Biochemistry, 1988 Jan 12, 27(1), 206 - 12 Inhibition of phosphatase and sulfatase by transition-state analogues; Stankiewicz PJ et al.; The inhibition constants for vanadate, chromate, molybdate, and tungstate have been determined with Escherichia coli alkaline phosphatase, potato acid phosphatase, and Helix pomatia aryl sulfatase . Vanadate was a potent inhibitor of all three enzymes . Inhibition of both phosphatases followed the order WO4(2-) greater than MoO4(2-) greater than CrO4(2-) . The Ki values for potato acid phosphatase were about 3 orders of magnitude lower than those for alkaline phosphatase . Aryl sulfatase followed the reverse order of inhibition by group VI oxyanions . Phenol enhanced inhibition of alkaline phosphatase by vanadate and chromate but did not affect inhibition of acid phosphatase . Phenol enhanced inhibition of aryl sulfatase by metal oxyanions in all cases following the order H2VO4- greater than CrO4(2-) greater than MoO4(2-) greater than WO4(2-), and N-acetyltyrosine ethyl ester enhanced inhibition of aryl sulfatase by H2VO4- and CrO4(2-) more strongly than did phenol . It is apparent that the effectiveness of metal oxyanions as inhibitors of phosphatases and sulfatases can be selectively enhanced in the presence of other solutes . The relevance of these observations to the effects of transition metal oxyanions on protein phosphatases in vivo is discussed. Biochemistry, 1988 Jan 12, 27(1), 142 - 50 NMR sequential assignment of Escherichia coli thioredoxin utilizing random fractional deuteriation; LeMaster DM et al.; All non-proline residues except for the N-terminal dipeptide have been assigned in the 108-residue protein Escherichia coli thioredoxin . Central to these experiments has been the use of protein samples in which all carbon-bound hydrogen positions are substituted to 75% with deuterium by bacterial growth on partially deuteriated carbon sources and media . The dilution of the local proton density gives rise to narrower line widths with little loss in sensitivity . In addition, passive or secondary coupling to protons not directly involved in the coherence transfer process of correlation experiments is largely suppressed, thus significantly improving the resolution for side-chain couplings . Simultaneous multiresidue-type assignments have been obtained by incorporation of several amino acids with differing selective alpha- and/or beta-deuteriation into a fractionally deuteriated background . Combined with several single residue type labeling experiments, these selective labelings have yielded direct residue type assignments for two-thirds of the protein . In addition to improved resolution, the amide to carbon-bound proton NOESY spectra offered equivalent sensitivity while the amide to amide NOESY spectra offered superior sensitivity to that observed for natural abundance samples . The resultant sequential assignment has an average number of nearest-neighbor NOE connectivities of 2.35 out of the possible 3 alpha-amide, beta-amide, and amide-amide connectivities. Biochemistry, 1988 Jan 12, 27(1), 436 - 45 Preferential location of bulged guanosine internal to a G.C tract by 1H NMR; Woodson SA et al.; A series of double-helical oligodeoxyribonucleotides of sequence corresponding to a frame-shift mutational hot spot in the lambda CI gene, 5'-dGATGGGGCAG, are compared by proton magnetic resonance spectroscopy at 500 MHz of the exchangeable protons . Duplexes containing an extra guanine in a run of two, three, and four G.C base pairs are compared to regular helices of the same sequence and to another sequence containing an isolated bulged G, 5'-dGATGGGCAG.dCTGCGCCATC . The imino proton resonances are assigned by one-dimensional nuclear Overhauser effect spectroscopy . Resonances assigned to the G tract in bulge-containing duplexes are shifted anomalously upfield and are very broad . Imino proton lifetimes are determined by T1 inversion-recovery experiments . The exchange rates of G-tract imino protons in bulged duplexes are rapid compared to those in regular helices and are discussed in terms of the apparent rate of solvent exchange for the isolated G bulge . Delocalization of a bulged guanosine in homopolymeric sequences can explain the observed changes in chemical shift and relaxation times across the entire G.C run, and the chemical shifts can be fit by a simple model of fast exchange between base-paired and unpaired states for the imino protons . This allows us to calculate the relative occupancies of each bulge site . In these sequences, we find the extra base prefers positions internal to the G tract over those at the edge. Biochemistry, 1988 Jan 12, 27(1), 481 - 6 Expression and properties of the regulatory subunit of Dictyostelium cAMP-dependent protein kinase encoded by lambda gt11 cDNA clones; Mutzel R et al.; lambda gt11 phages harboring five different cDNA fragments for the regulatory (R) subunit of Dictyostelium discoideum cAMP-dependent protein kinase (CAK) directed the synthesis of this protein in Escherichia coli cells . Crude bacterial extracts were probed with an antiserum against the Dictyostelium R subunit . The presence of specific epitopes for the R subunit in a given extract was compared with high-affinity cAMP-binding activity and with the ability to inhibit the catalytic (C) subunit through protein-protein interaction . The expression and the biochemical properties of these proteins were correlated with their cDNA nucleotide sequence . The results show that the Dictyostelium R subunit can be functionally expressed in E . coli cells either as a fusion protein with beta-galactosidase or as a nonfusion protein . In both cases, the products of cDNA clones containing the entire coding sequence retained high-affinity cAMP-binding activity and the capacity to interact with the catalytic subunit . One of the fusions, lacking the 94 N-terminal residues, failed to inhibit catalytic activity, although it bound cAMP with an affinity similar to that of the native R protein from D . discoideum. Nucleic Acids Res, 1988 Jan 11, 16(1), 61 - 76 Promoters of Mycoplasma capricolum ribosomal RNA operons: identical activities but different regulation in homologous and heterologous cells; Gafny R et al.; The 5' region of the rRNA operon, rrnA, of M . capricolum was cloned . Sequence analysis revealed two tRNA genes, tRNA(leu) and tRNA(lys), upstream to the promoter of the rRNA operon . The in vivo transcription start sites of the rRNA operon and of the tRNA genes were mapped . The same promoters used by M . capricolum RNA polymerase are also recognized by E . coli RNA polymerase both in vivo and in vitro . We find that high levels of ppGpp in E . coli, resulting from amino acid starvation or from spoT mutation, activate rather than repress the transcription of the mycoplasma rrnA operon. Nucleic Acids Res, 1988 Jan 11, 16(1), 265 - 77 Isolation of cloned Moloney murine leukemia virus reverse transcriptase lacking ribonuclease H activity; Kotewicz ML et al.; Retroviral reverse transcriptase possesses DNA polymerase and ribonuclease H (RNase H) activity within a single polypeptide . Chemical or proteolytic treatment of reverse transcriptase has been used in the past to produce enzyme that is missing DNA polymerase activity and retains RNase H activity . It has not been possible to obtain reverse transcriptase that lacks RNase H but retains DNA polymerase activity . We have constructed a novel deletion derivative of the cloned Moloney murine leukemia virus (M-MLV) reverse transcriptase gene, expressed the gene in E . coli, and purified the protein to near homogeneity . The purified enzyme has a fully active DNA polymerase, but has no detectable RNase H activity . These results are consistent with, but do not prove, the conclusion that the DNA polymerase and RNase H activities of M-MLV reverse transcriptase reside within separate structural domains. J Biol Chem, 1988 Jan 5, 263(1), 527 - 34 Interaction of T7 RNA polymerase with DNA in an elongation complex arrested at a specific psoralen adduct site; Shi YB et al.; We have probed the interaction of T7 RNA polymerase with DNA in an elongation complex arrested by a site specifically placed psoralen diadduct or furanside monoadduct using DNase I footprinting techniques . The psoralen derivative, HMT (4'-hydroxy-methyl-4,5',8-trimethylpsoralen), was first placed at a specific site in the middle of a chemically synthesized double-stranded DNA fragment containing a T7 RNA polymerase promoter at one end . The psoralen molecule was photochemically attached either to 2 adjacent thymidine residues on opposite strands as a diadduct or to only 1 thymidine residue on the coding strand as a furan-side monoadduct . Using these psoralen-modified DNAs as templates for transcription, we found that T7 RNA polymerase was blocked at the psoralen adduct site and that the arrested elongation complex protected about 15 nucleotides upstream from the adduct on the coding strand and 20 nucleotides around the adduct on the noncoding strand from DNase I digestion . The two psoralen-modified DNA templates yielded identical RNA transcripts and DNase I footprints . In contrast, T7 polymerase protected only the coding strand from -20 to +8 in the initiation complex . These results suggest that the RNA polymerase undergoes a marked conformational change upon converting from an initiation complex to an elongation complex. J Biol Chem, 1988 Jan 5, 263(1), 404 - 8 Characterization of the internal signal-anchor domain of Escherichia coli leader peptidase; Dalbey RE et al.; Leader peptidase, an integral transmembrane protein of Escherichia coli, is synthesized without a cleavable amino-terminal leader peptide . Of the five domains that participate in the membrane assembly of this protein, one is an internal "signal" region . We have used oligonucleotide-directed mutagenesis to examine the properties of the internal signal that are crucial for leader peptidase assembly . For this purpose, the net charge at the amino terminus of the internal signal was changed from +2 to +1 and -1 and, at the carboxyl terminus of the signal, from 0 to -1 or +1 . These mutations had no effect on the membrane assembly of leader peptidase, suggesting that the charges have little role in the signal function . The apolar core of this signal was disrupted by substitution of basic amino acids for apolar residues . Substitution of an arginyl residue at position 70, or two arginyl residues at position 67 and 69, prevented membrane assembly . However, substitution of an arginyl residue at position 66 or either arginyl or lysyl residue at position 68 was without effect . Thus, while the apolar character of the internal signal is important, the precise position of a charged residue determines its effect on assembly. J Biol Chem, 1988 Jan 5, 263(1), 344 - 9 Alterations to the signal peptide of an outer membrane protein (OmpA) of Escherichia coli K-12 can promote either the cotranslational or the posttranslational mode of processing; Freudl R et al.; The signal sequence of the precursor of the Escherichia coli outer membrane protein OmpA was altered by oligonucleotide insertions into the corresponding gene . In one case, OmpA-S1, the hydrophobic core of the signal peptide, is reduced from 12 to 10 residues, and one positive charge is added near the NH2-terminus . In another case, OmpA-P1, the hydrophobic core is extended from 12 to 16 residues . The pro-OmpA protein is normally processed partially co- and partially posttranslationally . Processing of the pro-OmpA-S1 protein was entirely posttranslational and that of the pro-OmpA-P1 protein strictly cotranslational . Evidence is presented which strongly suggests that posttranslational processing reflects posttranslational translocation across the plasma membrane . The generation times of cells expressing pro-OmpA-P1 or pro-OmpA-S1 were identical and the pro-OmpA-S1 polypeptide could be chased into the mature protein in the absence of protein synthesis . Hence, it does not matter which mode of processing, or rather translocation, is used . The same oligonucleotides were inserted into the ompA gene of plasmid pRD87; a plasmid which leads to overproduction of the protein and to massive accumulation of both the mature protein and the precursor . In the OmpA signal sequence encoded by pRD87-P2 the hydrophobic core is extended from 12 to 20 residues . This peptide was also rapidly removed . Therefore, regardless of whether the hydrophobic core contains 12, 16, or 20 lipophilic residues, not only does the signal sequence always function correctly to mediate export, but in each case, the cleavage site is always accessible to the signal peptidase. J Biol Chem, 1988 Jan 5, 263(1), 314 - 20 Facilitated diffusion of p-nitrophenyl-alpha-D-maltohexaoside through the outer membrane of Escherichia coli . Characterization of LamB as a specific and saturable channel for maltooligosaccharides; Freundlieb S et al.; LamB, an outer membrane protein of Escherichia coli, is a component of the maltose-maltooligosaccharide transport system . We used p-nitrophenyl-alpha-D-maltohexaoside, a chromogenic analog of maltohexaose, and a periplasmic amylase that hydrolyzes this compound to study the LamB-mediated diffusion of p-nitrophenyl-alpha-D-maltohexaoside into the periplasm . Using this approach, we were able to characterize LamB in vivo as a saturable channel for maltooligosaccharides . Permeation through LamB follows Michaelis-Menten kinetics, with a Km of 0.13 mM and a Vmax of 3.3 nmol/min/10(9) cells . Previous studies suggested that maltose-binding protein increases the rate of maltooligosaccharide diffusion through LamB . We show here that, at least in strains that are unable to transport maltooligosaccharides into the cytoplasm, maltose-binding protein does not influence the rate of substrate diffusion . The periplasmic amylase had been previously described as being of the alpha-type . We have now purified this protein and analyzed its mode of action using chromogenic maltooligosaccharides of varying length . Analysis of the hydrolytic products revealed that the enzyme recognizes its substrate from the nonreducing that the enzyme recognizes its substrate from the nonreducing end and preferentially liberates maltohexaose, in contrast to the behavior of classical alpha-amylases that are endohydrolases . Using p-nitrophenyl-alpha-D-maltohexaoside as a substrate, we determined a Km of 3 microM and a Vmax of 0.14 mumol/min/mg of protein. J Biol Chem, 1988 Jan 5, 263(1), 243 - 6 Increased binding of operator DNA by trp superrepressor EK49; Klig LS et al.; The mechanism of superrepression by the mutant trp repressor EK49 was examined . This superrepressor has a glutamic acid-to-lysine change at residue 49 . The purified EK49 trp repressor was found to have a 10-fold higher affinity than wild type repressor for trp operator DNA . This increased affinity was shown to be due to a decrease in dissociation rate . The binding of trp operator DNA by EK49 trp repressor in the filter binding assay was more sensitive to high salt concentrations than binding by wild type repressor. J Biol Chem, 1988 Jan 5, 263(1), 200 - 9 Reversibility of strand invasion promoted by recA protein and its inhibition by Escherichia coli single-stranded DNA-binding protein or phage T4 gene 32 protein; Chow SA et al.; When recA protein promotes homologous pairing and strand exchange involving circular single strands and linear duplex DNA, the protein first polymerizes on the single-stranded DNA to form a nucleoprotein filament which then binds naked duplex DNA to form nucleoprotein networks, the existence of which is independent of homology, but requires the continued presence of recA protein (Tsang, S . S., Chow, S . A., and Radding, C . M . (1985) Biochemistry 24, 3226-3232) . Further experiments revealed that within a few minutes after the beginning of homologous pairing and strand exchange, these networks began to be replaced by a distinct set of networks with inverse properties: their formation depended upon homology, but they survived removal of recA protein by a variety of treatments . Formation of this second kind of network required that homology be present specifically at the end of the linear duplex molecule from which strand exchange begins . Escherichia coli single-stranded DNA-binding protein or phage T4 gene 32 protein largely suppressed the formation of this second population of networks by inactivating the newly formed heteroduplex DNA, which, however, could be reactivated when recA protein was dissociated by incubation at 0 degrees C . We interpret these observations as evidence of reinitiation of strand invasion when recA protein acts in the absence of auxiliary helix-destabilizing proteins . These observations indicate that the nature of the nucleoprotein products of strand exchange determines whether pairing and strand exchange are reversible or not, and they further suggest a new explanation for the way in which E . coli single-stranded DNA-binding protein and gene 32 protein accelerate the apparent forward rate of strand exchange promoted by recA protein, namely by suppressing initiation of the reverse reaction. J Biol Chem, 1988 Jan 5, 263(1), 472 - 9 Contributions of RNA secondary structure and length of the thymidine tract to transcription termination at the thr operon attenuator; Lynn SP et al.; The thr operon of Escherichia coli is regulated by an attenuation mechanism in which regulated transcription termination occurs in response to the levels of charged tRNAthr and tRNAile . Transcription of the thr operon regulatory region in vitro produces a 162-base transcript that is terminated efficiently at the attenuator . The attenuator sequence is similar to other rho-independent terminators . It contains a G + C region of dyad symmetry followed by a run of 9 A + T residues . We have characterized in detail the sequence requirements for efficient transcription termination in vitro . Using a set of point mutations in the G + C region of dyad symmetry of the thr attenuator, we have characterized the effects of these mutations on the efficiency of transcription termination . The efficiency was reduced in all of the mutants analyzed with the greatest effect being an approximate 20% decrease in termination . In some instances the electrophoretic mobilities of the terminated transcripts on 8% polyacrylamide, 8 M urea gels were shifted substantially relative to the wild type-terminated transcript, but the sites of transcription termination were altered by only a few base pairs . We also constructed a set of deletions removing consecutive thymidines which follow the G + C-rich region of dyad symmetry . Removal of 1 or 3 of the 9 thymidine residues had no effect on termination efficiency in vitro or in vivo . Removal of four to six thymidines caused a linear decrease in the efficiency of termination . When only one or two thymidines were present in the template, termination was completely abolished . These results indicate that both the integrity of the RNA stem and the length of the consecutive thymidine residues are important signals recognized by RNA polymerase during transcription of the thr operon regulatory region. J Biol Chem, 1988 Jan 5, 263(1), 76 - 83 High salt activation of recA protein ATPase in the absence of DNA; Pugh BF et al.; The recA protein of Escherichia coli is a DNA-dependent ATPase . In the absence of DNA, the rate of recA protein-promoted ATP hydrolysis drops 2000-fold, exhibiting an apparent kcat of approximately 0.015 min-1 . This DNA-independent activity can be stimulated to levels approximating those observed with DNA by adding high concentrations (approximately 2M) of a wide variety of salts . The increase in ATP hydrolysis appears to require the minimal interaction of three to four ions with recA protein . The active species in ATP hydrolysis is an aggregate of recA protein . There appears to be little or no cooperativity with respect to ATP binding (Hill coefficient = 1.0) . The salt-stimulated ATP hydrolysis reaction is dependent upon Mg2+ ions and is optimal between pH 7.0 and 8.0 . In many respects, the high salt concentration appears to be functionally mimicking DNA in activating the recA protein ATPase. J Biol Chem, 1988 Jan 5, 263(1), 436 - 42 Simian virus 40 large T antigen DNA helicase . Characterization of the ATPase-dependent DNA unwinding activity and its substrate requirements; Wiekowski M et al.; The ATPase of SV40 large T antigen (T antigen) which is essential for the replication of SV40 minichromosomes was recently shown to be functionally related to a newly discovered DNA helicase activity . The T antigen helicase unwinds DNA duplices of several kilobase pairs in a reaction depending on the presence of hydrolyzable ribo- or deoxyribonucleoside triphosphates . The in vitro rate of movement through duplex DNA was found to be about 100 base pairs/min at 37 degrees C . For DNA unwinding, T antigen requires a 3'-single strand extension of a partially double-stranded substrate and invades the double strand section processively, in a 3' to 5' direction . The minimum length of the single-stranded tail was determined to be less than 5 nucleotides . Unwinding studies in the presence of the Escherichia coli single strand-specific DNA-binding protein and competition experiments indicate that T antigen helicase binds preferentially at the single-stranded/double-stranded DNA junction . This DNA structure is therefore proposed to serve as an entry site for the T antigen helicase . Previously reported data suggest that T antigen is the replicative helicase of the SV40 minichromosome . The results presented here are consistent with these findings and imply that T antigen migrates actively and processively along the template for the leading strand. J Biol Chem, 1988 Jan 5, 263(1), 383 - 92 The unwinding of duplex regions in DNA by the simian virus 40 large tumor antigen-associated DNA helicase activity; Goetz GS et al.; The DNA helicase activity associated with purified simian virus 40 (SV40) large tumor (T) antigen has been examined . A variety of DNA substrates were used to characterize this ATP-dependent activity . Linear single-stranded M13 DNA containing short duplex regions at both ends was used to show that SV40 T antigen helicase displaced the short, annealed fragment by unwinding in a 3' to 5' direction . Three different partial duplex structures consisting of 71-, 343-, and 851-nucleotide long fragments annealed to M13 single-stranded circular DNA were used to show that SV40 T antigen can readily unwind short and long duplex regions with almost equal facility . ATP and MgCl2 were required for this reaction . With the exception of GTP, dGTP, and CTP, the other common nucleoside triphosphates substituted for ATP with varied efficiency, while adenosine 5'-O-(thiotriphosphate) was inactive . The T antigen helicase activity was also examined using completely duplex DNA fragments (approximately 300 base pairs) with or without the SV40 origin sequence as substrates . In reactions containing small amounts (0.6 ng) of DNA, the ATP-dependent unwinding of duplex DNA fragments occurred with no dependence on the origin sequence . This reaction was stimulated 5- to 6-fold by the addition of the Escherichia coli single-stranded DNA-binding protein . When competitor DNA was added so that the ratio of SV40 T antigen to DNA was reduced 1000-fold, only DNA fragments containing a functional SV40 origin of replication were unwound . This reaction was dependent on ATP, MgCl2, and a DNA-binding protein, and was stimulated by inorganic phosphate or creatine phosphate . The origin sequence requirements for the unwinding reaction were the same as those for replication (the 64-base pair sequence present at T antigen binding site 2) . Thus, under specified conditions, only duplex DNA fragments containing an intact SV40 core origin were unwound . In contrast, unwinding of partially duplex segments of DNA flanked by single-stranded regions can occur with no sequence specificity. J Mol Biol, 1988 Jan 5, 199(1), 35 - 45 Effect of dam methylation on Tn5 transposition; Yin JC et al.; The effect of dam methylation on Tn5 transposition was investigated by analyses of mutations in the host (Escherichia coli) and the element . Wild-type elements transposed at a higher frequency and showed higher levels of transposase expression in a dam-host . Mutations were made in the promoter region of the transcript that codes for the transposase . Transposition and transposase levels from these mutants were independent of the host methylation system . Measurements of the amount of RNA support the hypothesis that dam methylation exerts its effect on Tn5 transposition by modulating the frequency of transcriptional initiation of the transposase gene . Since Tn5 transposition increases when the transposase levels increase, at normal concentrations the amount of transposase is a rate-limiting factor that determines the transposition frequency of Tn5 . Transposition of IS50, one of the insertion sequences that constitutes Tn5, is also sensitive to dam methylation by a second mechanism in addition to that of modulating transcriptional initiation . dam methylation, either directly or indirectly, inhibits the usage of IS50 sequences by the transposase . Thus, dam methylation can affect both the expression of the transposase and the DNA substrate upon which it acts. J Mol Biol, 1988 Jan 5, 199(1), 137 - 47 Electron microscopy and computer image averaging of ice-embedded large ribosomal subunits from Escherichia coli; Wagenknecht T et al.; Electron micrographs of frozen-hydrated, large ribosomal subunits from Escherichia coli have been analyzed by computer image processing . Images of subunits in the so-called "crown" orientation were analyzed by correlation alignment procedures developed for negatively stained specimens . Averages of the aligned images showed both similarities and differences to averages determined for negatively stained specimens . The L1 ridge is more dense and stalk-like in frozen-hydrated as compared with negatively stained subunits, possibly because it is associated with ribosomal RNA . The results show that it should be feasible to determine the three-dimensional structure of the large ribosomal subunit from micrographs of individual, frozen-hydrated subunits that have been tilted in the electron microscope. J Mol Biol, 1988 Jan 5, 199(1), 115 - 36 A detailed model of the three-dimensional structure of Escherichia coli 16 S ribosomal RNA in situ in the 30 S subunit; Brimacombe R et al.; A large body of intra-RNA and RNA-protein crosslinking data, obtained in this laboratory, was used to fold the phylogenetically and experimentally established secondary structure of Escherichia coli 16 S RNA into a three-dimensional model . All the crosslinks were induced in intact 30 S subunits (or in some cases in growing E . coli cells), and the sites of crosslinking were precisely localized on the RNA by oligonucleotide analysis . The RNA-protein crosslinking data (including 28 sites, and involving 13 of the 21 30S ribosomal were used to relate the RNA structure to the distribution of the proteins as determined by neutron scattering . The three-dimensional model of the 16 S RNA has overall dimensions of 220 A x 140 A x 90 A, in good agreement with electron microscopic estimates for the 30 S subunit . The shape of the model is also recognizably the same as that seen in electron micrographs, and the positions in the model of bases localized on the 30 S subunit by immunoelectron microscopy (the 5' and 3' termini, the m7G and m6(2)A residues, and C-1400) correspond closely to their experimentally observed positions . The distances between the RNA-protein crosslink sites in the model correlate well with the distances between protein centres of mass obtained by neutron scattering, only two out of 66 distances falling outside the expected tolerance limits . These two distances both involve protein S13, a protein noted for its anomalous behaviour . A comparison with other experimental information not specifically used in deriving the model shows that it fits well with published data on RNA-protein binding sites, mutation sites on the RNA causing resistance to antibiotics, tertiary interactions in the RNA, and a potential secondary structural "switch" . Of the sites on 16 S RNA that have been found to be accessible to chemical modification in the 30 S subunit, 87% are at obviously exposed positions in the model . In contrast, 70% of the sites corresponding to positions that have ribose 2'-O-methylations in the eukaryotic 18 S RNA from Xenopus laevis are at non-exposed (i.e . internal) positions in the model . All nine of the modified bases in the E . coli 16 S RNA itself show a remarkable distribution, in that they form a "necklace" in one plane around the "throat" of the subunit . Insertions in eukaryotic 18 S RNA, and corresponding deletions in chloroplast or mammalian mitochondrial ribosomal RNA relative to E . coli 16 S RNA represent distinct sub-domains in the structure.(ABSTRACT TRUNCATED AT 400 WORDS) Eur J Biochem, 1988 Jan 4, 170(3), 515 - 20 cDNAs for canavalin and concanavalin A from Canavalia gladiata seeds . Nucleotide sequence of cDNA for canavalin and RNA blot analysis of canavalin and concanavalin A mRNAs in developing seeds; Yamauchi D et al.; By a method of Escherichia coli expression-vector-primed cDNA synthesis, a cDNA expression library was constructed from total poly(A)-rich RNA that was prepared from immature embryos of Canavalia gladiata . Essentially full-length cDNA clones for two seed proteins, canavalin and concanavalin A, were selected from the library by immunological screening of the colonies and in vitro RNA synthesis and translation . The complete amino acid sequence of canavalin was determined from the nucleotide sequence of the corresponding cDNA and was found to be very homologous to 7S seed proteins of other legumes . The nucleotide sequence of the cDNA predicts a 26-amino-acid extension in the precursor at the amino terminus of the mature canavalin . Canavalin mRNA and concanavalin A mRNA levels at successive stages of the seed development were estimated by RNA blot hybridization and results indicated that the two mRNA levels are differently regulated. FEBS Lett, 1988 Jan 4, 226(2), 291 - 6 Binding of ras p21 to bands 4.2 and 6 of human erythrocyte membranes; Tanimoto T et al.; The direct binding protein(s) of ras p21 was (were) investigated in inside-out vesicles of human erythrocyte ghosts using the pure v-Kirsten (Ki)-ras p21 synthesized in E . coli . The bound ras p21 was detected immunochemically using an anti-v-Ki-ras p21 monoclonal antibody, ras p21 bound to vesicles . Prior digestion of the vesicles with trypsin reduced this binding significantly . When ras p21 was laid over vesicle proteins immobilized on a nitrocellulose sheet by transfer from the gel of SDS-polyacrylamide gel electrophoresis, ras p21 bound to bands 4.2 and 6 . ras p21 binding to these proteins was reduced by prior incubation of ras p21 with the purified band 4.2 or 6 protein . These results indicate that v-Ki-ras p21 can bind directly to bands 4.2 and 6 of human erythrocyte membranes as far as tested in an in vitro cell-free system. FEBS Lett, 1988 Jan 4, 226(2), 255 - 60 Does the channel for nascent peptide exist inside the ribosome? Immune electron microscopy study; Ryabova LA et al.; MS2 phage RNA-directed synthesis of an N-terminal polypeptide of the phage coat protein on Escherichia coli 70 S ribosomes was initiated in a cell-free system with the N-dinitrophenyl derivative of methionyl-tRNAFMet) and performed in the absence of tyrosine, lysine, cysteine and methionine . As a result, the translating ribosomes carried peptides up to 42 amino acid residues in length with the dinitrophenyl hapten at the N-ends . Using the immune electron microscopy technique the positions of the nascent peptide N-ends on the 70 S ribosomes have been visualized . It has been found that (i) the N-ends of nascent peptides of these lengths are accessible to antibodies, (ii) the exit site of a nascent peptide is the pocket between the base of the central protuberance and the L1 ridge on the 50 S subunit, i.e . presumably its peptidyl transferase center, and (iii) the further pathway of a nascent peptide seems to proceed along the groove on the external surface of the 50 S subunit. Eur J Biochem, 1988 Jan 4, 170(3), 637 - 42 ATP4, the structural gene for yeast F0F1 ATPase subunit 4; Velours J et al.; A plasmid containing the gene coding for the Saccharomyces cerevisiae F0F1 ATPase subunit 4 was isolated from a yeast genomic DNA library using the oligonucleotide probe procedure . The gene and the surrounding regions were cloned into M13 tg 130 and M13 tg 131 phage vectors . A 732-base-pair open reading frame encoding a 244-amino-acid polypeptide is described . The nucleotide sequence predicts that subunit 4 is probably derived from a precursor protein with a hydrophilic and basic 35-amino-acid leader sequence . Mature subunit 4 contains 209 amino acid residues and the predicted molecular mass is 23250 Da . This subunit presents amphiphilic behaviour with two distinct domains . A high alpha-helix content of 77% was predicted from the sequence . Subunit 4 shows homology with the b subunit of Escherichia coli ATP synthase. Eur J Biochem, 1988 Jan 4, 170(3), 627 - 30 F0 part of the ATP synthase from Escherichia coli . Influence of subunits a, and b, on the structure of subunit c; Steffens K et al.; Four different sets of proteoliposomes were prepared from F0, subunit c, a complex of subunits a and c (ac complex) and an ac complex supplemented with subunit b . Only liposomes containing intact F0 or all subunits of F0 were active in proton translocation and F1 binding {Schneider, E . and Altendorf, K . (1985) EMBO J . 4, 515-518} . The conformation of subunit c in the different preparations was analyzed by labelling the proteoliposomes with the hydrophobic photoactivatable reagent 3-(trifluoromethyl)-3-(m-{125I}iodophenyl)diazirine ({125I}TID) . Subsequent isolation and Edman degradation of this polypeptide revealed distinct radioactive labelling patterns over the entire amino acid sequence . In the F0 complex and in the ac complex subunit c retains a labelling pattern which is related to that found in TID-labelled membrane vesicles of Escherichia coli {Hoppe et al . (1984) Biochemistry 23, 5610-5616} . In the absence of subunit a, considerably more and different amino acid residues of subunit c are modified . The labelling data are discussed in relation to structural aspects of F0 and functional properties of proteoliposomes reconstituted with F0 or individual subunits. FEBS Lett, 1988 Jan 4, 226(2), 232 - 4 Influence of probe structure on unique (regiospecific) cleavage of RNA by RNase H; Metelev VG et al.; Chimeric oligo(ribo-deoxyribo)nucleotides with an internucleotide pyrophosphate bond are novel probes for regiospecific hydrolysis of RNA by RNase H . It has been shown that the use of d(TGTGTAT)ppGCCAU leads to unique hydrolysis of the TMV RNA fragment pAAUGGCAUACAC between C10 and A11. Proc Natl Acad Sci U S A, 1988 Jan, 85(2), 372 - 6 Trans-splicing in chloroplasts: the rps 12 loci of Nicotiana tabacum; Hildebrand M et al.; The rps12 gene in tobacco chloroplasts consists of three exons that code for a polypeptide with homology to Escherichia coli ribosomal protein S12 . C-terminal exons 2 and 3 of rps12 are located in the inverted repeat regions of the tobacco chloroplast genome . Exon 1 of rps12 is 29 kilobase pairs downstream of the nearest copy of exons 2 and 3 and 69 kilobase pairs away from the distal copy of exons 2 and 3 . RNA gel blot hybridization analysis and primer extension sequencing of cDNA to rps12 encoding RNAs indicate that exon 1 and exons 2 and 3 are encoded on separate transcripts . Exon 1 and exons 2 and 3 are covalently ligated in the correct reading frame in rps12 mRNA . These results indicate that a bimolecular (trans-) splicing event occurs during the formation of mature rps12 mRNA. Proc Natl Acad Sci U S A, 1988 Jan, 85(2), 319 - 23 Structure and expression of rat osteosarcoma (ROS 17/2.8) alkaline phosphatase: product of a single copy gene; Thiede MA et al.; Alkaline phosphatase {ALP; orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1} is a ubiquitous enzyme of unknown function expressed at high levels in cells of mineralizing tissues . To study the structure, function, and expression of ALP, a full-length cDNA of rat ALP (2415 bases) was isolated from a ROS 17/2.8 osteosarcoma cell lambda gt10 cDNA library . The predicted amino acid sequence spans 524 residues and includes an N-terminal signal peptide of 17 amino acids, the phosphohydrolase active site, a rather hydrophilic backbone with five potential N-glycosylation sites, and a short hydrophobic C-terminal sequence . ALP negative CHO cells transfected with an expression vector containing the ALP coding sequences express ALP . The rat bone, liver, and kidney ALP shows remarkable 90% homology with the corresponding human enzyme, the most divergent region being the C-terminal hydrophobic domain through which the enzyme may be anchored to the plasma membrane . The rat ALP also shows 50% homology with the human placental and intestinal ALP and 25% homology with the Escherichia coli ALP . The amino acids involved in catalysis show nearly complete homology among all known ALP sequences, suggesting that these enzymes evolved from a common ancestral gene . The rat ALP cDNA pRAP 54, used as a hybridization probe in RNA blot analysis of several tissues that express ALP, revealed the presence of an ALP mRNA of approximately equal to 2500 bases . Furthermore, hybridization patterns derived from Southern blot analysis of rat chromosomal DNA offered molecular evidence that the ALP expressed in ROS 17/2.8 osteosarcoma and various rat tissues, excluding the intestine, is the product of the same single copy gene. J Clin Lab Immunol, 1988 Jan, 25(1), 41 - 5 5-lipoxygenase inhibitor (AA-861) attenuates neutrophil-mediated oxidative stress on the venular endothelium in endotoxemia; Suematsu M et al.; Neutrophil-mediated oxidative stress on the rat mesenteric microcirculation was studied in the experimental model of endotoxin-induced disseminated intravascular coagulation (DIC) by using an intravital fluorescent microscope equipped with a Silicon Intensifier Target Image Tube camera and luminol-dependent chemiluminescence (ChL) analysis . Leukocytes adhering to the venules were visualized by the injection of acridine orange, a fluorochrome tracer which shows high affinity to white cells . Endotoxin (E . coli, O-111 B4) was administered intravenously at a dose of 2 mg/kg/hour . After starting the infusion of endotoxin, the number of adherent cells gradually increased in the venular endothelium and was followed by a transient neutropenia . ChL activities from neutrophils were also significantly elevated, which may reflect the enhanced ability to generate oxygen-radicals . To elucidate the role of 5-lipoxygenase products in the locomotive and metabolic changes of neutrophils, the effects of AA-861, a specific inhibitor of 5-lipoxygenase was tested . In addition prednisolone and indomethacin were evaluated . AA-861 and prednisolone reduced neutropenia, leukocyte adhesion to the venular walls and ChL activities from neutrophils . It was concluded that 5-lipoxygenase may modulate neutrophil-mediated oxidative stress on microvasculature in endotoxin-induced DIC. Bioorg Khim, 1988 Jan, 14(1), 43 - 7 {An improved chemico-enzymatic method of synthesizing long DNA segments}; Kobets ML et al.; A simple and economy method of the biochemical assembling of long double-stranded DNA segments is described . A single-stranded polydeoxynucleotide 122 bases long representing a fragment of synthetic gene of human beta-interferon was assembled from three synthetic fragments 36 (two) and 50 bases long on four complementary 12-mers as templates . This single-stranded polynucleotide was converted, in the presence of DNA polymerase 1 and a 12-meric primer, in to the full-length double-stranded DNA (the beta-interferon gene segment) . It was cloned into an E . coli plasmid vector pBR322 and its sequence confirmed. Proteins, 1988, 3(1), 18 - 31 Flexibility of the DNA-binding domains of trp repressor; Lawson CL et al.; An orthorhombic crystal form of trp repressor (aporepressor plus L-tryptophan ligand) was solved by molecular replacement, refined to 1.65 A resolution, and compared to the structure of the repressor in trigonal crystals . Even though these two crystal forms of repressor were grown under identical conditions, the refined structures have distinctly different conformations of the DNA-binding domains . Unlike the repressor/aporepressor structural transition, the conformational shift is not caused by the binding or loss of the L-tryptophan ligand . We conclude that while L-tryptophan binding is essential for forming a specific complex with trp operator DNA, the corepressor ligand does not lock the repressor into a single conformation that is complementary to the operator . This flexibility may be required by the various binding modes proposed for trp repressor in its search for and adherence to its three different operator sites. Mol Biol (Mosk), 1988 Jan-Feb, 22(1), 131 - 8 {A method of determining the primary structure of oligodeoxyribonucleotides}; Sviriaeva TV et al.; Sanger method was modified to fulfill the requirements of sequencing of oligodeoxyribonucleotides . E . coli DNA polymerase I Klenow fragment was used for all the reactions . The method consists of three steps made in succession in one tube: 1 . Optional hydrolysis of a 5'-labeled oligodeoxyribonucleotide primer in order to get a set of primers of different lengths . 2 . Elongation of the produced set of primers in the presence of a template, natural dNTPs and chain terminating dNTP analogs . 3 . Hydrolysis of the products of the previous step in order to remove the unterminated molecules . Change of steps in achieved just by varying the reaction conditions without any product purification . The method in insensitive to the presence of admixture of oligonucleotides which is not complementary to the primer or to the template. Folia Microbiol (Praha), 1988, 33(2), 101 - 7 Effect of detergents on aspartate ammonia-lyase activity in Escherichia alcalescens; Malanik V et al.; The effect of twelve detergents on aspartate ammonia-lyase activity of Escherichia alcalescens used for the production of L-aspartic acid was tested . Best permeabilization was found with Triton X-100, Slovafol 910 and Corona, a mixed commercial preparation . In contrast to Triton X-100 and Slovafol 910, a much narrower range of suitable concentrations was observed with Corona. Curr Genet, 1988, 13(1), 75 - 82 Nucleotide sequence and determination of the extremities of the 26S ribosomal RNA gene in wheat mitochondria: evidence for sequence rearrangements in the ribosomal genes of higher plants; Falconet D et al.; The nucleotide sequence of the wheat mitochondrial 26S ribosomal RNA gene and flanking regions was determined and compared with mitochondrial 26S rRNA genes from maize and Oenothera . All three genes exhibit a high degree of homology except within two variable regions . When the plant mitochondrial 26S rRNA genes are compared with Escherichia coli 23S rRNA and chloroplast 23S and 4.5S rRNA genes, a third variable region is apparent close to the 3' end of the gene . The 5' and 3' ends of the wheat mitochondrial gene were determined by S1 nuclease mapping . Computer analysis of the wheat mitochondrial gene revealed several small sequences present either in the 5' region of the 26S rRNA gene or in the 18S rRNA gene. Am J Vet Res, 1988 Jan, 49(1), 125 - 9 Synergistic effects of Fusobacterium necrophorum lipopolysaccharide, cytoplasmic, and culture supernatant fractions on induction of acute hepatic necrosis in rabbits; Nakajima Y et al.; Synergistic effects of toxic fractions of Fusobacterium necrophorum were examined for evaluation of the role of the toxin in inducing liver abscesses in rabbits . Cytoplasmic and culture supernatant fractions of F necrophorum had preparative activity for the Shwartzman reaction, and lipopolysaccharide of F necrophorum had preparative and provocative activities for the reaction . All 3 fractions were hepatotoxic . Inoculation into the bile duct with each fraction followed by intravenous inoculation with F necrophorum or Escherichia coli lipopolysaccharide had a greater synergistic effect in inducing severe hepatic necrosis than did inoculations with double doses of the cytoplasmic or supernatant fractions of F necrophorum . This synergism may have been attributable to the Shwartzman reaction. DNA, 1988 Jan-Feb, 7(1), 63 - 70 DNA amplification in vitro using T4 DNA polymerase; Keohavong P et al.; We have evaluated in vitro DNA amplification by polymerase chain reaction using either T4 DNA polymerase or Klenow fragment of Escherichia coli DNA polymerase I . Both polymerases under optimal salt conditions permit efficient amplification of exon 3 of the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene from human genomic DNA and from plasmid containing the HPRT cDNA . DNA sequences amplified from human genomic DNA, using two 20-nucleotide primers flanking the ends of the exon, showed a marked difference between the two polymerases . T4 DNA polymerase yielded only the expected amplified DNA fragment, whereas Klenow fragment produced many lower-molecular-weight bands in addition to the expected DNA fragment . On the basis of the reported fidelity of in vitro DNA synthesis using Klenow fragment and T4 DNA polymerase, it is expected that the latter will create substantially fewer errors during the amplification process . For these reasons, T4 DNA polymerase should be particularly valuable for amplification of sequences present at a very low frequency requiring many cycles of amplification to be detected. Am J Physiol, 1988 Jan, 254(1 Pt 2), H181 - 6 Improved techniques for cardiovascular monitoring in rats as applied during endotoxemia; Biber B et al.; We describe a new combination of techniques for measurements of systemic blood pressure, central venous pressure, pulmonary arterial (PA) pressure, PA wedge pressure, and cardiac output in the rat . Application of the method to the conscious rat in a septic shock (Escherichia coli endotoxin iv injection) model demonstrated a response pattern of decreased cardiac output and stroke volume, increased total peripheral vascular resistance and heart rate, and transiently decreased systemic arterial pressure . In the pulmonary circulation, a very brief hypertension and a sustained increase in pulmonary vascular resistance were observed, but changes in PA wedge pressure were small . The soft PA catheter (0.3 mm ID, 0.6 mm OD) had no undue effects on cardiovascular function . We suggest that this combined technique could be useful for many cardiovascular studies in the rat, not only as related to shock research. Am J Physiol, 1988 Jan, 254(1 Pt 1), E45 - 51 Regional lactate production in early canine endotoxin shock; van Lambalgen AA et al.; High serum lactate may not reflect the severity of endotoxin shock: the lactate load could even be formed immediately after the endotoxin challenge . During the first 30 min after endotoxin injection (Escherichia coli; 1.5 mg/kg iv) into anesthetized dogs (4 mg.kg-1.h-1 etomidate, n = 19) we studied arterial lactate concentration; contributions of portal and splanchnic (n = 6), renal and pulmonary (n = 7), and femoral (n = 6) vascular beds to the early lactate rise; and regional O2 extraction and blood flow (microspheres) . In control dogs (n = 5, no endotoxin), we found no significant hemodynamic and biochemical changes . Endotoxin caused an immediate decrease in blood pressure, cardiac output, and organ perfusion, followed by recovery after approximately 5 min to approximately 75% of preshock values at t = 30 min (except for renal blood flow, which remained low) . Arterial lactate concentration started to increase almost immediately after endotoxin and increased rapidly until t = 15 min (to 300%) and then leveled off, but in spite of the hemodynamic recovery it remained elevated . A major part of the early increase in lactate concentration can be explained by splanchnic lactate production . The total splanchnic bed released more lactate than the portal bed, indicating that the liver produces lactate . We conclude that the lactate concentration later in canine endotoxin shock depends on events that occur during early shock in which the liver may play a crucial role. J Surg Res, 1988 Jan, 44(1), 73 - 81 Naloxone requires circulating catecholamines to attenuate the cardiovascular suppression of endotoxic shock; Allgood SC et al.; The opiate antagonist naloxone (NAL) improves cardiovascular performance in canine hemorrhagic and endotoxic shock . If the release of neural and adrenal catecholamines is attenuated, NAL does not produce the expected improvement in cardiovascular function in canine hemorrhagic shock . This study tests the hypothesis that an endorphin-catecholamine interaction at the heart is responsible for a part of the cardiovascular depression of endotoxic shock . Two groups of five dogs were instrumented to measure mean arterial pressure (MAP), the first derivative of left ventricular pressure over time (LV dP/dt max), cardiac output, and heart rate (HR); they were then subjected to bilateral adrenalectomy and given chlorisondamine to produce ganglionic blockade . At t = 0 min the dogs were given Escherichia coli endotoxin at 1 mg/kg (LD80) . Group I animals received NAL at 2 mg/kg + 2 mg/kg.hr iv from t = 30 to t = 60 . At t = 45 these animals were treated with epinephrine (EPI) at 20 micrograms/kg.hr iv until t = 60 . Group II animals got EPI from t = 30 to t = 60 and NAL from t = 45 to t = 60 at the same doses as Group I . In Group I, NAL alone had no effect on MAP, LV dP/dt max, or HR . EPI significantly increased (P less than 0.002) cardiovascular parameters with MAP increasing from 52 +/- 7 to 159 +/- 14 mm Hg . In Group II, EPI produced a significant increase in all parameters, and the addition of NAL produced a further significant increase; MAP increased from 37 +/- 3 to 126 +/- 16 mm Hg with EPI and then to 175 +/- 11 mm Hg with NAL . These data support the above hypothesis and indicate that circulating catecholamines need to be present to allow naloxone to reverse the cardiovascular depression in endotoxic shock. J Bacteriol, 1988 Jan, 170(1), 203 - 12 Gene organization and structure of the Streptomyces lividans gal operon; Adams CW et al.; We present the gene organization and DNA sequence of the Streptomyces lividans galactose utilization genes . Complementation of Escherichia coli galE, galT, or galK mutants and DNA sequence analysis were used to demonstrate that the galactose utilization genes are organized within an operon with the gene order galT, galE, and galK . Comparison of the inferred protein sequences for the S . lividans gal gene products to the corresponding E . coli and Saccharomyces carlbergensis sequences identified regions of structural homology within each of the galactose utilization enzymes . Finally, we discuss a potential relationship between the gene organization of the operon and the functional roles of the gal enzymes in cellular metabolism. Infect Immun, 1988 Jan, 56(1), 278 - 82 Evidence for the phagocytic transport of intestinal particles in dogs and rats; Wells CL et al.; Fluorescent latex beads of two different colors were implanted into separate intestinal segments in individual dogs and rats . Mesenteric lymph node phagocytes subsequently contained multiple beads of one or the other color but rarely both colors, indicating that intestinal phagocytes transported the latex beads to the draining lymph node . Fluorescent labeled Escherichia coli was implanted into rat ligated intestinal segments, and rare mesenteric lymph node phagocytes subsequently contained fluorescent bacteria, suggesting that intestinal bacteria might be transported in the same manner as inert latex beads. Circ Res, 1988 Jan, 62(1), 25 - 30 Neuropeptide Y prevents the blood pressure fall induced by endotoxin in conscious rats with adrenal medullectomy; Evequoz D et al.; Neuropeptide Y (NPY) is a vasoconstrictor peptide known to be present in the adrenal medulla, the terminal nerve endings, and in plasma . This study was designed to test whether NPY could prevent the acute blood pressure fall induced by endotoxin administration . Normotensive rats were subjected to adrenal demedullation on the right side and were either adrenalectomized or sham-operated on the left side . Eight to ten days later, NPY (0.07 microgram/min i.v.) or its vehicle were infused for 95 minutes into these conscious, semirestrained rats . The same experiments were performed with rats that received an infusion of epinephrine (0.1 microgram/min) . These doses of NPY and epinephrine when given alone had no blood pressure effect . During the last 75 minutes of the 95-minute infusion, endotoxin (lipopolysaccharide Escherichia coli 0.111:B4, 10 micrograms/min i.v.) or its vehicle were administered . In rats with an intact adrenal gland, endotoxin failed to induce hypotension . In rats lacking a functioning adrenal medulla, however, endotoxin induced a pronounced mean blood pressure fall of 55 +/- 11.6 mm Hg (mean +/- SEM) . This blood pressure drop could be prevented equally well with NPY and with epinephrine infusion and averaged 11 +/- 2.3 and 16 +/- 2.4 mm Hg, respectively, at the end of the experiment . Additional rats were biadrenalectomized and supplemented with an excess of glucocorticoids and mineralocorticoids . In these rats also, NPY markedly attenuated the blood pressure fall resulting from endotoxemia . These data taken together indicate that in conscious rats with no adrenal medulla, the acute blood pressure fall induced by endotoxin administration is greatly enhanced.(ABSTRACT TRUNCATED AT 250 WORDS) Int J Tissue React, 1988, 10(1), 7 - 16 Studies on inflammation in mice: dynamics of inflammatory response induced by extract from Escherichia coli and influence of antiinflammatory drugs; Ishibashi Y et al.; The intraperitoneal injection of E . coli extract in the mouse resulted in a biphasic accumulation of leukocytes . The first phase of leukocyte accumulation was based on neutrophil influx in the peritoneal cavity, which occurred 1-2 days after E . coli extract administration . The second phase was based on macrophage influx, which occurred 12 days after E . coli extract administration . Preceding the influx of neutrophils in the first phase, the exudation of plasma proteins in the peritoneal fluid occurred at 4 h . A slight increase in plasma exudation accompanied the macrophage influx in the second phase of leukocyte accumulation . These kinetics of the inflammatory response to E . coli extract were similar to those of the inflammatory response to lipopolysaccharide (LPS), suggesting that the inflammatory response induced by E . coli extract administration was mainly due to the effect of LPS, a component of the E . coli extract . Indomethacin reduced the plasma exudation at 4 h but had no influence on the leukocyte accumulation . On the other hand, dexamethasone suppressed the leukocyte accumulation in both phases. CRC Crit Rev Biochem, 1988, 23 Suppl 1, S43 - 86 DNA strand exchanges; Griffith JD et al.; Biochemical and electron microscopic studies of the strand exchange reactions catalyzed by the RecA protein of Escherichia coli and the UvsX protein of T4 phage reveal that these reactions proceed in three distinct steps . The first step, termed joining, involves the assembly of RecA (or UvsX) protein onto a single-stranded DNA (ssDNA) molecule and the subsequent search for homology with a double-stranded DNA (dsDNA) partner and formation of a stable synapsis . In the second step (envelopment/exchange), the exchange of DNA strands occurs fueled by the hydrolysis of ATP . The third step (release of products) entails the resolution of the complexes and dissociation of the protein from the DNAs . The structure of the intermediates in the in vitro reactions catalyzed by the RecA and UvsX proteins is emphasized in this review . The results of pairing different DNA molecules in vitro (such as linear ssDNA pairing with linear or supertwisted dsDNA) are described . Paranemic joints represent a major pathway of joining between two DNA molecules which may involve, in some cases, most of the DNA substrate molecules . Since the nature of paranemic joints has only recently begun to be understood, the nature, role, and possible in vivo function of paranemic joining are considered. Protein Seq Data Anal, 1988, 1(4), 263 - 7 Periodic distribution of homologous genes or gene segments on the Escherichia coli K12 genome; Kunisawa T et al.; A computer search for homologous relationships among Escherichia coli proteins has been carried out with the use of databases . Homologous genes or gene segments thus identified at the amino acid sequence level have a tendency to lie on the genome separated by distances of multiples of 7 min . This relatively regular pattern of gene distribution on the E . coli genome is interpreted as reflecting the early history of multiple genome doublings . Evolutionary advantages of duplications of the total genome are discussed in relation to the acquisition of new multistep pathways such as the Krebs cycle and to the generation of a variety of proteins in existence today. Gene, 1988, 63(1), 65 - 74 Cloning of a phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tü494 and its expression in Streptomyces lividans and Escherichia coli; Strauch E et al.; Phosphinothricin-tripeptide (Ptt), also known as bialaphos, contains phosphinothricin (Pt), a potent inhibitor of glutamine synthetase . A 4.0-kb Bam HI fragment coding for Ptt resistance was cloned in Streptomyces lividans TK23 . The fragment was isolated from a Ptt-resistant mutant of Streptomyces viridochromogenes Tu494 . Subcloning experiments revealed that Ptt resistance can be assigned to a 0.8-kb Bg/II fragment . This fragment was shown to include the Ptt-resistance promoter . Subcloning this fragment downstream from the lacZ promoter conferred Ptt resistance to Escherichia coli JM83 in one of the two possible orientations . Biochemical investigations revealed that the Bg/II fragment codes for a Pt N-acetyltransferase. Gene, 1988, 63(1), 135 - 9 Promoter vectors with restriction-site banks; Bleicher F et al.; New vectors harboring the promoter for the chloramphenicol acetyl transferase gene (cat promoter) have been constructed . These vectors are all derived from pJRD184 {Heusterspreute et al., Gene 39 (1985) 299-304}, which contains a restriction-site bank . The cat promoter has been inserted at various positions and in reverse orientations so that almost all the restriction sites originally present on JRD184 can be used in cloning experiments . The expression of the aceK gene of Escherichia coli cloned under the control of the cat promoter has been tested . A large increase in the synthesis of the isocitrate dehydrogenase kinase, the aceK gene product, has demonstrated the efficiency of the newly constructed vectors. Arch Geschwulstforsch, 1988, 58(2), 73 - 8 Genotoxicity assessment of the plasmacytomagenic agent pristane (2.6.10.14-tetramethylpentadecane) and four related alkanes by the SOS chromotest; Janz S et al.; The most extensively studied model of plasmacytomagenesis is the induction of plasmacytomas in BALB/c mice by i.p . injections of mineral oil or, chemically more defined, by several branched alkanes such as pristane (2.6.10.14-tetramethylpentadecane), phytane (2.6.10.14-tetramethylhexadecane), and 7-n-hexyloctadecane . The available evidence suggests that the primary biologic action of these plasmacytomagenic agents is to induce the formation of a chronic granulomatous tissue, the histological matrix of plasmacytoma development . However, certain genotoxic effects caused by the presence of these substances can not be ruled out a priori . Pristane, 2-methyldodecane, and 1.3-di-tert-butyl-5-methyl-cyclohexane as well as perhydroanthracene and hexahydrodibenzsuberane were proofed as potential genotoxic agents by the SOS chromotest, a quantitative bacterial colorimetric assay for genotoxins . The substances tested did not express any sign of genotoxicity, but exerted toxic effects to the E . coli tester strain. Z Naturforsch {C}, 1988 Jan-Feb, 43(1-2), 91 - 8 Selective binding of amino acid residues to tRNA molecules detected by anticodon-anticodon interactions; Bujalowski W et al.; Anticodon-anticodon pairing of complementary tRNA's has been studied by fluorescence temperature jump measurements in the presence of different ligands as an approach for the evaluation of ligand binding to tRNA . This procedure is particularly useful for ligands which do not show spectroscopic changes upon binding, but affect the pairing potential of anticodons . Addition of phenylalanine-, tyrosine- and tryptophan-amide leads to a substantial decrease of the tRNAPhe.tRNAGlu pairing constant Kp, whereas Kp remains almost unaffected by addition of leucine amide and increases upon addition of glycine amide . The effects observed for the aromatic amino acid amides can be described quantitatively by a site binding model with preferential binding of the amides to tRNAPhe . The binding constants evaluated according to this model (Phe-amide 120 M-1, Tyr-amide 160 M-1 and Trp-amide 580 M-1) are consistent with values obtained independently by fluorescence titrations with tRNAPhe . Selective binding of these amino acid residues to tRNAPhe is deduced from the observed concentration dependence which is not compatible with a corresponding binding process to tRNAGlu . Addition of glutamic acid diamide induces an increase of the tRNAPhe.tRNAGlu pairing constant, which is however equivalent to that observed for tRNAPhe.tRNALys pairing and thus does not demonstrate a selective binding to tRNAGlu . The pairing of tRNAPhe with tRNAGlu is strongly enhanced by addition of Mg2+ or spermine . Evaluation of the Mg2+ data by a site model leads to constants of 360 M-1 for the binding of Mg2+ to monomer tRNA and 3000 M-1 for the binding of Mg2+ to the tRNAPhe.tRNAGlu dimer.(ABSTRACT TRUNCATED AT 250 WORDS) Vet Q, 1988 Jan, 10(1), 48 - 52 Therapeutic efficacy of doxycycline hyclate in experimental Escherichia coli infection in broilers; Goren E et al.; Treatment of experimentally induced colibacillosis in broilers with doxycycline hyclate through the drinking water was just effective at a dose of 200 mg/kg body weight (1000 ppm) . The achieved therapeutic effects were similar to those of tetracycline at the same dose and of flumequine at a dose of 19 mg/kg body weight (100 ppm). Mol Biol (Mosk), 1988 Jan-Feb, 22(1), 201 - 8 {Stability of normal, abnormal and recombinant proteins in Escherichia coli strains deficient for intracellular proteinase La--the product of the lon gene}; Ryzhavskaia AS et al.; Escherichia coli Lon-mutants deficient in intracellular protease La have been isolated . The rate of degradation of normal cellular proteins was 1.5-2-fold lower in Lon-mutants as compared with that of the wild type strain . The rate of degradation of canavanine-containing abnormal proteins, as well as foreign proteins was significantly higher in E . coli than that of normal proteins . Lon-mutants possessed 2-2.5-fold lower rates of degradation of abnormal proteins as compared with Lon+-strains . The rate of degradation of human interferon alpha-2 was 10-fold higher in E . coli than that of abnormal proteins . B . amyloliquefaciens alpha-amylase degraded in E . coli with the rate comparable with that of abnormal proteins, since chloramphenicolacetyltransferase from Tn9 was stable in E . coli . The rate of degradation of interferon alpha-2 was 2-fold lower in Lon-mutants (half-life 23-26 min) than in the initial strain (11-12 min) . Lon-mutants were effectively used as recipient strains for constructing strains-producers of several human alpha- and beta-interferons. Mol Biol (Mosk), 1988 Jan-Feb, 22(1), 187 - 94 {Interaction of tryptophanase with substrate analogs}; Zakomyrdina LN et al.; Tryptophanase from Escherichia coli was studied with respect to its interactions with L-alanine, beta-chloro-L-alanine, L-phenylalanine, L-methionine, L-threonine, beta-phenyl-DL-serine (threo form) and also with a new tryptophan analog oxindolyl-L-alanine . Slow transamination of L-alanine in the active site of the enzyme was observed . Some evidence is presented which indicates that the side transamination reaction occurs during incubation of tryptophanase with an adequate substrate, beta-chloro-L-alanine . Absorption and circular dichroism (CD) spectra of the enzyme-quasisubstrate complexes have been recorded . Addition of beta-phenylserine and threonine to the enzyme induces a decrease of absorbance at 337 nm and an increase of absorbance at 420 nm . The spectral changes are associated with inversion of the CD sign, i.e . with disappearance of positive CD in the 420 nm band and appearance of negative CD in this band . It is inferred that beta-phenylserine and threonine form an external coenzyme-substrate aldimine which undergoes slow conversion to give a keto acid and the free enzyme . Addition of oxindolylalanine to tryptophanase results in the formation of an intense narrow absorption band at 504 nm with a shoulder at about 475 nm . This band belongs to a quinonoid intermediate . A positive CD is seen in the 504 nm band; the dissymmetry factor (delta A/A) in this band is much smaller than that in the absorption bands of the free enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Biol (Mosk), 1988 Jan-Feb, 22(1), 111 - 6 {The role of in vivo interaction of proteins SSB with DNA polymerase II}; Fradkin GE et al.; In order to elucidate the role of in vivo interaction of single-strand DNA binding protein with DNA polymerase II isogenic strains of Escherichia coli were constructed combining the ssb+, ssb-1 alleles with DNA polymerases I or II mutations; their radiosensitivity and a level of UV-induced DNA degradation were studied . Received findings suggest a functional antagonism of SSB-proteins depending on intracellular conditions (from the balance of DNA and protein synthesis) . The SSB-proteins provide the stability of the genome or, vice versa, perturb the stability of the genome, by degrading of DNA macromolecules. Microbiol Immunol, 1988, 32(1), 115 - 7 Chemotaxis for methamphetamine in Escherichia coli; Fukunaga S et al.; Escherichia coli showed the chemotactic response for methamphetamine, a biogenic stimulative amine . The response was qualitatively detected by using a paper disk filter . The quantitative assay showed that E . coli responded to methamphetamine at 10(-5) M or more. J Dairy Sci, 1988 Jan, 71(1), 29 - 40 Molecular cloning and expression of bovine kappa-casein in Escherichia coli; Kang YC et al.; A cDNA library was constructed using poly(A) +RNA from bovine mammary gland . This cDNA library of 6000 clones was screened employing colony hybridization using 32P-labelled oligonucleotide probes and restriction endonuclease mapping . The cDNA from the selected plasmid, pKR76, was sequenced using the dideoxy-chain termination method . The cDNA insert of pKR76 carries the full-length sequence, which codes for mature kappa-casein protein . The amino acid sequence deduced from the cDNA sequence fits the published amino acid sequence with three exceptions; the reported pyroglutamic acid at position 1, tyrosine at position 35, and aspartic acid at position 81 are, respectively, a glutamine, a histidine, and an asparagine in the clone containing pKR76 . The MspI-, NlaIV-cleaved fragment (630 base pair) from the kappa-casein cDNA insert has been subcloned into expression vectors pUC18 and pKK233-2, which contain a lac promoter and a trc promoter, respectively . Escherichia coli cells carrying the recombinant expression plasmids were shown to produce kappa-casein protein having the expected mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and being recognized by specific antibodies raised against natural bovine kappa-casein. Gene, 1988, 62(1), 135 - 9 Site-directed mutagenesis with Escherichia coli DNA polymerase III holoenzyme; Tsurushita N et al.; Escherichia coli DNA polymerase III holoenzyme was used to synthesize double-stranded DNA from M13 single-stranded DNA hybridized to a phosphorylated synthetic oligodeoxynucleotide containing a nucleotide substitution . The resulting DNA was transfected into E . coli JM101 without further treatment . Sequence analysis of randomly chosen phage clones revealed that the efficiency of mutagenesis was nearly 50%, which is the theoretical maximum . Treatment with DNA ligase after DNA synthesis was not necessary to obtain high efficiency of mutagenesis . Thus, use of DNA polymerase III holoenzyme provides a simple and efficient procedure for site-directed mutagenesis. Vopr Virusol, 1988 Jan-Feb, 33(1), 37 - 44 {Immunodiagnosis of AIDS using preparations of the recombinant antigen of the surface protein of the HTLV-III virus}; Kovgan AA et al.; A technological scheme has been developed for purification of recombinant antigen of surface protein (ASP) of the causative virus of AIDS from Escherichia coli cells carrying plasmid pL2 which codes for the synthesis of a hybrid polypeptide consisting of phage lambda N protein (59 amino acid residues) and a fragment of SP (env) of HIV virus (569 a.r.) . Purification of ASP is based on two separation principles: fractionation of polypeptides of bacterial lysates by preparative isoelectrofocusing in a granulated gel layer and the method of preparative polyacrylamide gel electrophoresis in "Multiphor" apparatus (Pharmacia) . The ASP purified by these methods was used for construction of an immunodiagnostic preparation for AIDS, employing for this purpose solid-phase enzyme-immunoassay in two modifications: competitive sandwich method and analysis of the tested sera with direct sorption of ASP on nitrocellulose filters. Prikl Biokhim Mikrobiol, 1988 Jan-Feb, 24(1), 35 - 41 {Enzymatic synthesis of L-malic acid from fumaric acid using immobilized Escherichia coli cells}; Verevkin AN et al.; Optimal conditions were chosen for cultivation of Escherichia coli 85 cells with a rather high fumarate-hydratase activity on a cheap medium containing no edible raw material . An active biocatalyst for the synthesis of L-malic acid from fumaric acid was obtained based on E . coli 85 cells immobilized in carrageenan . The enzymatic synthesis of L-malic acid from potassium fumarate was kinetically studied and optimized . Some thermodynamic parameters of fumaric acid hydration into malic acid were determined . A technique for assaying the reaction mixture was developed that involved high performance liquid chromatography. Mol Microbiol, 1988 Jan, 2(1), 43 - 52 Fumarate reductase of Escherichia coli: an investigation of function and assembly using in vivo complementation; Condon C et al.; Recombinant plasmids which carried portions of the Escherichia coli frd operon were constructed and their expression examined by in vivo complementation of E . coli MI1443 . This strain lacked a chromosomal frd operon and was unable to grow anaerobically on glycerol and fumarate . Introduction of all four fumarate reductase subunits into E . coli MI1443 was essential for the restoration of growth . The FRD A, FRD B dimer (but neither subunit alone) was active in the benzyl viologen oxidase assay . Both FRD C and FRD D were required for membrane association of fumarate reductase and for the oxidation of reduced quinone analogues . Introduction into E . coli MI1443 of the frdABC and frdD genes on two separate plasmid vectors failed to restore anaerobic growth on glycerol and fumarate . Thus separation of the DNA coding for the FRD C and FRD D proteins affected the ability of fumarate reductase to assemble into a functional complex. Arch Microbiol, 1988 Jan, 149(3), 232 - 9 Glycine betaine reverses the effects of osmotic stress on DNA replication and cellular division in Escherichia coli; Meury J; The accumulation of glycine betaine to a high internal concentration by Escherichia coli cells in high osmolarity medium restores, within 1 h, a subnormal growth rate . The experimental results support the view that cell adaptation to high osmolarity involves a decrease in the initiation frequency of DNA replication via a stringent response; in contrast, glycine betaine transport and accumulation could suppress the stringent response within 1-2 min and restore a higher initiation frequency . High osmolarity also triggers the cells to lengthen, perhaps via an inhibition of cellular division; glycine betaine also reverses this process . It is inferred that turgor could control DNA replication and cell division in two separate ways . Glycine betaine action is not mediated by K+ ions as the internal level of K+ ions is not modified significantly following glycine betaine accumulation. Zh Mikrobiol Epidemiol Immunobiol, 1988 Jan, (1), 8 - 11 {Kinetic analysis of the action of cetyltrimethylammonium bromide on the membranes of the capsular variant of Escherichia coli CA189 recorded by ethidium bromide fluorescence}; Puchkov EO et al.; A mathematical model, describing the kinetics of ethidium bromide probe fluorescence in the suspension of E . coli CA 189 capsular antigen after introduction of cetyltrimethylammonium bromide (a detergent), has been developed . Experimental testing of this mathematical model has confirmed that it describes the kinetics of the probe fluorescence augmentation sufficiently accurately . The new model permits the quantitative characterization of the rate of surfactant diffusion through the bacterial capsule. Nippon Geka Gakkai Zasshi, 1988 Jan, 89(1), 6 - 20 {Experimental study on the pathophysiology of endotoxin shock as analysed by alterations in thromboxane B2 and 6-keto-PGF1 alpha levels}; Baba H; To evaluate the pathophysiological role of thromboxane A2 (TXA2) in endotoxin shock, plasma concentrations of TXA2 and PGI2 following E . coli endotoxin (ET) administration were measured in dogs and rats by radioimmunoassay of their stable metabolites TXB2 and 6-keto-PGF1 alpha, respectively . Also, the effects of TXA2 synthetase inhibitor (OKY046) on eicosanoid levels, haemodynamics and survival were assessed . The following results were obtained: 1) Survival rates of the rats given 50 mg/kg of ET were 31% at 12 hrs and 17% at 24 hrs . Pretreatment with OKY046 markedly improved the survival rates . 2) Plasma concentrations of TXB2 were rapidly elevated in untreated control dogs and rats following ET administration, whereas plasma 6-keto-PGF1 alpha levels were gradually elevated . TXB2/6-keto-PGF1 alpha ratio showed an early elevation at 15 minutes after ET administration . The ratio became lower than base line, thereafter . 3) In contrast to the controls, animals pretreated with OKY046 did not exhibit significant elevations in plasma TXB2 levels . On the other hand, plasma levels of 6-keto-PGF1 alpha were not altered by OKY046 treatment . 4) In the control dogs given ET, the early elevations in pulmonary artery pressure (PAP) and reduction in lung compliance correlated with the early elevation in plasma TXB2/6-keto-PGF1 alpha ratio . 5) In OKY046-treated dogs, the early elevation in TXB2/6-keto-PGF1 alpha ratio was not seen and PAP increase and lung compliance reduction were prevented . The results suggest that TXA2 plays an important pathophysiological role in the development of endotoxin shock. Folia Microbiol (Praha), 1988, 33(1), 59 - 67 Mutation analysis of the receptor for colicins E1-E7 . A pilot study; Smarda J et al.; Thirty eight mutant clones of the colicin indicator strain Escherichia coli K 12 ROW, selected by their insensitivity to any of the colicins E1-E7, were isolated . Comparison of their sensitivity-resistance patterns to colicins E1-E7 enabled us to draw a rough preliminary map of the receptor for E colicins . In this receptor, the highly specific binding site for colicin E1 partially overlaps with the domain shared by all colicins E2 through E7 . A specific binding site of this domain appears to be common for colicins E3 and E6; a part of the E3 and E6 binding site is also common for colicins E4 and E5 and a small, least specific, part also for colicins E2 and E7 . Using colicin assay experiments, the binding capacity of colicin E receptor mutants could be estimated . A decreased, but not completely lost ability of certain mutants to bind colicins E, correlated to their lowered sensitivity to them, was found . Thus the phenomenon of partial colicin resistance was established, showing that colicin sensitivity--resistance is not a qualitative but a quantitative marker. EMBO J, 1988 Jan, 7(1), 269 - 75 Contacts between the LexA repressor--or its DNA-binding domain--and the backbone of the recA operator DNA; Hurstel S et al.; Using hydroxyl radical footprinting and ethylation interference experiments, we have determined the backbone contacts made by the entire LexA repressor and its amino-terminal fragment with the recA operator DNA . These techniques reveal essentially the same contacts between both proteins and one side of the DNA helix if one assumes that the DNA stays in the normal B-conformation . This result is somewhat unexpected because protection of guanine bases against methylation suggested a somewhat twisted recognition surface . The backbone contacts revealed by both methods are symmetrically disposed with respect to the center of the operator, providing further evidence that the operator binds two LexA monomers . Each half-operator contains seven interfering phosphates . These phosphates are found on both sides of the 5'-CTGT sequence that is believed to be the principal recognition target . On the side close to the center of the operator are found two phosphates, whereas the other five are clustered on the side apart from the dyad axis . We are not aware of such an extended cluster of interfering phosphates for any other DNA-binding protein . A quantification of the hydroxyl radical footprints allowed us to compare further the affinity of the LexA repressor for the recA operator with that of its isolated DNA binding domain . We find an only 13-fold higher binding constant for LexA than for its amino-terminal domain, which is in good agreement with our earlier results for the uvrA operator using a completely different binding assay. Mol Gen Mikrobiol Virusol, 1988 Jan, (1), 33 - 6 {Recombinations during DNA cloning}; Gurevich AI et al.; RecA-independent recombinations accompanying the processes of plasmids preparation and cloning into Escherichia coli cells are induced within the short direct and inverted repeats of several types. Genetika, 1988 Jan, 24(1), 13 - 22 {Genetic mapping of the ilv7434 mutation providing threonine deaminase resistance to isoleucine inhibition in Escherichia coli}; Gavrilova OF et al.; Location of previously isolated ilv7434 mutation was determined by use of transductional shortening of the F'14 episome . The ilv7434 mutation causes resistance of threonine deaminase (coded for by ilvA gene) to feed-back inhibition by isoleucine . Another phenotype characteristics of the ilv7434 mutant is the ability to feed a lawn of isoleucine auxotrophs in the cross-streak test . The F'14 strain AB1206 carrying ilv7434 mutation was used as a donor for making transductionally shortened episomes in recA recipient . These shortened F'14 episomes containing variable segments of the ilv cluster were then tested for their ability to transfer ilv7434 phenotype by complementation with ilv recA recipients . The data of complementation test suggest that ilv7434 is situated between ilvD and ilvC genes . One of 20 tested shortened episomes carrying, as shown by complementation test, incomplete ilvA gene was found to transfer ilv7434 phenotype by recombination with ilvA527 recA+ recipient . These data allow to conclude that ilv7434 mutation is located within the ilvA gene. Artery, 1988, 15(2), 71 - 89 Time-dependent effects of endotoxin on the ultrastructure of aortic endothelium; Lee MM et al.; The aortic endothelium from control and Escherichia coli (E . coli) endotoxin-treated rats and rabbits was examined by transmission and scanning electron microscopy . Following the intravenous injection of endotoxin, the animals were sacrificed at intervals ranging from 1 min to 4 hr . As early as 1 min after endotoxin, there was a widening of the subendothelial space (SES) and an increase in tortuosity of the internal elastic lamina (IEL) . At 5 min, the tortuosity of the IEL increased to a peak value, and the SES showed an increase in the amount of smooth muscle cells (SMC) . Initial endothelial damage occurred 5 min after endotoxin: SEM showed some spindle-shaped endothelial cells starting to peel from the underlying SES, and TEM showed some endothelial cells protruding or arching into the lumen . The new findings in this study are that endotoxin injection a) has a very rapid (less than 15 min) effect on rat and rabbit aortic endothelium, including localized endothelial injuries in the intima, and b) induces ultrastructural alterations also in the SES, IEL and portions of the tunica media . These effects were largely reversed within 1 hr after endotoxin administration, thus indicating that the endothelium and other components of the arterial wall can recover with great speeds. Am J Vet Res, 1988 Jan, 49(1), 90 - 5 Hemodynamics, plasma eicosanoid concentrations, and plasma biochemical changes in calves given multiple injections of Escherichia coli endotoxin; Templeton CB et al.; Twelve male neonatal calves (39 to 50 kg) were allotted to 3 groups of 4 calves each . All calves were anesthetized with halothane, and then Escherichia coli endotoxin was given intravenously (3 times) and intraperitoneally (3 times) during a 6-hour period . Group-1 calves were untreated, group-2 calves were pretreated with a low dose of flunixin meglumine (1.1 mg/kg of body weight), and group-3 calves were pretreated with a high dose of flunixin meglumine (4.4 mg/kg) . In calves of group 1, the mean systemic arterial blood pressure (MABP) and cardiac output (CO) decreased, but pulmonary arterial pressure increased after the initial intravenous and intraperitoneal injections of endotoxin . In calves of this group, these changes were accompanied by increased plasma thromboxane B2 (TxB2) concentration . During this period, increased plasma TxB2 concentration or hemodynamic changes were not detected in calves of groups 2 and 3 . Only calves of group 1 had altered hemodynamics early in the experiment; however, after 6 hours, calves of all 3 groups had similarly decreased CO and MABP . In calves of the untreated group, plasma 6-keto-prostaglandin (PG)F1 alpha concentration increased steadily from the beginning of the experiment until 3 hours later . The CO and MABP were low at the time when serum 6-keto-PGF1 alpha concentration was high; however, these 2 measurements also were low in treated calves who did not have correspondingly high plasma 6-keto-PGF1 alpha concentration . Regional blood flow analysis did not reveal correlations between prostanoid concentrations and altered blood flow to selected tissues.(ABSTRACT TRUNCATED AT 250 WORDS) Am J Vet Res, 1988 Jan, 49(1), 77 - 81 Lidocaine treatment of dogs with Escherichia coli septicemia; Hardie EM et al.; Effects of lidocaine on organ localization of neutrophils and bacteria and on hemodynamic and metabolic variables were determined during septic shock in dogs . Twelve anesthetized dogs were infused with 10(10) Escherichia coli/kg of body weight through a portal vein catheter over a 1-hour period . Six of these 12 dogs were treated with 2 mg of lidocaine HCl/kg (6 mg/kg/h) 15 minutes after the bacterial infusion had begun . Six dogs not given E coli (surgical controls) were given saline solution at the same rate as the bacterial and lidocaine infusions . Over 4 hours, nontreated dogs with septicemia developed systemic hypotension, decreased cardiac output, increased portal pressure, increased serum alanine transaminase activity, increased liver extravascular water, increased liver glycogen depletion, and decreased PaO2, compared with control dogs . Accumulations of polymorphonuclear leukocytes and E coli were found in the liver and lungs of dogs with septicemia . Lidocaine treatment did not alter the hemodynamic measurements and resulted in metabolic acidosis and hypoalbuminemia . Decreased numbers of E coli were recovered from the liver of lidocaine-treated dogs, whereas increased numbers of organisms were recovered from the blood . Neutrophil sequestration was increased in the liver, but not the lungs of lidocaine-treated dogs. J Appl Bacteriol, 1988 Jan, 64(1), 89 - 98 Evaluation of the Anderson Baird-Parker direct plating method for enumerating Escherichia coli in water; Havelaar AH et al.; The direct plating (DP) method for enumerating Escherichia coli in food was adapted for water analysis by membrane filtration and a standardized protocol was described . The DP method was found to give equal or better recoveries of E . coli than a membrane filtration method using 0.1% sodium lauryl sulphate agar; the repeatability of the DP method was markedly better . The necessity to transfer membranes from the non-selective medium tryptone soy agar (TSA) to the selective medium tryptone bile agar (TBA) after pre-incubation for 4 h was considered disadvantageous for practical purposes . A double-layer method, where the membrane filter is placed on a layer of TSA poured over TBA, with incubation in an incubator that automatically switches from 37 degrees to 44 degrees C after 4 h, was found to be an acceptable alternative . Recovery of E . coli and inhibition of competitive flora were equal or only slightly less than for the standard DP method. Biol Chem Hoppe Seyler, 1988 Jan, 369(1), 39 - 46 Active and inactive forms of hemolysin (HlyA) from Escherichia coli; Wagner W et al.; The HlyA protein (Mr 110 kDa) which is the gene product of the hlyA gene encoded by the hemolysin determinant of Escherichia coli (Goebel, W . & Hedgpeth, J . (1982) J . Bacteriol . 151, 1290-1298) was observed to accumulate in the culture supernatant (in the presence of the three other Hly proteins HlyC, B and D) throughout the active growth cycle . However, the amount of extracellular HlyA protein did not correlate with the external hemolytic activity, which declined when the cells entered the stationary phase . External hemolytic activity was highly sensitive to phospholipase C and to ultrasonication . The size of the HlyA protein on SDS-PAGE was not changed by these treatments although the hemolytic activity was entirely abolished . On a polyacrylamide gel containing 2M urea but only 0.1% SDS hemolytically active HlyA migrated slightly ahead of the inactive HlyA suggesting that HlyA is more negatively charged than HlyA . Active hemolysin from unconcentrated hemolytic supernatants migrated on Sephacryl S-400 and on glycerol gradients as large complexes . Analysis of the hemolytically active fractions on SDS-PAGE yielded in both cases only HlyA (110 kDA) as major protein . An internal hemolytic activity appeared in most Escherichia coli K-12 strains in the stationary phase which was independent of the presence of HlyA or any other Hly gene product . This hemolytic activity which reached in some strains about 10% of the level determined by the hly genes was sensitive to proteinase K and disappeared upon shift of the cells to the logarithmic phase. Environ Mol Mutagen, 1988, 11(2), 241 - 55 Adaptive response of Escherichia coli to alkylation damage; Volkert MR; Treatment of cells with low levels of alkylating agents for extended periods of time causes them to become resistant to the lethal and mutagenic effects of subsequent high-level challenge treatments with alkylating agents . This increased resistance has been called the adaptive response to alkylation damage and results from the induction of an alkylation-specific DNA repair response . The adaptive response is most efficiently induced by methylating agents and is most effective against the lethal and mutagenic effects of methylation damage to DNA . Four genes have been identified as components of this response, ada, alkA, alkB and aidB . The functions of two of these genes are known . AlkA protein functions as a glycosylase that repairs N3-meA, N3-meG, O2-meT, and O2-meC residues in DNA, and Ada protein functions as an alkyltransferase that removes alkyl groups from O6-meG, O4-meT residues as well as methylphosphotriesters . After it interacts with methylated DNA, Ada protein functions as a positive regulatory element that controls the expression of the adaptive response by stimulating the expression of the ada-alkB operon, and the alkA and aidB genes. J Clin Microbiol, 1988 Jan, 26(1), 92 - 5 Alkaline phosphatase-conjugated oligonucleotide probes for enterotoxigenic Escherichia coli in travelers to South America and West Africa; Oprandy JJ et al.; Several studies have demonstrated the usefulness of 32P-labeled recombinant DNA probes for identifying enterotoxigenic Escherichia coli (ETEC) . The use of radioisotopes and X-ray development, however, severely handicaps the utility of DNA probes in most clinical laboratories . In this study, enzyme-labeled oligonucleotide probes for ETEC LT (heat-labile toxin) and ST (heat-stable toxin) genes were compared with the standard Y1 adrenal cell and suckling mouse assays for their ability to identify ETEC in a population of American adults experiencing acute episodes of diarrhea in South America and West Africa . The LT probe hybridized with 12% (64 of 529) of the E . coli colonies tested, whereas 11% (57 of 529) were positive by Y1 adrenal cell assay . DNA from 9% (47 of 529) of the E . coli colonies tested hybridized with the ST probe, whereas only 5% (28 of 529) produced ST as measured by the suckling mouse bioassay . For the patient samples tested, correlation between probe and bioassay for LT was 97%, or three discrepancies in 111 patients tested . Overall concordance of the ST probe and bioassay was 95%, or five discrepancies in 111 patients . Enzyme-labeled oligonucleotide probes represent a major advance in the diagnosis of ETEC-associated diarrheal disease and may be used in laboratories with minimal equipment. J Clin Microbiol, 1988 Jan, 26(1), 149 - 50 Predominance of the ac variant in K88-positive Escherichia coli isolates from swine; Westerman RB et al.; Monoclonal antibodies to K88ac and K88ab were used in enzyme-linked immunosorbent assays on Escherichia coli cultures known to produce K88 pili . A total of 415 K88-positive E . coli isolates from nine states were all found to be the K88ac variant . The cultures tested were isolated during the years 1976 to 1985. J Clin Microbiol, 1988 Jan, 26(1), 116 - 20 Comparison of Western blot (immunoblot) based on recombinant-derived p41 with conventional tests for serodiagnosis of human immunodeficiency virus infections; Hofbauer JM et al.; To evaluate the performance of a serological test for human immunodeficiency virus type 1 (HIV-1) infections based on the use of a recombinant envelope gene-derived protein as the antigen, we caused expression of a 1.4-kilobase fragment of HIV.DNA that codes for the complete gp41 transmembrane protein in an Escherichia coli expression vector and used Western blots (WB; immunoblots) prepared with recombinant material (pEX-41) to detect antibodies to HIV-1 . This test detected all 339 sera which were positive by a combination of conventional serodiagnostic assays and produced no false-positive results with 311 negative samples . Also no false-positive results were obtained with 20 sera from systemic lupus erythematosus patients which had high titers of cross-reactive autoantibodies . In six cases, the pEX-41 WB proved to be more sensitive than individual assays applied on their own, and in five cases it was even more sensitive than a combination of conventional assays . We tested 221 sera in both our pEX-41 WB and a commercially available recombinant enzyme immunoassay (EIA {Abbott}) . The results were identical in 188 cases . A total of 27 sera containing antibodies to gp41 as demonstrated in the pEX-41 WB, as well as the Abbott recombinant EIA, had no antibodies to the recombinant core antigen as measured in the Abbott EIA . However, 25 of these sera did stain the 24-kilodalton band on a WB with purified virus . Six sera that were positive in all of the conventional confirmatory assays and reacted strongly with the pEX-41 WB did not recognize the surface antigen used in the Abbott recombinant EIA . We conclude that the use of WB prepared with recombinant-derived p41 offers a very sensitive and specific method to detect antibodies to HIV. Proc Natl Acad Sci U S A, 1988 Jan, 85(2), 401 - 5 Disulfide bonds and thermal stability in T4 lysozyme; Wetzel R et al.; Disulfide bonds are thought to serve a stabilizing role in extracellular globular proteins, but little is known about the modes of stabilization or their mechanisms . Thermodynamic data presented here demonstrate that an engineered 3-97 disulfide bond previously shown to stabilize T4 lysozyme in vitro against irreversible thermal inactivation also stabilizes the molecule against reversible thermal unfolding . In this paper, we explore the relationship between the disulfide's thermodynamic contribution to protein folding and its role in providing resistance to irreversible thermal inactivation . In T4 lysozyme (C54V/C97S), a non-crosslinked mutant lacking the two cysteines found in the wild type, sensitivity toward irreversible thermal inactivation increases dramatically at temperatures above the melting temperature of the molecule . In addition, most of the lost activity can be restored by denaturation/renaturation with guanidine hydrochloride . In contrast, the crosslinked mutant T4 lysozyme (13C-97C/C54V) inactivates relatively slowly, even above its melting temperature, and the lost activity is not restored by denaturation/renaturation . These observations suggest that the predominant inactivation pathways for non-crosslinked T4 lysozymes are conformation related, while those for the crosslinked variant are insensitive to the conformational route and thus are susceptible only to slower processes of a chemical nature . We also show that multiple mutants, constructed to contain the 3-97 disulfide plus a temperature-sensitive lesion, are more stable than the wild type to irreversible inactivation even though they are less stable to reversible thermal unfolding . These findings together suggest that the 3-97 disulfide provides stability to irreversible inactivation primarily via a pathway that is independent of its thermodynamic contribution . The 3-97 disulfide may stabilize T4 lysozyme by restricting the unfolded state to a class of more compact structures with less exposed hydrophobic surface, compared to the unfolded states of non-crosslinked T4 lysozymes . The results have implications both for the use of the stabilizing potential of disulfide bonds in protein engineering and for their roles in protein function and evolution. Proc Natl Acad Sci U S A, 1988 Jan, 85(1), 21 - 5 Characterization of the TPR-MET oncogene p65 and the MET protooncogene p140 protein-tyrosine kinases; Gonzatti-Haces M et al.; The proteins encoded by the human TPR-MET oncogene (p 65tpr-met) and the human MET protooncogene (p140met) have been identified . The p65tpr-met and p140met, as well as a truncated TPR-MET product expressed in Escherichia coli, p50met, are autophosphorylated in vitro on tyrosine residues . Using the immunocomplex kinase assay, p140met activity was detected in various human tumor epithelial cell lines . In vivo, p65tpr-met is phosphorylated on both serine and tyrosine residues, while p140met is phosphorylated on serine and threonine . p140met is labeled by cell-surface iodination procedures, suggesting that it is a receptor-like transmembrane protein-tyrosine kinase. Int Arch Allergy Appl Immunol, 1988, 85(1), 127 - 9 Cloning and expression of DNA coding for the major house dust mite allergen Der p 1 in Escherichia coli; Thomas WR et al.; A cDNA clone coding for the major house dust mite allergen Der p 1 was isolated from a lambda gt 11 library . Its sequence correlates with known amino acid sequences of Der p 1 and it produces a fusion protein which reacts with rabbit anti-Der p 1 antiserum. Am J Public Health, 1988 Jan, 78(1), 64 - 5 Hemolytic-uremic syndrome: a population-based study in Washington, DC and Baltimore, Maryland; Kinney JS et al.; A population-based study of hemolytic-uremic syndrome (HUS) revealed that 20 child residents of Washington, DC and Baltimore, Maryland were hospitalized with HUS from January 1979 through September 1983 . The number of cases peaked during the summer and fall; none occurred during the winter . Incidence of hospitalized cases was higher in Whites and girls than in Blacks or boys, and the average annual incidence was 1.08 cases/100,000 children less than 5 year old . This study demonstrates that HUS is not unique to the West Coast, as previously suggested. J Surg Res, 1988 Jan, 44(1), 54 - 61 Effect of glucan on neutrophil dynamics and immune function in Escherichia coli peritonitis; Williams DL et al.; Previous studies from our laboratory have demonstrated that glucan, a nonspecific immunomodulator, modifies the course of murine Escherichia coli peritonitis . The protective effect of glucan was mediated, in part, by macrophages . In the present study, leukocyte dynamics in the peritoneal cavity and peripheral blood of glucan-treated mice following E . coli challenge was examined . Additional studies examined in vitro bone marrow proliferation, as well as phagocytosis and intracellular killing of E . coli by neutrophils following glucan administration . ICR/HSD mice were injected ip with glucan (150 mg/kg) or dextrose (5% w/v) on Days 5 and 3 prior to ip challenge with 1 X 10(8) E . coli . Glucan increased (P less than 0.05) total peritoneal neutrophil numbers prior to and following septic challenge . Examination of peripheral blood revealed that ip glucan treatment in E . coli peritonitis significantly (P less than 0.001) increased the number of circulating neutrophils . Additionally, neutrophils from glucan-treated mice showed increased phagocytosis of E . coli in vitro . Glucan therapy also increased bone marrow proliferation . We conclude that (1) glucan enhances peritoneal neutrophil levels, (2) peripheral blood neutrophils are increased following glucan and E . coli, (3) ip glucan increases bone marrow proliferation, and (4) neutrophils from glucan-treated mice showed enhanced phagocytosis of E . coli in vitro . Thus, the beneficial effect of glucan is mediated not only by activated macrophages, but also by the neutrophilic leukocyte. J Med Microbiol, 1988 Jan, 25(1), 33 - 9 Experimental Escherichia coli peritonitis in immunosuppressed mice: the role of specific and non-specific immunity; Vuopio-Varkila J; An experimental Escherichia coli septicaemia-peritonitis model was adapted to immunosuppressed mice . The mice were made neutropenic by a sublethal dose of cyclophosphamide, which resulted in a 100-fold increase in their susceptibility to intraperitoneal injection of E . coli O18:K1 . A lethal infection could be prevented by passive immunisation with anti-K1 capsular or anti-O18 LPS antibodies but not with anti-J5 bacterial antibodies . The anti-K1 and anti-O18 antisera were able to increase the LD50 of the E . coli challenge by factors of 50 and 5, respectively . The role of non-specific, lipopolysaccharide (LPS)-mediated resistance to infection was also investigated in this model, in which only long-living phagocytic cells such as macrophages are believed to be functional . Pretreatment of mice with LPS was shown to prevent growth of the bacterial challenge in the peritoneal cavity and blood and to result in a five-fold increase in the LD50 of the challenge strain . These findings suggest an important role for macrophages as effector cells in defence against E . coli infection. J Med Chem, 1988 Jan, 31(1), 129 - 37 Evaluation of the importance of hydrophobic interactions in drug binding to dihydrofolate reductase; Taira K et al.; The interaction of dihydrofolate reductase (DHFR) from Escherichia coli with drugs such as methotrexate (MTX) and 2,4-diamino-6,7-dimethylpteridine (DAM) has been studied by means of site-directed mutagenesis, fluorescence spectroscopy, and steady-state as well as transient kinetics . A strictly conserved residue at the dihydrofolate binding site of DHFR, phenylalanine-31, has been replaced with tyrosine or valine to ascertain the importance for binding of this hydrophobic amino acid, which interacts with both the pteridine ring and the p-aminobenzoyl moiety . The first mutation (Phe-31----Tyr) has a minimal effect on the binding of the classical inhibitor, DAM . On the other hand, the second mutation (Phe-31----Val) has increased the dissociation constant of DAM from the DHFR.NADPH.DAM ternary complex over 150-fold (greater than 3 kcal/mol) . The dissociation constant of DAM from the (Val31-DHFR).DAM binary complex was too large to be measured fluorometrically . More importantly, these mutations have decreased the overall tight binding of MTX, from 100- to 140-fold (corresponding to a loss of binding energy of 2.2-2.4 kcal/mol) for the Tyr-31 and Val-31 mutants, respectively . These results indicate that hydrophobic interactions between MTX and DHFR are at least as important as formation of the MTX.DHFR salt bridge in the tight binding of MTX. J Infect Dis, 1988 Jan, 157(1), 149 - 55 Reliable detection of individuals seropositive for the human immunodeficiency virus (HIV) by competitive immunoassays using Escherichia coli-expressed HIV structural proteins; Dawson GJ et al.; We molecularly cloned the gag and env genes of the human immunodeficiency virus (HIV) and expressed fragments of these genes in Escherichia coli . Using the recombinant core and envelope proteins, we developed two competitive immunoassays (CIAs) . Samples that recognized either the envelope or core proteins were considered positive for antibodies to HIV . This test system was comparable with western blot in detecting antibodies in patients with AIDS or AIDS-related complex that were repeatably reactive in the HIV screening test . All 360 individuals who were positive by western blot were positive by the CIA . A total of 844 samples repeatably reactive by an ELISA screening test were negative both by western blot and by the CIA; 48 samples positive by ELISA, but negative or indeterminate by western blot, were positive by the CIA . Alternate research procedures verified the positivity of these individuals . These data indicate that the CIA described here may be useful as an adjunct or alternative to the western blot. J Comput Assist Tomogr, 1988 Jan-Feb, 12(1), 118 - 21 CT detection of sacral osteomyelitis associated with pelvic abscesses; Merine D et al.; In three patients the diagnosis of sacral osteomyelitis was made when CT demonstrated intraosseous (two) and intraforaminal (one) gas . Two of the three patients also had radionuclide bone scans, one of which was unremarkable . In the other case, radionuclide scintigraphy greatly underestimated the extent of the disease process when compared with CT . All three patients had contiguous pelvic abscesses as a cause of the osteomyelitis . Although there was a high clinical suspicion for an intraabdominal process, the diagnosis of superimposed osteomyelitis of the sacrum was unsuspected . The detection of intraosseous gas is a pathognomonic, albeit uncommon, manifestation of osteomyelitis . Although the radionuclide bone scan is the method of choice for detecting osteomyelitis, CT should be used as a complementary study in certain patients. J Bacteriol, 1988 Jan, 170(1), 56 - 64 Nucleotide sequence of the iucD gene of the pColV-K30 aerobactin operon and topology of its product studied with phoA and lacZ gene fusions; Herrero M et al.; Gene iucD of the aerobactin operon of the Escherichia coli plasmid ColV-K30 encodes a membrane-bound enzyme synthesizing N6-hydroxylysine, the first product of the aerobactin biosynthesis pathway . The entire nucleotide sequence of the cloned iucD gene was determined, from which the primary and some aspects of the secondary structure of the encoded peptide were deduced . E . coli cells harboring multicopy plasmid pVLN12 (iucD+) hyperproduced an approximately 50-kilodalton peptide which was purified and identified as the product of the gene by examination of its amino-terminal sequence . Two iucD'-'lacZ gene fusions were constructed in vitro and four iucD'-'phoA gene fusions were generated in vivo by mutagenesis of iucD with transposon TnphoA (Tn5 IS50L::phoA) . Analysis of the corresponding fusion proteins suggested at least two domains of attachment of the IucD protein to the inner side of the cytoplasmic membrane . The first apparent membrane-bound domain was found within the first 25 amino acids of the protein and showed a sequence which resembled that of the signal peptides. J Bacteriol, 1988 Jan, 170(1), 485 - 8 Flagellin domain that affects H antigenicity of Escherichia coli K-12; Kuwajima G; Escherichia coli K-12 mutants with altered flagellum antigenicity were isolated by introducing random deletions into the flagellin gene . The deletions were identified in the central region of the gene . It is suggested that this region corresponds to the flagellin domain molecule which affects flagellum antigenicity. J Bacteriol, 1988 Jan, 170(1), 463 - 7 Genetic and molecular characterization of the guaC-nadC-aroP region of Escherichia coli K-12; Roberts RE et al.; The guaC (GMP reductase), nadC (quinolinate phosphoribosyltransferase), and aroP (aromatic amino acid permease) genes of Escherichia coli K-12 were located in the 2.5-min region of the chromosome (muT-guaC-nadC-aroP-aceE) by a combination of linkage analysis, deletion mapping, restriction analysis, and plasmid subcloning . The guaC locus expressed a product of Mr 37,000 with a clockwise transcriptional polarity, and the GMP reductase activities of guaC+ plasmid-containing strains were amplified 15- to 20-fold. J Bacteriol, 1988 Jan, 170(1), 456 - 8 Sufficiency of the Klenow fragment for survival of polC(Ts) pcbA1 Escherichia coli at 43 degrees C; Bryan SK et al.; Escherichia coli cells with the pcbA1 allele and temperature-sensitive DNA polymerase III mutations survived at a restrictive temperature if DNA polymerase I activity was present . The Klenow fragment of DNA polymerase I was capable of supporting cell survival as well. J Bacteriol, 1988 Jan, 170(1), 416 - 21 Metabolism of L-fucose and L-rhamnose in Escherichia coli: aerobic-anaerobic regulation of L-lactaldehyde dissimilation; Baldoma L et al.; L-Lactaldehyde is a branching point in the metabolic pathway of L-fucose and L-rhamnose utilization . Under aerobic conditions, L-lactaldehyde is oxidized to L-lactate by the enzyme lactaldehyde dehydrogenase, while under anaerobic conditions, L-lactaldehyde is reduced to L-1,2-propanediol by the enzyme propanediol oxidoreductase . Aerobic growth on either of the methyl pentoses induces a lactaldehyde dehydrogenase enzyme which is inhibited by NADH and is very stable under anaerobic conditions . In the absence of oxygen, the cell shifts from the oxidation of L-lactaldehyde to its reduction, owing to both the induction of propanediol oxidoreductase activity and the decrease in the NAD/NADH ratio . The oxidation of L-lactaldehyde to L-lactate is again restored upon a change to aerobic conditions . In this case, only the NAD/NADH ratio may be invoked as a regulatory mechanism, since both enzymes remain active after this change . Experimental evidence in the presence of rhamnose with mutants unable to produce L-lactaldehyde and mutants capable of producing but not further metabolizing it points toward L-lactaldehyde as the effector molecule in the induction of lactaldehyde dehydrogenase . Analysis of a temperature-sensitive mutation affecting the synthesis of lactaldehyde dehydrogenase permitted us to locate an apparently single regulator gene linked to the ald locus at 31 min and probably acting as a positive control element on the expression of the structural gene. J Bacteriol, 1988 Jan, 170(1), 330 - 4 DNA sequence of the D-serine deaminase activator gene dsdC; Palchaudhuri S et al.; We have determined the DNA sequence of dsdC, the gene that encodes the D-serine deaminase activator protein of Escherichia coli K-12 . The sequence contains a single open reading frame that terminates in a UGA codon . One the basis of the size of the protein, 33 kilodaltons, and the amino acid sequence encoded by the open reading frame, we identified a likely translation initiation codon 731 base pairs upstream of the translation initiation codon for the divergently transcribed D-serine deaminase gene . There is a broad range of codon usage, not surprising in view of the weak expression of the gene . The N-terminal two-thirds of the activator is arginine-lysine rich and quite polar; the remainder is more neutral . The segment of the protein that seems most likely to have potential to form the helix-turn-helix structure characteristic of DNA-regulatory proteins is located near the end of the polar region . The protein contains a region with significant homology to lambda attB. J Bacteriol, 1988 Jan, 170(1), 308 - 15 Isolation and characterization of an Escherichia coli K-12 mutant deficient in glutaredoxin; Kren B et al.; Mutants of Escherichia coli K-12 deficient in glutaredoxin were isolated and partially characterized . The mutants have detectable but significantly reduced glutaredoxin activity in assays of whole cells made permeable with ether as well as in assays of crude extracts coupled to ribonucleotide reductase . In vivo, the mutants appear to be deficient in both sulfate and ribonucleotide reduction, suggesting that in vivo glutaredoxin is the preferred cofactor for ribonucleotide reductase and adenosine 3'-phosphate 5'-phosphosulfate reductase . Complementation of the mutant phenotype by transformants was used to clone the wild-type glutaredoxin allele . The transformants had a high level of glutaredoxin activity and contained a plasmid with an insert that had a restriction endonuclease pattern identical to that predicted by the DNA sequence for glutaredoxin determined by Hoog et al . (J.-O . Hoog, H . von Bahr-Lindstrom, H . Jornvall, and A . Holmgren, Gene 43:13-21, 1986). Infect Immun, 1988 Jan, 56(1), 71 - 8 Genetic and physicochemical characterization of the recombinant DNA-derived 47-kilodalton surface immunogen of Treponema pallidum subsp . pallidum; Chamberlain NR et al.; Previous work has established the importance of the 47-kilodalton (kDa) surface immunogen of Treponema pallidum subsp . pallidum (T . pallidum) in the immunopathogenesis of syphilis; the 47-kDa immunogen gene was cloned and expressed in Escherichia coli (M . V . Norgard, N . R . Chamberlain, M . A . Swancutt, and M . S . Goldberg, Infect . Immun . 54:500-506, 1986) . To facilitate additional structural-functional analysis of this protein for immunopathogenesis studies, the recombinant DNA-derived molecule was examined with respect to its genetic expression and physicochemical properties . Subcloning of partial PstI digests of the original 47-kDa antigen-encoding DNA segment localized the 47-kDa antigen gene to a 1.3-kilobase (kb) T . pallidum DNA fragment . A 20- to 100-fold enhanced expression of the 47-kDa antigen was obtained when a 2.85-kb DNA insert containing the entire 1.3-kb structural gene was subcloned into a T7 RNA polymerase-dependent expression vector system . Under these conditions, several derivatives of the recombinant 47-kDa protein possessing different molecular m