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