sequence of the Azospirillum brasilense Sp7 glutamine synthetase structural gene; Bozouklian H et al.; The complete nucleotide sequence of the glnA gene, encoding the glutamine synthetase subunit of Azospirillum brasilense Sp7, was established . This is the first Azospirillum gene sequenced . The gene encodes a 468 residue polypeptide of MW 51,917 . The similarity coefficient (SAB) between the polypeptidic sequence of Azospirillum and Anabaena 7120, which is the only other glnA sequence available, is 58% . No significant homology with E . coli canonical and ntr promoters, or with the promoter region of the Anabaena glnA gene was found . When fused to an E . coli promoter, the gene could be translated in E . coli, despite a very biased codon usage and an atypical Shine-Dalgarno sequence.

Biochimie, 1986 Oct-Nov, 68(10-11), 1159 - 63
Mutation that affects pepN translation initiation in Escherichia coli; Gharbi S et al.; Mutants altered in their expression of the hybrid pepN-lacZ gene have been selected for resistance to p-nitrophenyl-beta-D-thiogalactopyranoside (a bacteriostatic compound that enters the cells via lac permease) . A unique mutation decreasing the level of pepN expression to 9% of that of the wild type has been studied in detail . This mutation controls in cis the expression of the pepN gene . The pepN region from a pepN-lacZ gene fusion has been cloned and sequenced . Comparison of the mutant and wild type sequences indicates that the mutation lies between the Shine-Dalgarno sequence (AGGT) and the initiation codon (AUG) . This mutation is a T----C transition which might allow the formation of a stable secondary structure in the region of translation initiation thus decreasing the level of pepN expression.

Eur J Biochem, 1986 Oct 1, 160(1), 77 - 82
Nucleotide sequence of the structural gene for dihydroorotase of Escherichia coli K12; Backstrom D et al.; The nucleotide sequence of the dihydroorotase structural gene, pyrC, of Escherichia coli K12 has been determined . The DNA sequence predicts a polypeptide chain of 347 amino acid residues corresponding in size and composition to the previously purified dihydroorotase subunit . Nuclease S1 mapping indicated that transcription of pyrC is initiated around 40 base pairs upstream from the translational start . The transcriptional leader region contains a region of dyad symmetry, which allows a stable hairpin to be formed . This sequence may have regulatory functions since similar structures are found in other pyr genes . The nucleotide sequence also contains a 186-codon open reading frame in front of pyrC . Nuclease Bal31-deletion derivatives of pyrC plasmids indicate that this gene does not affect the expression of pyrC . The predicted polypeptide chain shows a putative signal sequence . Downstream from the structural gene a sequence similar to a rho-independent transcriptional terminator is found . This unknown gene may thus encode a membrane protein of unknown function.

Eur J Biochem, 1986 Oct 1, 160(1), 169 - 74
Conformational interactions between alpha and beta subunits in the F1 ATPase of Escherichia coli as shown by chemical modification of uncA401 and uncD412 mutant enzymes; Stan-Lotter H et al.; In contrast to wild-type F1 adenosine triphosphatase, the beta subunits of soluble ATPase from Escherichia coli mutant strains AN120 (uncA401) and AN939 (uncD412) were not labeled by the fluorescent thiol-specific reagents 5-iodoacetamidofluorescein, 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid or 4-{N-(iodoacetoxy)ethyl-N-methyl}amino-7-nitrobenzo-2-oxa-1,3-diazole . The mutation in the alpha subunit (uncA401) of F1 ATPase thus influences the accessibility of the single cysteinyl residue in the beta subunit . Following reaction of ATPase with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole or N,N'-dicyclohexylcarbodiimide, the alpha and beta subunits of the uncA401, but not of the uncD412 mutant F1 ATPase were intensely labeled by a fluorescent thiol reagent . The mutation in the beta subunit (uncD412) thus influences the accessibility of the cysteinyl residues in the alpha subunit . In other work {Stan-Lotter, H . and Bragg, P.D . (1986) Arch . Biochem . Biophys . 248} we have shown that 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid react with a different beta subunit from that labeled by N,N'-dicyclohexylcarbodiimide . This asymmetry with respect to modification by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and N,N'-dicyclohexylcarbodiimide was seen in both mutant enzymes . In addition, the modification of one beta subunit of the uncA401 F1 ATPase induced the previously unreactive sulfhydryl group of another beta subunit to react with 2-(4'-iodoacetamidoanilino-naphthalene-6-sulfonic acid . These results provide evidence for at least three types of conformational interactions of the major subunits of F1 ATPase: from alpha to beta, from beta to alpha, and from beta to beta . As in wild-type ATPase, labeling of membrane-bound unc mutant ATPase by a fluorescent thiol reagent modified the alpha subunits . This suggests that a conformational change of yet a different type occurs when the enzyme binds to the membrane.

J Bacteriol, 1986 Oct, 168(1), 22 - 30
Large, unstable inserts in the chromosome affect virulence properties of uropathogenic Escherichia coli O6 strain 536; Knapp S et al.; The hemolytic, uropathogenic Escherichia coli 536 (O6:K15:H31) contains two inserts in its chromosome (insert I and insert II), both of which carried hly genes, were rather unstable, and were deleted spontaneously with a frequency of 10(-3) to 10(-4) . These inserts were not found in the chromosome of two nonhemolytic E . coli strains, whereas the chromosomal sequences adjacent to these inserts appeared to be again homologous in the uropathogenic and two other E . coli strains . Insert I was 75 kilobases in size and was flanked at both ends by 16 base pairs (bp) (TTCGACTCCTGTGATC) which were arranged in direct orientation . For insert I it was demonstrated that deletion occurred by recombination between the two 16-bp flanking sequences, since mutants lacking this insert still carried a single copy of the 16-bp sequence in the chromosome . Both inserts contained a functional hemolysin determinant . However, the loss of the inserts not only affected the hemolytic phenotype but led to a considerable reduction in serum resistance and the loss of mannose-resistant hemagglutination, caused by the presence of S-type fimbriae (sfa) . It is shown that the Sfa-negative phenotype is due to a block in transcription of the sfa genes . Mutants of strain 536 which lacked both inserts were entirely avirulent when tested in several animal model systems.

Infect Immun, 1986 Oct, 54(1), 104 - 8
Interaction of a 60-kilodalton D-mannose-containing salivary glycoprotein with type 1 fimbriae of Escherichia coli; Babu JP et al.; A 60-kilodalton glycoprotein previously isolated and purified from human saliva (J . B . Babu, E . H . Beachey, D . L . Hasty, and W . A . Simpson, Infect . Immun . 51: 405-413, 1986) was found to interact with type 1 fimbriae and prevent adhesion of type 1 fimbriated Escherichia coli to animal cells in a D-mannose-sensitive manner . Purified salivary glycoprotein agglutinated type 1 fimbriated E . coli and, at subagglutinating concentrations, blocked the ability of type 1 fimbriated E . coli to attach to human buccal epithelial cells or agglutinate guinea pig erythrocytes . Both interactions were inhibited by alpha-methyl-D-mannoside but not by alpha-methyl-D-glucoside . Complexing of the glycoprotein to type 1 fimbriae was demonstrated by molecular sieve chromatography and modified Western blots . When mixed with type 1 fimbriae, the radiolabeled salivary glycoprotein coeluted with type 1 fimbriae from a column of Sepharose 4B . When blotted from a sodium dodecyl sulfate gel to nitrocellulose sheets, the glycoprotein interacted directly with type 1 fimbriae applied to the blots . Both of the latter interactions also were blocked by alpha-methyl-D-mannoside but not by alpha-methyl-D-glucoside . Chemical modification of the glycoprotein with sodium metaperiodate abolished its ability to interact with isolated type 1 fimbriae or type 1 fimbriated E . coli . These results suggest that the carbohydrate moiety of the 60-kilodalton glycoprotein serves as a receptor for type 1 fimbriae in the oral cavity, and we postulate that the interaction may cause agglutination and early removal of E . coli, thereby preventing colonization by these organisms of oropharyngeal mucosae and dental tissues.

Mikrobiyol Bul, 1986 Oct, 20(4), 266 - 77
{Immunoelectrophoretic analysis of Escherichia coli strains (EPEC, ETEC, EIV and UPEC) (1)}; Cicioglu R et al.; Water extracts of Escherichia coli "O" and "K" antigen test strains (EPEC, ETEC, EIEC, UPEC) were examined in immunodiffusion and immunoelectrophoresis tests . The precipitation arcs corresponding to the O-antigen specificity and to the thermostable polysaccharide K antigen were easy to identify . All strains gave an O antigen precipitation arc found either on the anodic or the cathodic side of application basin and close to this . The so-called enteropathogenic types (from infantile diarrhoea) had a cathodic O antigen arc type; from dysentery-like disease had a negatively charged O-antigen, but no special thermostable K-antigen . Thus E . coli strains which may invade the tissues when conditions allow have a negatively charged surface antigen, either O-antigen lipopolysaccharide or both . Acidic components were found in the anodic O-antigen.

J Inorg Biochem, 1986 Oct-Nov, 28(2-3), 227 - 38
The role of calcium in the regulation of free radical reduction by thioredoxin reductase at the surface of the skin; Schallreuter KU et al.; Membrane associated thioredoxin reductase has been previously shown to reduce free radicals on the outer plasma membranes of human keratinocytes and melanocytes to provide a possible first line of defense against free radical damage at the surface of the skin . Preliminary experiments with cell cultures of human keratinocytes and melanocytes grown in serum-free medium showed that the enzyme activity depends on extracellular calcium concentration in the medium . Thioredoxin reductase activity at the surface of the skin, at the surface of human keratinocytes and melanocytes, and purified thioredoxin reductase from E . coli and adult human keratinocytes all exhibited calcium-dependent allosteric control . Since thioredoxin reductase contains two extremely reactive thiolate groups at the active site with pK values close to neutrality, both of these anions can form covalent complexes with N-ethylmaleimide by nucleophilic attack on the double bond . In our experiments we used spin-labeled maleimide {4-maleimido-tempo} to examine the local environment in the active site of thioredoxin reductase in the presence and absence of calcium . Both spin-labeled thioethers are distinguishable by EPR spectroscopy, with one site being significantly more immobilized than the other . Hence, it has been possible to observe direct evidence for active site closure by calcium . These results suggest that extracellular calcium may play an important role in regulation of thioredoxin reductase activity for the defense mechanism against UV-mediated free radical damage at the surface of human skin.

EMBO J, 1986 Oct, 5(10), 2539 - 44
Endothelial plasminogen activator inhibitor (PAI): a new member of the Serpin gene family; Pannekoek H et al.; A human endothelial cDNA expression library, based on the Escherichia coli plasmid pUC9, was screened with a heterologous antibody raised against purified bovine aortic endothelial plasminogen activator inhibitor (PAI) . A synthetic oligonucleotide, derived from a partial PAI cDNA expression clone, was used to select a full-length PAI cDNA, the size of which coincides with the length of PAI mRNA (approximately 2350 nucleotides) as determined by Northern blot analysis . The authenticity of full-length PAI cDNA is demonstrated by the expression of biologically active PAI both in lysates of transformed E . coli cells and in conditioned media of mouse Ltk- cells, transfected with PAI cDNA inserted into vector pSV2 . Analysis of the de novo synthesized anti-plasminogen activator activity, employing reverse fibrin autography, shows that transfected mouse Ltk- cells synthesize a polypeptide with a mol . wt identical to that of the native PAI glycoprotein (Mr 52,000), whereas in E . coli an unglycosylated, active product with a mol . wt of 43,000 is made . The amino acid sequence, derived from the determined nucleotide sequence, shows that pre-PAI consists of 402 amino acids . It is proposed that the mature PAI is preceded by a signal peptide of 23 amino acid residues . The amino acid sequence of mature PAI includes three potential asparagine-linked glycosylation sites and lacks cysteine residues . The predicted amino acid sequence reveals significant homology with members of the serine protease inhibitor (Serpin) family, e.g . alpha 1-proteinase inhibitor and antithrombin III.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Pharmacol, 1986 Oct 1, 35(19), 3267 - 75
Cellular targets of adriamycin-induced damage in Escherichia coli; Gelvan D et al.; The cellular targets of adriamycin (ADR) activity were studied in Escherichia coli by following colony forming ability and various cellular functions . The parameter exhibiting the best correlation with mortality was inhibition of RNA synthesis . Total DNA synthesis was inhibited to a lesser extent, but may reflect a concurrent inhibition of replication and stimulation of DNA repair activity . Protein synthesis, membrane function and rate of oxygen consumption were affected later . No extensive DNA fragmentation was observed . The inhibition of RNA synthesis was independent of the stringent response and of inhibition of DNA synthesis induced by nalidixic acid . ADR activated the SOS repair system, and the lesions induced by the drug could be repaired by recA dependent functions . These results indicate that the primary activity of ADR was directed against the DNA and interfered with the DNA template function.

Proc Natl Acad Sci U S A, 1986 Oct, 83(20), 7731 - 5
Exonuclease III and endonuclease IV remove 3' blocks from DNA synthesis primers in H2O2-damaged Escherichia coli; Demple B et al.; Escherichia coli deficient in exonuclease III (xth gene mutants) are known to be hypersensitive to hydrogen peroxide . We now show that such mutants accumulate many more DNA single-strand breaks than do wild-type bacteria upon exposure to H2O2 . DNA isolated from H2O2-treated xth- cells contains strand breaks that do not efficiently support synthesis by E . coli DNA polymerase I, indicating the presence of blocking groups at the DNA 3' termini . Purified E . coli exonuclease III activates this blocked DNA to allow substantial synthesis by polymerase I in vitro . Another E . coli enzyme, endonuclease IV, also activates primers for DNA polymerase . Exonuclease III accounts for greater than 95% of the total activity in E . coli crude extracts for removal of 3'-terminal phosphoglycolaldehyde esters from model DNA substrates . Purified exonuclease III and endonuclease IV can each efficiently remove 3'-terminal phosphoglycolaldehyde in vitro . An important physiological function for exonuclease III is thus the activation of blocked 3' ends for DNA repair synthesis . Endonuclease IV can also initiate the repair of ruptured 3'-deoxyribose in DNA.

Proc Natl Acad Sci U S A, 1986 Oct, 83(20), 7648 - 52
Computer analysis of retroviral pol genes: assignment of enzymatic functions to specific sequences and homologies with nonviral enzymes; Johnson MS et al.; A computer analysis of the amino acid sequences from the putative gene products of retroviral pol genes has revealed a 150-residue segment that is homologous with the ribonuclease H of Escherichia coli . The segment occurs at the carboxyl terminus of the region assigned to the 90-kDa reverse transcriptase polypeptide . In contrast, a section nearer the amino terminus of this sequence can be aligned with nonretroviral polymerases . The order of activities in the pol gene is thus: polymerase-ribonuclease-endonuclease . On another note, all retroviral endonuclease sequences contain a consensus zinc-binding "finger." This should not be confused with the well-known zinc requirement of reverse transcriptases.

Proc Natl Acad Sci U S A, 1986 Oct, 83(19), 7419 - 23
Identification and chemical synthesis of a ribosomal protein antigenic determinant in systemic lupus erythematosus; Elkon K et al.; The characteristics of eukaryotic ribosomal proteins P0, P1, and P2 (P proteins) and their antigenic determinants were studied using the sera of patients with systemic lupus erythematosus (SLE) . P0, P1, and P2 were isolated as a macromolecular complex by preparative isoelectric focusing and anion-exchange chromatography in the presence of 6 M urea . The apparent molecular size of the complex was 140 kDa as determined by gel filtration on a Sephadex G-200 column . P0 may, therefore, be the eukaryotic equivalent of Escherichia coli ribosomal protein L10 . In addition, all three P proteins were detected in the postribosomal supernatant of HeLa cells, and P0 and P1 were found to be more acidic than their ribosome-bound counterparts . Partial proteolysis experiments revealed that SLE anti-P sera recognized one or both ends of the P2 equivalent protein from Artemia salina (eL12) . Sixteen SLE sera containing antibodies to P0, P1, and P2 reacted with a carboxyl-terminal peptide 22 amino acids in length of eL12 and not with an amino-terminal peptide of 20 amino acids . Even though the carboxyl-terminal peptide completely inhibited the ability of the antiserum to react with all three proteins on an immunological blot, the same peptide produced only small decreases in binding of the SLE antibody to the native, nondenatured P proteins . These findings indicate that SLE anti-P antibodies react with a single sequential (linear) antigenic determinant on all three P proteins, but that additional antibodies recognize a conformational determinant(s).

J Bacteriol, 1986 Oct, 168(1), 192 - 8
Two monoclonal antibodies specific for different epitopes within the amino-terminal region of F pilin; Frost LS et al.; Two murine monoclonal antibodies (JEL 92 and 93) specific for adjacent epitopes on F pilin were purified and characterized . JEL 93 immunoglobulin G (IgG) and its Fab fragments were specific for the amino-terminal region and were completely reactive with a synthetic peptide representing the first eight amino acids of F pilin . The acetyl group was demonstrated to be an important part of the epitope, since an unacetylated version of the amino-terminal peptide was 100-fold less reactive with JEL 93 IgG . JEL 92 IgG reacted with the region of F pilin surrounding Met-9, represented by a tryptic peptide derived from the first 17 amino acids . This reactivity was completely abolished by cleavage of the peptide with cyanogen bromide . As shown by electron microscopy, both monoclonal antibodies bound to a vesiclelike structure at one end of purified free pili and did not bind to the sides of the pili, nor did they appear to bind to the tip . When sonication was used to break pili into shorter fragments, the number of binding sites for JEL 92 but not JEL 93 IgG increased as measured by a competitive enzyme-linked immunosorbent assay.

J Virol, 1986 Oct, 60(1), 312 - 6
An immunodominant domain in adenovirus type 2 early region 1A proteins; Tsukamoto AS et al.; Six independent rat hybridoma cell lines producing monoclonal antibodies to human subgroup C adenovirus early region 1A (E1A) proteins were isolated . Competition binding experiments revealed that each of the monoclonal antibodies was directed against the same epitope or overlapping cluster of epitopes on the E1A proteins . Viral E1A deletion mutants and deleted forms of E1A proteins expressed in Escherichia coli were used to localize the antibody recognition sites to sequences between amino acids 23 and 120, encoded within the first exon of the E1A gene . Similarly, polyclonal antisera raised against the trpE-E1A fusion protein, as well as against the native, biologically active E1A protein, were also directed primarily against this immunodominant region.

J Virol, 1986 Oct, 60(1), 267 - 74
Murine leukemia virus pol gene products: analysis with antisera generated against reverse transcriptase and endonuclease fusion proteins expressed in Escherichia coli; Hu SC et al.; The organization of the murine leukemia virus (MuLV) pol gene was investigated by expressing molecular clones containing AKR MuLV reverse transcriptase or endonuclease or both gene segments in Escherichia coli and generating specific antisera against the expressed bacterial proteins . Reaction of these antisera with detergent-disrupted virus precipitated an 80-kilodalton (kDa) protein, the MuLV reverse transcriptase, and a 46-kDa protein which we believe is the viral endonuclease . A third (50-kDa) protein, related to reverse transcriptase, was also precipitated . Bacterial extracts of clones expressing reverse transcriptase and endonuclease sequences competed with the viral 80- and 46-kDa proteins, respectively . These results demonstrate that the antisera are specific for viral reverse transcriptase and endonuclease . Immunoprecipitation of AKR MuLV with antisera prepared against a bacterial protein containing only endonuclease sequences led to the observation that reverse transcriptase and endonuclease can be associated as a complex involving a disulfide bond(s).

FEBS Lett, 1986 Sep 29, 206(1), 142 - 6
Template-free ribosomal synthesis of polypeptides from aminoacyl-tRNA . Polyphenylalanine synthesis from phenylalanyl-tRNALys; Yusupova GZ et al.; Misacylated phenylalanyl-tRNALys, just as lysyl-tRNALys, but not phenylalanyl-tRNAPhe, have been shown to serve as substrates for ribosomal synthesis of polypeptides (polyphenylalanine and polylysine, respectively) in the absence of a template polynucleotide (poly(A)) . The conclusion was made that it is the structure of tRNA that determines the ability of the aminoacyl-tRNALys to participate in peptide elongation on ribosomes without codon-anticodon interactions.

Vet Rec, 1986 Sep 27, 119(13), 319 - 21
Cow milk yield and composition before development of Escherichia coli mastitis; Jones TO et al.; High milk yield, low milk fat and low milk protein were considered as possible predisposing factors to bovine Escherichia coli mastitis . Morning and afternoon milk yields were recorded in 46 Friesian cows later developing E coli mastitis and compared with 92 uninfected controls . Animals developing E coli mastitis gave a significantly higher milk yield than controls . The overall morning: afternoon ratio was (mean +/- se) 1.66 +/- 0.41, with no difference in ratio for the two groups . Further studies on 85 animals later developing E coli mastitis, and 192 controls, in four Friesian herds did not reveal differences in milk fat content (except as related to yield), milk protein or in the interrelationship of days of lactation, milk protein or in the interrelationship of days of lactation, milk fat and milk protein in the two groups . Again there was a correlation between high milk yield and a tendency to develop E coli mastitis but this may have been an age effect in both investigations . No correlation between milk yield and mastitis severity was detected . High yielders which succumbed to E coli mastitis in three herds were producing less milk than mastitis-free controls in the fourth herd which suggests that the correlation is not with yield per se.

Science, 1986 Sep 26, 233(4771), 1403 - 8
A genetic approach to analyzing membrane protein topology; Manoil C et al.; Fusions of the secreted protein alkaline phosphatase to an integral cytoplasmic membrane protein of Escherichia coli showed different activities depending on where in the membrane protein the alkaline phosphatase was fused . Fusions to positions in or near the periplasmic domain led to high alkaline phosphatase activity, whereas those to positions in the cytoplasmic domain gave low activity . Analysis of alkaline phosphatase fusions to membrane proteins of unknown structure may thus be generally useful in determining their membrane topologies.

Cell, 1986 Sep 26, 46(7), 1023 - 8
Transcription termination at lambda tR1 is mediated by interaction of rho with specific single-stranded domains near the 3' end of cro mRNA; Chen CY et al.; To determine whether E . coli rho protein mediates termination of transcription by interacting with specific segments of the nascent transcript, DNA oligonucleotides were used to sequester segments of phage lambda cro mRNA in hybrid helices . Formation of hybrids was demonstrated with RNAase H assays . Oligonucleotides complementary to either of two distinct, single-stranded sequences near the 3' end inhibited rho action at tR1, while oligonucleotides complementary to the sequence between those segments or to more 5' segments did not . The inhibitory oligonucleotides did not affect the elongation of cro mRNA or rho action on other transcripts . The results indicate that termination of transcription at tR1 is dependent upon contact of rho factor with specific, single-stranded domains near the 3' end of cro mRNA.

Nucleic Acids Res, 1986 Sep 25, 14(18), 7379 - 90
The 52-protein subunit of T4 DNA topoisomerase is homologous to the gyrA-protein of gyrase; Huang WM; T4 gene 52 encodes one of the three subunits of T4 DNA topoisomerase . The T4 enzyme is required for normal phage DNA replication . I have cloned the entire gene, and it is expressed in uninfected E . coli cells . The sequence of 1966 nucleotides of T4 deletion delta sa9 surrounding gene 52 has been determined . The reading frame of the gene was established by identifying the first ten amino acids in the large open reading frame derived from the DNA sequence as those at the amino-terminus of the purified 52-protein . Based on the DNA sequence, 52-protein has 441 amino acids and a calculated peptide molecular weight of 50,583 daltons . This T4 topoisomerase subunit shares significant amino acid sequence homology with the gyrA subunit of bacterial gyrases and the carboxyl-half of yeast topoisomerase II in spite of the large differences in their sizes, confirming their functional equivalence in type II enzyme directed DNA topoisomerization . Amino acid sequence homology is highest in the amino-terminal portions of the equivalent peptides . The homology alignment suggests a consensus sequence organization surrounding the reactive tyrosine which is used to form the transient protein-DNA intermediate in the double-stranded DNA passing reaction . The delta sa9 deletion in T4 brings gene 52 to a location 30 nucleotides 3' from the rIIB gene whose C-terminal 167 codons are also reported here.

Nucleic Acids Res, 1986 Sep 25, 14(18), 7325 - 40
RecA independent recombination of poly{d(GT)-d(CA)} in pBR322; Murphy KE et al.; Short sequence tracts composed of alternating guanosine and thymidine nucleotide residues poly{d(GT)-d(CA)} carried in a derivative of pBR322 were recombinogenic in a recA host . Recombination brought about by poly{d(GT)-d(CA)} tracts displayed two interesting properties: (i) the reaction was quasi-sequence-specific in that while recombination usually occurred between two poly{d(GT)-d(CA)} tracts, recombination also occurred between sequences bordering the dinucleotide repeats . (ii) recombination was enhanced when two poly{d(GT)-d(CA)} tracts were clustered within 250 base pairs of each other, but not when the repeats were separated by 3 kilobase pairs . The mechanism by which poly{d(GT)-d(CA)} stimulated recombination remains to be determined, but the behavior of these sequences is consistent with the idea that general recombination in E . coli may involve formation of Z-DNA.

Nucleic Acids Res, 1986 Sep 25, 14(18), 7213 - 26
Site-specific recombination and topoisomerization by Tn21 resolvase: role of metal ions; Castell SE et al.; The resolvase from the transposon Tn21 catalyses site-specific recombination between the two res sites on its DNA substrate both in the absence and presence of Mg2+ ions . This contrasts with reports on the resolvase from gamma-delta (Tn1000) and on other recombinational proteins that are homologous to Tn21 resolvase but which need Mg2+ for their activity . Magnesium ions could enhance the activity of Tn21 resolvase, as did a number of other cations but some metal ions such as Ni2+ inhibit recombination . The metal ions are not directly involved in the catalytic process but probably affect recombination by altering the conformation of the DNA . Tn21 resolvase relaxes its DNA substrate in the presence and in the absence of Mg2+, and also in ionic conditions that inhibit recombination . Hence, the topoisomerization reflects an activity of resolvase that is distinct from recombination . However, the two activities are functions of the same DNA-protein complex . The complex contains about 6 molecules of the resolvase dimer per molecule of DNA.

J Biol Chem, 1986 Sep 25, 261(27), 12472 - 6
Iron mediates paraquat toxicity in Escherichia coli; Korbashi P et al.; The role of iron ions in paraquat toxicity was studied in bacterial system . We show that addition of ferrous iron led to an enhancement of the bacterial killing, whereas addition of chelating agents, such as nitrilotriacetate and desferrioxamine, markedly reduced, up to a total abolishment, the toxic effects . The calculated rates of bacterial killing are proportional to both paraquat and iron concentrations, and conform to the rate equation: dN/dt = -k{paraquat} {Fe2+} . The killing constant for iron, k, is 24-fold smaller than the corresponding value for copper . Mannitol, an OH . scavenger, has a partial protective effect: 15-35% at concentrations range of 1-50 mM, respectively . Histidine, on the other hand, provided a more efficient protection that may be due to a combination of various effects . Induction of endogenous superoxide dismutase and catalase provided partial protection (about 25%) . These findings, together with an earlier study on the role of copper in paraquat toxicity (Kohen, R., and Chevion, M . (1985) Free Rad . Res . Commun . 1, 79-88) indicate that transition metals play a central catalytic role in the production of the deleterious effects of paraquat, probably by redox cycling and producing OH . via the site-specific Fenton reaction.

J Biol Chem, 1986 Sep 25, 261(27), 12477 - 85
The systematic characterization by aqueous column chromatography of solutes which affect protein stability; Washabaugh MW et al.; We have systematically characterized, by aqueous column chromatography on a size exclusion cross-linked dextran gel (Sephadex G-10), 12 solutes, 11 of which are known to affect protein stability . Six are chaotropes (water structure breakers) and destabilize proteins, while five are polar kosmotropes (polar water structure makers) and stabilize proteins . Analysis of the chromatographic behavior of these neutral (ethylene glycol, urea), positively charged (Tris, guanidine, as the hydrochloride salts) and negatively charged (SO2-4, HPO2-4, F-, Cl-, Br-, Cl3CCO-2, I-, SCN-, as the sodium salts, in order of elution) solutes at pH 7 as a function of sample concentration (up to 0.6 M), supporting electrolyte, and temperature yields four conclusions, based largely on the behavior of the anions . Chaotropes adsorb to the gel according to their position in the Hofmeister series, with the most chaotropic species adsorbing most strongly . ++Chaotropes adsorb to the gel less strongly in the presence of chaotropes (a salting in effect) and more strongly in the presence of polar kosmotropes (a salting out effect) . Polar kosmotropes do not adsorb to the gel, and are sieved through the gel according to their position in the Hofmeister series, with the most kosmotropic species having the largest relative hydrodynamic radii . The hydrodynamic radii of polar kosmotropes is increased by chaotropes and decreased by polar kosmotropes . These results suggest that a chaotrope interacts with the first layer of immediately adjacent water molecules somewhat less strongly than would bulk water in its place; a polar kosmotrope, more strongly.

Biochemistry, 1986 Sep 23, 25(19), 5825 - 30
Specific substitution into the anticodon loop of yeast tyrosine transfer RNA; Bare LA et al.; The aminoacylation kinetics of 19 different variants of yeast tRNATyr with nucleotide substitutions in positions 33-35 were determined . Substitution of the conserved uridine-33 does not alter the rate of aminoacylation . However, substitution of the anticodon position 34 or position 35 reduces Km from 2- to 10-fold and Vmax as much as 2-fold, depending on the nucleotide inserted . The ochre and amber suppressor tRNAsTyr both showed about a 7-fold reduction in Vmax/Km . Data from tRNATyr with different modified nucleotides at position 35 suggest that specific hydrogen bonds form between the synthetase and both the N1 and N3 hydrogens of psi-35 . The effect of simultaneous substitutions at positions 34 and 35 can be predicted reasonably well by combining the effects of single substitutions . These data suggest that yeast tyrosyl-tRNA synthetase interacts with positions 34 and 35 of the anticodon of tRNATyr and opens the possibility that nonsense suppressor efficiency may be mediated by the level of aminoacylation.

Biochemistry, 1986 Sep 23, 25(19), 5780 - 6
Pressure dissociation and conformational drift of the beta dimer of tryptophan synthase; Silva JL et al.; Micromolar solutions of tryptophan synthase beta 2 dimer dissociate into monomers in the pressure range of 800-1600 bars as shown by studies of the spectral shift of the intrinsic fluorescence and of the fluorescence polarization of dansyl conjugates . At 25 degrees C the standard change in volume on dissociation (dV0) of the holoprotein was -162 mL mol-1, and the dissociation constant at 1 bar was K0 = 3.7 10(-10) M . Pyridoxal-reduced holoprotein and apoprotein had, within 10%, the same dV0, but K0 was decreased in the reduced protein (6 X 10(-11) M) and increased in the apoprotein (3.6 X 10(-9) M) . At 4 degrees C the free energy of association of the holoprotein was reduced by 1.4 kcal mol-1, but dV0 was unchanged . In all the protein forms the decompression curves differed from the respective compression curves, indicating the loss of some free energy of association following separation of the monomers . This hysteretic behavior was largest in the apoprotein and amounted to a loss of 2.6 kcal mol-1 in the free energy of association . When the pressure was rapidly raised to 2.2 kbars, half-dissociation of the reduced pyridoxal beta 2 dimer took approximately 12 min . Upon return to atmospheric pressure reassociation was complete in 2-3 min and half of the enzyme activity was regained in 10 min; pyridoxal fluorescence recovered more slowly with a biphasic course . The independent return of these properties and the hysteretic behavior indicate that subunit separation is followed by a conformational drift like that observed in lactate dehydrogenase dissociated by either pressure or temperature or in enolase dissociated by dilution.

Biochemistry, 1986 Sep 23, 25(19), 5766 - 74
Secondary structure of acyl carrier protein as derived from two-dimensional 1H NMR spectroscopy; Holak TA et al.; Sequence-specific assignments of 1H NMR resonances were obtained for the backbone protons in acyl carrier protein (ACP) from Escherichia coli, a protein of 77 residues . The observations, in the NOESY spectra, of 1H-1H sequential and medium-range connectivities indicate the presence of three or four alpha-helical segments joined by short sequences of mixed conformations . The observations are used to refine a secondary structure model previously proposed on the basis of a Chou-Fasman algorithm {Rock, C . O., & Cronan, J . E., Jr . (1979) J . Biol . Chem . 254, 9778-9785}.

Biochemistry, 1986 Sep 23, 25(19), 5736 - 44
Imino proton exchange in the 5S RNA of Escherichia coli and its complex with protein L25 at 490 MHz; Leontis NB et al.; Imino proton exchange has been examined by NMR in the 5S RNA of Escherichia coli, its principal RNase A resistant fragment, fragment 1 (bases 1-11, 69-120), and complexes between that fragment and ribosomal protein L25 by using both real-time and relaxation techniques . Fragment 1 RNA imino protons exchange at rates between 0.5 and 15 s-1 at 303 K in 5 mM cacodylate buffer, pH 7.4 . In contrast with many tRNAs, intact 5S RNA contains no imino protons with exchange lifetimes as great as 1 min . Consistent with the results of Gueron and his colleagues {Leroy, J . L., Bolo, N., Figueroa, N., Plateau, P., & Gueron, M . (1985) J . Biomol . Struct . Dyn . 2,915-939; Leroy, J . L., Broseta, D., & Gueron, M . (1985) J . Mol . Biol . 184, 165-178} with tRNA, exchange in 5S RNA is catalyst-limited under conditions generally used for imino proton spectroscopy, such as those given above . Using Gueron's catalyst saturation technique, base pair opening rates have been measured for several AU and GU base pairs in fragment 1 . They range from 50 to 300 s-1 at 303 K and depend on base pair type and also to some degree on context . Similar studies have been done on complexes of L25 and fragment 1 . The binding of L25 to fragment 1 reduces the exchange rate of many imino protons within the region to which it binds, consistent with the hypothesis that its binding stabilizes the secondary structure of 5S RNA.

Biochemistry, 1986 Sep 23, 25(19), 5726 - 35
Identification of form III conformers in tRNAPhe from Escherichia coli by intramolecular photo-cross-linking; Dubreuil YL et al.; In the absence of divalent cations, at neutral pH, low ionic strength, and low to moderate temperature, tRNAs are known to be in a denatured form, designated form III in the tRNA phase diagram by Cole et al . {Cole, P . E., Yang, S . R., & Crothers, D . M . (1972) Biochemistry 11, 4358-4368} . Form III tRNAPhe from Escherichia coli has been studied at pH 7, 5 mM Na+, and 10 degrees C . As judged from ethidium bromide intercalation, it exhibits extensive secondary structure . 4-Thiouridine in position 8 of the tRNAPhe sequence was used as a built-in photoaffinity probe . Spectroscopic and spectrofluorometric analysis in the near-UV range of form III tRNAPhe irradiated with broad-band near-UV light to completion of the reaction before or after reduction with NaBH4 revealed that the Pdo(4-5)Cyt (8-C) and Pdo(4-5)Urd (8-U) adducts form in equimolar yield . In different experiments, the overall yield of s4U conversion to these adducts varies between 20 and 40% . The remaining s4U is photolyzed to weakly absorbing product(s) in the near-UV range . The disappearance of s4U follows biexponential kinetics while the 8-C adduct formation follows monoexponential kinetics, indicating the presence of at least two tRNA classes of conformers, not in equilibrium on the time scale of the reaction . Migration on a denaturing polyacrylamide gel of irradiated form III tRNAPhe revealed three main bands, D1, D2, and D3, and no slowly migrating tRNA dimers . D1 migrates at the control position and presumably contains the photolysis product(s) P . The fast-migrating D2 and D3 bands contain 8-Pyr cross-links which were identified by sequence analysis as 8-(66-68) in D2 and 8-(40-43) and 8-(59-62) in D3 . On the basis of these data, it is proposed that the minor poorly photoreactive class II conformers are the cloverleaf and close variants, whereas the major class I cross-linkable conformers are essentially long-extended secondary structures . Clearly, our data demonstrate the polymorphism of form III tRNAPhe.

Biochemistry, 1986 Sep 23, 25(19), 5699 - 709
Fluorine-19 nuclear magnetic resonance studies of the structure of 5-fluorouracil-substituted Escherichia coli transfer RNA; Hardin CC et al.; 19F nuclear magnetic resonance has been used to study fully active Escherichia coli tRNA1Val in which 5-fluorouracil has replaced more than 90% of all uracil and uracil-derived modified bases . The 19F spectrum of the native tRNA contains resolved resonances for all 14 incorporated 5-fluorouracils . These are spread over a 6 ppm range, from 1.8 to 7.7 ppm downfield of the standard free 5-fluorouracil . The 19F resonances serve as sensitive monitors of tRNA conformation . Removal of magnesium or addition of NaCl produces major, reversible changes in the 19F spectrum . Most affected is the lowest field resonance (peak A) in the spectrum of the native tRNA . This shifts 2-3 ppm upfield as the Mg2+ concentration is lowered or the NaCl concentration is raised . Thermal denaturation of the tRNA results in a collapse of the spectrum to a single broad peak centered at 4.7 ppm . Study of the pH dependence of the 19F spectrum shows that five incorporated fluorouracils with 19F signals in the central, 4-5.5 ppm, region of the spectrum, peaks C, D, E, F, and H, are accessible to titration in the pH 4.5-9 range . All have pKa's close to that of free 5-fluorouridine (ca . 7.5) . Evidence for a conformation change in the tRNA at mildly acidic pHs, ca . 5.5, is also presented . Four of the titratable 5-fluorouracil residues, those corresponding to peaks D, E/F, and H in the 19F spectrum of fluorine-labeled tRNAVal1, are essentially completely exposed to solvent as determined by the solvent isotope shift (SIS) on transfer of the tRNA from H2O to 2H2O . These are also the 5-fluorouracils that readily form adducts with bisulfite, a reagent that reacts preferentially with pyrimidines in single-stranded regions . On the basis of these results, resonances D, E, F, and H in the middle of the 19F spectrum are attributed to 5-fluorouracils in non-base-paired (loop) regions of the tRNA . Evidence from the ionic strength dependence of the 19F spectrum and arguments based on other recent studies with fluorinated tRNAs support earlier suggestions {Horowitz, J., Ofengand, J., Daniel, W . E., & Cohn, M . (1977) J . Biol . Chem . 252, 4418-4420} that the resonances at lowest field correspond to tertiary hydrogen-bonded 5-fluorouracils . Consideration of ring-current effects and the preferential perturbation of upfield 19F resonances by the cyclophotoaddition of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen, which is known to react most readily with pyrimidines in double-stranded regions, permits initial assignment of upfield resonances to 5-fluorouracils in helical stems.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemistry, 1986 Sep 23, 25(19), 5576 - 82
Carbamoyl-phosphate synthetase: an example of effects on enzyme properties of shifting an equilibrium between active monomer and active oligomer; Anderson PM; Carbamoyl-phosphate synthetase from Escherichia coli is subject to allosteric activation by ornithine, allosteric inhibition by uridine 5'-phosphate (UMP), and reversible concentration-dependent self-association . Positive allosteric effectors, magnesium adenosine 5'-triphosphate (MgATP), K+, and inorganic phosphate facilitate association . The purpose of this study was to determine the state of association of carbamoyl-phosphate synthetase in the presence and absence of different substrates and effectors and to consider the basis for the observed effects of enzyme concentration on specific activity . Studies employing gel filtration chromatography have shown that when the concentration of carbamoyl-phosphate synthetase is low (less than 0.01 mg/mL), the enzyme exists as monomer under all conditions, including the presence of UMP in phosphate buffer and the presence of all substrates plus ornithine (conditions that support maximal catalytic activity) . At higher enzyme concentrations (e.g., greater than 0.01 mg/mL) the specific activity increases with increasing enzyme concentration when MgATP is nonsaturating but is independent of enzyme concentration when MgATP is saturating or when ornithine is present with MgATP being either saturating or nonsaturating . These results indicate that the catalytic activity of this enzyme is not directly linked to oligomer formation . The theoretical properties and possible significance of a generalized model of enzyme association-dissociation in which the active monomeric form, in equilibrium with another monomeric form, is specifically subject to self-association but the different states of association have the same specific activity, are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1986 Sep 23, 25(19), 5479 - 84
Allosteric regulation of inducer and operator binding to the lactose repressor; Daly TJ et al.; The effects of cysteine modification and variations in pH on the equilibrium parameters for inducer and operator binding to the lactose repressor protein were examined . Operator binding affinity was minimally affected by increasing the pH from 7.5 to 9.2, whereas inducer binding was decreased for both the unliganded protein and the repressor-operator complex over the same range . Inducer binding to the repressor became more cooperative at high pH . The midpoint for the change in inducer affinity and cooperativity was pH 8.3; this value correlates well with cysteine ionization . The differential between repressor-operator affinity in the presence and absence of inducer was significantly decreased by modification of the protein with methyl methanethiosulfonate (MMTS) . In contrast to unreacted protein, the inducer binding parameters for MMTS-modified repressor were largely unaffected by pH variation . The free energy for formation of the completely liganded protein was calculated for two pathways; the delta G values for these two independent routes were equivalent only for stoichiometries of four inducers and two operators per repressor molecule . All of the binding data were analyzed quantitatively by using a Monod-Wyman-Changeux two-state model for allosteric regulation . The observed dependences of the isopropyl beta-D-thiogalactoside binding curves on pH, DNA concentration, and MMTS modification were fitted by varying only the equilibrium constant between the two conformational states of the protein . With this analysis, high pH favors the T (high operator/low inducer affinity) state, while modification of cysteine-281 with MMTS elicits a shift into the R (high inducer/low operator affinity) state.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1986 Sep 23, 25(19), 5474 - 8
Characterization and modification of a monomeric mutant of the lactose repressor protein; Daly TJ et al.; A monomeric mutant lactose repressor protein (T-41), containing serine at position 282 in place of tyrosine {Schmitz, A., Schmeissner, U., Miller, J . H., & Lu, P . (1976) J . Biol . Chem . 251, 3359-3366}, has been purified by a series of chromatographic and precipitation methods . The molecular weight of the mutant as determined by gel filtration was approximately 40,000 . The inducer equilibrium binding constant for the mutant was comparable to that of the tetrameric wild-type repressor at pH 7.5, whereas operator DNA binding was not detectable . In contrast to wild-type repressor, equilibrium and kinetic rate constants for inducer binding to the monomer were largely independent of pH; thus, the quaternary structure of the wild-type repressor is required for the pH-associated effects on inducer binding . Although ultraviolet absorbance difference spectra indicated that inducer binding to T-41 protein elicited different changes in the environment of aromatic residues from those generated in wild-type repressor, the shift in the fluorescence emission maximum in response to inducer binding was similar for T-41 and wild-type repressors . Similarity in 1-anilinonaphthalene-8-sulfonic acid binding to monomer and tetramer suggests that this fluorophore does not bind at subunit interfaces . Modification of Cys-281 with methyl methanethiosulfonate was observed at low molar ratios of reagent per T-41 monomer (4-fold) . This result is in contrast to data observed for tetrameric wild-type repressor which requires high molar ratios for this cysteine to react . We conclude that Cys-281, adjacent to the site of the T-41 mutation, is located on the surface of the monomer in this region crucial for subunit interaction.

Biochemistry, 1986 Sep 23, 25(19), 5468 - 74
Formation of mixed disulfide adducts at cysteine-281 of the lactose repressor protein affects operator and inducer binding parameters; Daly TJ et al.; The lactose repressor protein has been modified with three sulfhydryl-specific reagents which form mixed disulfide adducts . Methyl methanethiosulfonate (MMTS) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) completely reacted with all three cysteine residues, whereas only partial reaction was observed with didansylcystine . Cysteines-107 and -140 reacted stoichiometrically with MMTS and DTNB, while Cys-281 was modified only at higher molar ratios . Didansylcystine reacted primarily with cysteines-107 and -140 . Affinity of MMTS-modified repressor for 40 base pair operator DNA was decreased 30-fold compared to unmodified repressor, and this decrease correlated with modification of cysteine-281 . DTNB-modified repressor bound operator DNA with a 50-fold weaker affinity than unmodified repressor . Modification of the lac repressor with didanylcystine decreased operator binding only 4-fold, and nonspecific DNA binding increased 3-fold compared to unmodified repressor . No change in the inducer equilibrium binding constant was observed following modification with any of these reagents . In contrast, inducer association and dissociation rate constants were decreased approximately 50-fold for repressor completely modified with MMTS or DTNB, while didansylcystine had minimal effect on inducer binding kinetics . Correlation between modification of Cys-281 and the observed decrease in rate constants indicates that this region of the protein regulates the accessibility of the sugar binding site . The parallel between the increase in the Kd for repressor binding to operator, the altered rate constant for inducer binding, and modification of cysteine-281 suggests that this region of the protein is crucially involved in the function of the repressor protein.

Biochemistry, 1986 Sep 23, 25(19), 5445 - 52
Folding of dihydrofolate reductase from Escherichia coli; Touchette NA et al.; The urea-induced equilibrium unfolding transition of dihydrofolate reductase from Escherichia coli was monitored by UV difference, circular dichroism (CD), and fluorescence spectroscopy . Each of these data sets were well described by a two-state unfolding model involving only native and unfolded forms . The free energy of folding in the absence of urea at pH 7.8, 15 degrees C is 6.13 +/- 0.36 kcal mol-1 by difference UV, 5.32 +/- 0.67 kcal mol-1 by CD, and 5.42 +/- 1.04 kcal mol-1 by fluorescence spectroscopy . The midpoints for the difference UV, CD, and fluorescence transitions are 3.12, 3.08, and 3.18 M urea, respectively . The near-coincidence of the unfolding transitions monitored by these three techniques also supports the assignment of a two-state model for the equilibrium results . Kinetic studies of the unfolding and refolding reactions show that the process is complex and therefore that additional species must be present . Unfolding jumps in the absence of potassium chloride revealed two slow phases which account for all of the amplitude predicted by equilibrium experiments . Unfolding in the presence of 400 mM KCl results in the selective loss of the slower phase, implying that there are two native forms present in equilibrium prior to unfolding . Five reactions were observed in refolding: two slow phases designated tau 1 and tau 2 that correspond to the slow phases in unfolding and three faster reactions designated tau 3, tau 4, and tau 5 that were followed by stopped-flow techniques . The kinetics of the recovery of the native form was monitored by following the binding of methotrexate, a tight-binding inhibitor of dihydrofolate reductase, at 380 nm.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1986 Sep 23, 25(19), 5420 - 5
Active enzyme sedimentation, sedimentation velocity, and sedimentation equilibrium studies of succinyl-CoA synthetases of porcine heart and Escherichia coli; Wolodko WT et al.; Succinyl-CoA synthetases from Escherichia coli and porcine heart muscle have been viewed as prototypes of two classes of the enzyme . The bacterial enzyme has been reported to be an alpha 2 beta 2 tetramer, with many suggestions in the literature for cooperative interactions between active sites that may contribute to its catalytic efficacy . In contrast, gel filtration experiments of others have indicated that the heart enzyme is a simple alpha beta dimer, with no evidence of dimerization or interaction between like sites . All previous estimates of molecular size of these enzymes have been carried out at concentrations that are much higher than those that are used during activity measurements . The present study was carried out to confirm the differences in the quaternary structures of these two species of succinyl-CoA synthetase and to extend our knowledge of these structures to very low concentrations to enable correlation of their subunit structures with their catalytic properties . Conventional sedimentation velocity centrifugation with both enzymes indicates behavior typical of noninteracting globular proteins with no evidence of size heterogeneity . The sedimentation coefficients at infinite dilution (s20,w) have been determined to be 7.04 S and 4.55 S for the E . coli and porcine heart enzymes, respectively . Sedimentation velocity measurements have been extended to very low enzyme concentrations (typical of those used in activity measurements) by active enzyme centrifugation experiments, in which we have determined the rate of sedimentation of a zone of active enzyme through a chromogenic substrate solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1986 Sep 23, 25(19), 5583 - 9
Galactose-1-phosphate uridylyltransferase . Purification of the enzyme and stereochemical course of each step of the double-displacement mechanism; Arabshahi A et al.; A convenient new procedure for purifying galactose-1-phosphate uridylyltransferase from Escherichia coli is described . It departs from earlier methods by introducing the use of a Cibacron Blue-agarose (Bio-Rad Affi-Gel Blue) at an early stage . Purification is completed by ion-exchange chromatography using DEAE-Sephadex A-50 . The procedure is substantially shorter than earlier methods and reproducibly yields enzyme of high specific activity suitable for use in structural work such as characterization of the intermediate uridylyl-enzyme . The first step of the galactose-1-P uridylyltransferase reaction is the transfer of the uridylyl group from UDP-glucose to N3 of a histidine residue in the enzyme to form the covalent uridylyl-enzyme and glucose-1-P . The uridylyl-enzyme intermediate then reacts in a second step with galactose-1-P to form UDP-galactose . The enzyme accepts (RP)-UDP alpha S-glucose as a good substrate, converting it to (RP)-UDP alpha S-galactose, i.e., with overall retention of configuration . In this paper we show that reaction of the enzyme with (RP)-{2-14C}UDP alpha S-glucose produces a {2-14C}uridylyl alpha S-enzyme that can be converted by base-catalyzed cyclization to (RP)-{2-14C}cUMPS . Inasmuch as cyclization must have proceeded with inversion of configuration at phosphorus, the corresponding configuration in the intermediate must have been the inverse of that in the substrate . Therefore, formation of uridylyl alpha S-enzyme from (RP)-UDP alpha S-glucose proceeds with inversion of configuration, and overall retention arises from inversion in each of the two steps . The results support the authenticity of the isolated uridylyl-enzyme as the true reaction intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1986 Sep 23, 25(19), 5539 - 46
Role of a second catabolite activator protein molecule in controlling initiation of transcription at the galactose operon of Escherichia coli; Shanblatt SH et al.; The molecular mechanisms whereby RNA polymerase, catabolite activator protein (CAP), and cyclic AMP (cAMP) participate in transcriptional regulation at the galactose operon have been probed by a variety of in vitro techniques . Interactions between purified proteins and promoter-containing DNA fragments were assayed by gel electrophoresis, by resistance to restriction endonuclease digestion, and by monitoring runoff transcripts . The data bear on the multiple functions that CAP performs in gal control . A CAP-cAMP complex can exclude RNA polymerase from one of the two overlapping promoter regions (P2), thereby targeting the enzyme to the other (P1); this process is markedly influenced by the cAMP level . In addition, a second CAP molecule is involved in a cooperative process, which, at low cAMP, is required for efficient formation of transcriptionally competent complexes at P1 . This second CAP may serve to stabilize the 1:1:1 CAP-polymerase-gal DNA intermediate under physiological conditions, thus enhancing initiation from P1 relative to P2 . Kinetic analysis reveals that the modest effect of CAP on the rate of P1 open complex formation can be resolved into about a 4-fold increase in the binding of RNA polymerase to the P1 region, plus a 1.5-fold elevation in the rate of isomerization of enzyme-promoter complexes to the open state.

Biochemistry, 1986 Sep 23, 25(19), 5371 - 7
Expression of bovine intestinal calcium binding protein from a synthetic gene in Escherichia coli and characterization of the product; Brodin P et al.; Intestinal calcium binding proteins (ICaBP's) constitute a group of small vitamin D inducible proteins considered to play an important role in the absorption of dietary calcium . The mammalian ICaBP's are representatives of the "EF-hand" family of calcium binding proteins . As a first step in the application of protein engineering techniques to the study of structure-function relationships in mammalian ICaBP's, we have synthesized a gene encoding the minor A form (the native form lacking the two N-terminal amino acids) of bovine ICaBP employing a rapid, microscale gene synthesis technique based on "shotgun ligation" of sets of oligonucleotides . Expression of the synthetic gene from a plasmid containing the tac promoter in a lon protease deficient strain of Escherichia coli yielded the desired product at a level of about 1-2 wt % of total protein . During the purification of the ICaBP expressed in E . coli, a contaminant was strongly adhering to it but was efficiently removed by gel filtration after denaturation with urea . The minor A form of ICaBP produced in E . coli was characterized by its mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by its total amino acid composition, partial amino acid sequence, UV absorption spectrum, and 360-MHz 1H NMR spectrum, showing beyond reasonable doubt its identity with the minor A form of ICaBP obtained from bovine intestines.

Eur J Pharmacol, 1986 Sep 23, 129(1-2), 65 - 76
The release of a neutrophil chemotactic factor from peritoneal macrophages by endotoxin: inhibition by glucocorticoids; Cunha FQ et al.; Glucocorticoids inhibit the migration of neutrophils induced by endotoxin (E . coli lipopolysaccharide, LPS) in vivo . Macrophage monolayers stimulated by LPS showed a dose-dependent release into the supernatant of a chemotactic factor for neutrophils, MNCF, which was active in vivo and in vitro . Dexamethasone reduced the neutrophil migration into the abdominal cavities of rats that was induced by LPS, but did not affect the migration induced by MNCF . The release of MNCF by LPS-pretreated macrophage monolayers was inhibited by dexamethasone and hydrocortisone but not by indomethacin and BW755C . MNCF was stable at 56 degrees C and did not release histamine from mast cells . Thus, MNCF activity seems not to be due to arachidonic acid metabolites or C5/C5a . MNCF shares some properties with interleukin-I, such as the blockade of its release by glucocorticoids, the association of its activity with a material of a molecular weight greater than 10 000 Daltons and the abolition of its activity by incubation with phenylglyoxal . However MNCF liberation was not blocked by cycloheximide and the time course of its release by macrophage monolayers stimulated by LPS differed from that described for interleukin-1 . In addition human interleukin-1 when tested in dexamethasone-treated test rats failed to induce neutrophil migration . It is suggested that inhibition by glucocorticoids of LPS-induced neutrophil migration in vivo is not due to a direct effect upon the migrating cells but rather to an indirect one, i.e . through blockade of MNCF release by macrophages.

J Mol Biol, 1986 Sep 20, 191(2), 301 - 2
Preliminary X-ray data for aspartate aminotransferase from Escherichia coli; Smith DL et al.; Crystals of the aspartate aminotransferase from Escherichia coli (aspC gene product) have been examined by X-ray analysis . The crystals grow as elongated rectangular prisms, with the symmetry of space group C2221 . Unit cell dimensions are a = 156 A, b = 87.6 A, c = 80.6 A and alpha = beta = gamma = 90 degrees . There is one protein subunit of molecular weight 43,600 per asymmetric unit.

J Mol Biol, 1986 Sep 20, 191(2), 177 - 80
Mutagenesis originating in site-specific DNA damage; Mitchell N et al.; Covalent monoadducts are the major types of gene damage after exposure to chemical mutagens and carcinogens . These and other damage structures together give rise to the spectrum of mutations that includes base-pair substitutions, insertions and deletions . In this study we introduced the bulky adduct guanine-8-aminofluorene into defined sites on one or both strands of the lactose operator . After insertion into plasmid pBR322 and replication in Escherichia coli COEC40, operator mutants were recognized on 5-bromo,4-chloro,3-indolyl-beta-D-galactoside plates and by hybridization probing . Out of ten randomly selected mutants, nine were single-base deletions and one was a two-base deletion . All mutations were at the site of modification or immediately adjacent to that site . If modifications were placed into both plasmid strands, preventing excision repair, operator mutants comprised close to 100% of operator-containing plasmids.

J Mol Biol, 1986 Sep 20, 191(2), 181 - 9
Role of Escherichia coli IHF protein in lambda site-specific recombination . A mutational analysis of binding sites; Gardner JF et al.; The phage lambda attachment site, attP, contains three binding sites for an Escherichia coli protein, IHF, that is needed for efficient integrative recombination . We have used synthetic oligodeoxyribonucleotides to direct multiple base changes at each of these three sites . Alteration by two base-pairs of the consensus sequence for the leftmost binding site specifically interferes with IHF binding to that site and modestly depresses recombination in vitro . For each of the three binding sites, alteration of the consensus sequence by four base-pairs strongly depresses recombination in vitro, indicating that all three sites are important for attP function . The mutated attP sites are also depressed for recombination in vivo but some of the mutants are less affected than they are in vitro . The disparity between effects in vivo and in vitro for some mutants but not others suggests that the three binding sites are not functionally equivalent and that at some sites additional E . coli factors may replace or assist IHF . The non-equivalence of the three IHF sites is also indicated by the behavior of prophage attachment sites carrying mutations in the binding sites.

J Mol Biol, 1986 Sep 20, 191(2), 163 - 75
Site-directed mutagenesis of M1 RNA, the RNA subunit of Escherichia coli ribonuclease P . The effects of an addition and small deletions on catalytic function; Lawrence NP et al.; One addition mutation and several small deletion mutations have been created in vitro at a unique site in the gene coding for M1 RNA, the RNA subunit of Escherichia coli RNase P . The mutant genes exhibit a wide range of efficiencies in complementing another mutant that is thermosensitive for RNase P function in vivo . The transcripts of the mutated genes cleave a precursor tRNA in vitro with efficiencies that parallel their ability to function in the complementation assay in vivo . The secondary structures in solution of the mutant gene transcripts are shown to be different from the parent molecule by probing the structure of the transcripts with ribonuclease T1 . A local region of secondary structure, between nucleotides 275 and 295, must be maintained for normal function of M1 RNA.

Nature, 1986 Sep 18-24, 323(6085), 259 - 62
Immunization of Aotus monkeys with recombinant proteins of an erythrocyte surface antigen of Plasmodium falciparum; Collins WE et al.; Recent studies have identified and characterized a ring-infected erythrocyte surface antigen (RESA) of the human malaria parasite Plasmodium falciparum with a relative molecular mass (Mr) of approximately 155,000 (refs 1-7) . RESA is localized in the micronemes of merozoites and also the membrane of red cells infected with ring-stage parasites . It is thought to be released through the apical pore from the rhoptry at the time of merozoite invasion . Because antibodies directed against this antigen strongly inhibit parasite growth in vitro, RESA may be useful in developing a vaccine against this parasite Here we describe an immunization trial using Aotus monkeys and Escherichia coli-derived fused polypeptides corresponding to various regions of the RESA molecule . Some monkeys in all test groups, but not in the control group, were protected against overwhelming infection . Strikingly, protection correlated with antibody responses to either of two different repetitive sequences in RESA.

Eur J Biochem, 1986 Sep 15, 159(3), 479 - 83
Aminoacylaminonucleoside inhibitors of protein synthesis . A new approach for evaluating their potency; Theocharis DA et al.; In a model system derived from Escherichia coli, Ac{3H}Phe-puromycin is produced in a pseudo-first-order reaction between the preformed Ac{3H}Phe-tRNA-poly(U)-ribosome complex (complex C) and excess puromycin {Kalpaxis et al . Eur . J . Biochem . 154, 267, 1986} . Amicetin and gougerotin inhibit this reaction to various degrees depending on whether or not complex C is allowed to interact with the inhibitor (I) prior to the addition of puromycin (S) . The kinetic analysis shows a phase where competitive inhibition can be observed provided that S and I are added simultaneously . After preincubating C with I, the inhibition becomes of the mixed non-competitive type . The Ki (the dissociation constant of the CI complex), calculated from the competitive plot, is 20.0 microM for amicetin and 15.0 microM for gougerotin . This inhibition constant (Ki) cannot distinguish amicetin from gougerotin . Its acceptance as a criterion of potency does not explain why after preincubation amicetin proves to be a stronger inhibitor than gougerotin . The determination of the apparent catalytic rate constants of peptidyltransferase at various inhibitor concentrations and the appropriate replotting of these rate constants distinguish amicetin from gougerotin . A new approach for evaluating the potency of these inhibitors is proposed . The familiar Ki is supplemented with an apparent kinetic constant obtained from a replot in which the intercepts of the double-reciprocal plots (1/kobs versus 1/{S}) are plotted versus the inhibitor concentration.

Biochem Pharmacol, 1986 Sep 15, 35(18), 3039 - 43
Possible role of endogenous histamine in mediation of LPS-induced secretion of corticosterone in mice; Suzuki S et al.; Escherichia coli lipopolysaccharide (LPS) induced a strong secretion of corticosterone in C3H/HeN mice with a concomitant increase in the splenic histidine decarboxylase activity . Treatment of the mice with alpha-fluoromethyl histidine, a suicide substrate for the enzyme, markedly attenuated both the secretion and the increase . In C3H/HeJ mice, LPS provoked little corticosterone release and induction of the enzyme . However, these mice responded to tetradecanoyl phorbol acetate with a large increase in both this secretion and enzyme activity . Injection of LPS produced a comparable increase in the serum histamine and corticosterone level and activity of histidine decarboxylase in various tissues of genetically mast-cell-deficient W/WV mice and in closely related +/+ mice . These results suggest that secretion of corticosterone caused by LPS is mediated by histamine produced through induction of histidine decarboxylase in non-mast cells.

J Biol Chem, 1986 Sep 15, 261(26), 12310 - 6
An auxiliary protein for DNA polymerase-delta from fetal calf thymus; Tan CK et al.; An auxiliary protein which affects the ability of calf thymus DNA polymerase-delta to utilize template/primers containing long stretches of single-stranded template has been purified to homogeneity from the same tissue . The auxiliary protein coelutes with DNA polymerase-delta on DEAE-cellulose and phenyl-agarose chromatography but is separated from the polymerase on phosphocellulose chromatography . The physical and functional properties of the auxiliary protein strongly resemble those of the beta subunit of Escherichia coli DNA polymerase III holoenzyme . A molecular weight of 75,000 has been calculated from a sedimentation coefficient of 5.0 s and a Stokes radius of 36.5 A . A single band of 37,000 daltons is seen on sodium dodecyl sulfate gel electrophoresis, suggesting that the protein exists as a dimer of identical subunits . The purified protein has no detectable DNA polymerase, primase, ATPase, or nuclease activity . The ability of DNA polymerase-delta to replicate gapped duplex DNA is relatively unaffected by the presence of the auxiliary protein, however, it is required to replicate templates with low primer/template ratios, e.g . poly(dA)/oligo(dT) (20:1), primed M13 DNA, and denatured calf thymus DNA . The auxiliary protein is specific for DNA polymerase-delta; it has no effect on the activity of calf thymus DNA polymerase-alpha or the Klenow fragment of E . coli DNA polymerase I with primed homopolymer templates . Although the auxiliary protein does not bind to either single-stranded or double-stranded DNA, it does increase the binding of DNA polymerase-delta to poly(dA)/oligo(dT), suggesting that the auxiliary protein interacts with the polymerase in the presence of template/primer, stabilizing the polymerase-template/primer complex.

J Biol Chem, 1986 Sep 15, 261(26), 12074 - 8
15N-labeled tRNA . Identification of 4-thiouridine in Escherichia coli tRNASer1 and tRNATyr2 by 1H-15N two-dimensional NMR spectroscopy; Griffey RH et al.; Uridine is uniquely conserved at position 8 in elongator tRNAs and binds to A14 to form a reversed Hoogsteen base pair which folds the dihydrouridine loop back into the core of the L-shaped molecule . On the basis of 1H NMR studies, Hurd and co-workers (Hurd, R . E., Robillard, G . T., and Reid, B . R . (1977) Biochemistry 16, 2095-2100) concluded that the interaction between positions 8 and 14 is absent in Escherichia coli tRNAs with only 3 base pairs in the dihydrouridine stem . We have taken advantage of the unique 15N chemical shift of N3 in thiouridine to identify 1H and 15N resonances for the imino units of S4U8 and s4U9 in E . coli tRNASer1 and tRNATyr2 . Model studies with chloroform-soluble derivatives of uridine and 4-thiouridine show that the chemical shifts of the protons in the imino moieties move downfield from 7.9 to 14.4 ppm and from 9.1 to 15.7 ppm, respectively; whereas, the corresponding 15N chemical shifts move downfield from 157.5 to 162.5 ppm and from 175.5 to 180.1 ppm upon hydrogen bonding to 5'-O-acetyl-2',3'-isopropylidene adenosine . The large difference in 15N chemical shifts for U and s4U allows one to unambiguously identify s4U imino resonances by 15N NMR spectroscopy . E . coli tRNASer1 and tRNATyr2 were selectively enriched with 15N at N3 of all uridines and modified uridines . Two-dimensional 1H-15N chemical shift correlation NMR spectroscopy revealed that both tRNAs have resonances with 1H and 15N chemical shifts characteristic of s4UA pairs . The 1H shift is approximately 1 ppm upfield from the typical s4U8 resonance at 14.8 ppm, presumably as a result of local diamagnetic anisotropies . An additional s4U resonance with 1H and 15N shifts typical of interaction of a bound water or a sugar hydroxyl group with s4U9 was discovered in the spectrum of tRNATyr2 . Our NMR results for tRNAs with 3-base pair dihydrouridine stems suggest that these molecules have an U8A14 tertiary interaction similar to that found in tRNAs with 4-base pair dihydrouridine stems.

J Biol Chem, 1986 Sep 15, 261(26), 12238 - 43
3-Hydroxyacyl-CoA epimerases of rat liver peroxisomes and Escherichia coli function as auxiliary enzymes in the beta-oxidation of polyunsaturated fatty acids; Yang SY et al.; The beta-oxidation of 2-trans,4-cis-decadienoyl-CoA, an assumed metabolite of linoleic acid, by purified enzymes from mitochondria, peroxisomes, and Escherichia coli was studied . 2-trans,4-cis-Decadienoyl-CoA is an extremely poor substrate of the beta-oxidation system reconstituted from mitochondrial enzymes . The results of a kinetic evaluation lead to the conclusion that in mitochondria 2-trans,4-cis-decadienoyl-CoA is not directly beta-oxidized, but instead is reduced by NADPH-dependent 2,4-dienoyl-CoA reductase prior to its beta-oxidation . Hence, the mitochondrial beta-oxidation of 2-trans,4-cis-decadienoyl-CoA does not require 3-hydroxyacyl-CoA epimerase, a conclusion which agrees with the finding that 3-hydroxyacyl-CoA epimerase is absent from mitochondria (Chu, C.-H., and Schulz, H . (1985) FEBS Lett . 185, 129-134) . However, 2-trans,4-cis-decadienoyl-CoA can be slowly oxidized by the bifunctional beta-oxidation enzyme from rat liver peroxisomes, as well as by the fatty acid oxidation complex from E . coli . The observed rates of 2-trans,4-cis-decadienoyl-CoA degradation by these two multi-functional proteins were significantly higher than the values calculated according to steady-state velocity equations derived for coupled enzyme reactions . This is attributed to the direct transfer of L-3-hydroxy-4-cis-decenoyl-CoA from the active site of enoyl-CoA hydratase to that of 3-hydroxyacyl-CoA dehydrogenase on the same protein molecule . All observations together lead to the suggestion that the chain shortening of 2-trans,4-cis-decadienoyl-CoA in peroxisomes and in E . coli occurs simultaneously by two different pathways . The major pathway involves the NADPH-dependent 2,4-dienoyl-CoA reductase, whereas 3-hydroxyacyl-CoA epimerase functions in the metabolism of D-3-hydroxyoctanoyl-CoA which is formed via the minor pathway.

FEBS Lett, 1986 Sep 15, 205(2), 241 - 5
Evidence that TraT interacts with OmpA of Escherichia coli; Riede I et al.; The OmpA protein is one of the major outer membrane proteins of Escherichia coli . Among other functions the protein serves as a receptor for several phages and increases the efficiency of F-mediated conjugation when present in recipient cells . TraT is an F-factor-coded outer membrane lipoprotein involved in surface exclusion, the mechanism by which E . coli strains carrying F-factors become poor recipients in conjugation . To determine a possible interaction of TraT with OmpA, the influence of TraT on phage binding to cells was measured . Because TraT inhibits inactivation of OmpA-specific phages it is suggested that TraT interacts directly with OmpA . Sequence homology of TraT with proteins 38, the phage proteins recognizing outer membrane proteins, supports this finding . A model of protein interactions is discussed.

Eur J Biochem, 1986 Sep 15, 159(3), 605 - 9
Characterization of a beta-galactosidase hybrid protein carrying the catalytic domain of Escherichia coli adenylate cyclase; Crenon I et al.; A hybrid protein of Escherichia coli, exhibiting both adenylate cyclase and beta-galactosidase activities, was purified and characterized . This protein, obtained by genetic engineering, contained the first 556 amino acids of adenylate cyclase connected to the eighth-residue of beta-galactosidase through a pentapeptide Val-Gly-Asp-Pro-Val . The fusion protein was less stable than the native beta-galatosidase . Trypsin cleaved preferentially the adenylate cyclase moiety of the hybrid protein at a ratio of 1/50 (w/w) . The kinetic properties of the hybrid protein were comparable, with a few exceptions, to those of native adenylate cyclase and beta-galactosidase . 'Truncated' adenylate cyclase was no longer sensitive to inhibition by excess ATP, which seems to indicate a second nucleotide binding site of wild-type adenylate cyclase . Photoirradiation of the hybrid protein with 8-azidoadenosine 5'-triphosphate inactivated the adenylate cyclase activity, leaving intact the beta-galactosidase activity . A radiolabeled ATP analog was incorporated after photoirradiation into the adenylate cyclase moiety of the fusion protein as shown by limited digestion with trypsin.

FEBS Lett, 1986 Sep 15, 205(2), 200 - 4
Purification and characterization of recombinant murine immune interferon; Nagata K et al.; The recombinant murine immune interferon (rMu-IFN-gamma) was purified to homogeneity from Escherichia coli harboring the expression vector of murine IFN-gamma . The purified rMu-IFN-gamma showed an Mr of 15 000 in SDS-polyacrylamide gel electrophoresis . Results of amino acid analysis, amino- and carboxyl-terminal analyses and peptide mapping of rMu-IFN-gamma suggest that it has the complete protein sequence predicted on the basis of cDNA except for lack of four amino acid residues from the mature carboxyl-terminus.

J Biol Chem, 1986 Sep 15, 261(26), 12179 - 83
The nucleotide sequence of the serA gene of Escherichia coli and the amino acid sequence of the encoded protein, D-3-phosphoglycerate dehydrogenase; Tobey KL et al.; The nucleotide sequence of serA, the structural gene of Escherichia coli which codes for D-3-phosphoglycerate dehydrogenase, has been determined . The structural gene contains 1233 nucleotides which code for the 409 amino acids of the subunit of the tetrameric enzyme, as well as the initiator methionine, which is cleaved from the mature protein, and the termination codon . The majority of the primary structure of the enzyme has been confirmed by automated Edman degradation of peptide fragments produced by a variety of cleavage agents . Comparison of the amino acid sequence of phosphoglycerate dehydrogenase with other NAD-dependent oxidoreductases reveals less than 20% homology, although conservation of certain specific residues in the coenzyme binding domain appears to be evident.

J Biol Chem, 1986 Sep 15, 261(26), 12128 - 33
Differences in the kinetic properties of BamHI endonuclease and methylase with linear DNA substrates; Nardone G et al.; BamHI endonuclease and methylase were found to exhibit a kinetic preference for linear pBR322 DNA substrates containing the recognition site in a central location . The Km values for substrates having the recognition site in a terminal location were approximately 3-fold greater than those with a centrally located site . This phenomenon may be partially due to facilitated transfer of the enzymes to the recognition site over nonspecific flanking sequences . The exploitation of facilitated transfer by these enzymes has been inferred from studies demonstrating kinetic preferences for longer DNA substrates . The reaction rates of the endonuclease were 9-fold greater with full-length pBR322 DNA than with a 74-base pair derivative . The methylase exhibits a kinetic preference for longer substrates but only under conditions of comparatively higher DNA concentrations . In addition, the methylase has the property of increasing long chain preference with increasing salt concentrations up to 120 mM . Increasing salt concentrations decreased the endonuclease's preference for longer substrates . Nonspecific inhibition studies revealed qualitative and quantitative differences between the two enzymes under catalytic conditions . These studies suggest that BamHI endonuclease and methylase interact with nonspecific DNA in different ways.

Biochem Biophys Res Commun, 1986 Sep 14, 139(2), 845 - 51
Biosynthesis of reovirus-specified polypeptides . Molecular cDNA cloning and nucleotide sequence of the reovirus serotype 1 Lang strain s3 mRNA which encodes the nonstructural RNA-binding protein sigma NS; George CX et al.; Human reovirus serotype 1 Lang strain s3 mRNA, which encodes the nonstructural RNA-binding polypeptide sigma NS, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13 . A complete consensus nucleotide sequence was determined . The Lang strain s3 mRNA is 1198 nucleotides in length and possesses an open reading frame with a coding capacity of 366 amino acids, sufficient to account for a sigma NS polypeptide of 41,179 daltons . Comparison of the serotype 1 (Lang) s3 sequence with the serotype 3 (Dearing) s3 sequence reveals 86.8 percent homology at the nucleotide level . The predicted sigma NS polypeptides of the Lang and Dearing strains display 97 percent homology at the amino acid level.

Biochem Biophys Res Commun, 1986 Sep 14, 139(2), 771 - 9
Biochemical and biological activities of N-ras proteins; Geis AM et al.; Recombinant N-ras proteins, expressed and produced from synthetic genes cloned into E . coli, have been tested in vitro for GTPase and autophosphorylation activity . The genes corresponding to the assayed proteins were tested for their ability to transform NIH 3T3 cells . Mutations of glutamine to lysine at amino acid position 61 and glycine to valine at position 12 were both found to activate the ability of the N-ras gene to transform NIH 3T3 cells while significantly reducing the GTPase activity of the corresponding protein . N-ras proteins were also found to autophosphorylate in the presence of GTP when a threonine acceptor amino acid is provided at position 59.

Lancet, 1986 Sep 13, 2(8507), 605 - 7
Polymyositis and molecular mimicry, a mechanism of autoimmunity; Walker EJ et al.; The amino acid sequences of Escherichia coli histidyl-tRNA synthetase and alanyl-tRNA synthetase, two proteins recently identified as autoantigens in polymyositis, were compared by a computer alignment procedure with those of the 3600 proteins tabulated in the National Biomedical Research Foundation protein sequence database . Both proteins contain sequences long enough to function as epitopes that match sequences on viral and muscle proteins . The homology thus revealed not only lends strong support to mechanisms of autoimmunity that invoke the theory of molecular mimicry of viral proteins, but also suggests a rationale for the skeletal muscle target of polymyositis.

Cell, 1986 Sep 12, 46(6), 895 - 904
Self-assembly of purified polyomavirus capsid protein VP1; Salunke DM et al.; The polyomavirus major capsid protein VP1, purified after expression of the recombinant gene in E . coli, was isolated as oligomers resembling the dissociated capsomeres derived from viral capsids . Image analysis of low-dose electron micrographs demonstrates that these VP1 oligomers are exclusively pentamers . The purified VP1 pentamers associated to form capsid-like assemblies and polymorphic aggregates at high ionic strength . The capsid-like assemblies were stabilized at low ionic strength by the addition of calcium . Self-assembly of the unmodified, recombinant DNA-generated VP1 implies that the posttranslational charge modifications of VP1 and the minor virion protein components, VP2 and VP3, are not essential for capsid formation . The nonequivalently related subunits of the penta- and hexavalent capsomeres therefore must spontaneously switch their bonding specificity during assembly.

Nucleic Acids Res, 1986 Sep 11, 14(17), 7115 - 23
Nucleotide sequence of the gene responsible for D-xylose uptake in Escherichia coli; Kurose N et al.; The nucleotide sequence of the cloned DNA, 363 bp in length, has been determined . It can complement the mutation of Escherichia coli having a decreased activity of D-xylose uptake at low temperature . Nucleotide sequence analysis found one possible reading frame coding for a polypeptide consisting of 61 amino acids . Several signal sequences conserved in the promoter regions of E . coli were found in the upstream regions of the open frame . This included the Shine-Dalgarno sequence, the Pribnow box, and the sequence conserved in the "-35 region" with a preferable spacing from each other for an efficient transcription . Downstream from the termination codon, the inverted repeat sequence was present, followed by 3 successive T's.

Nucleic Acids Res, 1986 Sep 11, 14(17), 6857 - 70
Promoter selectivity of Escherichia coli RNA polymerase: alteration by fMet-tRNAfMet; Nomura T et al.; An in vitro mixed transcription system was employed to examine the possible alteration of the promoter selectivity of Escherichia coli RNA polymerase by specific tRNAs . Transcription in vitro was inhibited by most of the tRNAs examined, although the extent of the inhibition differed with the tRNA species . The inhibition by tRNAs was due to competition with DNA for binding RNA polymerase . This inhibitory effect remained after charging of the tRNAs with amino acids . The charging of tRNAfMet with fMet, but not with Met, abolished its inhibitory effect, and instead gave a stimulatory effect on the transcription from some promoters . These observations suggest that fMet-tRNAfMet plays a specific regulatory role in the coupling of transcription to translation.

Nucleic Acids Res, 1986 Sep 11, 14(17), 6821 - 33
Control of cell division in Escherichia coli . DNA sequence of dicA and of a second gene complementing mutation dicA1, dicC; Bejar S et al.; A mutation in a gene dicA of Escherichia coli leads to temperature-sensitive cell division, by allowing expression of a nearby division inhibition gene dicB (1) . We have now established the sequence of the DicA region and identified DicA as a 15.5 KD protein . A second gene dicC transcribed divergently from dicA and coding for an 8.5 KD protein can also complement mutation dicA1 when provided on a multicopy plasmid.

Nucleic Acids Res, 1986 Sep 11, 14(17), 6965 - 81
Structure of the DNA distal to the gene for ribosomal protein S20 in Escherichia coli K12: presence of a strong terminator and an IS1 element; Mackie GA; The sequence of nucleotides extending over 2.3 kb distal to the gene for ribosomal protein S20 of E . coli has been determined . Included in the sequence is an efficient rho-independent terminator 50 b.p . distal to the coding sequence for S20, a complete copy of IS1 which lacks, however, flanking direct repeats, and finally, an open reading frame capable of encoding a 28 kDa polypeptide of unknown function . Several lines of evidence suggest that the IS1 sequence described here must represent one of the copies resident in the bacterial chromosome rather than a newly transposed copy . Northern blotting experiments show that the gene for S20 is functionally monocistronic under all conditions tested in several genetic backgrounds . Thus it seems unlikely that the distal copy of IS1 plays any role in the termination or stability of mRNA transcribed from the gene for S20.

Biochim Biophys Acta, 1986 Sep 10, 851(2), 223 - 8
Energetic consequences of multiple K+ uptake systems in Escherichia coli; Mulder MM et al.; The energetics of growth of Escherichia coli FRAG 1 under potassium-limited growth conditions and with glucose as sole carbon and energy source were studied in the chemostat and compared with those of a mutant, FRAG 5, defective in the high-affinity potassium uptake system . The steady-state concentration of biomass decreased with increasing growth rate and was the same in both parent and mutant . For each growth rate, the rate of production of ATP was higher in the parent than the mutant strain . Under potassium-limited conditions, FRAG 1 has at least two potassium uptake systems, an inducible high-affinity uptake system and a constitutive low-affinity uptake system (Rhoads, D.B., Waters, F.B . and Epstein, W . (1976) J . Gen . Physiol . 67, 325-341) . Apparently, the presence of the high-affinity uptake system in the parent leads to an energy drain . We suggest that this energy drain is due to futile cycling of potassium ions . On the basis of a mosaic non-equilibrium thermodynamic description of bacterial growth, it is concluded that the growth behaviour under potassium limitation corresponds to that expected for a catabolite limitation.

Biochemistry, 1986 Sep 9, 25(18), 5083 - 91
Synthesis and properties of oligodeoxyribonucleotides containing an ethylated internucleotide phosphate; Weinfeld M et al.; Internucleotide phosphotriesters comprise an important class of DNA lesions produced by carcinogenic alkylating agents . To avoid confusion resulting from the presence of other DNA lesions, synthetically prepared oligonucleotides containing ethylated internucleotide phosphates as the sole form of damage were employed to investigate several chemical and biochemical properties of DNA alkyl phosphotriesters . A total of four oligonucleotides were synthesised for this study, the dimers Tp(Et)T and pTp(Et)T and the decamer d-TpTpTp(Et)TpCpTpApTpTpT together with its unmodified analogue . The dimers were characterized by UV and phosphorus NMR spectroscopy and the decamers by two-dimensional homochromatography, alkali hydrolysis, and variable-temperature circular dichroism (CD) . Alkali hydrolysis of the ethylated decamer produced strand breaks in approximately 75% of the molecules . This is in close agreement with data previously obtained for dinucleoside ethyl phosphotriesters and triesters in alkylated cellular DNA . Results from the CD study suggest that the ethyl substituent does not disrupt base stacking within the oligomer . The interactions of two enzymes with the alkylated oligonucleotides were examined . First, it was found that ethylation of the internucleotide phosphate renders TpT inactive as a substrate for T4 polynucleotide kinase, implying that a negative charge is required on the 3'-phosphate group of the nucleotide to be phosphorylated . Hence, postlabeling assays of DNA damage that depend upon enzymatic phosphorylation of modified 3'-nucleotides cannot be applied to dinucleoside alkyl phosphotriesters . Second, both decamers, when annealed to a single-stranded plasmid template, were able to prime DNA synthesis, catalyzed by Escherichia coli DNA polymerase I, with equal effectiveness . The use of this reaction as a means of site-specifically incorporating phosphotriesters into viral vectors is recognized.

Biochemistry, 1986 Sep 9, 25(18), 5131 - 45
NMR studies of conformations and interactions of substrates and ribonucleotide templates bound to the large fragment of DNA polymerase I; Ferrin LJ et al.; The large fragment of DNA polymerase I (Pol I) effectively uses oligoribouridylates and oligoriboadenylates as templates, with kinetic properties similar to those of poly(U) and poly(A), respectively, and has little or no activity in degrading them . In the presence of such oligoribonucleotide templates, nuclear Overhauser effects (NOE's) were used to determine interproton distances within and conformations of substrates bound to the large fragment of Pol I, as well as conformations and interactions of the enzyme-bound templates . In the enzyme-oligo(rU)54 +/- 11-Mg2+dATP complex, the substrate dATP has a high anti-glycosidic torsional angle (chi = 62 +/- 10 degrees) and an O1'-endo/C3'-endo sugar pucker (delta = 90 +/- 10 degrees) differing only slightly from those previously found for enzyme-bound dATP in the absence of template {Ferrin, L.J., & Mildvan, A.S . (1985) Biochemistry 24, 4680-4694} . Both conformations are similar to those of deoxynucleotidyl units of B DNA but differ greatly from those of A or Z DNA . The conformation of the enzyme-bound substrate analogue AMPCPP (chi = 50 +/- 10 degrees, delta = 90 +/- 10 degrees) is very similar to that of enzyme-bound dATP and is unaltered by the binding of the template oligo(rU)54 +/- 11 or by the subsequent binding of the primer (Ap)9A . In the enzyme-oligo(rA)50-Mg2+TTP complex, the substrate TTP has an anti-glycosidic torsional angle (chi = 40 +/- 10 degrees) and an O1'-endo sugar pucker (delta = 100 +/- 10 degrees), indistinguishable from those found in the absence of template and compatible with those of B DNA but not with those of A or Z DNA . In the absence of templates, the interproton distances on enzyme-bound dGTP cannot be fit by a single conformation but require a 40% contribution from a syn structure (chi = 222 degrees) and a 60% contribution from one or more anti structures . The presence of the template oligo(rU)43 +/- 9 simplifies the conformation of enzyme-bound dGTP to a single structure with an anti-glycosyl angle (chi = 32 +/- 10 degrees) and an O1'-endo/C3'-endo sugar pucker (delta = 90 +/- 10 degrees), compatible with those of B DNA, possibly due to the formation of a G-U wobble base pair . However, no significant misincorporation of guanine deoxynucleotides by the enzyme is detected with oligo(rU) as template.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemistry, 1986 Sep 9, 25(18), 5126 - 31
Transfer RNA contains sites of localized positive charge: carbon NMR studies of {13C}methyl-enriched Escherichia coli and yeast tRNAPhe; Agris PF et al.; The possibility of positively charged nucleosides in tRNA has been suspected because certain posttranscriptional methylations produce quaternary nitrogens . To investigate this possibility and the importance of such methylations to tRNA structure, we have continued our studies of {13C}methyl-enriched phenylalanine tRNA of Escherichia coli {Kopper, R.A., Schmidt, P.G., & Agris, P.F . (1983) Biochemistry 22, 1307-1401} and yeast {Smith, C., Petsch, J., Schmidt, P.G., & Agris, P.F . (1985) Biochemistry 24, 1434-1440} . E . coli and yeast tRNA were 13C-enriched in their methyl groups in vivo, and phenylalanine-specific tRNA was isolated . Methyl proton and carbon signal assignments were confirmed and correlated for the purified tRNAs under native conditions via the first application of two-dimensional carbon-proton correlation NMR spectroscopy to a native nucleic acid . The methyl proton chemical shift of the 7-methylguanosine (m7G) signal from tRNA was easily determined, although by conventional 1H NMR spectroscopy it would have been hidden by ribose resonances and H2O . The chemical shift for 1-methyladenosine (m1A) protons was shown to be 3.01 ppm . Resolution of close or overlapping peaks was greatly enhanced by the two-dimensional experiment especially for the proton methyl resonances . In addition, proton-carbon chemical shift correspondence has been determined for the two 5-methylcytidines (m5C's), the methyl esters of wybutosine (Y), and the two ribose methyl groups, Gm and Cm, of yeast tRNAPhe . Thermal denaturation and Mg2+ depletion affect the methyl carbon NMR chemical shifts of tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1986 Sep 9, 25(18), 5051 - 7
High-resolution analysis of lac transcription complexes inside cells; Borowiec JA et al.; A new primer extension analysis is used to determine the methylation pattern over the lac UV5 promoter when dimethyl sulfate is added to growing Escherichia coli . The high-resolution analysis reveals altered methylation of 15 bases when the transcription machinery occupies the promoter inside the cell and shows a striking dichotomy in the distribution of methylated bases . Four protected guanosines lie on the side of the helix shown previously to be closely bound by RNA polymerase in vitro {Siebenlist, U., Simpson, R . B., & Gilbert, W . (1980) Cell (Cambridge, Mass.) 20, 269-281} . By contrast, the 11 hyperreactive bases lie on the side of the DNA directly opposite from that bound by protein . Those not in the melted region form two distinct "back-side" patches near -35 and -16 . We suggest that such hyperreactive patches can be caused by proteins bending the DNA toward themselves to allow a full range of contacts, thus distorting the helix grooves on the "back" side and facilitating attack by the methylating reagent . This leads to a proposal for the formation of transcription complexes in which RNA polymerase interacts with deformed and torsionally stressed DNA.

J Biol Chem, 1986 Sep 5, 261(25), 11798 - 807
Specific contacts between the FLP protein of the yeast 2-micron plasmid and its recombination site; Bruckner RC et al.; Contact points between the FLP protein of the yeast 2-micron plasmid and its recombination site have been defined . Important features of the region previously defined as the minimal recombination site in vitro include a pair of 13-base pair inverted repeats separated by an 8-base pair spacer . The two FLP protein-binding sites within this region are 12 base pairs in length . In each case they include the internal 11 base pairs of one of the 13-base pair repeats, as well as the adjacent base pair within the spacer . The internal 6 base pairs within the spacer are not involved in binding or recognition by FLP protein . When the size of the spacer is increased or decreased by one base pair, the distance between the cleavage points is also increased or decreased correspondingly by one base pair . Points of cleavage are unaffected by changes in the spacer sequence . Specific contact points involving purine residues, identified by methylation protection and recombination interference experiments, are located in both the major and minor grooves of the DNA . Additional contact points between FLP protein and phosphate groups in the phosphate-deoxyribose backbone are clustered near the cleavage sites.

J Biol Chem, 1986 Sep 5, 261(25), 11765 - 9
Site-specific alteration of cysteine 176 and cysteine 234 in the lactose carrier of Escherichia coli; Brooker RJ et al.; In the present study, Cys-176 and Cys-234 in the lactose carrier have been modified to serine residues via site-specific mutagenesis . The resultant mutants have been characterized with regard to galactoside transport activity and sulfhydryl reagent sensitivity . The mutant proteins (in which Cys-176 or Cys-234 had been replaced with serine) are able to effectively transport galactosides, although the transport rates for lactose and methyl-beta-D-galactopyranoside are slightly reduced compared to the normal lactose carrier . In addition, both mutants are less sensitive than the wild-type to high concentrations of two different sulfhydryl reagents, N-ethylmaleimide and p-hydroxymercuribenzoate . Overall, the data are consistent with the idea that Cys-176 and Cys-234 are close to the substrate recognition site . However, neither residue appears to be essential for galactoside transport by providing an ionizable group near the active site or by forming a disulfide bond.

J Biol Chem, 1986 Sep 5, 261(25), 11460 - 5
Chemical characterization and purification of the beta subunit of the DNA polymerase III holoenzyme from an overproducing strain; Johanson KO et al.; We have purified the beta subunit of the DNA polymerase III holoenzyme to homogeneity from an overproducing strain (Blanar, M., Sandler, S., Armengod, M., Ream, L., and Clark, A . (1984) Proc . Natl . Acad . Sci . U.S.A . 81, 4622-4626) . From this procedure we can obtain 100 mg quantities of protein . The beta isolated from the overproducer is indistinguishable from that isolated from wild-type cells in terms of its activity and molecular weight . Partial amino acid sequence analysis has confirmed the DNA sequence of the dnaN gene (Ohmori, H., Kimura, M., Nagata, T., and Sakakibara, Y . (1984) Gene (Amst.) 28, 159-170) and established the sites for initiation and termination of translation . No processing that removes amino acid residues from beta occurs since the active protein begins with the initiating methionine and terminates at the position predicted from the DNA sequence . Our knowledge of the precise amino acid composition has been used to determine the extinction coefficient of beta to be 17,900 and 18,700 cm-1 M-1 at 280 and 277 nm, respectively . The extinction coefficient at 280 nm is reduced to 14,700 cm-1 M-1 under denaturing conditions in guanidine HCl . Conditions have been optimized so that 1 N-ethylmaleimide residue can be incorporated per beta monomer with full preservation of activity.

J Mol Biol, 1986 Sep 5, 191(1), 75 - 84
Evidence for replicative transposition of Tn5 and Tn9; Ahmed A; Two basic types of models, conservative and replicative, have been proposed to account for the mechanism of transposition in bacteria . A method was developed to test these models by positive selection of various transposon-promoted events as galactose-resistant colonies from plasmid-containing cells . The results show that recA plays an important role in the transposition of Tn5 and Tn9 in Escherichia coli . All Tn5-promoted events (cointegrates, deletions and transpositions) are suppressed in recA-, and restored in recA+ . In the case of Tn9, however, only transpositions (but not cointegrates or deletions) are diminished in recA- . Therefore, the recA function is required for cointegrate formation by Tn5, and for cointegrate resolution by Tn9 . Both Tn5 and Tn9 cointegrates segregate transpositions (which can be seen as sectors on indicator plates) in recA+ hosts . In recA-, the unresolved Tn9 cointegrates undergo a second round of cointegrate formation to excise plasmids bearing galactose-resistant deletions . In growing cultures, the proportion of cointegrates declines steadily while transpositions increase so that, in late stages, cultures are rich in transpositions and contain few cointegrates . This explains the failure of previous workers to identify cointegrates as essential intermediates in transposition . Hence, with the exception of the recA requirement, the mechanism of transposition of these composite transposons is not very different from simple transposons like Tn3 . It is concluded that transposition of Tn5 and Tn9 is normally a replicative process.

J Biol Chem, 1986 Sep 5, 261(25), 11906 - 17
Escherichia coli topoisomerase I can segregate replicating pBR322 daughter DNA molecules in vitro; Minden JS et al.; The reconstituted pBR322 DNA replication system has been used to identify a mechanism for the processing and segregation of daughter DNA molecules by Escherichia coli topoisomerase I (Topo I) during the terminal stages of DNA replication . At low concentrations of Topo I (sufficient to confer specificity to the replication system for DNA templates containing a ColE1-type origin of DNA replication), the major products of the replication reaction were: multigenome-length, linear, double-stranded DNA molecules (an aberrant product); multiply interlinked, catenated, supercoiled DNA dimers; and a last Cairns-type replication intermediate . Thirty- to fifty-fold higher concentrations of Topo I led to the appearance of form II and form I pBR322 DNA as the only synthetic products . A model was developed in which Topo I, bound to a single-stranded gap on the parental H strand DNA just upstream of the origin of DNA replication, catalyzed the decatenation of the intermolecular linkages between the two daughter DNA molecules that were generated by primosome-catalyzed unwinding of the residual nonreplicated parental duplex DNA in the last Cairns-type intermediate . At low concentrations of Topo I, however, the intermolecular linkages persisted and, within the context of this replication system, were not removed by DNA gyrase . In support of this model it was demonstrated that: there was a single-stranded gap between the nonreplicated parental duplex region and the 5' end of the nascent leading-strand DNA; the number of intermolecular linkages in the catenated supercoiled DNA dimers was inversely related to the concentration of Topo I; the supercoiled DNA dimers did not serve as a precursor of the final form I DNA product; and maturation of the last Cairns-type replication intermediate to form I DNA was not affected by the presence of coumermycin, a potent inhibitor of the activities of DNA gyrase.

J Biol Chem, 1986 Sep 5, 261(25), 11866 - 71
Deletion and insertion mutants in the structural gene for ribosomal protein S1 from Escherichia coli; Schnier J et al.; Mutants have been constructed by deleting regions of the gene rpsA for ribosomal protein S1, which had been cloned in plasmid pACYC184 . The mutant genes were analyzed for their ability to complement an S1 amber mutant containing a temperature-sensitive suppressor . Another series of mutants was constructed using the tac promoter plasmid pKK223-3, and the effect of the mutant proteins was analyzed in a strain wild type for rpsA . The gene products of all mutants were identified by the immunoblotting technique . Plasmids with a mutant rpsA gene which do not or only poorly complement the S1 amber mutation cause drastic growth reduction, whereas the overall protein synthesis is affected to different extents depending on the site of the deletion . Mutants which express S1 fragments comprising at least the NH2-terminal 100 amino acids stimulate or inhibit the synthesis of certain cellular proteins . The amount of chromosomal coded S1 was reduced by each mutant plasmid . Our data suggest that S1 has a general regulatory role during protein biosynthesis.

J Biol Chem, 1986 Sep 5, 261(25), 11645 - 9
Properties of cyclic AMP-independent catabolite gene activator proteins of Escherichia coli; Blazy B et al.; The protein products of two crp alleles encoding mutationally altered catabolite gene activator proteins CAP and CAPc, which are functionally active in vivo in the absence of cAMP, were purified by an immunoaffinity purification procedure . These proteins bind cAMP with the same affinity as does the wild-type catabolite gene activator protein . From their susceptibility to the proteolytic enzyme subtilisin, we conclude that the two mutationally altered proteins adopt structural features adequate for biological activity and similar to the conformation that cAMP elicits or stabilizes in wild-type catabolite gene activator protein . We note, however, that their conformation is not unique and can be modulated by cAMP . The two altered proteins, CAP and CAPc, bind to the lactose promoter, giving rise to specific DNA-protein complexes in the absence of cAMP and promote initiation of specific lac messenger RNA synthesis.

J Biol Chem, 1986 Sep 5, 261(25), 11448 - 51
Stimulation of Escherichia coli adenylate cyclase activity by elongation factor Tu, a GTP-binding protein essential for protein synthesis; Reddy P et al.; A unique feature of eucaryotic adenylate cyclases is their interaction with GTP-binding proteins that mediate hormonal responses . Until now, there has been no evidence for regulation of Escherichia coli adenylate cyclase by a GTP-binding protein . We describe here that the most abundant protein in E . coli, the GTP-binding protein EF-Tu, which is important as an elongation factor in protein synthesis, also serves as a stimulator of adenylate cyclase activity . Homogeneous EF-Tu specifically increased the activity of purified adenylate cyclase as much as 70%; other E . coli GTP-binding proteins had no effect on enzyme activity . A study of the guanine nucleotide specificity for EF-Tu-mediated stimulation of adenylate cyclase activity suggested that the preferred activator is EF-Tu X GDP . To account for the GTP-specific stimulation of adenylate cyclase activity observed in intact cells, we propose that the nucleotide specificity for EF-Tu-dependent activation of adenylate cyclase is governed by other factors in the cell.

J Mol Biol, 1986 Sep 5, 191(1), 135 - 8
Investigation of the tertiary folding of Escherichia coli ribosomal RNA by intra-RNA cross-linking in vivo; Stiege W et al.; "In vivo" cross-links were introduced into ribosomal RNA by direct ultraviolet irradiation of intact Escherichia coli cells, during growth in a 32P-labelled medium . Ribosomes were isolated from the irradiated cultures, dissociated into subunits and subjected to partial digestion with cobra venom nuclease . The intra-RNA cross-linked fragments were separated by two-dimensional gel electrophoresis and the sites of cross-linking determined, using our published methodology . A comparison with the data previously obtained by this procedure, after irradiation of isolated 30 S and 50 S subunits, showed that in the case of the 50 S subunit nine out of the ten previous cross-links in the 23 S RNA could be identified in the "in vivo" experiments, and correspondingly in the 30 S subunit five out of the six previous cross-links in the 16 S RNA were identified . Some new cross-links were found, as well as two cross-links in the 16 S RNA, which had hitherto only been observed after partial digestion of irradiated 30 S subunits with ribonuclease T1 . The relevance of these data to the tertiary folding of the rRNA in situ is discussed, with particular reference to the work of other authors, in which "naked" RNA was used as the substrate for cross-linking and model-building studies.

J Biol Chem, 1986 Sep 5, 261(25), 11859 - 65
Release of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase depends mainly on time elapsed after the start of initiation, not on length of product RNA; Shimamoto N et al.; To elucidate the mechanism of sigma release in the transcript by Escherichia coli DNA-dependent RNA polymerase, we obtained the time courses of sigma release and elongation of product RNA by a rapid kinetic technique; transcription was synchronously initiated from A1 promoter on T7 DNA by the addition of four substrates to a stoichiometric mixture of holoenzyme and template DNA, and then quenched by the addition of EDTA . The elongation rate was changed by limiting the concentration of one of four substrates, GTP . At reduced GTP concentration, elongation was decelerated, but the time course of sigma release was unchanged . No connection between sigma release and length of RNA product was found . The results lead to the conclusion that sigma is released depending only on time elapsed after initiation, not on the length of RNA product . We propose a two-step model for sigma release with a rapid triggering and a slow dissociation of about 5 s . This dissociation, the rate-determining step of sigma release, is independent of the rate of elongation.

Isr J Med Sci, 1986 Sep, 22(9), 690 - 5
Association between Legionella pneumophila and amoebae in water; Henke M et al.; The presence of amoebae and Legionella pneumophila in ground-water, drinking water supplies and whirlpools was investigated . Volumes of 10 to 1,000 ml were concentrated by membrane filtration . L . pneumophila was detected on buffered charcoal yeast extract (BCYE) agar, and amoebae by inverting filters on nonnutrient agar plates seeded with Escherichia coli that were incubated at 37 C for up to 12 days . In 65% of the samples positive for L . pneumophila amoebae were also detected . L . pneumophila and amoebae were detected together in 38% of warm drinking water samples . The highest isolation temperature for amoebae was 57 C, but fewer amoebae were detected above than below 50 C . In cold drinking water, amoebae were found in 88% of samples . The presence of L . pneumophila and amoebae in whirlpool waters (42%) presents a risk for man . Fresh environmental isolates of an Acanthamoeba species and L . pneumophila serogroup 4 were used for laboratory experiments . The amoebae supported intracellular multiplication of L . pneumophila in Chang's medium and autoclaved tap water, as shown by colony-forming unit (CFU) counts, direct fluorescent antibody test and Gimenez staining . Results confirmed that interaction between L . pneumophila and amoebae could occur in nature, and that the latter could act as hosts for legionellae and support their growth.

Anal Biochem, 1986 Sep, 157(2), 199 - 207
N4-(6-aminohexyl)cytidine and -deoxycytidine nucleotides can be used to label DNA; Gillam IC et al.; Bisulfite is known to catalyze transamination between cytidine derivatives and amines . Using 1,6-diaminohexane we describe the synthesis and recovery of the 5'-triphosphates of N4-(6-aminohexyl)cytidine and -deoxycytidine (dahCTP) . Both may be incorporated into DNA by nick translation with DNA polymerase I of Escherichia coli to provide reactive sites for the attachment of immunological or other labels . Biotinyl dahCTP is actively incorporated into DNA by the same system and can be detected by the binding of streptavidin complexed to an indicator enzyme such as acid phosphatase . Such labeled DNA is a suitable nonradioactive probe for detection of related sequences by hydridization.

J Vet Pharmacol Ther, 1986 Sep, 9(3), 254 - 63
Pharmacokinetics and bioavailability of chloramphenicol in normal and febrile goats; Kume BB et al.; The effect of induced fever on the plasma concentrations and disposition kinetics of chloramphenicol (CHPC) was studied in adult goats . Fever was induced and maintained for 12 h by injecting Escherichia coli endotoxin (0.1 microgram/kg, i.v.) and repeating it at half the dose (0.05 microgram/kg) 8 h later . Pharmacokinetics of CHPC was studied in both normal (n = 6) and febrile (n = 6) animals following intravenous administration of CHPC Na-succinate (25 mg/kg) . Intramuscular bioavailability of the drug was also investigated in both normal and febrile animals . Pharmacokinetics of CHPC following intravenous administration could be described by a two-compartment open model in both normal and febrile animals . In normal animals the half-life of CHPC was 73.0 +/- 4.95 min and the volume of distribution was 2.217 +/- 0.24 l/kg . These and other pharmacokinetic parameters did not differ significantly (P less than 0.05) between normal and febrile animals, except for Cop and A . Absorption of CHPC following intramuscular administration was good as indicated by its high bioavailability in both normal (83.34%) and febrile (81.98%) animals . Volume of distribution is usually expected to change when the febrile state is induced . Lack of such an effect in the present study could be due to high individual variation, or to the fact that CHPC already has a relatively large volume of distribution, which is less likely to be altered by a febrile state of short duration.

Vet Res Commun, 1986 Sep, 10(5), 399 - 405
Thermostable factor(s) in soya producing a net excess of secretion in the ligated gut test in pigs; Nabuurs MJ; The ligated gut test (LGT) is the standard method for the examination of Escherichia coli strains for enterotoxin production in pigs . As solid pig feed has been associated with diarrhea, soya products (the main protein source for piglets) were investigated with the same test as E . coli strains . After injection of different soya products into ligated segments of the small intestine fluid accumulation was observed, indicating a net excess of secretion . The factor in soya products responsible for this effect was found to be thermostable, as its effect was unaltered after heating at 120 degrees C during an hour . No indications of a possible allergic phenomenon accounting for the fluid accumulation were found . From the results of this study it is concluded that soyabean products can produce results in the LGT similar to those produced by enterotoxigenic E . coli strains.

Pediatr Res, 1986 Sep, 20(9), 825 - 7
Biosynthetic somatomedin C (SM-C/IGF-I) increases the length and weight of Snell dwarf mice; van Buul-Offers S et al.; An Escherichia coli derived somatomedin-C/IGF-I preparation (rec-IGF-I) with an amino acid sequence identical to the natural IGF-I derived from human plasma, increases body length and weight, as well as the growth of several organs of Snell dwarf mice, when administered for 4 wk . After 2 wk of treatment rec-IGF-I (22.2 micrograms/day) induced a significant increase over buffer treated controls, to a comparable degree as obtained with bacterially synthesized human growth hormone (bhGH; 8.4 micrograms/day) . The weight/length ratio of rec-IGF-I and bhGH-treated dwarf mice after 4 wk of treatment were not significantly different . A significant increase over controls was obtained with both preparations . Organs with increased weights after bhGH treatment (brain; submandibular salivary glands; heart, liver, kidneys, thymus, and spleen) were also heavier after rec-IGF-I . Significance was only reached for the kidneys and the spleen and the musculus quadriceps femoris . Organ weights expressed as a percentage of body weight of bhGH and rec-IGF-I treated dwarfs were similar except for the relative weight of the heart of the bhGH group, which was significantly increased compared to the controls and the rec-IGF-I group . These data resolve the issue as to whether or not pure SM-C/IGF-I will induce growth in length and demonstrate the usefulness of recombinant IGF-I in the studies of growth regulation.

Mutat Res, 1986 Sep, 162(2), 153 - 63
Site-specific mutagenesis in vivo by single methylated or deaminated purine bases; Hill-Perkins M et al.; The technique of site-directed mutagenesis has been used to investigate the mutagenicity of O6-methylguanine (O6-MeG) or hypoxanthine introduced as a single lesion at a specific locus in an M13mp9 RF molecule constructed in vitro . Following transformation of O6-MeG-containing RF molecules into E . coli JM101, mutant progeny phage were produced at a frequency not significantly different from that observed with wild-type M13mp9 RF . The mutant yield was greatly enhanced by exhausting cellular O6-MeG DNA-methyltransferase before transformation . In contrast, hypoxanthine exhibited miscoding mutagenesis in the absence of interference with cellular repair mechanisms . This indicates that cellular hypoxanthine-DNA glycosylase acts inefficiently in the removal of hypoxanthine from DNA in vivo . The precise mutational changes induced by hypoxanthine were determined by DNA sequence analysis.

Mutat Res, 1986 Sep, 162(2), 145 - 51
Sensitivity of Deinococcus radiodurans to near-ultraviolet radiation; Caimi P et al.; Although Dienococcus radiodurans is notoriously resistant to far-ultraviolet radiation (FUV; 254 nm), it is highly sensitive to near-ultraviolet radiation (NUV; 300-400 nm), thus demonstrating that the mechanisms of damage (and/or recovery) by the two types of irradiation are different . This observed difference between FUV and NUV effects in D . radiodurans agrees with previous studies with Escherichia coli . Near-ultraviolet radiation produces DNA damage which is presumed to be single-strand breaks (SSB) in the DNA of D . radiodurans . Unique lesions, such as DNA-protein crosslinks could not be demonstrated in this study . Cells that were pre-irradiated with a small dose of NUV were subsequently protected against inactivating doses of NUV . The data presented are consistent with induced DNA repair following NUV damage in D . radiodurans; this is in contrast to FUV damage where DNA repair is constitutive but not induced.

Rev Fr Transfus Immunohematol, 1986 Sep, 29(4), 287 - 98
Antiprotease targeting: altered specificity of alpha 1-antitrypsin by amino acid replacement at the reactive centre; Jallat S et al.; Alpha-1 antitrypsin (alpha 1AT) is an efficient inhibitor of the human neutrophil proteases, elastase and cathepsin G . The reactive centre P1 residue (Met358) of alpha 1AT is important in defining the specificity of inhibition; furthermore, oxidation of this residue results in a loss of inhibitor activity . There is evidence that oxidative inactivation of alpha 1AT may be involved in the pathogenesis of pulmonary emphysema associated with cigarette smoking . We have studied the effect of a series of amino acid replacements at the active centre on the inhibition properties of alpha 1AT . The mutant proteins were produced in E . coli following in vitro mutagenesis of the alpha 1AT cDNA . Alpha-1-AT (Ile358), (Ala358) and (Val358) were efficient inhibitors of both neutrophil and pancreatic elastase, but not cathepsin G . Alpha-1-AT (Ala356, Val358) and alpha 1AT (Phe358) were specific for pancreatic elastase and cathepsin G respectively . Alpha-1-AT (Leu358) inhibited both neutrophil elastase and cathepsin G . These data show that, for effective inhibition, a potential cleavage site for the protease must be displayed at the alpha 1AT active centre . In each case, replacement of Met358 led to resistance to oxidative inactivation . Since alpha 1AT (Leu358) inhibits both neutrophil proteases and is resistant to oxidation, this variant may be of increased potential for the therapy of destructive lung disorders.

Biochem J, 1986 Sep 1, 238(2), 475 - 83
The overexpression and complete amino acid sequence of Escherichia coli 3-dehydroquinase; Duncan K et al.; The enzyme 3-dehydroquinase was purified in milligram quantities from an overproducing strain of Escherichia coli . The amino acid sequence was deduced from the nucleotide sequence of the aroD gene and confirmed by determining the amino acid composition of the overproduced enzyme and its N-terminal amino acid sequence . The complete polypeptide chain consists of 240 amino acid residues and has a calculated subunit Mr of 26,377 . Transcript mapping revealed that aroD is a typical monocistronic gene.

J Pharmacobiodyn, 1986 Sep, 9(9), 722 - 8
Characterization of the pyrogenicity of two different lipopolysaccharides and their lipid A-bovine serum albumin complexes; Ogawa Y et al.; In order to elucidate the dependency of pyrogenicity of lipopolysaccharide (LPS) on the lipid A structure, we investigated the pyrogenicity of both LPS and lipid A prepared from Mima polymorpha var . oxidans which is deficient in 3-hydroxymyristic acids linked to the 3-hydroxy group of other fatty acids . LPS and lipid A were also prepared as reference compounds from Escherichia coli UKT-B . Furthermore, the establishment of reliable indices for pyrogenicity was undertaken . The following results were obtained . The correlation in linearity was demonstrated between maximal increase in body temperature (delta Tmax) and dose of LPS or lipid A complexed with bovine serum albumin (BSA) . The dose-response curves based on delta Tmax were more reliable statistically than the Fever Index-4h representing the area under fever curves for 4 h . The minimum pyrogenic dose (MPD) of E . coli LPS was 1.6 X 10(-3) micrograms/kg i.v . In contrast, the MPD of M . polymorpha LPS was 7.0 X 10(-3) micrograms/kg i.v . By intracisternal injection, the MPD of E . coli LPS was 2.5 X 10(-6) micrograms/kg and that of M . polymorpha LPS 1.0 X 10(-4) micrograms/kg . The end points of Limulus amoebocyte lysate gelation were 10(-5) micrograms/ml in E . coli LPS and 10(-3) micrograms/ml in M . polymorpha LPS . The MPDs of lipid A/BSA complexes by i.v . injection were 0.15 micrograms/kg in E . coli and 2.5 micrograms/kg in M . polymorpha . The rabbits immunized with E . coli lipid A/BSA complex acquired pyrogenic tolerance to the parent LPS but the cross tolerance to M . polymorpha LPS was not observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Int, 1986 Sep, 13(3), 433 - 8
Molecular cloning of cDNA for argininosuccinate lyase of rat liver; Amaya Y et al.; A cDNA expression library constructed from poly(A)+ RNA of rat liver was screened immunologically using an antibody against argininosuccinate lyase (EC 4.3.2.1), a urea cycle enzyme, of rat liver . A cDNA clone was isolated and identified by hybrid-selected translation . The clone contained an insert approximately 1.5 kilobase pairs in length . In the bacterial clone, a specific protein of Mr = about 25,000 was expressed . The argininosuccinate lyase mRNA of about 2.1 kilobases long was detected in the liver and in a lesser amount in the kidney and spleen, but not in the small intestine and heart of the rats.

Arch Microbiol, 1986 Sep, 145(4), 334 - 41
Overproduction of the proFhuA outer membrane receptor protein of Escherichia coli K-12: isolation, properties, and immunocytochemical localization at the inner side of the cytoplasmic membrane; Hoffmann H et al.; The fhu operon of Escherichia coli K-12 comprises four genes, termed fhuA,C,D,B, which are involved in the uptake of iron-hydroxamate compounds . The fhuA gene encodes the outer membrane receptor protein . Cells that contained three copies of the fhuACD fragment on the thermoamplifiable plasmid pHK232 accumulated at 37 degrees C large amounts of the proFhuA protein . Most of the overproduced proFhuA protein was not translocated into the outer membrane but instead precipitated at the cytoplasmic side of the inner membrane, presumably at the sites of synthesis . Despite inhibition of export proFhuA synthesis continued . The precipitate formed was sedimented by centrifugation at 8,000 x g . The proFhuA protein could be solubilized in 1% sodium dodecyl sulfate . Replacement of sodium dodecyl sulfate by Triton X-100 resulted in a proFhuA protein which exhibited 10% of the phage T5 binding activity of renatured mature FhuA protein . Binding of the phage T5 was inhibited by the FhuA-specific ligands ferrichrome, albomycin and colicin M . Limited proteolysis of the isolated pro- and mature form of the FhuA protein with trypsin yielded similar oligopeptide patterns . Addition of ferrichrome affected trypsin cleavage of both proteins in the same way . The common proteolytic intermediates together with phage inactivation indicate a similar conformation of the pro- and mature form.

Acta Pathol Jpn, 1986 Sep, 36(9), 1335 - 46
Experimental study on secondary amyloidosis associated with chronic arthritis; Aoki S et al.; Various organs from rabbits immunized with heat-killed E . coli 0:14 to induce chronic polyarthritis resembling rheumatoid arthritis (RA) were examined for amyloid deposition since amyloidosis is a frequent feature of long-standing RA . Amyloids were confirmed mainly by polarizing as well as nonpolarizing light microscopy after Congo red staining and partly by electron microscopy and immunoperoxidase technique . Amyloid deposits after an additional 2 months from the first appearance of arthritis were most frequently found in the spleen and kidneys suggesting secondary amyloidosis . Amyloidosis was observed in 36% of 75 arthritic rabbits . The incidence of amyloidosis was significantly higher in the arthritic rabbits than in the non-arthritic rabbits (p less than 0.05) . This suggests that prolonged sensitization with heat-killed E . coli 0:14 containing endotoxic substance participates in the formation of both arthritis and amyloidosis.

Tsitol Genet, 1986 Sep-Oct, 20(5), 323 - 6
{Ultrastructural characteristics of the biological action of an endotoxin on sensorimotor cortical neurons}; Povilaitite PE et al.; The work is devoted to the ultrastructural investigation of the state of the sensomotor cortex neurons after intravenous and intracisternal administration of endotoxin . The evidences are presented concerning ultrastructural alterations in mitochondria and the presence of compensatory reaction in them after intravenous injection of endotoxin . Besides, coated vesicles and subsurface cisternae whose formation is induced by the endotoxin action have been studied.

Mol Cell Biol, 1986 Sep, 6(9), 3050 - 8
Escherichia coli aspartate transcarbamylase: a novel marker for studies of gene amplification and expression in mammalian cells; Ruiz JC et al.; Eucaryotic expression vectors containing the Escherichia coli pyrB gene (pyrB encodes the catalytic subunit of aspartate transcarbamylase {ATCase}) and the Tn5 phosphotransferase gene (G418 resistance module) were transfected into a mutant Chinese hamster ovary cell line possessing a CAD multifunctional protein lacking ATCase activity . G418-resistant transformants were isolated and analyzed for ATCase activity, the ability to complement the CAD ATCase defect, and the ability to resist high concentrations of the ATCase inhibitor N-(phosphonacetyl)-L-aspartate (PALA) by amplifying the donated pyrB gene sequences . We report that bacterial ATCase is expressed in these lines, that it complements the CAD ATCase defect in trans, and that its amplification engenders PALA resistance . In addition, we derived rapid and sensitive assay conditions which enable the determination of bacterial ATCase enzyme activity in the presence of mammalian ATCase.

J Biochem (Tokyo), 1986 Sep, 100(3), 727 - 33
Cloning and expression of human lymphotoxin mRNA derived from a human T cell hybridoma; Kobayashi Y et al.; We cloned human lymphotoxin (LT) cDNA from a cDNA library prepared from phorbol myristate acetate (PMA) and Con A-stimulated human T cell hybridoma (AC5-8) cells, The nucleotide sequence of the cDNA insert in the plasmid, pLT13, was determined and compared with those of peripheral blood mononuclear cell derived LT cDNA and the LT gene . Four stretches, containing 14 nucleotides in total, were different among the three sequences . The differences included one base change from C to A in the coding region . Because this change was expected to result in a change in the amino acid at position 26 from Thr to Asn, we constructed an E . coli expression plasmid (pLT13tac6-8.2) and a mammalian cell expression plasmid (pSV2-LT) in order to confirm that pLT13 contains the coding sequence of human LT . Both plasmids were found to synthesize active LT molecules after transfection into JM105 and COS-1 cells, respectively, and their lytic activities were found to be completely neutralized by anti human LT serum . Using an insertion mutant and a deletion mutant, we examined the role of the C terminal domain in the LT activity . The results obtained strongly suggested that the intactness of the C terminal less than 10 residues is required for the LT activity.

J Biochem (Tokyo), 1986 Sep, 100(3), 679 - 85
Effects of temperature and monovalent cations on activity and quaternary structure of tryptophanase; Honda T et al.; Effects of temperature and monovalent cations on the activity and the quaternary structure of tryptophanase of Escherichia coli were studied . The conversion of the apoenzyme into the active holoenzyme was attained at 30 degrees C in Tris-HCl buffer (pH 8.0) containing pyridoxal-P and K+, while no conversion occurred at 5 degrees C . The active holoenzyme thus formed was stable even at 5 degrees C, as long as the cation was present . When K+ was absent, however, the active enzyme gradually lost the activity upon chilling to 5 degrees C . The HPLC gel filtration analysis of the active holoenzyme and the low temperature-inactivated enzyme species revealed that the tetrameric holoenzyme dissociated into the dimeric apoenzyme concomitant with the low temperature-induced inactivation at 5 degrees C . The results of HPLC experiments together with other available evidence also suggest that the inactive tetrameric holoenzyme was first formed from the dimeric apoenzyme and pyridoxal-P prior to the formation of the active holoenzyme and that the cation promoted the conversion of the inactive holoenzyme into the active holoenzyme rather than being involved in the conversion of the apoenzyme and pyridoxal-P into the holoenzyme . Among various cations tested for the above effects, NH4+ exhibited the largest effect and K+ the second.

Biofizika, 1986 Sep-Oct, 31(5), 810 - 3
{31P NMR study of changes in the intracellular pH of Escherichia coli after freezing-thawing}; Naryshkina EP et al.; Influence of two types of freezing, at -196 and -50 degrees C with following thawing of Escherichia coli cells at 37 degrees C on the value of intracellular pH has been studied by means of 31P NMR spectroscopy . All the cycles of freeze-thawing have been shown to result in acidification of intracellular medium on 1.0 unit pH apart from the freezing type . The extracellular medium (pHex) was acidified too, but the degree of pHex changes after freeze-thawing depended on the freezing depth.

Vet Microbiol, 1986 Sep, 12(3), 241 - 53
Dysentery in calves caused by an atypical strain of Escherichia coli (S102-9); Chanter N et al.; Dysentery lasting 4-8 days was produced in five 4-day-old colostrum-fed calves, after inoculation with an atypical strain of Escherichia coli S102-9; peak excretion of S102-9 occurred during the period of dysentery . Two calves were killed when clinical signs were most severe and bacteria were seen attached to the surfaces of enterocytes in the large intestine; microscopic lesions were seen in these areas . The lesions were identical to those previously reported in a natural outbreak of dysentery in calves, from which E . coli S102-9 was isolated, and to those seen in gnotobiotic calves experimentally infected with S102-9 . Reinfection of the three surviving calves 16-20 days later with S102-9 and primary infection of two calves aged 24 and 51 days did not cause dysentery . Four of 659 coliforms isolated from field outbreaks of calf diarrhoea resembled the atypical strain S102-9 . These four isolates and S102-9 did not produce heat-stable enterotoxin, but all produced a toxin cytopathic for Vero and HeLa cells . Two of the four isolates were inoculated alone into 4-day-old gnotobiotic calves deprived of colostrum; neither calf developed dysentery but microscopic lesions identical to those produced by S102-9 were detected in the large intestines of both animals.

Toxicol Lett, 1986 Sep, 32(3), 235 - 41
L-asparaginase effects on inhibition of protein synthesis and lowering of the glutamine content in cultured rat hepatocytes; Villa P et al.; Primary cultures of rat hepatocytes were exposed to various concentrations of L-asparaginase derived from Escherichia Coli . Protein synthesis was inhibited by about 33% and cellular glutamine was reduced proportionally to the enzyme concentration . However, protein synthesis was inhibited only by amounts of enzyme able to reduce glutamine to critical levels below 10 nmol/mg cell protein . These data suggest that the glutaminase activity which probably contaminates E . coli asparaginase may be responsible for reduced liver protein synthesis.

Scand J Gastroenterol, 1986 Sep, 21(7), 819 - 23
Colicinogeny in chronic inflammatory bowel disease; Bures J et al.; There are several indirect arguments for a possible role of colicins in chronic inflammatory bowel disease (IBD) . Colicinogeny was therefore investigated in 54 patients with ulcerative colitis, 39 patients with Crohn's disease, and 160 clinically healthy controls . No significant difference was found among the examined groups . The leukocyte migration inhibition test (with colicins as antigens) was performed to estimate cellular hypersensitivity to colicins . Migration indices not exceeding the normal range in controls contrasted with abnormal values found in 36% of ulcerative colitis and 80% of Crohn's disease patients . The results are believed to be proof of cellular hypersensitivity of IBD patients to colicins of their own Escherichia coli strains . The importance of this finding must be further clarified.

Res Commun Chem Pathol Pharmacol, 1986 Sep, 53(3), 381 - 98
Endotoxin-induced changes in nitrogen metabolism; Kilpatrick-Smith L et al.; Intraperitoneal administration of a sublethal dose of E . coli endotoxin to fasted rats produced within 12 hrs a rise in plasma ammonia, urea, glutamine, glutamate and aspartate as compared to controls . At this time, hepatic ureagenesis also increased as did the levels of glutamate, glutamine, aspartate, alanine and asparagine . However, liver glucose and ketone levels were unchanged . Hepatic {ATP}/{ADP} ratios declined in endotoxin-treated animals and there was an oxidation of mitochondrial pyridine nucleotides and a reduction of cytosolic pyridine nucleotides . The results suggest that impairment of nitrogen metabolism is an early effect of endotoxemia.

Mol Biol (Mosk), 1986 Sep-Oct, 20(5), 1399 - 408
{Non-canonical nucleotide exchange catalyzed by Escherichia coli DNA-polymerase I and the two-center model of the mechanism of action of this enzyme}; Chidzhavadze ZG et al.; It is shown, that DNA hydrolysis catalyzed by E . coli DNA polymerase I is inhibited, when a reaction mixture contains one type of deoxynucleoside 5'-triphosphate (dNTP) . When the reaction mixture contains {32P}dNTP, then {32P} is incorporated into DNA and v . v . (32P) from DNA is transferred into dNTP . The nucleotide exchange between DNA and dNTP in the assay mixture is observed only in the case, when the chemical nature of nucleotide residue of dNTP and that of the 3'-terminus of DNA is the same . Analysis of products of DNA hydrolysis in the presence of one type of dNTP using electrophoresis in polyacrylamide gel shows that most of the DNA molecules are terminated at the 3'-termini by the dNMP residue of the same chemical nature as the dNTP in the assay mixture . However, in some cases DNA molecules contain one additional nucleotide residue . This phenomenon can be explained by incorporation of one additional dNMP residue originating from dNTP only in those cases, when a non-typical base pairing of this nucleotide residue with a template residue readily takes place . The above-mentioned facts can be interpreted within the model for DNA hydrolysis with involvement of two intermediate covalent forms of dNMP residues with DNA polymerase I; one dNMP-intermediate should be placed at the elongation center and the other--at the hydrolysis center . The DNA hydrolysis by 3'----5' exonuclease activity of DNA polymerase I proceeds through these two covalent forms . DNA polymerases alpha from calf thymus and T4 phage do not catalyze the nucleotide exchange between DNA and dNTP from the reaction media.

Mol Biol (Mosk), 1986 Sep-Oct, 20(5), 1390 - 8
{Statistical characteristics of primary structures of the functional regions of the Escherichia coli genome . III . Computer recognition of coding regions}; Borodovskii MIu et al.; We have presented the method for recognition of structural domains of DNA . This method uses statistical description of coding and non-coding regions in the form of stationary or nonstationary Marcov chain, which was introduced in our previous papers . Calculation of the probability that the given fragment of the DNA appears part of the coding region, is the main operation of this algorithm . The results, obtained for the number of E . coli DNA sequences showed the ability of the method to find the structural domains and correct reading frame, so as to give the estimation of the extent of protein expressivity . Provided necessary statistical data are available, the proposed method may be used for the analysis of DNA of other organisms.

Mol Biol (Mosk), 1986 Sep-Oct, 20(5), 1299 - 304
{Sense codon in Escherichia coli are translated in context}; Shpaer EG; The nucleotide frequencies 5' and 3' to the sense codons in highly and weakly expressed genes have been investigated by the chi-squares method . A comparison between the experimental and computer-generated random nucleotide sequences (in which each codon is substituted by a random synonymous one) was made . It was shown that the choice of a particular codon among the synonymous ones in a given position of the gene depends on the three nucleotides 3' and 5' adjacent to the codon in highly expressed genes (the triplet 3' and a single nucleotide 5' to the codons in weakly expressed genes) . Concrete patterns for the preferable choice of synonymous codons depending on their contexts are presented . It is suggested that these constraints are related to the efficiency of messenger translation . The constraints on the amino acid sequences of encoded proteins also lead to statistically significant bases in nucleotide frequencies around the sense codons . The biological role of these constraints is discussed.

Mol Biol (Mosk), 1986 Sep-Oct, 20(5), 1290 - 8
{Effect of mutation in phoB and phoM genes for the positive control of alkaline phosphatase biosynthesis on Escherichia coli envelope proteins}; Tsfasman IM et al.; The suppression of some envelope proteins, localized in both the periplasm and the outer and inner membranes was shown in phoB and phoM phoR mutants of E . coli . Among these proteins are the proteins of the phosphate regulon and also those not pertaining them . As a result of phoB and phoM phoR mutations, the cytoplasmic membrane was found to be lacking in minor protein of 28,000 Mr, which belongs to the phosphate regulon . Besides, the phoM phoR mutation leads to the loss of protein of 55,000 Mr of the outer membranes, whereas phoB mutation causes loss of protein 37 000 Mr, identified as outer membrane protein OmpT . A damage in the phoB mutant of the protein proteolytic modification, probably determining the suppression of the biosynthesis of E . coli envelope secreted proteins is suggested.

J Antimicrob Chemother, 1986 Sep, 18(3), 375 - 80
In-vitro uptake of gentamicin and tobramycin by rat renal tubules in the presence or absence of Escherichia coli endotoxin; Bergeron MG et al.; Renal tubules of rats were incubated with aminoglycoside (gentamicin or 3H-tobramycin, 10 mg/l) in the presence or absence of Escherichia coli endotoxin (10 mg/l) . The kinetics of aminoglycoside uptake by the tubule were only slightly affected by endotoxin . The percentage of serum in the medium affected the tobramycin uptake . This uptake decreased from a mean ratio of concentration in the tubules/concentration in the medium (T/M) of 1.63 in 5% serum to a mean T/M of 0.86 in 10% serum (P less than 0.01).

Farmakol Toksikol, 1986 Sep-Oct, 49(5), 105 - 7
{Pharmacologic correction of the cellular immunity disorders in infants in the 1st 3 months of life with escherichiosis}; Vasiunin AV et al.; Seventy-five children in the first three months of life with escherichiosis were studied . It was found that levamisole to a lesser degree and methyluracyl decrease total duration of the disease by shortening periods of disorders of the gastrointestinal tract functions and bacterial excretions; the drugs improved indices of the condition of the cellular link of the immunity system.

Curr Eye Res, 1986 Sep, 5(9), 629 - 34
Time course for prostaglandin synthesis by rabbit lens during endotoxin-induced ocular inflammation; Fleisher LN et al.; Three hours to 14 days following the intravitreal injection of 10 ng of E . coli endotoxin into the vitreal chamber of one eye of the New Zealand white rabbit, ocular inflammation was evaluated by clinical and biochemical criteria and prostaglandins were measured in the intraocular fluids and in the incubation medium of the intact lens . Increased synthesis of PGE2 was detected for lenses from inflamed eyes beginning at 18 h post-endotoxin injection . Lenticular PGE2 synthesis remained above control levels for the duration of the time course . Lenses also exhibited increased PGF2 alpha synthesis, which began at 18 h and returned to control levels by day 7 . At the times of peak production, aqueous humor PGE2 concentration correlated with lenticular PGE2 synthesis and with aqueous humor leukocyte number . No correlations were found for lenticular PGE2 vs . cell number, or vitreous humor PGE2 vs . aqueous humor PGE2 . These results suggest that during ocular inflammation, aqueous humor PGE2 may be derived, at least in part, from the lens and leukocytes.

Appl Environ Microbiol, 1986 Sep, 52(3), 479 - 83
Use of geostatistics to predict virus decay rates for determination of septic tank setback distances; Yates MV et al.; Water samples were collected from 71 public drinking-water supply wells in the Tucson, Ariz., basin . Virus decay rates in the water samples were determined with MS-2 coliphage as a model virus . The correlations between the virus decay rates and the sample locations were shown by fitting a spherical model to the experimental semivariogram . Kriging, a geostatistical technique, was used to calculate virus decay rates at unsampled locations by using the known values at nearby wells . Based on the regional characteristics of groundwater flow and the kriged estimates of virus decay rates, a contour map of the area was constructed . The map shows the variation in separation distances that would have to be maintained between wells and sources of contamination to afford similar degrees of protection from viral contamination of the drinking water in wells throughout the basin.

Am J Pathol, 1986 Sep, 124(3), 367 - 72
The role of interleukin-1 in neutrophil leukocyte emigration induced by endotoxin; Cybulsky MI et al.; Chemotactic factors induce neutrophil emigration into tissues . Interleukin-1 (IL-1) was found to be several log times more potent in this respect than C5a des Arg, leukotriene B4, and f-Met-Leu-Phe and of comparable potency to endotoxin . Kinetic studies revealed a rapid and transient neutrophil influx, with the peak rate at 30-90 minutes . Cross tachyphylaxis was observed between IL-1 and endotoxin; and this, together with its high potency and rapid onset of action, suggest that IL-1 mediates endotoxin-induced neutrophil emigration.

Mol Gen Genet, 1986 Sep, 204(3), 463 - 8
Transient suppression of F-plasmid incompatibility in a strain of Escherichia coli; Warrier R et al.; The incompatibility between F plasmids is transiently suppressed in Escherichia coli strain CSH54 . As a result this strain is able to maintain two F' factors or an F' factor and a mini-F plasmid for considerably longer periods than normal strains . When selective pressure for two markers carried by two separate F's (or an F' and mini-F) is imposed on normal strains, the two plasmids tend to form a cointegrate structure which can be detected genetically by the joint transfer of both the markers upon mating . This does not happen in CSH54; instead, the two plasmids are maintained and transferred independently . Physical evidence for the maintenance of an F' and a mini-F plasmid is provided by agarose gel electrophoresis.

J Clin Microbiol, 1986 Sep, 24(3), 368 - 71
Comparison of beta-glucuronidase-based substrate systems for identification of Escherichia coli; Edberg SC et al.; Methods based on the measurement of beta-glucuronidase have been shown to be specific and inexpensive for the identification of Escherichia coli from bacterial colonies within 1 h . Recently, commercial systems incorporating beta-glucuronidase substrates were introduced . Rapid Identification Method E . coli (Austin Biological Laboratories, Curtin Matheson Scientific, Inc., Houston, Tex.) and Rapid Detect E . coli (Organon Teknika, Morris Plains, N.J.) are single-tube test combinations to simultaneously measure beta-glucuronidase (fluorescence at 366 nm), o-nitrophenyl-beta-D-galactopyranoside (yellow), and indole (red) . To determine the accuracy and utility of these two systems, we used them to test 169 E . coli and 150 non-E . coli and compared them with conventional substrate tests . The Rapid Detect test was more efficient than the Rapid Identification Method in demonstrating beta-glucuronidase activity, but the commercial systems were equal to each other and to the conventional tests for o-nitrophenyl-beta-D-galactopyranoside and indole . There were no false reactions by either system.

Eur J Biochem, 1986 Sep 1, 159(2), 227 - 37
Characterization of the phosphoproteins of Escherichia coli cells by electrophoretic analysis; Cortay JC et al.; The phosphorylated proteins of Escherichia coli, radioactively labeled with {32P}orthophosphate, have been analyzed by the O'Farrell gel technique and autoradiography . The effects of various culture conditions on the pattern of protein phosphorylation have been studied, including growth on different carbon sources in either exponential or stationary phase, treatment of cells with ethanol, heat shock and amino acid starvation . A total number of 128 different phosphoproteins, labeled to a varying extent, have been detected and each of them has been characterized by both its molecular mass and isoelectric point . These proteins are located mainly in the cytosolic fraction of cells, none of them being present within either ribosomes or nucleoids, and only three being associated with membranes . Analysis of their phosphoamino acid content has shown that they are phosphorylated mostly at serine residues and, less frequently, at threonine and tyrosine residues.

Arch Biochem Biophys, 1986 Sep, 249(2), 579 - 87
Conformational properties of membrane-bound fumarate reductase of Escherichia coli; Fronticelli C et al.; Anaerobically grown cells of Escherichia coli harboring the plasmid pFRD63 over-produce fumarate reductase, a membrane-bound complex localized in the inner membrane of the cell, where this enzyme represents at least 90% of the total membrane proteins (B . D . Lemire, J . J . Robinson, and J . H . Weiner (1982) J . Bacteriol . 152, 1126-1131) . Preparations of inner membrane fractions suspended in 40% sucrose are optically clear, allowing optical spectroscopic measurements . Circular dichroism spectra showed that between pH 6 and 11 the secondary structure of the enzyme is at least 55% in alpha helix and that above pH 11 the structure abruptly changes to a beta-like conformation . The same phenomenon is observed in samples solubilized in the nonionic detergent C12E9 . Absorption spectra of the enzyme either membrane bound or solubilized in detergents or exposed to alkaline pH showed that the accessibility of the active site to solvent components is modulated by the interaction of the protein with the membrane . Solubilization of the membrane-bound enzyme with 1% Triton X-100 or C12E9 produced a decrease in ellipticity and in enzymatic activity.

Am Rev Respir Dis, 1986 Sep, 134(3), 471 - 5
Effect of varying the time interval between intratracheal administration of eglin-c and human neutrophil elastase on prevention of emphysema and secretory cell metaplasia in hamsters . With observations on the fate of eglin-c and the effect of repeated instillations; Lucey EC et al.; Eglin-c is a naturally occurring polypeptide of 70 amino acids with a molecular mass of 8,100 daltons . It is a strong inhibitor of human neutrophil elastase (HNE) and cathepsin-G, and, when given intratracheally to hamsters 1 h before human neutrophil elastase, it can prevent or ameliorate the emphysema produced by HNE . The present experiments were designed to determine the duration of the effectiveness of eglin-c, prepared by DNA technology from Escherichia coli, in preventing the emphysema and secretory cell metaplasia induced by HNE . Eglin-c (2,000 micrograms in 0.5 ml saline) was effective in ameliorating emphysema, as determined histologically and physiologically, when it was given intratracheally to hamsters 1, 2, 4, and 8 h before the intratracheal instillation of 300 micrograms of HNE . Eglin-c ameliorated bronchial secretory cell metaplasia when given 1 h before HNE but not when the time intervals were 2 h or longer . The clearance of {3H}eglin-c from the lungs was assessed . Four h after intratracheal instillation of 446 micrograms of {3H}eglin-c, 33% of the tritium was found in the lung tissue and bronchoalveolar lavage fluid; 83% of the radioactivity in the lavage fluid supernatant was associated with functionally active eglin-c . No evidence of bronchopulmonary toxicity was seen in hamsters given 4 intratracheal instillations of 2,000 micrograms of eglin-c at 1-wk intervals.

Am J Physiol, 1986 Sep, 251(3 Pt 2), R591 - 7
Enhancement of slow-wave sleep by endotoxin and lipid A; Krueger JM et al.; Some muramyl peptides derived from bacterial peptidoglycan enhance slow-wave sleep (SWS) . The purpose of this study was to test whether another cell wall component, lipopolysaccharide (LPS), and its lipid A moiety also have an effect on sleep . When injected intravenously, both LPS and lipid A enhanced the duration of SWS, increased electroencephalogram delta-wave amplitudes, suppressed rapid eye movement (REM) sleep, and induced biphasic fevers . The effects of intravenously administered lipid A and LPS on SWS were present primarily during the first 3 h postinjection . Intraventricular lipid A administration enhanced SWS, did not suppress REM, and induced a monophasic fever; the SWS effect had a 3-h latency, whereas temperature started to rise during the second hour . Regardless of the route of administration, within the dose range used here, sleep was normal by the following criteria: sleep was episodic, animals could be easily aroused, and brain temperature, although elevated to "febrile" levels, continued to fluctuate during sleep-state transitions indistinguishably from control conditions . We conclude that LPS and lipid A are capable of modulating sleep.

Proc Natl Acad Sci U S A, 1986 Sep, 83(18), 6959 - 63
Heat shock response in Escherichia coli influences cell division; Tsuchido T et al.; Analysis of a mutant in fam, a pleiotropic gene affecting cell division in Escherichia coli, revealed that this gene is probably identical to the heat shock regulatory gene htpR . The fam-715 mutant and different htpR mutants were found to share the following three characteristics: temperature-sensitive growth, faulty cell division, and inability to induce the normal cellular heat shock response . These defects were all corrected in fam and htpR mutants by complementation with plasmids carrying intact htpR+ or by recombination between these mutant alleles and a plasmid carrying only a portion of htpR . These results implicate the E . coli heat shock system in the regulation of cell division and raise the question of a similar role in other organisms.

Proc Natl Acad Sci U S A, 1986 Sep, 83(18), 6945 - 9
The C-C (6-4) UV photoproduct is mutagenic in Escherichia coli; Glickman BW et al.; Mutation induced by ultraviolet light is predominantly targeted by UV photoproducts . Two primary candidates for the premutagenic lesion are the cyclobutane pyrimidine dimer and the less frequent (by a factor of 10) pyrimidine-pyrimidone (6-4) photoproduct . Methylation of the 3'-cytosine in the sequence 5' CCAGG 3' reduces the yield of (6-4) lesions, but not of cyclobutane dimers, at these sites . By taking advantage of mutants deficient in cytosine methylation, we show here that at the three sites in the lacI gene of Escherichia coli having this sequence, the specific increase in the formation of the (6-4) photoproducts is accompanied by a concomitant increase in mutation . At each site, a G X C to A X T transition results in an amber mutation . In the unmethylated state, these sites become among the most frequent nonsense mutations recovered . We conclude that the (6-4) photoproduct constitutes a major premutagenic lesion in E . coli.

Proc Natl Acad Sci U S A, 1986 Sep, 83(17), 6548 - 52
Construction of two Escherichia coli amber suppressor genes: tRNAPheCUA and tRNACysCUA; Normanly J et al.; Amber suppressor genes corresponding to Escherichia coli tRNAPhe and tRNACys have been constructed for use in amino acid substitution studies as well as protein engineering . The genes for either tRNAPheGAA or tRNACysGCA both with the anticodon 5' CTA 3' were assembled from four to six oligonucleotides, which were annealed and ligated into a vector . The suppressor genes are expressed constitutively from a synthetic promoter, derived from the promoter sequence of the E . coli lipoprotein gene . The tRNAPhe suppressor (tRNAPheCUA) is 54-100% efficient in vivo, while the tRNACys suppressor (tRNACysCUA) is 17-50% efficient . To verify that the suppressors insert the predicted amino acids, both genes were used to suppress an amber mutation in a protein coding sequence . NH2-terminal sequence analysis of the resultant proteins revealed that tRNAPheCUA and tRNACysCUA insert phenylalanine and cysteine, respectively . To demonstrate the potential of these suppressors, tRNAPheCUA and tRNACysCUA have been used to effect amino acid substitutions at specific sites in the E . coli lac repressor.

Proc Natl Acad Sci U S A, 1986 Sep, 83(17), 6297 - 301
Regulatory mechanisms for induction of synthesis of repair enzymes in response to alkylating agents: ada protein acts as a transcriptional regulator; Nakabeppu Y et al.; Expression of the ada and alkA genes, both of which are involved in the adaptive response of Escherichia coli to alkylating agents, is positively controlled by Ada protein, the product of the ada gene . Large amounts of ada- and alkA-specific RNA were formed in cells treated with a methylating agent, whereas little such RNA was produced in untreated cells . The in vivo transcription-initiation sites for the two genes were determined by primer-extension cDNA synthesis . In an in vitro reconstituted system, both ada and alkA transcripts were formed in an Ada protein-dependent manner . However, responses of the two transcription reactions to methylating agents differed; ada transcription was stimulated by methylnitrosourea, while alkA transcription was suppressed . We prepared a methylated form of Ada protein by an in vitro reaction and compared the activity with that of the normal, unmethylated form . The methylated form was more effective in promoting ada transcription than was the unmethylated form, but the effects of both forms were much the same with regard to alkA transcription . Based on these findings, we propose a model for the molecular mechanism of adaptive response.

Mutat Res, 1986 Sep, 175(1), 17 - 21
SOS induction of the gene sulA is partly inhibited in Escherichia coli K12 cells overproducing the RecA protein; Koukalova B et al.; A study was made of the SOS induction of the gene sulA of Escherichia coli K12 in relation to the gene dosage of the gene recA . In experiments the sulA::lacZ fusion strain PQ37 and derivatives of PQ37 with the multi-copy plasmids pDR1453 or pBR322 were used . The SOS response was induced with nitrofurantoin, SOS induction of the gene sulA was determined on the basis of the amount of beta-galactosidase synthesized, i.e . by the SOS chromotest (Quillardet et al., 1982a) . It was found in this work that cells with the plasmid pDR1453, which contain the gene recA of E . coli K12 (Sancar and Rupp, 1979), have a decreased SOS induction of the gene sulA . Cells with the plasmid pBR322 do not exhibit this decrease . Inactivation of the gene recA in the plasmid pDR1453 with preservation of the functional gene recA in the chromosome leads to a restoration of 'standard' SOS induction of the gene sulA . The results show that the amount of the gene product of the gene recA affects the SOS induction of the gene sulA.

Mutat Res, 1986 Sep, 162(2), 187 - 99
Nickel(II) genotoxicity: potentiation of mutagenesis of simple alkylating agents; Dubins JS et al.; Many metals have been shown to alter the function of a wide range of enzyme systems, including those involved in DNA repair and replication . To assess the impact in vivo of such metal actions a "Microtitre" fluctuation assay was used to examine the ability of Ni(II) to act as a comutagen with simple alkylating agents . In E . coli, Ni(II) chloride potentiated the mutagenicity of methyl methanesulfonate (MMS) in polymerase-proficient strains (WP2+ and WP2-), but not in polA- strains (WP6 and WP67) or in lexA- (CM561) or recA- (CM571) strains . The absence of UV excision repair (WP2- and WP67) had little, if any, effect . An extended lag phase was seen at 2-4 h in the polA- strains following treatment with Ni(II) chloride and MMS, but normal growth resumed thereafter . Results suggested that mutations induced by MMS were fixed during log phase growth and that more than 2 h of exposure were necessary for potentiation by Ni(II) to be observed . Thus, the extended lag phase probably cannot explain the lack of potentiation . RecA-dependence of the comutagenic effect was corroborated with S . typhimurium TA1535 and TA100 . Only in the pKM101 containing strain, TA100, was potentiation of ethyl methanesulfonate (EMS) and MMS by Ni(II) chloride evident . The mucAB genes carried on pKM101 increase the sensitivity of TA100 to a variety of mutagens, providing there is a functional recA gene product . Taken together, the data suggest that Ni(II) acts indirectly, as a comutagen, in bacterial systems, possibly affecting processes involving recA- and/or polA-dependent function(s).

J Exp Med, 1986 Sep 1, 164(3), 932 - 7
Cytotoxic T lymphocytes against disease-associated determinant(s) in ankylosing spondylitis; Geczy AF et al.; Cytotoxic T lymphocytes, induced by stimulating the PBMC of an HLA-B27+ normal individual (B27+, AS-) with the PBMC of an HLA-identical sibling suffering from ankylosing spondylitis (AS) (B27+, AS+), specifically lyse B27+, AS+ PBMC but not PBMC from HLA-27+ or B27-, AS- normal controls, or from HLA-B27- AS patients (B27-,AS+) . CTL of similar specificity can also be raised by immunizing in vitro B27+,AS- cells with autologous cells modified by cross-reactive bacterial antigens . These results suggest that CTL can recognize certain bacterial antigens in association with HLA-B27 and that this interaction may lead to an inflammatory episode during the initial stages of the disease.

J Cell Physiol, 1986 Sep, 128(3), 421 - 31
In vitro actions on hemopoietic cells of recombinant murine GM-CSF purified after production in Escherichia coli: comparison with purified native GM-CSF; Metcalf D et al.; Recombinant murine GM-CSF produced in Escherichia coli was purified to homogeneity and tested in parallel with purified native GM-CSF . Both recombinant and native GM-CSF stimulated granulocyte and/or macrophage colony formation by adult and fetal mouse progenitor cells, and with adult marrow cells the specific activity of the recombinant GM-CSF (25 X 10(8) U/mg) was similar to that of the native form (15 X 10(8) U/mg) . At high concentrations (greater than 200 U/ml), both forms of GM-CSF also stimulated eosinophil colony formation by adult marrow cells and, at very high concentrations (greater than 800 U/ml), megakaryocyte and some erythroid and mixed-erythroid colony formation . Recombinant GM-CSF was as effective in stimulating the proliferation of the GM-CSF-dependent cell line FD as the native molecule . Both recombinant and native GM-CSF were able to induce partial differentiation in colonies of WEHI-3B myeloid leukemic cells . Recombinant GM-CSF competed effectively for the binding of 125I-labeled native GM-CSF to hemopoietic cells, and antiserum to recombinant GM-CSF also neutralized the biological activity of native GM-CSF . The bacterially synthesized GM-CSF was a slightly more effective stimulus for megakaryocyte colony formation than the native molecule . The demonstration that purified bacterially synthesized GM-CSF is biologically active in vitro now permits studies to be undertaken on the in vivo effects of this material.

J Bacteriol, 1986 Sep, 167(3), 935 - 9
Electron probe analysis, X-ray mapping, and electron energy-loss spectroscopy of calcium, magnesium, and monovalent ions in log-phase and in dividing Escherichia coli B cells; Chang CF et al.; The elemental composition of individual cells of rapidly frozen and cryosectioned Escherichia coli B was measured with electron optical microanalytic methods . The Ca content was high (26.2 mmol/kg) in a 10-nm-wide region of the cell envelope . Amounts of cytoplasmic Ca in actively dividing cells were significantly higher (32.6 mmol/kg {dry weight}) than in the log-phase (1.5 mmol/kg) cells . Cellular Mg was 205 mmol/kg (dry weight) and it was uniformly distributed throughout the cell . Cells washed in distilled water before freezing lost monovalent ions (Na, Cl, and K), but the membrane-bound Ca and cellular Mg were not reduced, indicating that cellular Mg and membrane Ca are more tightly bound.

J Bacteriol, 1986 Sep, 167(3), 928 - 34
Nucleotide sequence of the btuCED genes involved in vitamin B12 transport in Escherichia coli and homology with components of periplasmic-binding-protein-dependent transport systems; Friedrich MJ et al.; The products of the btuCED region of the Escherichia coli chromosome participate in the transport of vitamin B12 across the cytoplasmic membrane . The nucleotide sequence of the 3,410-base-pair HindIII-HincII DNA fragment carrying a portion of the himA gene and the entire btuCED region was determined . Comparison of the location of the open reading frames with the gene boundaries defined by transposon insertions allowed the assignment of polypeptide products to gene sequences . The btuC product is a highly nonpolar integral membrane protein of molecular weight 31,683 . The distribution of hydrophobic regions suggests the presence of numerous membrane-spanning domains . The btuD product is a relatively polar but membrane-associated polypeptide of Mr 27,088 and contains segments bearing extensive homology to the ATP-binding peripheral membrane constituents of periplasmic binding protein-dependent transport systems . Other regions of this protein are similar to portions of the outer membrane vitamin B12 receptor . The btuE product (Mr 20,474) appears to have a periplasmic location . It has the mean hydropathy of a soluble protein but lacks an obvious signal sequence . The cellular locations and structural and sequence homologies of the Btu polypeptides point to the similarity of these three proteins to components of binding protein-dependent transport systems . However, the dependence on a periplasmic vitamin B12-binding protein has not yet been demonstrated.

J Bacteriol, 1986 Sep, 167(3), 920 - 7
Identification of the btuCED polypeptides and evidence for their role in vitamin B12 transport in Escherichia coli; de Veaux LC et al.; Passage of vitamin B12 across the outer and cytoplasmic membranes of Escherichia coli occurs in two steps, each involving independent transport systems . Since the vitamin accumulated in btuC or btuD mutants is readily released from the cell by chase or osmotic shock and does not undergo the usual metabolic conversions, the products of these genes might participate in transport across the cytoplasmic membrane . Mutations in btuC and btuD are complemented by recombinant plasmids carrying a 3,410-base-pair HindIII-HincII DNA fragment . Transposon Tn1000 mutagenesis and subcloning defined the location of these two genes and showed that they are separated by approximately 800 base pairs . The polypeptides elicited by this fragment and its derivatives were identified by using a maxicell system . The apparent molecular weight of the btuC product was approximately 26,000, that of the btuD product was 29,000 . Both polypeptides were associated with the cell membrane . Transposon insertions in the region between btuC and btuD, as well as those in the two genes, conferred a deficiency in vitamin B12 utilization and transport when they were crossed onto the chromosome . This region, termed btuE, encoded a 22,000-Mr polypeptide and lesser amounts of a 20,000-Mr species . A portion of the BtuE protein was released from maxicells by osmotic shock or spheroplast formation . The relative production of BtuE and BtuD in response to plasmids carrying transposon insertions suggested that the three genes are arranged in an operon in the order btuC-btuE-btuD and that internal promoters exist since polarity was incomplete . Substantial elevation of transport activity was engendered by plasmids carrying the intact btu region, but not when any of the btu genes was disrupted . The btuCED region thus may encode a transport system for passage of vitamin B12 across the cytoplasmic membrane . This system bears similarities to periplasmic binding protein-dependent transport systems, although the putative periplasmic component is not required for its function.

J Bacteriol, 1986 Sep, 167(3), 850 - 4
Immediate entrance to the export pathway after synthesis as a requirement for export of the sak gene product in Escherichia coli; Sako T; Export through the cytoplasmic membrane and processing of the sak product in Escherichia coli cells were investigated with E . coli strains carrying pTS301, which produce large amounts of staphylokinase at 42 degrees C . High-level synthesis of the sak product caused transient accumulation not only of the staphylokinase precursor (pSAK) but also of the maltose-binding protein and outer membrane protein A precursors . Thus it was concluded that the sak product shares the export pathway with E . coli secreted proteins at least at a certain step . During high-level synthesis of the sak product, a significant amount of the newly synthesized pSAK remained unprocessed after a chase period, possibly causing the observed accumulation of pSAK . Accumulating pSAK did not mature for a long period, whereas the newly synthesized sak product was exclusively detected in the mature form . These results suggest that it is necessary for the sak product to enter the export pathway during or immediately after synthesis to be exported and processed normally.

J Bacteriol, 1986 Sep, 167(3), 809 - 17
Further evidence for overlapping transcriptional units in an Escherichia coli cell envelope-cell division gene cluster: DNA sequence and transcriptional organization of the ddl ftsQ region; Robinson AC et al.; A 1.2-kilobase-pair BamHI fragment from a cell envelope-cell division gene cluster of Escherichia coli containing ddl and part of ftsQ was cloned and sequenced, and the sequence was interpreted with the aid of genetic complementation and promoter fusion data for the region . Both ddl and ftsQ were transcribed in the same direction (clockwise on the genetic map) . ddl was shown to be capable of independent expression from a promoter of its own, and a promoter was identified within the ddl structural gene . The structural gene of ddl consisted of 918 nucleotides, encoding a 306-residue polypeptide of molecular weight 32,840; the synthesis of a protein of this molecular weight was shown to be directed from the 1.2-kilobase-pair BamHI fragment in minicells . Analysis of the DNA sequence further showed that the termination codon of ddl is separated from the initiation codon of ftsQ by one base, which suggests that these two genes may be translationally coupled when transcription is initiated upstream of ddl . This represents a second instance of potential translational coupling within this gene cluster and also indicates that the ddl and ftsQ transcriptional units must overlap (as has been reported earlier for ftsQ and ftsA and for ftsA and ftsZ).

J Bacteriol, 1986 Sep, 167(3), 1077 - 80
Induction of autolysis in nongrowing Escherichia coli; Tuomanen E et al.; Unless relaxation of the stringent response is achieved, all nongrowing bacteria rapidly develop resistance to autolysis induced by a variety of agents, including all classes of cell wall synthesis inhibitors . We now describe inhibitors of cell wall synthesis which were unusual in that they could continue to effectively induce autolysis in relA+ Escherichia coli even after prolonged amino acid starvation . The process of cell wall degradation seems to be catalyzed by similar hydrolytic enzymes in nongrowing and growing cells, yet the activity of these new agents capable of inducing autolysis in the nongrowing relA+ cells did not involve relaxation of RNA or peptidoglycan synthesis . We propose that the suppression of autolysis characteristic of nongrowing cells can be bypassed by a novel mechanism of autolytic triggering which is independent of the relA locus.

J Bacteriol, 1986 Sep, 167(3), 1055 - 7
ATP hydrolysis during SOS induction in Escherichia coli; Barbe J et al.; Changes in cellular ATP concentration during SOS induction in strains of Escherichia coli with different levels of RecA and LexA proteins were studied . UV irradiation of RecA+ strains induced a twofold increase in the ATP concentration around the first 20 min, followed by a decrease to the values of nonirradiated cells . On the other hand, mutants defective in RecA protein or with either deficient RecA protease activity or cleavage-resistant LexA repressor did not show any decrease, suggesting that ATP consumption is related to LexA repressor hydrolysis . Furthermore, strains presenting a constitutive synthesis of RecA protein showed the same changes in ATP concentration as the wild-type strain . Likewise, the presence in a RecA+ strain of a LexA(Def) protein, which is defective in its capacity for binding specifically to SOS operators, did not disturb the changes in ATP when compared with the LexA+ RecA+ strain . Moreover, after UV irradiation, a LexA(Def) RecA- double mutant showed an important increase in ATP concentration, which remained elevated for at least 120 min after UV treatment.

J Bacteriol, 1986 Sep, 167(3), 1048 - 54
Molecular cloning, expression, and characterization of the gene for the surface (HPI)-layer protein of Deinococcus radiodurans in Escherichia coli; Peters J et al.; The HPI protein of Deinococcus radiodurans belongs to the class of surface layer proteins which form crystalline two-dimensional arrays on bacterial cell envelopes . We have cloned and expressed the gene for this protein of Mr about 100,000 by using plasmid pUC8 in Escherichia coli . As judged by immunoreaction with monospecific antibodies, apparent Mr, and limited proteolysis, a single clone contained the gene encoding the complete polypeptide on an 8.9-kilobase (kb) insert . The insert was reduced to a 5.7-kb HindIII fragment, cloned in pUC18 in both orientations, and subjected to unilateral processive deletion with exonuclease III . The library of deletion derivatives was mapped and, in conjunction with protein immunoblotting of expressed polypeptides, was used to locate the positions of the structural gene and the Deinococcus promoter region that was responsible for expression of the HPI polypeptide . The HPI gene was confined to a stretch 2.95 kb in length.

Crit Care Med, 1986 Sep, 14(9), 802 - 6
Effects of Escherichia coli endotoxin on pulmonary vascular resistance in intact dogs; D'Orio V et al.; The effects of endotoxin on pulmonary hemodynamics were studied in seven intact dogs . The distribution of pulmonary vascular resistance was estimated by the effective pulmonary capillary pressure, which was derived from the pressure transient recorded while the pulmonary artery catheter was rapidly wedged . After the injection of endotoxin, cardiac output and aortic pressure consistently fell . Pulmonary artery occlusion (wedge) pressure also decreased, but not significantly . Although pulmonary artery pressure did not necessarily rise, total pulmonary vascular resistance increased in every dog . The absolute increase in pulmonary artery resistance was greater (142 mm Hg/L X min/kg); than in venous resistance (111 mm Hg/L X min/kg); however, the relative increase in venous resistance was higher (410% for venous resistance vs . 220% for pulmonary artery resistance) . As a result of venoconstriction, there was a consistent increase in effective pulmonary capillary pressure (from 2.5 to 6.3 mm Hg) . Our data indicate that the pulmonary vascular response to endotoxin injection is characterized by constriction of both pulmonary arteries and pulmonary veins . The capillary wedge pressure did not reflect the pulmonary microvascular pressure, since it varied in the opposite direction to the effective capillary pressure.

Eur J Immunol, 1986 Sep, 16(9), 1099 - 103
Structural requirements for inducing in vitro B lymphocyte activation by chemically synthesized derivatives related to the nonreducing D-glucosamine subunit of lipid A; Kumazawa Y et al.; Mitogenic and polyclonal B cell activation (PBA) activities of 16 synthetic compounds related to the nonreducing D-glucosamine (GlcN-II) subunit of lipid A were investigated . Among compounds possessing the GlcN backbone, a 4-O-phosphorylated GlcN derivative carrying N-linked 3-tetradecanoyloxytetradecanoyl {C14-O-(C14)} and 3-O-linked tetradecanoyl (C14) groups, GLA-27, expressed the highest degree of both activities . Omission of the 3-O-linked C14 group from GLA-27 and transfer of the C14 group to the C-6 position induced critical changes in expression of activities . Both 4-O-phosphorylated compounds carrying an N-linked C14 or 3-hydroxytetradecanoyl (C14OH) group instead of the C14-O-(C14) group in GLA-27 showed no detectable activity . Substituting a 3-O-linked C14 group in GLA-27 for the C14-O-(C14) group also markedly decreased mitogenic and PBA activities . Change of phosphorylation site from the C-4 to the C-6 position and bisphosphorylation at the C-4 and C-6 positions induced somewhat weak depression . Much weaker activities were observed in a compound carrying N-linked 3-dodecanoyloxydodecanoyl {C12-O-(C12)} and 3-O-dodecanoyl (C12) as fatty acid substituents . No detectable activity was seen in a compound carrying N-linked 3-hexadecanoyloxyhexadecanoyl {C16-O-(C16)} and 3-O-hexadecanoyl (C16), indicating that the most suitable carbon chain length for expressing the activities is C14 . Regarding structural change of the GlcN backbone, a 1-deoxy derivative of GLA-27 exhibited stronger activity than did GLA-27 itself . Mitogenic and PBA activity of GLA-27 were stronger than those of lipid X, which corresponds to the reducing D-GlcN (GlcN-I) subunit of Escherichia coli lipid A and is a 1-O-phosphorylated GlcN derivative carrying N- and 3-O-linked C14OH groups . These results indicate that N-linked acyloxyacyl and 3-O-linked acyl groups and phosphorylation are critical for expressing both mitogenic and PBA activities.

Br J Anaesth, 1986 Sep, 58(9), 1027 - 30
Effects of coronary bypass surgery under high-dose fentanyl anaesthesia on granulocyte chemiluminescence; Perttila J et al.; Phagocytosis provides one of the body's first-line defences against invading bacteria . The present study evaluated granulocyte microbicidal-related oxidative mechanisms by measurement of chemiluminescence responses in the phagocytosis of zymosan, S . aureus, E . coli and N-formyl-methionyl-leucylphenylalanine (FMLP) in 12 patients undergoing coronary bypass surgery under high-dose fentanyl anaesthesia . With preoperative values as a baseline, a significant depression of in vitro responses to zymosan was seen on days 1, 3-4 after operation and to S . aureus and E . coli on days 3-4 after operation, with recovery by days 6-7 . Responses to FMLP were unaffected.

Jpn J Antibiot, 1986 Sep, 39(9), 2519 - 24
{Pharmacokinetic and clinical studies of cefotiam during the perinatal period in pregnant women}; Ninomiya K et al.; Pharmacokinetic and clinical studies of cefotiam (CTM) were carried out in pregnant women . The results obtained are summarized below . The concentration of CTM in amniotic fluid increased gradually up to 14.7 micrograms/ml at 4.5 hours after administration and gradually declined thereafter . This amniotic fluid concentration was sufficiently higher than reported MIC90's of CTM against E . coli strains . Passages of CTM to embryo, fetus and fetal appendages were minimal . The passage of CTM to milk was minimal . The CTM was used in the treatment of 6 pregnant patients with pyelonephritis and unknown fever and 1 with puerperal pyelonephritis . Clinical responses were positive in 85.7% (6/7) . The CTM was used 7 patients with rupture of the membrane and 2 patients with operation for the purpose of prophylaxis and it was effective in 77.8% (7/9) . Neither noteworthy adverse reactions nor abnormal laboratory data in our patients or neonates were observed throughout the studies.

J Bacteriol, 1986 Sep, 167(3), 774 - 83
Organization and characterization of genes essential for symbiotic nitrogen fixation from Bradyrhizobium japonicum I110; Noti JD et al.; A total of 96 independent Tn5 insertions within a 39-kilobase-pair (kbp) segment of chromosomal DNA containing the three structural genes for nitrogenase (nifH, nifD, and nifK) from Bradyhizobium japonicum I110 were obtained in Escherichia coli and transferred to the wild-type strain by marker exchange . Individual transconjugants containing a Tn5 insertion were inoculated onto Glycine max cv . Wilkin (soybeans) and analyzed for their effect on symbiotic nitrogen fixation . In addition to the three structural genes, genes essential for nitrogen fixation (fix genes) were located in three separate regions: 9 kbp upstream of the nifDK operon; 1.5 kbp downstream of the nifDK operon; 4.5 kbp upstream of nifH . All of the fix::Tn5 insertion strains formed nodules which contained low or undetectable levels of nitrogenase activity . Bacteroids isolated from these nodules had approximately the same levels of the nifDK and nifH transcripts as those detectable from nodules formed by the wild-type strain . Western blot analysis of bacteroid proteins from nodules formed by the fix::Tn5 mutants or the wild-type strain showed the presence of similar levels of the nitrogenase protein subunits . The region upstream of nifH was characterized further by DNA sequence analysis and was shown to contain the nifB gene . The coding sequence of the nifB gene consisted of 1,494 nucleotides and was preceded by putative promoter (5' GTGG-10 base pairs {bp} TTGCA 3') and upstream activator (5' TGT-4 bp-T-5 bp-ACA 3') sequences.

Proteins, 1986 Sep, 1(1), 66 - 73
A domain of the Klenow fragment of Escherichia coli DNA polymerase I has polymerase but no exonuclease activity; Freemont PS et al.; The Klenow fragment of DNA polymerase I from Escherichia coli has two enzymatic activities: DNA polymerase and 3'-5' exonuclease . The crystal structure showed that the fragment is folded into two distinct domains . The smaller domain has a binding site for deoxynucleoside monophosphate and a divalent metal ion that is thought to identify the 3'-5' exonuclease active site . The larger C-terminal domain contains a deep cleft that is believed to bind duplex DNA . Several lines of evidence suggested that the large domain also contains the polymerase active site . To test this hypothesis, we have cloned the DNA coding for the large domain into an expression system and purified the protein product . We find that the C-terminal domain has polymerase activity (albeit at a lower specific activity than the native Klenow fragment) but no measurable 3'-5' exonuclease activity . These data are consistent with the hypothesis that each of the three enzymatic activities of DNA polymerase I from E . coli resides on a separate protein structural domain.

J Gen Microbiol, 1986 Sep, 132 ( Pt 9), 2643 - 6
Investigation of the regulation of the Escherichia coli btuB gene using operon fusions; Fletcher AJ et al.; Operon fusions were isolated between Mu dX (lac CmR ApR) and btuB, the gene encoding the multivalent vitamin B12 outer membrane receptor . Using these fusions, vitamin B12-mediated repression of btuB in Escherichia coli was demonstrated . Mutations in metH, metE and ompR as well as exogenous methionine, membrane pertubants, high osmolar conditions and temperature had no major effect on the expression of the btuB gene.

Mol Cell Biol, 1986 Sep, 6(9), 3291 - 4
rasH mutants deficient in GTP binding; Der CJ et al.; Single amino acid substitutions were introduced into a region of the rasH protein (residues 116, 117, and 119) homologous to a variety of diverse GTP-binding proteins . Each of the mutant p21 proteins displayed a significant reduction (10- to 5,000-fold) in GTP binding affinity . Activated rasH proteins deficient in GTP binding were unaltered in their ability to morphologically transform NIH 3T3 cells.

Biochimie, 1986 Sep, 68(9), 1071 - 8
Sequence similarities among the family of aminoacyl-tRNA synthetases; Hountondji C et al.; Recent affinity labeling studies have led to the identification of lysine residues at the CCA binding site of tRNA in Escherichia coli methionyl- and tyrosyl-tRNA synthetases . The comparison of the labeled peptides to the known primary structures of the aminoacyl-tRNA synthetases reveals new sequence similarities among this family of enzymes . These similarities include a 'constant' lysine residue whose functional significance is discussed . Moreover, a systematic computer analysis was conducted to search for similarities between the aminoacyl-tRNA synthetases taken as pairs.

Vet Med (Praha), 1986 Sep, 31(9), 565 - 75
{The complex etiology of epidemic diseases in calves on large-capacity farms and their clinical and epizootic characteristics}; Mensik J et al.; It is not easy to exactly diagnose the etiology of the mass infections of new-born calves on large farms where considerable losses are suffered . On the basis of the complex epizootological, clinical and laboratory examination in four large calf-rearing facilities, rotaviruses, coronaviruses, the infectious bovine rhinotracheitis (IBR) virus and the bovine viral diarrhoea (BVD) virus, and in some cases also the enteropathogenic E . coli, were found to be etiologically involved in the mass rise of diarrhoea, complicated by respiratory symptoms already during the first days after birth . The clinical picture of the disease, therapeutically difficult and reminding of "pneumoenteritis", has often been observed in stocks where, in addition to rotaviruses and coronaviruses in the faeces, the IBR or BVD viruses (sometimes both at the same time) were detected and identified in the respiratory and enteral tract . The serological examination of a higher number of animals in the stocks of calves under study confirmed the considerable rate of spreading of all the four viruses in the cattle population and, at the same time, demonstrated the very unfavourable immunological profile of the herds . The high percentage of animals low in antibody titres and the serologically negative animals constitute the infection-sensitive part of population in the affected herds . With the high culling rate and with the open herd turnover it is impossible to reach the required immunity through natural disinfection . Loss-free rearing of healthy calves will be achieved on the basis of a well-oriented vaccination programme with a good combination of inoculants.

Proc Natl Acad Sci U S A, 1986 Sep, 83(18), 6776 - 80
Cloning and sequence of a cDNA coding for the human beta-migrating endothelial-cell-type plasminogen activator inhibitor; Ny T et al.; A lambda gt11 expression library containing cDNA inserts prepared from human placental mRNA was screened immunologically using an antibody probe developed against the beta-migrating plasminogen activator inhibitor (beta-PAI) purified from cultured bovine aortic endothelial cells . Thirty-four positive clones were isolated after screening 7 X 10(5) phages . Three clones (lambda 1.2, lambda 3, and lambda 9.2) were randomly picked and further characterized . These contained inserts 1.9, 3.0, and 1.9 kilobases (kb) long, respectively . Escherichia coli lysogenic for lambda 9.2, but not for lambda gt11, produced a fusion protein of 180 kDa that was recognized by affinity-purified antibodies against the bovine aortic endothelial cell beta-PAI and had beta-PAI activity when analyzed by reverse fibrin autography . The largest cDNA insert was sequenced and shown to be 2944 base pairs (bp) long . It has a large 3' untranslated region {1788 bp, excluding the poly(A) tail} and contains the entire coding region of the mature protein but lacks the initiation codon and part of the signal peptide coding region at the 5' terminus . The two clones carrying the 1.9-kb cDNA inserts were partially sequenced and shown to be identical to the 3.0-kb cDNA except that they were truncated, lacking much of the 3' untranslated region . Blot hybridization analysis of electrophoretically fractionated RNA from the human fibrosarcoma cell line HT-1080 was performed using the 3.0-kb cDNA as hybridization probe . Two distinct transcripts, 2.2 and 3.0 kb, were detected, suggesting that the 1.9-kb cDNA may have been copied from the shorter RNA transcript . The amino acid sequence deduced from the cDNA was aligned with the NH2-terminal sequence of the human beta-PAI . Based on this alignment, the mature human beta-PAI is 379 amino acids long and contains an NH2-terminal valine . The deduced amino acid sequence has extensive (30%) homology with alpha 1-antitrypsin and antithrombin III, indicating that the beta-PAI is a member of the serine proteinase inhibitor (serpin) superfamily.

Proc Natl Acad Sci U S A, 1986 Sep, 83(17), 6357 - 61
p21 ras proteins and guanine nucleotides modulate the phosphorylation of 36- and 17-kilodalton mitochondria-associated proteins; Backer JM et al.; We have found that, when isolated rat liver mitochondria are incubated with {gamma-32P}ATP, there is phosphorylation of 36- and 17-kDa proteins . These proteins together with their protein kinase(s) are released as a complex by incubation of the isolated rat liver mitochondria at 20 degrees C for 30 min with 10 mM glucose 6-phosphate, 0.5 mM inositol phosphate, or 0.01 mM inositol triphosphate . Phosphorylation of the 36- and 17-kDa proteins in this soluble protein fraction is modulated by p21 proteins encoded by ras oncogenes and synthesized in Escherichia coli via recombinant DNA methods . A normal p21 ras protein stimulates phosphorylation of the 36-kDa protein and inhibits phosphorylation of the 17-kDa protein, whereas two transforming p21 ras proteins inhibit phosphorylation of both the 36- and 17-kDa proteins . Although GDP and 5'-guanylyl imidodiphosphate also influence the phosphorylation of these proteins, we present evidence that the effects of p21 ras protein are not simply due to their bound GDP . This novel system may be useful for further studies on the biochemical functions of the p21 ras proteins.

Clin Chem, 1986 Sep, 32(9), 1637 - 41
CEDIA, a new homogeneous immunoassay system; Henderson DR et al.; Genetic engineering of beta-galactosidase (EC 3.2.1.23) has led to the development of a new homogeneous assay system, CEDIA . The Z gene of the lac operon of Escherichia coli encodes a large enzymatically inactive polypeptide that spontaneously aggregates and folds to form active beta-galactosidase . Using recombinant DNA techniques, we have been able to engineer beta-galactosidase protein into a large polypeptide (an enzyme acceptor, EA) and a small polypeptide (an enzyme donor, ED) . The EAs and EDs are both enzymatically inactive, but spontaneously associate to form enzymatically active tetramers . In the assay, hapten or analyte is attached to an ED, and an analyte-specific antibody is used to inhibit the spontaneous assembly of active enzyme . Analyte in a patient's serum competes with the analyte in the analyte-ED conjugate for antibody, modulating the amount of beta-galactosidase formed . The signal generated by enzyme substrates is directly proportional to the analyte concentrations in the patient's serum . We describe quick (5-15 min) colorimetric tests for digoxin, requiring no serum pretreatments or predilutions and suitable for use with centrifugal and random-access analyzers.

Mol Cell Biol, 1986 Sep, 6(9), 3200 - 6
A consensus sequence polymer inhibits in vivo expression of heat shock genes; Xiao H et al.; Promoter function for hsp70 gene expression in Drosophila melanogaster was studied with an in vivo competition assay . A polymer of 40 tandem copies of the pair of regulatory elements of the hsp70 gene was constructed and cloned into a plasmid vector . Various marked genes were cotransfected with the polymer plasmid into Schneider line 2 cells, and their expression was determined by enzyme activity measurements . The polymer dramatically inhibited expression of cotransfected hsp70, hsp26, and hsp83 genes, but not cotransfected copia and histone genes . Our results indicate that in vivo, a trans-acting, positive regulatory factor, which can be titrated by heat shock consensus sequences, is required for activation of heat shock genes and is specific for these genes; the coordinate induction of different heat shock genes appears to be mediated by similar, but not identical, interactions of the trans-acting induction factor and the cis-acting heat shock consensus sequences; and the uninduced or basal level expression of the transfected hsp70 gene is also due to interaction of the consensus sequence with a positively acting factor.

Mol Cell Biol, 1986 Sep, 6(9), 3059 - 67
Introduction of UAG, UAA, and UGA nonsense mutations at a specific site in the Escherichia coli chloramphenicol acetyltransferase gene: use in measurement of amber, ochre, and opal suppression in mammalian cells; Capone JP et al.; We have used oligonucleotide-directed site-specific mutagenesis to convert serine codon 27 of the Escherichia coli chloramphenicol acetyltransferase (cat) gene to UAG, UAA, and UGA nonsense codons . The mutant cat genes, under transcriptional control of the Rous sarcoma virus long terminal repeat, were then introduced into mammalian cells by DNA transfection along with UAG, UAA, and UGA suppressor tRNA genes derived from a human serine tRNA . Assay for CAT enzymatic activity in extracts from such cells allowed us to detect and quantitate nonsense suppression in monkey CV-1 cells and mouse NIH3T3 cells . Using such an assay, we provide the first direct evidence that an opal suppressor tRNA gene is functional in mammalian cells . The pattern of suppression of the three cat nonsense mutations in bacteria suggests that the serine at position 27 of CAT can be replaced by a wide variety of amino acids without loss of enzymatic activity . Thus, these mutant cat genes should be generally useful for the quantitation of suppressor activity of suppressor tRNA genes introduced into cells and possibly for the detection of naturally occurring nonsense suppressors.

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