Mol Gen Genet, 1989 Jun, 217(2-3), 317 - 23 High levels of manganese-containing superoxide dismutase and thermally induced DNA disruption in a dnaK7(Ts) mutant of Escherichia coli K12; Taniguchi H et al.; In dnaK7(Ts) mutant cells, scission of DNA strands occurred after temperature shift up . When cells at 30 degrees C were labeled with {3H}-thymidine and then shifted to 46 degrees or 49 degrees C for 20 min, the profiles of sedimentation of their cellular DNA in an alkaline sucrose gradient revealed a decrease in the size of DNA to a quarter of that at 30 degrees C in the mutant, but not in wild-type cells . The level of manganese-containing superoxide dismutase (MnSOD) in the mutant was about twice that in wild-type cells, even at the permissive temperature, implying increased production of superoxide radical anion, which may cleave DNA strands directly or indirectly in the mutant . Moderate increase in the MnSOD level on temperature shift up was observed in both strains . These results indicated that some components of the DnaK protein participate in protection of cellular membrane functions from thermal damage resulting from elevated production of the superoxide anion radical. Mol Gen Genet, 1989 Jun, 217(2-3), 301 - 8 Vitamin B12 transport in Escherichia coli K12 does not require the btuE gene of the btuCED operon; Rioux CR et al.; Transport of vitamin B12 across the cytoplasmic membrane of Escherichia coli requires the products of btuC and btuD, two genes in the btuCED operon . The role of btuE, the central gene of this operon, was examined . Deletions within btuE were constructed by removal of internal restriction fragments and were crossed onto the chromosome by allelic replacement . In-frame deletions that removed 20% or 82% of the btuE coding region did not affect expression of the distal btuD gene . These nonpolar deletions had little effect on vitamin B12 binding (whole cells or periplasmic fraction) and transport . They did not affect the utilization of vitamin B12 or other cobalamins for methionine biosynthesis, even in strains with decreased outer membrane transport of vitamin B12 . The btuE mutations did not impair adenosyl-cobalamin dependent catabolism of ethanolamine or repression of btuB expression . Thus, despite its genetic location in the transport operon, the btuE product plays no essential role in vitamin B12 transport. Mol Gen Genet, 1989 Jun, 217(2-3), 289 - 93 Cloning and molecular characterization of the gene rimL which encodes an enzyme acetylating ribosomal protein L12 of Escherichia coli K12; Tanaka S et al.; The rimL gene of Escherichia coli K12 encodes an enzyme catalyzing the acetylation of the N-terminal serine of ribosomal protein L12, thereby converting it into L7 . Using a mutant strain defective in this acetylation reaction, we cloned the rimL gene into cosmid pHC79 and characterized it at the molecular level . From analysis by SDS-polyacrylamide gel electrophoresis of the proteins synthesized in maxi-cells containing derivatives of the rimL-harboring plasmid into which transposon gamma delta had been inserted at various sites, the product of this gene was identified as a protein with an apparent molecular weight of 20.3 kDa . The nucleotide sequence of the gene and the amino acid sequence deduced from the nucleotide sequence were compared with those of two other ribosomal protein acetylases encoded by the rimI and rimJ genes (Yoshikawa et al . 1987) . A considerable degree of overall similarity was seen between rimL and rimJ, but the degree of similarity between rimL and rimI was very low . In addition, a short stretch of similar amino acid sequence was found in all three rim acetylases . The significance of these results with respect to other acetylating enzymes, in particular those involved in the acetylation of aminoglycoside antibiotics is discussed. Mol Gen Genet, 1989 Jun, 217(2-3), 278 - 80 Dam methylated and hemimethylated oriC plasmids are replicated symmetrically; a novel and general test of replication symmetry; Hughes P et al.; Deoxyadenosine methylation (dam) of the numerous GATC sequences present in the Escherichia coli origin of chromosomal replication (oriC) has been shown to be important both in vivo and in vitro for efficient initiation of DNA synthesis . Recent in vivo data suggest that initiation is only inefficient when these sequences are hemimethylated . This raises the interesting possibility that initiation may be inefficient because it only takes place on one strand of the template, i.e., replication is asymmetric on hemimethylated DNA . We tested this possibility by a novel and rapid approach which relies on the specificities of the restriction endonucleases MboI, MboII and DpnI . Although we show that replication takes place equally well on both strands of methylated and hemimethylated oriC DNA templates, the method should be applicable to the analysis of replication symmetry on most DNA templates which contain methylated deoxyadenosine GATC sequences as part of MboII restriction sites. Mol Gen Genet, 1989 Jun, 217(2-3), 254 - 6 Genetic requirements for hyper-recombination by very short patch mismatch repair: involvement of Escherichia coli DNA polymerase I; Dzidic S et al.; It has been established that very short patch (VSP) mismatch repair, depending in Escherichia coli on MutL, MutS and Dcm functions, is responsible for the hyper-recombinogenic effect of a class of genetic markers . We show that VSP repair requires the presence of the complete DNA polymerase I enzyme . The absence of endonuclease activities involved in the repair of base-loss sites, Nth, Nfo and Xth, does not affect VSP repair . Implications for the mechanism of the VSP repair are discussed. Genetics, 1989 Jun, 122(2), 269 - 78 Genetic and physical analysis of plasmid recombination in recB recC sbcB and recB recC sbcA Escherichia coli K-12 mutants; Luisi-DeLuca C et al.; The effect of mutations in known recombination genes (recA, recB, recC, recE, recF, recJ, recN, recO, recQ and ruv) on intramolecular recombination of plasmids was studied in recB recC sbcB and recB recC sbcA Escherichia coli mutants . The rate of recombination of circular dimer plasmids was at least 1000-fold higher in recB recC sbcB or recB recC sbcA mutants as compared to wild-type cells . The rate was decreased by mutations in recA, recF, recJ, recO, ruv or mutS in recB recC sbcB mutants, and by mutations in recE, recN, recO, recQ, ruv or mutS in recB recC sbcA mutants . In addition to measuring the recombination rate of circular dimer plasmids, the recombination-mediated transformation of linear dimer plasmids was also studied . Linear dimer plasmids transformed recB recC sbcB and recB recC sbcA mutants 20- to 40-fold more efficiently than wild-type cells . The transformation efficiency of linear dimer plasmids in recB recC sbcB mutants was decreased by mutations in recA, recF, recJ, recO, recQ or lexA (lexA3) . In recB recC sbcA mutants the transformation efficiency of linear dimers was decreased only by a recE mutation . Physical analysis of linear dimer- or circular dimer-transformed recB recC sbcB mutants revealed that all transformants contained recombinant monomer genotypes . This suggests that recombination in recB recC sbcB cells is very efficient. Can J Microbiol, 1989 Jun, 35(6), 670 - 3 Binding specificities of heat-labile enterotoxins isolated from porcine and human enterotoxigenic Escherichia coli for different gangliosides; Sugii S et al.; The binding specificities of heat-labile enterotoxins (LTp and LTh) isolated from porcine and human enterotoxigenic Escherichia coli on human erythrocytes were studied by competitive binding assays using different gangliosides as inhibitors . The binding of 125I-labeled LTp to neuraminidase-treated human type A erythrocytes was most effectively inhibited by ganglioside GM1 . Ganglioside GM1 was 11 and 105 times more potent than gangliosides GD1b and GM2, respectively . Gangliosides GD1a, GT1b, and GM3 were much less potent . Similar results were also obtained in competitive binding assays with the 125I-labeled B subunit of LTh and neuraminidase-treated human type B erythrocytes, and in those with 3H-labeled ganglioside GM1 and LTp-coupled Sepharose 4B . The binding of 3H-labeled ganglioside GM1 to LTp was not effectively inhibited by galactose-beta(1----3)N-acetyl-D-galactosamine at the highest concentration used . These findings suggest that the combining sites of LTp and LTh may be specific for at least the galactose-N-acetyl-D-galactosamine-galactose (N-acetyl-neuraminic acid) portion of ganglioside GM1. Trends Biochem Sci, 1989 Jun, 14(6), 233 - 7 Molecular dissection of a transfer RNA and the basis for its identity; Hou YM et al.; The recognition of transfer RNAs (tRNAs) by aminoacyl tRNA synthetases establishes the connection between amino acids and trinucleotides . However, for E . coli alanine tRNA the trinucleotide sequence which specifies alanine is not important for recognition . Instead a single base pair is a major determinant for the identity of this tRNA . Even a synthetic RNA microhelix with seven base pairs can be aminoacylated if it includes the major determinant. J Virol Methods, 1989 Jun, 24(3), 321 - 6 Detection of antibodies against hepatitis B core antigen using the avidin-biotin system; Korec E et al.; An enzyme avidin-biotin assay for the detection of anti-HBcAg antibody in human sera was developed . The assay uses genetically engineered HBcAg . HBcAg is immobilized on the surface of the wells of microtitre plates and the test serum sample, biotin-labelled HBcAg and streptavidin-labelled horseradish peroxidase are added . The assay was found to be specific and was compared with a commercial radioimmunoassay kit for sensitivity by testing 96 human clinical sera for anti-HBcAg antibody . Both assays gave identical results. J Virol Methods, 1989 Jun, 24(3), 275 - 83 Discrimination of HIV-1 and HIV-2 infection based on recombinant immunoblots; Baur A et al.; Recombinant polypeptides representing various parts of structural proteins of HIV-1 and HIV-2 were expressed in E . coli . Fragments of the transmembrane proteins gp41 of HIV-1 (HTLV-IIIB) and gp38 of HIV-2 (LAV-2 ROD) proved to be highly antigenic in the immunoblot test system . Each protein was found to be suitable for detection and differentiation of antibodies in sera of patients infected with HIV-1 or HIV-2 . Sera of 100 patients from West-Germany, which were confirmed as positive for HIV-1 antibodies, reacted clearly with the HIV-1 recombinant antigen (fpOE-6); no or only weak immune reactions were seen when the analogous recombinant HIV-2 antigen (fpOE-T) was used in the same immunoblot test . An inverse reaction pattern was found with 7 sera from Africa which, by conventional means, were proved to be HIV-2 antibody positive . These sera specifically reacted with the homologous HIV-2 fusion protein and no or only weakly stained bands were identified on the analogous strip with the HIV-1 antigen (fpOE-6). FEMS Microbiol Lett, 1989 Jun, 50(3), 319 - 23 Prevalence of CS31A and F165 surface antigens in Escherichia coli isolates from animals in France, Canada and India; Contrepois M et al.; Bovine and porcine enterotoxigenic and non-enterotoxigenic Escherichia coli isolates from France, Canada, and India were characterized with respect to serogroup and production of fimbrial antigens CS31A and F165 . Of 231 bovine isolates from the 3 countries, 20.5% produced CS31A alone, 17.7% produced F165 alone, and 17.3% produced both CS31A and F165 . On the other hand, of 84 porcine isolates from Canada, 1.2% produced CS31A alone, 14.3% produced F165 alone, and no isolate produced both CS31A and F165 . CS31A was found together with F5 (K99) in 7 of 16 bovine enterotoxigenic E . coli isolates of serogroups 08, 09, 020, and 023, but was not found in any of 20 F4 (K88)- or 5 F6 (987P)-positive porcine enterotoxigenic E . coli isolates . F165 was not found in enterotoxigenic E . coli . Among non-enterotoxigenic isolates, CS31A and F165 were mainly associated with serogroups 08, 09, 011, 015, 017, 023, 025, 078, 0101, 0115, 0117, 0141, and 0153. Alcohol Clin Exp Res, 1989 Jun, 13(3), 407 - 12 Ethanol administration diminishes the endotoxin-induced increase in glucose metabolism; Molina PE et al.; The metabolism of ethanol (ETOH) is known to increase the cytosolic NADH/NAD ratio and consequently impairs hepatic glucose output in the fasted state . In contrast, one of the characteristic alterations in glucose metabolism produced by the administration of endotoxin is an increase in the de novo synthesis of glucose . Therefore, the present study tests the hypothesis that the acute administration of ETOH will prevent the endotoxin-induced increase in glucose production . In vivo glucose kinetics were determined by the infusion of {6-3H, U-14C}glucose in catheterized conscious rats . The intravenous infusion of tracer glucose, and ETOH (100 mg/100 g b.w./hr) or saline were started at the same time and both continued throughout the experiment . Two hours later the ETOH infusion rate was decreased to maintain the blood ETOH levels between 100 and 160 mg/dl . At 140 min, endotoxin (100 micrograms/100 g b.w.) was injected . ETOH alone did not alter basal values of plasma glucose (5 mM), glucose rate of appearance (Ra; 35 mumols/min/kg) or metabolic clearance (MCR; 7 ml/min/kg) . Endotoxin alone increased plasma glucose (80%) and lactate (140%) concentrations, glucose Ra (60%) and recycling (40%) in saline-infused rats, whereas in ETOH-infused animals, plasma glucose and lactate levels were only elevated 40% and glucose Ra and recycling were unchanged . The results show that acute ETOH administration diminishes the increased glucose production and utilization seen in endotoxemia . The attenuation of the endotoxin effect by ethanol is due to inhibition of hepatic glucose production and peripheral glucose utilization. Mol Microbiol, 1989 Jun, 3(6), 839 - 41 The ribonucleoside diphosphate reductase gene (nrdA) of Escherichia coli carries a repetitive extragenic palindromic (REP) sequence in its 3' structural terminus; Merino E et al.; A computer search for repeated sequences led us to identify five REP (repetitive extragenic palindromic) sequences in the 3'-terminal region of the Escherichia coli ribonucleoside diphosphate reductase gene (nrdA) . These REP sequences are located within a putative duplicated DNA region, the first of them being part of the carboxy-terminal coding region of the nrdA gene . This is the first report of a REP sequence within a structural gene and also the first example of a REP sequence apparently generated by DNA duplication. Mol Microbiol, 1989 Jun, 3(6), 825 - 38 The Azorhizobium caulinodans nitrogen-fixation regulatory gene, nifA, is controlled by the cellular nitrogen and oxygen status; Ratet P et al.; The nucleotide sequence of the Azorhizobium caulinodans ORS571 nifA locus was determined and the deduced NifA amino acid sequence compared with that of NifA from other nitrogen-fixing species . Highly conserved domains, including helix-turn-helix and ATP-binding motifs, and specific conserved residues, such as a cluster of cysteines, were identified . The nifA 5' upstream region was found to contain DNA sequence motifs highly homologous to promoter elements involved in nifA/ntr-mediated control and a consensus element found in the 5' upstream region of the Bradyrhizobium japonicum 5-aminolevulinic acid synthase (hemA) gene and of Escherichia coli genes activated during anaerobiosis via the fnr (fumarate nitrate reduction) control system . A nifA-lac fusion was constructed using miniMu-lac and its activity measured in different genetic backgrounds and under various physiological conditions (in culture and in planta) . NifA expression was found to be negatively autoregulated, repressed by rich nitrogen sources and high oxygen concentrations, and controlled (partially) by the ntrC gene, both in culture and in planta . DNA supercoiling was also implicated in nifA regulation, since DNA gyrase inhibitors severely repressed nifA-lac expression. J Interferon Res, 1989 Jun, 9(3), 295 - 304 Production of two human 2',5'-oligoadenylate synthetase enzymes in Escherichia coli; Mory Y et al.; We have isolated and characterized two types of cDNA clones corresponding to interferon (IFN)-induced 1.6- and 1.8-kb mRNAs, as encoding two different forms of the 2',5'-oligoadenylate (2'-5')A synthetase enzyme . Direct expression of the two cDNAs was obtained in Escherichia coli under the control of a trp-lac hybrid promoter strongly inducible in E . coli by IPTG . Bacterial extracts were tested for 2'-5'A synthetase activity after adsorption to immobilized poly(I).poly(C) or in solution . With either one of the cDNA constructions, IPTG induced 2'-5'A synthetase activity in the bacteria to levels 10 times higher per microgram of protein than those in SV80 cells treated by 500 U/ml of IFN-beta1 for 24 h . Both bacterially produced enzymes bind to double-stranded (ds)RNA and are maximally active at 100 micrograms/ml of poly(I).poly(C) . Both enzymes synthesized similar 2'-5'(Ap)nA oligomers of 2 to 8 residues in length . Antibodies against a synthetic peptide common to the two enzymes were used to characterize the bacterial products on immunoblots and confirmed that the 1.6-kb RNA produces a 39-kD protein, whereas the 1.8-kb RNA encodes a 45- to 46-kD protein . The E . coli enzyme coded by the 1.6-kb mRNA was purified to nearly homogeneity . When immobilized on poly(I).poly(C) agarose, the enzyme produces, per milliliter of poly(I).poly(C), 10(3) times more 2'-5'(Ap)nA oligomer than the most active cellular extracts . Moreover, the immobilized enzyme remains stable for several months at 4 degrees C. J Appl Physiol, 1989 Jun, 66(6), 2553 - 8 Critical O2 delivery to skeletal muscle at high and low PO2 in endotoxemic dogs; Bredle DL et al.; An ischemic canine limb model was used to determine whether endotoxin reduces the ability of resting skeletal muscle to extract O2 and whether increasing the arterial PO2 would increase its O2 extraction . Isolated limbs were pump perfused via an extracorporeal circuit with membrane oxygenator at three progressively lower flows and PO2 of both 60 and 200 Torr, whereas the rest of the body remained normoxic and normotensive . Six anesthetized, paralyzed dogs were injected with endotoxin (4 mg/kg, ENDO), and another six were controls (CONT) . Limb critical O2 delivery was higher (P less than 0.05) in ENDO than CONT (8.3 vs . 6.1 ml.kg-1.min-1) . Critical venous PO2 was also higher (P less than 0.05) in ENDO than CONT (38 vs . 30 Torr) . Critical O2 extraction ratio was lower (P less than 0.05) in ENDO than CONT (0.60 vs . 0.73) . There were no differences in these variables between low and high arterial PO2 . We concluded that 1) endotoxin can cause a small but significant O2 extraction defect in skeletal muscle, 2) increasing arterial PO2 did not correct such a defect, nor did it improve O2 uptake in ischemic, but otherwise healthy, muscle, and 3) skeletal muscle may contribute to the peripheral O2 extraction defect in adult respiratory distress syndrome insofar as endotoxin effects model those found in adult respiratory distress syndrome. Helv Paediatr Acta, 1989 Jun, 43(5-6), 443 - 8 Authentic recombinant human growth hormone . Results of a multicenter clinical trial in patients with growth hormone deficiency; Rasmussen LH et al.; 197 patients with growth hormone deficiency (144 boys, 53 girls, age 1.5-19.5 {mean 11 +/- 3.6}, bone age 0.3-15 {mean 8.9 +/- 3.5} years) were treated for one year with authentic recombinant human growth hormone (r-hGH, Norditropin} in several European paediatric centers . 107 patients were newly treated (group A), and 90 transferred from pituitary or methionine hGH (groups B and C) . In 19 of the latter, treatment was interrupted for 6 months (group B), in the others, it was changed without interruption (group C) . The dosage was 0.45 +/- 0.2 IU/kg/week given s.c . 6-7 times a week . Height velocity increased from 4.1 +/- 2.4 to 8.3 +/- 2.5 (group A), 2.6 +/- 1.8 to 8.3 +/- 2.3 (group B), and 6.2 +/- 2.7 to 6.8 +/- 2.2 cm/year (group C) . Few patients complained of local discomfort at the injection site, but this disappeared after changing metacresol in the solvent to 0.9% benzyl alcohol . Only 3 patients developed antibodies to hGH in low titers, which did not interfere with growth . No changes in E . coli protein antibodies were observed . It is concluded that r-hGH is an efficient and safe treatment for children with growth hormone deficiency. Circ Shock, 1989 Jun, 28(2), 89 - 100 Plasma proteolysis and circulating cells in relation to varying endotoxin concentrations in porcine endotoxemia; Naess F et al.; Ten juvenile pigs receiving a continuous infusion of 0.01 mg/kg of endotoxin over 3 hr and seven animals infused with sterile saline (serving as controls) were studied for 5 hr . Endotoxin concentrations in plasma as determined with a chromogenic Limulus amoebocyte lysate (LAL) test reached a steady state of about 1,000 ng/liter after 1 hr and declined rapidly as the infusion was discontinued . Preinfusion values were reached at the end of the observation period . Endotoxin concentrations found during the infusion period were comparable with those seen in humans with septicemia . The endotoxin infusion was followed by hemoconcentration, leukocytopenia, and thrombocytopenia . Using chromogenic peptide substrate assays, activation of the plasma kallikrein-kinin, fibrinolytic, and coagulation systems was detected . Although the endotoxin concentrations reached preinfusion values within the last 2 hr of the observation period, changes found in circulating cells and components of the plasma cascade systems did not normalize, and the hemodynamic situation did not change. Blut, 1989 Jun, 58(6), 287 - 90 Adult respiratory distress syndrome in neutropenic leukemia patients; Vansteenkiste JF et al.; Seven episodes of adult respiratory distress syndrome, occurring in leukemic patients with longstanding (average 11 days) and severe neutropenia (less than 0.1 x 10(9)/1) are described . Pathological and clinical data give further support to the view that the complement-neutrophil pathway is not the only mechanism in generating clinical ARDS in leukemia patients. Proc Natl Acad Sci U S A, 1989 Jun, 86(12), 4465 - 9 T5 DNA polymerase: structural--functional relationships to other DNA polymerases; Leavitt MC et al.; T5 DNA polymerase, a highly processive single-polypeptide enzyme, has been analyzed for its primary structural features . The amino acid sequence of T5 DNA polymerase has a high degree of homology with that of DNA polymerase I from Escherichia coli and retains many of the amino acid residues that have been implicated in the 3'----5' exonuclease and DNA polymerase activities of that enzyme . Alignment with sequences of polymerase I and T7 DNA polymerase was used to identify regions possibly involved in the high processivity of this enzyme . Further, amino acid sequence comparisons of T5 DNA polymerase with a large group of DNA polymerases previously shown to exhibit little similarity to polymerase I indicate certain sequence segments are shared among distantly related DNA polymerases . These shared regions have been implicated in the 3'----5' exonuclease function of polymerase I, which suggests that the proofreading domains of all these enzymes may be evolutionarily related. J Med Microbiol, 1989 Jun, 29(2), 139 - 44 4-quinolones and the SOS response; Lewin CS et al.; The SOS DNA repair system is induced in bacteria treated with 4-quinolones . However, whether the response exacerbates or repairs the damage caused by these drugs is still unclear . The recA13 and the recB21 mutations impair recombination repair and render bacteria unable to induce the SOS response when treated with nalidixic acid or other agents that affect DNA synthesis . However, UV treatment induces the SOS response in recB21 mutants but not in recA13 mutants . Both these mutants are hypersensitive to nalidixic acid and, therefore, either recombination repair or SOS repair would appear to repair DNA damage caused by the drug . However, since the lexA3 mutation (which also renders bacteria incapable of inducing the SOS response without affecting recombination repair) had no effect on the susceptibility of bacteria to nalidixic acid, the SOS response neither contributes to nor repairs DNA damage caused by the drug . Consequently, it would seem that the hypersensitivity of the recA13 and recB21 mutants to nalidixic acid is due to their deficiency in recombination repair . This view was confirmed by testing a recA430 mutant that is recombination-repair proficient but SOS repair-deficient and finding it to be no more sensitive to nalidixic acid than its parent . Thus it would appear that, although induced by nalidixic acid treatment, the SOS DNA repair system does not play any role in bacterial responses to the damage caused by the drug . In contrast, the recombination repair system does repair damage caused by nalidixic acid. J Gen Virol, 1989 Jun, 70 ( Pt 6), 1337 - 45 The transcription termination region of the adenovirus 2 major late transcript contains multiple functional elements; Dressler GR et al.; In order to understand the process of transcription termination by eukaryotic RNA polymerase II, the transcription termination region of the advenovirus 2 major late transcription unit was analysed in a transient transfection system . Previously, it had been demonstrated that the entire sequence from map units (m.u.) 97.1 to 100 of the adenovirus 2 genome terminates transcription when inserted into the 5' or 3' untranslated sequences of the chloramphenicol acetyltransferase gene . Using subclones and Bal 31 deletion mutants of the termination region, we have shown that the termination region consists of multiple elements each capable of inhibiting gene expression independently . A DNA sequence analysis reveals the presence of a highly repetitive A-rich sequence motif throughout the entire termination region . The data suggest that the A-rich motif may mediate the transcription termination process. Eur J Biochem, 1989 Jun 1, 182(1), 125 - 8 Covalent cofactor binding to flavoenzymes requires specific effectors; Brandsch R et al.; Modification by covalent FAD attachment to a histidine residue via an 8 alpha-(N3-histidyl)-riboflavin linkage occurs in several flavoenzymes . Among them is 6-hydroxy-D-nicotine oxidase (6-HDNO) of Arthrobacter oxidans and the flavoprotein subunits of the fumarate reductase and succinate dehydrogenase complex of Escherichia coli and other bacterial and eukaryotic cells . We found that 6-HDNO holoenzyme formation from apo-6-HDNO, monitored by {14C}FAD incorporation and increase in enzyme activity, can be mediated not only by phosphoenolpyruvate {Nagursky, H., Bichler, V . and Brandsch, R . (1988) Eur . J . Biochem . 177, 319-325}, but also by one of the glycolytic intermediates glyceraldehyde-3-P, glycerate-3-P, or the intermediate in glycerol utilization by bacteria, glycerol-3-P . Apoflavoprotein of fumarate reductase and succinate dehydrogenase was obtained in an E . coli riboflavin-requiring strain (E . coli RR28rf) overexpressing the frdABCD or the sdhCDAB operon from the recombinant plasmids pGS39 and pGS141, respectively . In extracts obtained from these cells, flavoprotein flavinylation, analyzed as covalent {14C}FAD incorporation into the apoflavoprotein polypeptide by polyacrylamide gel electrophoresis and fluorography, was stimulated severalfold by the citric acid cycle intermediates citrate, isocitrate, succinate and fumarate . Our results suggest that covalent modification and thus activation of these enzymes is dependent on specific metabolic intermediates which may act as allosteric effectors in the reaction. Clin Chem, 1989 Jun, 35(6), 946 - 52 Development of a confirmatory enzyme-linked immunosorbent assay for HIV-1 antibodies; Chiang CS et al.; We subcloned six discrete protein-coding regions representing the gag (Kp24 and Kp55), env (Kp41, Kp120N, and Kp120CC), and pol (Kp66/31) gene products of the human immunodeficiency virus type 1 (HIV-1) and expressed them in Escherichia coli as fusion proteins with the first 56 residues of galactokinase . An enzyme-linked immunosorbent assay for confirming the presence of HIV-1 antibodies was developed by coating the six purified antigens on individual wells of a microtiter plate (the HIVAGEN assay) . This assay yielded no false-negative results and fewer indeterminate results than the Western blot assay for 143 specimens from patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC) . Analysis of 1016 specimens from seronegative donors by the HIVAGEN assay yielded no false-positive results, and the rate of indeterminate results was substantially lower than for the Western blot assay . The HIVAGEN assay is well suited for routine confirmation of the presence of HIV-1 antibodies because it is objective, quantitative, rapid, precise, and readily automatable. Arch Surg, 1989 Jun, 124(6), 727 - 32 The effect of endotoxin on glucose metabolism in skeletal muscle requires the presence of plasma; Amaral JF et al.; The administration of endotoxin in vivo results in an increase in glucose utilization through an as yet undetermined mechanism . This study evaluated (1) the contribution of blood to the increased glucose utilization noted following endotoxemia, (2) the direct action of endotoxin on skeletal muscle glucose uptake in an isolated hindlimb perfusion system and in incubated muscle, and (3) the possibility that the increased glucose uptake in skeletal muscle mediated by endotoxin requires the presence of plasma . Incubation of blood with 50 and 100 mg/L of endotoxin increased glucose uptake and lactate production in a dose-dependent manner . Muscle incubations and perfusions in the absence of plasma and white blood cells showed that glucose uptake and lactate production were not affected by the presence of 50 to 250 mg/L of endotoxin, while 500 mg/L of endotoxin produced a 26.2% decrease in glucose uptake . In contrast, incubation of muscle in the presence of plasma and endotoxin increased glucose uptake by 37% . These findings suggest that (1) the increased glucose utilization of endotoxemia is only partially explained by increased glucose metabolism by blood, (2) endotoxin does not have a direct effect on the glucose uptake of skeletal muscle, and (3) an interaction of endotoxin with a component of plasma is required for an endotoxin-mediated increase in glucose utilization by skeletal muscle. Virology, 1989 Jun, 170(2), 362 - 9 Proteolytic activity of the plum pox potyvirus NIa-like protein in Escherichia coli; Garcia JA et al.; The nucleotide sequence of the small nuclear inclusion protein (NIa)-like cistron of plum pox potyvirus (PPV) has been determined . Viral proteolytic activity was expressed in Escherichia coli cells harboring plasmids with a PPV cDNA insert approximately 7000 nt long . Free PPV capsid protein was detected in these cells, but it was not produced when a mutation was introduced in the PPV cDNA insert which induced a Gln to Pro substitution at the large nuclear inclusion protein (NIb)-capsid protein junction . By mutational analysis, the NIa-like protein was determined to be responsible for the proteolytic activity . A Gln to Ser substitution at the presumed NIa-NIb junction, which inhibited proteolytic processing at the carboxyl end of the protease, had no effect on proteolytic cleavage at the NIb-capsid protein junction . In contrast with the high efficiency of proteolytic processing at the NIb-capsid protein cleavage site, processing at the ends of the PPV protease was not complete, suggesting that the PPV polyprotein, like that of other potyviruses, contains cleavage sites with different properties. Proc Natl Acad Sci U S A, 1989 Jun, 86(11), 4147 - 51 Single adduct mutagenesis: strong effect of the position of a single acetylaminofluorene adduct within a mutation hot spot; Burnouf D et al.; 2-Acetylaminofluorene (AAF), a potent rat liver carcinogen that binds covalently to the C-8 position of guanine residues in DNA, is an effective frameshift mutagen . The mutations are distributed nonrandomly, in that most are located at a few specific DNA sequences (i.e., mutation hot spots) . Among these hot spots, the Nar I sequence (GGCGCC) is especially susceptible to the induction of -2 frameshift mutations (GGCGCC----GGCC) . Due to the nature of the Nar I sequence, G1G2CG3CC, three different molecular events, each involving the deletion of two contiguous base pairs (i.e., G2C, CG3, G3C), can give rise to the observed end point (GGCC) . To compare the potential role of each of the three possible guanine-AAF adducts in the Nar I site to induce the -2 frameshift mutation, we constructed double-stranded plasmid molecules containing a single-AAF adduct bound to one of the three guanine positions . Using these plasmids, we found that only the adduct in the G3 position induces the -2 frameshift mutation . This strong effect of the position of the -AAF adduct within the Nar I site is discussed in relation to the possible involvement of an unusual DNA conformation in the mutagenic processing. Proc Natl Acad Sci U S A, 1989 Jun, 86(11), 4052 - 5 Site-directed mutagenesis of the phosphocarrier protein . IIIGlc, a major signal-transducing protein in Escherichia coli; Presper KA et al.; The glucose-specific phosphocarrier protein (IIIGlc) of the bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) is a major signal transducer that mediates the intricate interplay among extracellular signals (PTS and non-PTS sugars), cytoplasmic and membrane proteins (PTS and non-PTS transporters), and adenylate cyclase . To further define the central role of IIIGlc in these multiplex signaling mechanisms, we have used site-directed mutagenesis to construct three mutant IIIGlc proteins containing single amino acid changes; Phe-3 was replaced with tryptophan {( Trp3}IIIGlc), and His-75 and the active-site His-90 were replaced with glutamine {( Gln75}IIIGlc and {Gln90}IIIGlc, respectively) . {Trp3}IIIGlc resembles the wild-type protein in most properties and should be valuable for spectrophotometric experiments . In contrast, clear differences between mutant and wild-type proteins were observed with both {Gln75}IIIGlc and {Gln90}IIIGlc in in vitro sugar phosphorylation assays . As predicted, {Gln90}IIIGlc with a modified active site cannot be phosphorylated . Unexpectedly, {Gln75}IIIGlc accepts but cannot transfer phosphoryl groups, suggesting His-75 may also be a critical amino acid for IIIGlc-mediated signaling mechanisms . The physiological effects of these mutations are briefly described. Proc Natl Acad Sci U S A, 1989 Jun, 86(11), 3992 - 6 A five-residue sequence near the carboxyl terminus of the polytopic membrane protein lac permease is required for stability within the membrane; Roepe PD et al.; The lac permease (lacY gene product) of Escherichia coli contains 417 amino acid residues and is predicted to have a short hydrophilic amino terminus on the inner surface of the cytoplasmic membrane, multiple transmembrane hydrophobic segments in alpha-helical conformation, and a 17-amino acid residue hydrophilic carboxyl-terminal tail on the inner surface of the membrane . To assess the importance of the carboxyl terminus, the properties of several truncation mutants were studied . The mutants were constructed by site-directed mutagenesis such that stop codons were placed at specified positions, and the altered lacY genes were expressed at a relatively low rate from plasmid pACYC184 . Permease truncated at position 407 or 401 retains full activity, and a normal complement of molecules is present in the membrane, as judged by immunoblot analyses . Thus, it is apparent that the carboxyl-terminal tail plays no direct role in membrane insertion of the permease, its stability, or in the mechanism of lactose/H+ symport . In marked contrast, when truncations are made at residues 396 (i.e., 4 amino acid residues from the carboxyl terminus of putative helix XII), 389, 372, or 346, the permease is no longer found in the membrane . Remarkably, however, when each of the mutated lacY genes is expressed at a high rate by means of the T7 RNA polymerase system {Tabor, S . & Richardson, C . C . (1985) Proc . Natl . Acad . Sci . USA 82, 1074-1079}, all of the truncated permeases are present in the membrane, as indicated by {35S}methionine incorporation studies; however, permease truncated at residue 396, 389, 372, or 346 is defective with respect to lactose/H+ symport . Finally, pulse-chase experiments indicate that wild-type permease or permease truncated at residue 401 is stable, whereas permease truncated at or prior to residue 396 is degraded at a significant rate . The results are consistent with the notion that residues 396-401 in putative helix XII are important for protection against proteolytic degradation and suggest that this region of the permease may be necessary for proper folding. Proc Natl Acad Sci U S A, 1989 Jun, 86(11), 3982 - 6 Incision by UvrABC excinuclease is a step in the path to mutagenesis by psoralen crosslinks in Escherichia coli; Sladek FM et al.; 4,5',8-Trimethylpsoralen (psoralen) plus near UV light produces interstrand crosslinks and monoadducts in DNA, both of which are mutagenic . In Escherichia coli, crosslinks are incised by UvrABC excinuclease, an event that can lead to homologous recombination and repair . To determine whether UvrABC incision of crosslinks is a step in the path to mutagenesis as well as repair, the effect of DNA homologous to a target gene on a plasmid was determined . pSV2-gpt DNA was treated with psoralen and transformed into a pair of hosts: one was gpt+, the other was delta (gpt-lac)5 . The DNA was extracted and transformed into a tester strain {delta (gpt-lac)5} in which Gpt- mutations in the plasmid were scored . The results show that psoralen-induced mutations were reduced to background levels by the presence of the gpt+ homolog in the host chromosome . delta gpt hosts that were constitutively induced for the SOS response yielded point mutations, whereas noninduced hosts yielded almost exclusively large deletions . Since crosslinks were estimated to be responsible for most of the mutations observed, we conclude that the premutagenic lesion of psoralen crosslinks is recombinagenic and therefore very likely to be the product of UvrABC incision. Proc Natl Acad Sci U S A, 1989 Jun, 86(11), 3973 - 7 Activation of prophage P4 by the P2 Cox protein and the sites of action of the Cox protein on the two phage genomes; Saha S et al.; Phage P2 induces the unrelated prophage P4 . In this paper we show that this is due to the activation of the P4 late promoter PII by the P2 Cox protein . This is in contrast to the effects of Cox on P2, for which it is known from previous work that it acts as a repressor of the promoter Pc, which is responsible for expression of the immunity repressor C . The activator role of Cox was revealed by its effect on replication of P4 DNA and on the formation of chloramphenicol acetyltransferase when a promoterless cat gene was inserted downstream of the P4 PII promoter . DNase I protection studies revealed that the Cox protein binds to the repressor promoter Pc of phage P2 and to the promoter PII of phage P4 . In the latter case the Cox protein binds upstream of the -35 region, in analogy to several other activators of promoters . A weak binding was found in the promoters Pe of phage P2 and Ple of phage P4 . The Cox protein is a case of viral transactivation of the replication genes of one phage by a control protein of the other . However, the effects of the Cox protein are totally different in the two phages, repressive in one case and activating in the other. Proc Natl Acad Sci U S A, 1989 Jun, 86(11), 3949 - 52 A specific role of MutT protein: to prevent dG.dA mispairing in DNA replication; Akiyama M et al.; Occurrence of the transversion mutation A.T to C.G is specifically enhanced in Escherichia coli mutT mutants . With the aid of the cloned mutT gene, the MutT protein, which has a molecular mass of 15 kilodaltons, was overproduced and purified to near homogeneity . The protein catalyzes hydrolysis of dGTP to dGMP . dGDP and GTP were also hydrolyzed by the protein, but at a lower rate than seen with dGTP . No other deoxynucleoside triphosphates were hydrolyzed . Using poly(dA).(dT)20 as a template-primer, we investigated the misincorporation of dGMP, dCMP, and dAMP by the alpha subunit and the core of E . coli DNA polymerase III . When the polymerization reaction was performed with the alpha subunit, both dCMP and dGMP were misincorporated . The core, composed of alpha, epsilon, and theta subunits, misincorporated only dGMP . This would imply that the proofreading function of the epsilon subunit of DNA polymerase III may correct the dC.dA mispair but not the dG.dA mispair . Misincorporation of dAMP was not observed in reactions with the alpha subunit or core . The misincorporation of dGMP, but not dCMP, was almost completely suppressed by adding purified MutT protein to the reaction mixture . Under these conditions, only a portion of dGTP present in the reaction mixture was degraded . It is therefore likely that the MutT protein may prevent dGMP misincorporation by degrading a specific form of dGTP, probably the syn form, which can pair with deoxyadenosine. J Virol, 1989 Jun, 63(6), 2457 - 60 Artificial cleavage site recognized by plum pox potyvirus protease in Escherichia coli; Garcia JA et al.; A synthetic plum pox virus (PPV) NIb-CP cleavage site was recognized by a PPV protease in an in vivo Escherichia coli expression system . The presence of the natural NIb-CP cleavage site did not affect processing at the artificial one . However, although both the proteases and the cleavage sites of PPV and tobacco etch virus show high sequence homology, a similar cassette from the tobacco etch virus NIb-CP junction was not efficiently recognized by the PPV protease. J Bacteriol, 1989 Jun, 171(6), 3583 - 5 Intergeneric conjugation between Escherichia coli and Streptomyces species; Mazodier P et al.; We have constructed Escherichia coli-Streptomyces shuttle plasmids which are capable of conjugal transfer from E . coli to Streptomyces spp . These plasmids contained the pBR322 and pIJ101 origins of replication and the RK2 (IncP) origin of transfer . The transfer of plasmid was specifically dependent the presence of a 760-base-pair, cis-acting, oriT-containing fragment and on RP4 (IncP) functions supplied in trans . Conditions of mating and selection of exconjugants were analyzed with Streptomyces lividans as recipient . Plasmid transfer to other Streptomyces species was also demonstrated. J Bacteriol, 1989 Jun, 171(6), 3494 - 503 Nucleotide sequence of the FNR-regulated fumarase gene (fumB) of Escherichia coli K-12; Bell PJ et al.; The nucleotide sequence of a 3,162-base-pair (bp) segment of DNA containing the FNR-regulated fumB gene, which encodes the anaerobic class I fumarase (FUMB) of Escherichia coli, was determined . The structural gene was found to comprise 1,641 bp, 547 codons (excluding the initiation and termination codons), and the gene product had a predicted Mr of 59,956 . The amino acid sequence of FUMB contained the same number of residues as did that of the aerobic class I fumarase (FUMA), and there were identical amino acids at all but 56 positions (89.8% identity) . There was no significant similarity between the class I fumarases and the class II enzyme (FUMC) except in one region containing the following consensus: Gly-Ser-Xxx-Ile-Met-Xxx-Xxx-Lys-Xxx-Asn . Some of the 56 amino acid substitutions must be responsible for the functional preferences of the enzymes for malate dehydration (FUMB) and fumarate hydration (FUMA) . Significant similarities between the cysteine-containing sequence of the class I fumarases (FUMA and FUMB) and the mammalian aconitases were detected, and this finding further supports the view that these enzymes are all members of a family of iron-containing hydrolyases . The nucleotide sequence of a 1,142-bp distal sequence of an unidentified gene (genF) located upstream of fumB was also defined and found to encode a product that is homologous to the product of another unidentified gene (genA), located downstream of the neighboring aspartase gene (aspA). J Bacteriol, 1989 Jun, 171(6), 3458 - 64 Cloning and expression of Thiobacillus ferrooxidans mercury ion resistance genes in Escherichia coli; Shiratori T et al.; A search of various domestic isolates of Thiobacillus ferrooxidans revealed that some were fairly resistant to mercury ion . A proportion of mercury-resistant clones were able to volatilize mercury, and their corresponding gene was localized not in the plasmid DNA but in chromosomal DNA . This mercury ion resistance gene was cloned in Escherichia coli . E . coli carrying the recombinant plasmid was able to grow in the presence of more than 40 micrograms of HgCl2 per ml . Deletion analysis of the recombinant plasmid showed that the entire coding sequence of the mercury ion resistance gene was located within a 2.3-kilobase fragment of the chromosomal DNA from strain E-15 . At least two polypeptides (molecular mass, 56 and 16 kDa, respectively) were coded by this fragment. J Bacteriol, 1989 Jun, 171(6), 3373 - 8 Two proteins encoded at the chlA locus constitute the converting factor of Escherichia coli chlA1; Pitterle DM et al.; Molybdopterin (MPT) is not produced by the Escherichia coli mutants chlA1, chlM, or chlN or by the Neurospora crassa mutant nit-1 . Extracts of E . coli chlA1 contain an activity, the converting factor, which is functionally defined by its ability to convert a low-molecular-weight precursor present in crude extracts of N . crassa nit-1 into molybdopterin in vitro . In this study, it has been shown that the converting factor consists of two associative proteins (10 and 25 kilodaltons {kDa}) which can be separated by using either anion-exchange or gel filtration chromatography . Neither protein is able to complement extracts of nit-1 by itself . Analysis of chlA Mu insertion mutants has shown that the two proteins are distinct gene products encoded at the chlA locus . Twelve chlA Mu insertion strains which lacked converting factor activity were deficient in one or both of the proteins . Converting factor activity could be generated by mixing extracts from strains having the 25-kDa protein with those having the 10-kDa protein but not those lacking both proteins . Finally, it was shown that the chlM mutant lacks the 10-kDa protein while the chlN mutant, which contains both the 10- and 25-kDa proteins, lacks a function required to activate the 10-kDa protein. J Bacteriol, 1989 Jun, 171(6), 3348 - 53 Alp, a suppressor of lon protease mutants in Escherichia coli; Trempy JE et al.; Escherichia coli lon mutants lack a major ATP-dependent protease, are sensitive to UV light and methylmethane sulfonate (MMS), and overproduce capsular polysaccharide . Evidence is presented that an activity (Alp), cloned on a multicopy plasmid, can suppress the phenotypes of lon mutants . The sensitivity to UV and MMS is a reflection of the stabilization of the cell division inhibitor SulA, while the capsule overproduction arises through the stabilization of a transcriptional activator of capsule biosynthetic genes, RcsA . Multicopy alp (pAlp) suppressed capsule formation in delta lon cells, and delta lon cells containing the pAlp plasmid were resistant to MMS treatment . The MMS resistance of delta lon pAlp+ cells correlates with an increase in the degradation of SulA to that found in lon+ cells . Lon-directed degradation of SulA was energy dependent, as was the increase in degradation of SulA in delta lon pAlp+ cells . alp maps close to pheA, at 57 min on the E . coli chromosome . Although pAlp can substitute for Lon, cells lacking alp activity did not have the phenotype on a lon mutant . This study demonstrates that at least one activity, when overproduced in the cell, can substitute for Lon protease. J Bacteriol, 1989 Jun, 171(6), 3337 - 42 Multiple control mechanisms for pyrimidine-mediated regulation of pyrBI operon expression in Escherichia coli K-12; Liu CG et al.; Expression of the pyrBI operon of Escherichia coli K-12, which encodes the subunits of the pyrimidine biosynthetic enzyme aspartate transcarbamylase, is negatively regulated over a several-hundredfold range by pyrimidine availability . This regulation occurs, at least in large part, through a UTP-sensitive attenuation control mechanism in which transcriptional termination at the pyrBI attenuator, a rho-independent transcriptional terminator located immediately upstream of the pyrB structural gene, is regulated by the relative rates of transcription and translation within the pyrBI leader region . There is suggestive evidence that an additional, attenuator-independent control mechanism also contributes to this regulation . To measure the level of regulation that occurs through the attenuation and attenuator-independent control mechanisms, we constructed a mutant strain in which a 9-base-pair deletion was introduced into the attenuator of the chromosomal pyrBI operon . This deletion, which removes the run of thymidine residues at the end of the attenuator, completely abolishes rho-independent transcriptional termination activity . When the mutant strain was grown under conditions of pyrimidine excess, the level of operon expression was 51-fold greater than that of an isogenic pyrBI+ strain . Under conditions of pyrimidine limitation, operon expression was increased an additional 6.5-fold in the mutant . These results demonstrate that the attenuation control mechanism is primarily responsible for pyrimidine-mediated regulation but that there is a significant contribution by an attenuator-independent control mechanism. J Bacteriol, 1989 Jun, 171(6), 3282 - 7 Variations in UDP-N-acetylglucosamine and UDP-N-acetylmuramyl-pentapeptide pools in Escherichia coli after inhibition of protein synthesis; Mengin-Lecreulx D et al.; The pool levels of the nucleotide precursors of peptidoglycan were analyzed after inhibition of protein synthesis in various Escherichia coli strains . In all cases UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pentapeptide) cell pools increased upon treatment with chloramphenicol or tetracycline . Similar results were observed after the treatment of K-12 strains with valine . Since the intermediate nucleotide precursors did not accumulate after the arrest of protein synthesis and since a feedback mechanism was unlikely, the increases of the UDP-MurNAc-pentapeptide pool appeared as a consequence of that of the UDP-GlcNAc pool by the unrestricted functioning of the intermediate steps of the pathway . The highest increase (sixfold) of UDP-GlcNAc was observed with strain K-12 HfrH growing in minimal medium and treated with chloramphenicol . When a pair of isogenic Rel+ and Rel- strains were considered, both the UDP-GlcNAc and UDP-MurNAc-pentapeptide pools increased upon treatment with chloramphenicol or valine . However, the UDP-GlcNAc pool of the Rel+ strain was at a high natural level, which increased only moderately (20%) after the addition of valine . The increase of the UDP-GlcNAc pool after the various treatments could be due to an effect on some upstream step by an unknown mechanism . The possible correlations of the variations of the precursor pools with the rate of synthesis and extent of cross-linking of peptidoglycan were also considered. J Bacteriol, 1989 Jun, 171(6), 3233 - 46 Genetic and physiological relationships among the miaA gene, 2-methylthio-N6-(delta 2-isopentenyl)-adenosine tRNA modification, and spontaneous mutagenesis in Escherichia coli K-12; Connolly DM et al.; The miaA tRNA modification gene was cloned and located by insertion mutagenesis and DNA sequence analysis . The miaA gene product, tRNA delta 2-isopentenylpyrophosphate (IPP) transferase, catalyzes the first step in the biosynthesis of 2-methylthio-N6-(delta 2-isopentenyl)-adenosine (ms2i6A) adjacent to the anticodon of several tRNA species . The translation start of miaA was deduced by comparison with mod5, which encodes a homologous enzyme in yeasts . Minicell experiments showed that Escherichia coli IPP transferase has a molecular mass of 33.5 kilodaltons (kDa) . Transcriptional fusions, plasmid and chromosomal cassette insertion mutations, and RNase T2 mapping of in vivo miaA transcription were used to examine the relationship between miaA and mutL, which encodes a polypeptide necessary for methyl-directed mismatch repair . The combined results showed that miaA, mutL, and a gene that encodes a 47-kDa polypeptide occur very close together, are transcribed in the same direction in the order 47-kDa polypeptide gene-mutL-miaA, and likely form a complex operon containing a weak internal promoter . Three additional relationships were demonstrated between mutagenesis and the miaA gene or ms2i6A tRNA modification . First, miaA transcription was induced by 2-aminopurine . Second, chromosomal miaA insertion mutations increased the spontaneous mutation frequency with a spectrum distinct from mutL mutations . Third, limitation of miaA+ bacteria for iron, which causes tRNA undermodification from ms2i6A to i6A, also increased spontaneous mutation frequency . These results support the notion that complex operons organize metabolically related genes whose primary functions appear to be completely different . In addition, the results are consistent with the idea that mechanisms exist to increase spontaneous mutation frequency when cells need to adapt to environmental stress. J Bacteriol, 1989 Jun, 171(6), 3053 - 9 Genetic reconstitution of the high-affinity L-arabinose transport system; Horazdovsky BF et al.; Expression plasmids containing various portions of araFGH operon sequences were assayed for their ability to facilitate the high-affinity L-arabinose transport process in a strain lacking the chromosomal copy of this operon . Accumulation studies demonstrated that the specific induction of all three operon coding sequences was necessary to restore high-affinity L-arabinose transport . Kinetic analysis of this genetically reconstituted transport system indicated that it functions with essentially wild-type parameters . Therefore, L-arabinose-binding protein-mediated transport appears to require only two inducible membrane-associated components (araG and araH) in addition to the binding protein (araF). J Bacteriol, 1989 Jun, 171(6), 3046 - 52 Gene conversion in Escherichia coli: the recF pathway for resolution of heteroduplex DNA; Fishel R et al.; The independent repair of mismatched nucleotides present in heteroduplex DNA has been used to explain gene conversion and map expansion after general genetic recombination . We have constructed and purified heteroduplex plasmid DNAs that contain heteroallelic 10-base-pair insertion-deletion mismatches . These DNA substrates are similar in structure to the heteroduplex DNA intermediates that have been proposed to be produced during the genetic recombination of plasmids . These DNA substrates were transformed into wild-type and mutant Escherichia coli strains, and the fate of the heteroduplex DNA was determined by both restriction mapping and genetic tests . Independent repair events that yielded a wild-type Tetr gene were observed at a frequency of approximately 1% in both wild-type and recB recC sbcB mutant E . coli strains . The independent repair of small insertion-deletion-type mismatches separated by 1,243 base pairs was found to be reduced by recF, recJ, and ssb single mutations in an otherwise wild-type genetic background and reduced by recF, recJ, and recO mutations in a recB recC sbcB genetic background (the ssb mutation was not tested in the latter background) . Independent repair of small insertion-deletion-type mismatched nucleotides that were as close as 312 nucleotides apart was observed . There was no apparent bias in favor of the insertion or deletion of mutant sequences. J Bacteriol, 1989 Jun, 171(6), 2975 - 80 Escherichia coli dnaT gene function is required for pBR322 plasmid replication but not for R1 plasmid replication; Masai H et al.; Plasmid pBR322 was unable to replicate in a temperature-sensitive dnaT1 strain at a nonpermissive temperature, whereas a pBR322-derived plasmid carrying the wild-type dnaT+ gene was able to replicate under the same conditions . In contrast to pBR322, plasmid R1 could replicate in the dnaT1 strain at a nonpermissive temperature . In keeping with this finding, in vitro replication of plasmid R1 did not require DnaT protein. J Bacteriol, 1989 Jun, 171(6), 2963 - 9 Cloning of a Thermomonospora fusca xylanase gene and its expression in Escherichia coli and Streptomyces lividans; Ghangas GS et al.; Thermomonospora fusca chromosomal DNA was partially digested with EcoRI to obtain 4- to 14-kilobase fragments, which were used to construct a library of recombinant phage by ligation with EcoRI arms of lambda gtWES . lambda B . A recombinant phage coding for xylanase activity which contained a 14-kilobase insert was identified . The xylanase gene was localized to a 2.1-kilobase SalI fragment of the EcoRI insert by subcloning onto pBR322 and derivatives of pBR322 that can also replicate in Streptomyces lividans . The xylanase activity produced by S . lividans transformants was 10- to 20-fold higher than that produced by Escherichia coli transformants but only one-fourth the level produced by induced T . fusca . A 30-kilodalton peptide with activity against both Remazol brilliant blue xylan and xylan was produced in S . lividans transformants that carried the 2.1-kilobase SalI fragment of T . fusca DNA and was not produced by control transformants . T . fusca cultures were found to contain a xylanase of a similar size that was induced by growth on xylan or Solka Floc . Antiserum directed against supernatant proteins isolated from a Solka Floc-grown T . fusca culture inhibited the xylanase activity of S . lividans transformants . The cloned T . fusca xylanase gene was expressed at about the same level in S . lividans grown in minimal medium containing either glucose, cellobiose, or xylan . The xylanase bound to and hydrolyzed insoluble xylan . The cloned xylanase appeared to be the same as the major protein in xylan-induced T . fusca culture supernatants, which also contained at least three additional minor proteins with xylanase activity and having apparent molecular masses of 43, 23, and 20 kilodaltons. J Bacteriol, 1989 Jun, 171(6), 2919 - 24 Identification of the enzymatic basis for delta-aminolevulinic acid auxotrophy in a hemA mutant of Escherichia coli; Avissar YJ et al.; The hemA mutation of Escherichia coli K-12 confers a requirement for delta-aminolevulinic acid (ALA) . Cell extract prepared from the hemA strain SASX41B was incapable of producing ALA from either glutamate or glutamyl-tRNA, whereas extract of the hem+ strain HB101 formed colorimetrically detectable amounts of ALA and transferred label from 1-{14C}glutamate and 3,4-{3H}glutamyl-tRNA to ALA . Extracts of both strains converted glutamate-1-semialdehyde to ALA and were capable of aminoacylating tRNAGlu . Glutamyl-tRNA formed by extracts of both strains could be converted to ALA by the extract of hem+ cells . The extract of hemA cells did not convert glutamyl-tRNA formed by either strain to ALA . However, the hemA cell extract, when supplemented in vitro with glutamyl-tRNA dehydrogenase isolated from Chlorella vulgaris cells, formed about as much ALA as did the unsupplemented hem+ cell extract . We conclude from these observations that the enzyme activity that is lacking in the ALA auxotrophic strain carrying the hemA mutation is that of glutamyl-tRNA dehydrogenase. Gynecol Oncol, 1989 Jun, 33(3), 392 - 4 Infectious pneumoperitoneum as an uncommon presentation of endometrial carcinoma: report of two cases; Douvier S et al.; Two patients with pneumoperitoneum are reported; in both cases, the cause was severe infection of the upper genital tract . Investigation led to the finding of an underlying endometrial carcinoma . Literature review of the etiology of pneumoperitoneum, nature of the usual infecting organisms, and therapeutic principles are presented . Endometrial carcinoma should be considered in the differential diagnosis of infectious pneumoperitoneum, especially where patient risk factors are present. Biochim Biophys Acta, 1989 Jun 1, 1008(1), 45 - 51 In vitro replication and mutagenesis of ColE1 plasmid DNA in extracts from repair deficient Escherichia coli mutants; Casaregola S et al.; We have investigated conditions in vitro for the analysis of replication of ultraviolet-irradiated ColE1 DNA in cell extracts from Escherichia coli . In wild-type extracts substantial replication was obtained; however, this could be greatly reduced when the irradiated plasmid was incubated in extracts prepared from a uvrA recB strain . Modest stimulation of DNA replication was then obtained by addition of extracts from the same strain previously ultraviolet-irradiated . However, this stimulating activity proved to be highly unstable and has so far proved unsuitable as a basis for purification of specific factors involved in replication on irradiated templates . We also investigated the mutagenesis of pBR325 DNA replicated in cell extracts from a strain expressing the SOS response constitutively . Conditions for efficient recovery and transformation by plasmid DNA replicated in vitro were determined and, using this system, a more than 10-fold increase in reversion frequency of a mutation in the tet gene, compared to that with wild-type extracts, was obtained . This mutagenesis appeared to be independent of replication, indicating the presence of an error-prone repair system in the extract . This effect was not enhanced by the presence of the muc gene products in the extracts . This suggests that the observed mutagenesis is also independent of the lexA-controlled umuCD genes. Mol Gen Genet, 1989 Jun, 217(2-3), 281 - 8 Characterization of the gene rimK responsible for the addition of glutamic acid residues to the C-terminus of ribosomal protein S6 in Escherichia coli K12; Kang WK et al.; Ribosomal protein S6 of wild-type strains of Escherichia coli contains up to six glutamic acid residues at its C-terminus . The first two residues are encoded by the structural gene for this protein (rpsF) and the rest are added post-translationally . Mutants deficient in this modification were isolated and characterized genetically and biochemically . The S6 protein in these mutants appeared to contain only two glutamic acid residues at the C-terminus as expected . The mutated gene was termed rimK and was mapped at 18.7 min between cmlA and aroA . The rimK gene was cloned into a cosmid vector and its nucleotide sequence determined . Analysis of the transcriptional and translational products of this gene indicates that it encodes a protein with an Mr of 31.5 kDa and that it forms an operon with a gene encoding a 24 kDa protein . An rpsF mutant containing a Glu to Lys replacement in the second residue from the C-terminus of protein S6 was isolated . The S6 protein of this mutant was apparently inaccessible to the RimK modification system . This indicates that the RimK modification system requires the wild-type amino acid sequence at least in the C-terminal region of ribosomal protein S6. Am J Vet Res, 1989 Jun, 50(6), 822 - 6 Influence of bovine intestinal fluid on the expression of K99 pili by Escherichia coli; Francis DH et al.; A study was conducted to determine whether intestinal fluid collected from various portions of bovine intestine differed in its effect on production of K99 pili by Escherichia coli . The small and large intestines of 7 calves, euthanatized 4 hours after a final feeding of milk, were divided into 6 to 9 segments from which intraluminal fluids were collected . Depending on the amount of fluid collected, up to 20 E coli strains that express K99 pili were grown on media prepared from the content of each specimen and then were tested for K99 pilus expression . In general, intestinal fluid from the most proximal small intestinal segments were more suppressive to K99 pilus expression than was fluid from more distal segments of small intestine . Only about 20% of the E coli test strains expressed K99 pili when grown on medium prepared from proximal small intestinal segmental fluid, whereas greater than 90% did when grown on medium prepared from distal small intestinal segmental fluid . Fluid from the large intestine varied considerably from calf to calf in its effect on K99 pilus expression . A correlation was found between K99 pilus expression and pH of the intestinal fluid, with the lower pH values (characteristic of proximal intestinal segmental fluid) being suppressive . The correlation between K99 pilus production and the pH of the medium was verified, using defined laboratory media adjusted to various pH values . Strains of E coli grown in medium at or below pH 5.5 failed to express K99 pili, whereas the same strains when grown in medium at or above pH 6.5 expressed K99 pili in abundance. Infect Immun, 1989 Jun, 57(6), 1731 - 9 Cloning and temperature-dependent expression in Escherichia coli of a Legionella pneumophila gene coding for a genus-common 60-kilodalton antigen; Hoffman PS et al.; All Legionella species express a 60-kilodalton (kDa) protein which contains a genus-specific epitope recognized by murine monoclonal antibody GW2X4B8B2H6 . A genomic cosmid library of Legionella pneumophila chromosomal DNA was constructed in pHC79 and screened for 60-kDa antigen-expressing clones with the monoclonal antibody . A 3.2-kilobase EcoRI fragment from cosmid 14B11 expressing a 60-kDa protein was subcloned into pUC19 (pSH16), and deletion of a 1.2-kilobase HindIII fragment (pSH16A) generated a 33-kDa truncated polypeptide no longer reactive with the monoclonal antibody . Southern blot analysis of chromosomal DNA from selected Legionella species restricted with EcoRI and probed with the 1.2-kilobase fragment coding for the carboxyl region of the protein revealed DNA homology which was not observed with DNA from Escherichia coli . Maxicell analysis of pSH16 identified a second polypeptide of approximately 15 kDa expressed from a gene (htpA) upstream of the gene coding the 60-kDa protein (htpB) . Both proteins were preferentially synthesized by L . pneumophila following heat shock (temperature shift from 25 to 42 degrees C), and under steady-state growth conditions the relative level of 60-kDa protein was unaffected by temperature . In E . coli, expression of a 60-kDa protein from pSH16 also increased following heat shock (25 to 42 degrees C), but under steady-state conditions expression was temperature dependent . Temperature-dependent expression from pSH16 was not observed in an rpoH (htpR) mutant strain of E . coli . The Legionella 60-kDa protein appears to be a heat shock protein which shares cross-reactive epitopes with the GroEL homolog of E . coli . In addition, a region of htpB encoding the 27-kDa carboxyl portion of the protein containing the monoclonal antibody-reactive epitope also contains DNA sequences unique to and conserved within the genus. Wei Sheng Wu Xue Bao, 1989 Jun, 29(3), 180 - 6 {Regulation in the expression of alpha-galactosidase gene in raf operon in Escherichia coli}; Su TZ et al.; The alpha-galactosidase, coded for by the first structural gene rafA in the plasmid determined raf operon was an inducible enzyme . In contrast to lac or mel operon, raf operon has more strict structural specificity for inducers . The enzyme can be induced by melibiose and raffinose, or weakly by D-galactose, but not by structurally related sugars such as lactose, PNPG etc. . The alpha-galactosidase forming capacity as function of growth curve reached a single peak at the end of the logarithmic phase of the growth . The structure and regulation of raf operon is similar to those of lac operon . The repressormor-mediated negative control plays a major role in the regulation of raf operon, and cAMP-CAP mediated positive control is also involved in the regulation . When 0.4% glucose was added into the medium with other carbon sources, the expression of the enzyme was repressed by 2-3 fold . Transient catabolite repression has been observed neither in inducible nor constitutive alpha-galactosidase expression . Based on alpha-galactosidase assay, in mutant strains CA8306(cya) and CA8445 (cya, crp) the expression level of raf operon was only 9% and 2.5% of that in wild type strain respectively . The glucose effect or the repression in cya mutant can be abolished by 1-5 mmol cAMP . The constitutive alpha-galactosidase expression in cya and cry double mutant (CA8445) remains repressible by glucose, but irreversible by cAMP, suggesting cAMP-CAP complex is not the exclusive mediator of the catablite repression.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Gen Genet, 1989 Jun, 217(2-3), 269 - 77 Nucleotide sequences from the colicin E5, E6 and E9 operons: presence of a degenerate transposon-like structure in the ColE9-J plasmid; Lau PC et al.; The nucleotide sequences of 1288 bp of plasmid ColE5-099, 1609 bp of ColE6-CT14 and 2099 bp of ColE9-J were determined . These sequences encompass the structural genes for the C-terminal receptor-binding and nuclease domains of colicins E5, E6 and E9, their cis- or trans-acting immunity proteins and four lysis proteins including an atypical one of non-lipoprotein nature (Lys) present in the ColE9-J plasmid . The ColE6 gene organisation, in the order col-imm-E8imm-lys, is identical to that found in the previously described double-immunity gene system of ColE3-CA38 (an RNase producer) . The corresponding genes in the two plasmids are 87%-94% homologous . In ColE9-J, the genes are organised as col-imm-lys-E5imm-lys . The E9 col-imm gene pair is homologous to the colicin E2-P9 type (a DNase producer) . Downstream from E9imm is an E5imm (designated E5imm{E9}) which is trans-acting . Neither the predicted structures of E5Imm{E9} nor the cis-acting Imm resident in the ColE5-099 plasmid which differs by a single amino acid shows any resemblance to other immunity structures which have been sequenced . Furthermore, the E5col sequences differ from those predicted previously for other colicins except for the conserved btuB-specified receptor-binding domain . A novel 205 nucleotide long insertion sequence is found in the ColE9-J plasmid . This insertion sequence, which we named ISE9, has features reminiscent of the degenerate transposon IS101 previously found in plasmid pSC101 . One effect of ISE9 is the presence of the atypical lysis gene, lys.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Gen Genet, 1989 Jun, 217(2-3), 233 - 9 Iron-hydroxamate transport into Escherichia coli K12: localization of FhuD in the periplasm and of FhuB in the cytoplasmic membrane; Koster W et al.; The fhuB, fhuC and fhuD genes encode proteins which catalyze transport of iron(III)-hydroxamate compounds from the periplasm into the cytoplasm of Escherichia coli . The fhuB, C, D genes were cloned downstream of a strong phage T7 promoter and transcribed by T7 RNA polymerase . The overexpressed FhuD protein appeared in two forms of 31 and 28 kDa and was released upon conversion of vegetative cells into spheroplasts, suggesting synthesis of FhuD as a precursor and export into the periplasm . The very hydrophobic FhuB protein was found in the cytoplasmic membrane . These properties, together with the previously found homologies in the FhuC protein to ATP-binding proteins, display the characteristics of a periplasmic binding protein dependent transport system across the cytoplasmic membrane . The molecular weight of FhuB and the sequence of fhuC, as previously published by us, was confirmed . FhuB exhibited double the size of most hydrophobic proteins of such systems and showed homology between the amino- and carboxy-terminal halves of the protein, indicating duplication of an original gene and subsequent fusion of the two DNA fragments. J Mol Evol, 1989 Jun, 28(6), 545 - 52 Two new members of the OmpR superfamily detected by homology to a sensor-binding core domain; Timme TL et al.; The OmpR superfamily includes proteins that act as transcriptional regulators of operons that respond to environmental stimuli . A homologous domain near the N-terminus, termed a sensor-binding core domain, is thought to play a role in recognition of a signal transduction protein . We have identified two previously unrecognized members of this regulator family of proteins: a 23.8-kd protein transcribed from the uvrC transcription unit and the PgtA gene product, which is a phosphoglycerate transport regulatory protein . The sensor-binding core domain is also present in four proteins that regulate bacterial sporulation and chemotaxis . The 23.8-kd protein also has sequence similarity to elongation factor Tu and two regulatory proteins: HtpR, the heat-shock regulatory protein, and TraJ, a regulator of expression of genes involved in conjugation . There is a 77-amino acid region near the C-terminus of the 23.8-kd protein that has 30% similarity with a 28.1-kd protein coded for by an open reading frame 5' to the reading frame of the 23.8-kd protein in the uvrC transcription unit . Genetic distance analysis of amino acid sequences of proteins with a sensor-binding core domain suggests that the 23.8-kd protein and the chemotaxis regulatory proteins are distantly related to the other regulatory proteins in the OmpR superfamily. Genetics, 1989 Jun, 122(2), 279 - 96 Genetic and physical mapping of the mcrA (rglA) and mcrB (rglB) loci of Escherichia coli K-12; Raleigh EA et al.; We have genetically analyzed, cloned and physically mapped the modified cytosine-specific restriction determinants mcrA (rglA) and mcrB (rglB) of Escherichia coli K-12 . The independently discovered Rgl and Mcr restriction systems are shown to be identical by three criteria: 1) mutants with the RglA- or RglB- phenotypes display the corresponding McrA- or McrB- phenotypes, and vice versa; 2) the gene(s) for RglA and McrA reside together at one locus, while gene(s) for RglB and McrB are coincident at a different locus; and 3) RglA+ and RglB+ recombinant clones complement for the corresponding Mcr-deficient lesions . The mcrA (rglA) gene(s) is on the excisable element e14, just clockwise of purB at 25 min . The mcrB (rglB) gene(s), at 99 min, is in a cluster of restriction functions that includes hsd and mrr, determinants of host-specific restriction (EcoK) and methyladenine-specific restriction respectively . Gene order is mcrB-hsdS-hsdM-hsdR-mrr-serB . Possible models for the acqusition of these restriction determinants by enteric bacteria are discussed. Appl Environ Microbiol, 1989 Jun, 55(6), 1386 - 90 Differential depuration of poliovirus, Escherichia coli, and a coliphage by the common mussel, Mytilus edulis; Power UF et al.; The elimination of sewage effluent-associated poliovirus, Escherichia coli, and a 22-nm icosahedral coliphage by the common mussel, Mytilus edulis, was studied . Both laboratory-and commercial-scale recirculating, UV depuration systems were used in this study . In the laboratory system, the logarithms of the poliovirus, E . coli, and coliphage levels were reduced by 1.86, 2.9, and 2.16, respectively, within 52 h of depuration . The relative patterns and rates of elimination of the three organisms suggest that they are eliminated from mussels by different mechanisms during depuration under suitable conditions . Poliovirus was not included in experiments undertaken in the commercial-scale depuration system . The differences in the relative rates and patterns of elimination were maintained for E . coli and coliphage in this system, with the logarithm of the E . coli levels being reduced by 3.18 and the logarithm of the coliphage levels being reduced by 0.87 . The results from both depuration systems suggest that E . coli is an inappropriate indicator of the efficiency of virus elimination during depuration . The coliphage used appears to be a more representative indicator . Depuration under stressful conditions appeared to have a negligible affect on poliovirus and coliphage elimination rates from mussels . However, the rate and pattern of E . coli elimination were dramatically affected by these conditions . Therefore, monitoring E . coli counts might prove useful in ensuring that mussels are functioning well during depuration. FEMS Microbiol Lett, 1989 Jun, 50(3), 253 - 8 Synthesis of Rhodobacter sphaeroides cytochrome c2 in Escherichia coli; McEwan AG et al.; The cytochrome c2 structural gene, cycA, from Rhodobacter sphaeroides was expressed in Escherichia coli . CycA-specific mRNA was detected in E . coli both under aerobic and anaerobic conditions with trimethylamine-N-oxide as electron acceptor . However mature holocytochrome c2 was only detected in anaerobically-grown cells . The mature form of cytochrome c2 (Mr = 12,500) was secreted into the periplasm of E . coli suggesting that the signal polypeptide was processed . The cytochrome c2 synthesized in E . coli exhibited absorbance maxima in the reduced form at 550 nm (alpha-band) and 521 nm (beta-band) and contained covalently attached haem c . The results indicate that a foreign c-type cytochrome can be secreted and assembled in E . coli under anaerobic conditions. J Clin Microbiol, 1989 Jun, 27(6), 1167 - 73 Development of an enzyme-linked immunosorbent assay for equine infectious anemia virus detection using recombinant Pr55gag; Archambault D et al.; To provide more sensitive and convenient methods for the detection of equine infectious anemia virus (EIAV), we developed an enzyme-linked immunosorbent assay (ELISA) employing the EIAV gag precursor (Pr55gag) produced by using recombinant DNA techniques . The antigenic reactivity of the recombinant EIAV Pr55gag was found to be equivalent to that of the virion p24gag and elicited high-titered antiserum in rabbits . When a large number of horse sera were analyzed for the presence of antibodies to EIAV by this ELISA, a radioimmunoassay for EIAV p15gag, or the standard agar gel immunodiffusion test, there was 98.7% concordance among the assays . By using the ELISA it was possible to specifically detect antibodies earlier after experimental infection of horses with EIAV than with the other two tests . A competition ELISA developed in order to detect EIAV gag antigens was found to be approximately 15 times more sensitive than the radioimmunoassay for EIAV p15gag . Antigens of other animal lentiviruses as well as those of the prototype oncovirus failed to compete in this assay. Mol Microbiol, 1989 Jun, 3(6), 797 - 805 Analysis of a cloned sequence of Legionella pneumophila encoding a 38 kD metalloprotease possessing haemolytic and cytotoxic activities; Quinn FD et al.; The DNA encoding the zinc metalloprotease of Legionella pneumophila Philadelphia 1 has been isolated and expressed in Escherichia coli . This protein, which is 38,000 Daltons in size, possesses immunological and biochemical properties identical to those previously described for the purified L . pneumophila protease . Periplasmic extracts of E . coli clones expressing the recombinant protease are also capable of causing the haemolysis of canine erythrocytes and the cytotoxic destruction of CHO cells . Using transposon mutagenesis, it was determined that a maximum of 1.2 kb of DNA encoded all three biological activities . Inactivation of proteolytic activity by transposon insertion occurred concomitantly with losses of the haemolytic and cytotoxic phenotypes . A putative regulatory sequence approximately 200-500 bp upstream of the gene's coding region was identified . A 4.0 kb fragment encoding these activities hybridized to the chromosomal DNA of the parent strain of L . pneumophila Philadelphia 1 as well as clinical isolates of L . pneumophila. Mol Microbiol, 1989 Jun, 3(6), 723 - 32 Identification, molecular cloning and sequence analysis of a gene cluster encoding the class II fructose 1,6-bisphosphate aldolase, 3-phosphoglycerate kinase and a putative second glyceraldehyde 3-phosphate dehydrogenase of Escherichia coli; Alefounder PR et al.; To investigate a possible chromosomal clustering of glycolytic enzyme genes, the complete nucleotide sequence of the 8029 bp insert of Escherichia coli DNA in the ColE1 plasmid pLC33-5 of the Clarke and Carbon collection (Clark and Carbon, 1976) was determined . Genes (pgk, fda) encoding the phosphoglycerate kinase and Class II fructose 1,6-bisphosphate aldolase, respectively, of E . coli were identified . The phosphoglycerate kinase was found to be highly homologous in primary structure to the same enzyme from eukaryotic organisms . A further large open reading frame, designated gapB, was also identified, which on the basis of sequence homology, appears to encode another glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase . This putative gene differs significantly from that (designated gapA) already identified as coding for this enzyme in E . coli and which maps elsewhere on the chromosome . The products, if any, of several other open reading frames remain to be identified. J Appl Physiol, 1989 Jun, 66(6), 2805 - 10 Exercise training attenuates the myocardial dysfunction induced by endotoxin; DeBlieux PM et al.; The purpose of this study was to determine whether exercise training protected against endotoxin-induced myocardial dysfunction . After a 12-wk treadmill training period, carotid catheters were implanted 24 h before saline or endotoxin administration into four groups of animals: trained saline-injected (TS), trained endotoxin-injected (TE), sedentary saline-injected (SS), and sedentary endotoxin-injected (SE) . Heart rate and mean arterial pressure were monitored 4 h after in vivo endotoxin or saline injection . Mean arterial pressure decreased an average of 32 +/- 3 mmHg 1 h after endotoxin administration but was normal (109 +/- 6 mmHg) 2 h later . Plasma catecholamines, in vitro myocardial performance, and isolated myocyte adenosine 3',5'-cyclic monophosphate (cAMP) production in response to isoproterenol were assessed 4 h after endotoxin injection . Plasma catecholamine levels were 5- to 15-fold higher in SE compared with the other groups . These data suggest that myocardial protection may be related to the lowered catecholamine levels elicited in TE compared with SE in response to endotoxin administration . The product of cardiac output and peak systolic pressure, an index of cardiac work, was 24-32% greater in TS compared with SS . Cardiac work was decreased 32% in TE compared with a 45% decrease in SE . cAMP was reduced in myocytes from SE in response to isoproterenol (-28%) and to forskolin (-44%) but not in myocytes from TE, compared with TS and SS . The difference in cAMP accumulation suggests that training maintains the integrity of the beta-adrenergic receptor adenylate cyclase system, which can be depressed by in vivo endotoxin administration. Circ Shock, 1989 Jun, 28(2), 149 - 58 Equine peritoneal macrophage production of thromboxane and prostacyclin in response to platelet activating factor and its receptor antagonist SRI 63-441; Morris DD et al.; The formation of eicosanoids may be a primary route through which platelet activating factor (PAF) exerts its effects during endotoxemia . Since endotoxemia is a common cause of death in horses, a study was conducted to determine whether PAF could stimulate equine macrophage release of thromboxane A2 (TxA2) and prostacyclin (PGI2) and whether a PAF-receptor antagonist would alter macrophage eicosanoid synthesis . Equine peritoneal macrophages were cultured from clinically normal horses and exposed to various concentrations of PAF, the PAF-receptor antagonist SRI 63-441, endotoxin, or a combination of these . The supernatant concentrations of TxB2 and 6-keto-prostaglandin F1 alpha were determined after 6 hr incubation . The media concentration of TxB2 was increased significantly above baseline after treatment of macrophages with PAF (10(-7) to 10(-5) M), and the magnitude was similar to that induced by endotoxin . This TxB2 increase was not prevented by prior treatment of macrophages with SRI 63-441 . SRI 63-441 (greater than or equal to 5 x 10(-5) M) significantly enhanced macrophage TxA2 synthesis, as well as its production of PGI2, similar to the effects of endotoxin . Media concentrations of 6-keto-prostaglandin F1 alpha were not increased significantly above baseline after treatment of macrophages with PAF (10(-8) to 10(-5) M) . These results suggest that PAF may cause increased TxA2 release during endotoxemia, which may not be preventable by use of the PAF-receptor antagonist SRI-63-441, which is capable of inhibiting PAF-induced aggregation of equine platelets. Proc Natl Acad Sci U S A, 1989 Jun, 86(12), 4609 - 13 Frameshifting is required for production of the transposase encoded by insertion sequence 1; Sekine Y et al.; Insertion sequence IS1 has two coding frames, insA and insB, which are essential for its transposition . Here, we show that a frameshifting event in the -1 direction from the 3' end region of the insA frame to an open reading frame (B' frame), extending from the 5' end of the insB frame, is involved in production of the InsA-B'-InsB fusion protein that has IS1 transposase activity . The frameshifting event is likely to have occurred at the sequence AAAAAC where the insA frame overlaps the B' frame . Interestingly, this sequence is also present in one of the two sequences identified in retroviruses as frameshift signals for production of transframe polyproteins from the overlapping genes gag-pro or gag-pro-pol. Proc Natl Acad Sci U S A, 1989 Jun, 86(12), 4430 - 4 Escherichia coli DNA helicase II (uvrD gene product) catalyzes the unwinding of DNA.RNA hybrids in vitro; Matson SW; DNA helicase II is a well-characterized Escherichia coli enzyme capable of unwinding duplex DNA and known to be involved in both methyl-directed mismatch repair and excision repair of pyrimidine dimers . Here it is shown that this enzyme also catalyzes the ATP-dependent unwinding of a DNA.RNA hybrid consisting of a radioactively labeled RNA molecule annealed on M13 single-stranded DNA . The DNA.RNA unwinding reaction required less protein to unwind more base pairs than the corresponding unwinding of duplex DNA . In addition, the rate of unwinding of the DNA.RNA hybrid was more than an order of magnitude faster than unwinding of a DNA partial duplex of similar length . The unwinding of the DNA.RNA hybrid is a property unique to helicase II since helicase I, Rep protein, and helicase IV failed to catalyze the reaction . In light of these results it seems likely that helicase II is involved in some previously unrecognized aspect of nucleic acid metabolism, in addition to its known roles in DNA repair reactions. Proc Natl Acad Sci U S A, 1989 Jun, 86(12), 4407 - 11 Methionine synthesis in Escherichia coli: effect of the MetR protein on metE and metH expression; Cai XY et al.; Studies by Urbanowski et al . {Urbanowski, M . L., Stauffer, L . T., Plamann, L . S . & Stauffer, G . V . (1987) J . Bacteriol . 169, 1391-1397} have identified a regulatory locus, called metR, required for the expression of the metE and metH genes . We recently purified the MetR protein from Escherichia coli and showed that it could stimulate the in vitro expression of the metE gene and autoregulate its own synthesis . In the present study, the purified MetR protein has been shown to stimulate the in vitro expression of the metH gene . Also, the in vitro synthesized MetE, MetH, and MetR proteins were shown to be biologically active . The transcription start sites for the metE and metR genes have been determined, and DNA footprinting experiments have identified regions in the metE-metR intergenic sequence that are protected by either the MetR or MetJ proteins. Proc Natl Acad Sci U S A, 1989 Jun, 86(12), 4367 - 71 Dual affinity fusion approach and its use to express recombinant human insulin-like growth factor II; Hammarberg B et al.; A dual affinity fusion concept has been developed in which the gene encoding the desired product is fused between two flanking heterologous genes encoding IgG- and albumin-binding domains . Using sequential IgG and serum albumin affinity chromatography, a full-length tripartite fusion protein is obtained . This approach was used to recover a full-length fusion product in Escherichia coli containing the human insulin-like growth factor II (IGF-II) . Surprisingly, the recombinant IGF-II showed increased stability against proteolytic degradation in E . coli when produced as a dual affinity fusion protein, as compared to an N-terminal fusion protein . After site-specific cleavage of the tripartite fusion protein, IGF-II molecules with immunological and receptor binding activity were obtained without renaturation steps . The results demonstrate that proteins can fold into biologically active structures, even if provided with large flanking heterologous protein domains . The concept was further used to characterize the specific degradation of recombinant IGF-II in this heterologous host. J Gen Virol, 1989 Jun, 70 ( Pt 6), 1571 - 8 The central segment of herpes simplex virus type 1 glycoprotein C (gC) is not involved in C3b binding: demonstration by using monoclonal antibodies and recombinant gC expressed in Escherichia coli; Huemer HP et al.; Three monoclonal antibodies (MAbs) have been raised against cell membrane-derived herpes simplex virus type 1 glycoprotein C (gC-1) . By using different DNA constructs of gC-1 expressed in Escherichia coli the sites recognized by these antibodies could be assigned to a peptide in the more hydrophobic and probably non-glycosylated middle third of the gC-1 molecule . This peptide segment corresponds to a 571 bp segment on the gC-1 gene located between the NcoI and the NruI restriction sites . None of the three MAbs interfered with the binding of the human serum complement component C3b to gC, which has been shown to be rather glycosylation-dependent . Since the data of other groups have suggested that antibodies directed against all regions of gC-1 inhibit C3b binding to gC-1, whereas our results suggest that the central part of gC-1 is not actually involved in C3b-binding activity, it can be inferred that the N- and C-terminal segments are involved in C3b binding by gC-1. AIDS Res Hum Retroviruses, 1989 Jun, 5(3), 259 - 68 Inhibition of bacterially expressed HIV protease activity determined by an in vitro cleavage assay with MuLV Pr65gag; Bu M et al.; HIV protease is a virally coded enzyme that cleaves gag as well as gag-pol precursor polyproteins into functional products needed for virus assembly . A pUC plasmid containing an HIV insert starting at the 5' end of the pol gene and ending just inside the intergrase coding sequence was expressed in E . coli . It provided an 11 kD gene product (protease) that specifically cleaved the Gazdar MuLV Pr65gag precursor into Pr40gag (p30 + p10) and Pr27gag (p15 + p12) intermediates, as well as lower molecular weight gag-encoded products . These were detected by immunoblotting with either MuLV anti-p30 or p12 sera . Using cleavage of MuLV Pr65gag as an assay system, pepstatin A, fusidic acid, and cerulenin were observed to inhibit HIV protease cleavage by greater than 50% at concentrations of 0.1, 0.2-0.5, and 0.5 mM, respectively. Arch Biochem Biophys, 1989 Jun, 271(2), 323 - 31 An electron spin resonance study of oxyradical generation in superoxide dismutase- and catalase-deficient mutants of Escherichia coli K-12; Schellhorn HE et al.; The response of superoxide dismutase- and catalase-deficient strains of Escherichia coli to redox active compounds was examined by electron spin resonance . Levels of radicals formed in response to pyocyanine in situ were extremely low and were found to be predominantly extracellular, even in a strain completely deficient in both superoxide dismutase and catalase . In cell-free extracts of superoxide dismutase-minus strains incubated with NADPH and pyocyanine, the primary accumulating radical was the superoxide anion (O2-), although low levels of the hydroxyl radical (.OH) were also detected . In contrast, extracts from strains lacking catalase were found to accumulate higher levels of hydroxyl radicals. Proc Natl Acad Sci U S A, 1989 Jun, 86(11), 3939 - 43 Simian virus 40 (SV40) large tumor antigen causes stepwise changes in SV40 origin structure during initiation of DNA replication; Roberts JM; We have studied structural changes in the simian virus 40 (SV40) replication origin induced by SV40 large tumor antigen (T antigen) . T-antigen-induced changes in origin DNA conformation can be visualized as specific and discrete topologic changes in origin DNA minicircles . We discovered three origin-T-antigen complexes defined by changes in DNA linking number . These complexes probably reflected essential early steps in the initiation of DNA replication since their formation required DNA sequences that are necessary for DNA replication but do not affect T-antigen binding . There are striking parallels between the T antigen-origin interactions uncovered by this assay and the interactions between the DnaA, -B, and -C proteins and the Escherichia coli replication origin, suggesting a significant evolutionary conservation in the mechanisms that initiate DNA replication. J Virol, 1989 Jun, 63(6), 2737 - 45 Regulated expression of the feline panleukopenia virus P38 promoter on extrachromosomal FPV/EBV chimeric plasmids; Clemens DL et al.; Feline panleukopenia virus/Epstein-Barr virus (FPV/EBV) chimeric expression plasmids were constructed to study regulation of the structural protein gene of the parvovirus, FPV, in a homologous cell culture system . Detection and quantitation of activity from the native FPV promoter, P38, was facilitated by fusing the Escherichia coli lacZ gene with the FPV structural protein gene . Feline cell lines which stably maintained these plasmids extrachromosomally were established . Constitutive beta-galactosidase activity was low but increased up to 40-fold after infection with FPV . Expression of beta-galactosidase was only detected when the FPV/lacZ gene was oriented in the same transcriptional direction as the Epstein-Barr virus gene coding for EBNA-1 . When a small open reading frame upstream of the FPV/lacZ initiation codon was deleted, beta-galactosidase expression increased another 4.7- to 26-fold . These changes in beta-galactosidase activity indicate that expression of the FPV structural protein gene is regulated both transcriptionally and posttranscriptionally. J Bacteriol, 1989 Jun, 171(6), 3557 - 9 Overproduction of transposon Tn10-encoded tetracycline resistance protein results in cell death and loss of membrane potential; Eckert B et al.; High-level expression of the Tn10 tetracycline resistance protein TetA in Escherichia coli caused partial collapse of the membrane potential, arrest of growth, and killing of the cells . Since alpha-methylglucoside transport was not affected, the overproduced TetA protein may cause not destruction of membrane structure but rather unrestricted translocation of protons and/or ions across the membrane. J Bacteriol, 1989 Jun, 171(6), 3518 - 22 Translational coupling in the threonine operon of Escherichia coli K-12; Little S et al.; In an attempt to express the two distal genes of the Escherichia coli threonine operon, the majority of the first gene in the operon, thrA, was removed and a series of transcriptional fusions were constructed placing the thrB and thrC genes downstream of either the trp or hybrid tac promoter . Analysis of the proteins produced by cells containing these fusions revealed that although the distal gene, thrC, was efficiently expressed, the proximal gene, thrB, was not expressed at a detectable level . A translational fusion was constructed which fused the cat gene in phase to the last 800 base pairs of thrA followed by thrB and thrC . Cells containing this fusion produced high levels of both the thrB and thrC gene products, showing that translation of thrB requires translation through thrA; thus, thrA and thrB are translationally coupled . In addition, it was found that a sequence between 220 and 57 base pairs before the start of thrB was necessary to allow translational coupling to occur. J Bacteriol, 1989 Jun, 171(6), 3228 - 32 The thiM locus and its relation to phosphorylation of hydroxyethylthiazole in Escherichia coli; Mizote T et al.; A mutant of Escherichia coli lacking hydroxyethylthiazole kinase (EC 2.7.1.50) was produced by a further mutation of a temperature-sensitive, auxotrophic mutant for hydroxyethylthiazole . The parent cells possessed two distinct enzymes capable of phosphorylating hydroxyethylthiazole: one was hydroxyethylthiazole kinase, and the other was a phosphotransferase species that required p-nitrophenylphosphate as a phosphoryl donor . Osmotic shock fluid prepared from the mutant cells phosphorylated hydroxyethylthiazole to an extent comparable to that observed with shock fluid from the parent cells, whereas extracts from shocked cells were unable to catalyze the kinase reaction . Shock fluid from a mutant of the other type obtained as a reduced phosphatase activity against p-nitrophenylphosphate did not show any appreciable activity for the phosphotransferase reaction, while extracts from shocked cells showed full kinase activity . The former mutant had lost its ability to grow on hydroxyethylthiazole at high temperature, but the latter mutant still responded to it . It thus appears that the kinase is an enzyme which might play a role in the biosynthesis of thiamine PPi in situ . By conjugation and P1 transduction, a gene governing hydroxyethylthiazole kinase activity, for which we propose the designation thiM, was mapped on the chromosome close to thiD, a gene specifying phosphomethylpyrimidine kinase activity. J Bacteriol, 1989 Jun, 171(6), 3158 - 61 Characterization of a purF operon mutation which affects colicin V production; Fath MJ et al.; A mini-Tn10-kan insertion mutation identified a gene in the chromosome of Escherichia coli required for colicin V production from plasmid pColV-K30 . With the complete restriction map of E . coli, the mutation was rapidly mapped to 50.0 min, within the purF operon . Sequence analysis showed that the insertion occurred in a gene with no previously known function which is located directly upstream of purF . We designated this gene cvpA for colicin V production . The mutant requires adenine for growth, probably because of a polar effect on purF expression . However, an adenine auxotroph showed no defect in colicin V production, suggesting that the cvpA mutation is responsible for the effect on colicin V production . Two possible models of cvpA1 allele function are discussed. J Bacteriol, 1989 Jun, 171(6), 3144 - 51 Presence of the dnaQ-rnh divergent transcriptional unit on a multicopy plasmid inhibits induced mutagenesis in Escherichia coli; Foster PL et al.; In Escherichia coli the dnaQ+ gene, which encodes epsilon, a fidelity subunit of DNA polymerase III, and the rnh+ gene, which encodes RNase H, share a promoter region but are transcribed in opposite directions . The presence of this divergent transcriptional unit on a multicopy plasmid inhibited by as much as 10-fold mutations induced by the SOS-dependent mutagens methyl methanesulfonate and UV light . Mutations in either gene eliminated the effect, suggesting that both genes contribute either directly or indirectly to the antimutagenic phenotype . Neither survival to mutagen exposure nor induction of the SOS response was comparably affected by the presence of the genes . Although the antimutagenic phenotype was partially suppressed by excess UmuDC proteins, which are required for SOS mutagenesis, the presence of the dnaQ+-rnh+ clone also reduced the induction of mutations by N-methyl-N'-nitro-N-nitrosoguanidine in cells deficient for SOS mutagenic processing . The results suggest that the presence of the dnaQ+-rnh+ divergent transcriptional unit interferes with an underlying mutagenic mechanism that is normally facilitated by the proteins induced as part of the SOS response. J Bacteriol, 1989 Jun, 171(6), 3139 - 43 Suppression of dnaE nonsense mutations by pcbA1; Maki H et al.; DNA polymerase III has been recognized as the required replication enzyme in Escherichia coli . The synthesis subunit of DNA polymerase III holoenzyme (alpha subunit) is encoded by the dnaE gene . We have reported that E . coli cells can survive and grow in the absence of a functional dnaE gene product if DNA polymerase I and the pcbA1 mutation are present . Existing mutations in the dnaE gene have been conditionally defective thermolabile mutations . We report here construction of nonsense mutations in the dnaE gene by use of a temperature-sensitive suppressor mutation to permit survival at the permissive temperature (32 degrees C) . Introduction of the pcbA1 mutation eliminated the temperature-sensitive phenotype . We confirmed by immunoblotting the lack of detectable alpha subunit at 43 degrees C. J Bacteriol, 1989 Jun, 171(6), 3074 - 9 Activity of CMP-2-keto-3-deoxyoctulosonic acid synthetase in Escherichia coli strains expressing the capsular K5 polysaccharide implication for K5 polysaccharide biosynthesis; Finke A et al.; The activity of the cytoplasmic CMP-2-keto-3-deoxyoctulosonic acid synthetase (CMP-KDO synthetase), which is low in Escherichia coli rough strains such as E . coli K-12 and in uncapsulated strains such as E . coli O111, was significantly elevated in encapsulated E . coli O10:K5 and O18:K5 . This enzyme activity was even higher in an E . coli clone expressing the K5 capsule . This and the following findings suggest a correlation between elevated CMP-KDO synthetase activity and the biosynthesis of the capsular K5 polysaccharide . (i) Expression of the K5 polysaccharide and elevated CMP-KDO synthetase activity were observed with bacteria grown at 37 degrees C but not with cells grown at 20 degrees C or below . (ii) The recovery kinetics of capsule expression of intact bacteria, in vitro K5 polysaccharide-synthesizing activity of bacteria, and CMP-KDO synthetase activity of bacteria after temperature upshift from 18 to 37 degrees C were the same . (iii) Chemicals which inhibit capsule (polysaccharide) expression also inhibited the elevation of CMP-KDO synthetase activity . The chromosomal location of the gene responsible for the elevation of this enzyme activity was narrowed down to the distal segment of the transport region of the K5 expression genes. Cancer Res, 1989 Jun 1, 49(11), 2905 - 8 Nucleoside uptake and membrane fluidity studies on N-trifluoroacetyladriamycin-14-O-hemiadipate-treated human leukemia and lymphoma cells; Lameh J et al.; N-Trifluoroacetyladriamycin-14-O-hemiadipate (AD 143), a DNA nonbinding derivative of Adriamycin, was studied for its effect on the uptake of labeled nucleosides into human leukemia (ML-1) and lymphoma (P3HR-1) cells in culture . After preincubation with AD 143 at concentrations as low as 5.2 microM (ML-1) or 13 microM (P3HR-1), the ability of the cells to take up extracellular labeled nucleosides was decreased by more than 50% . Similar experiments with Escherichia coli cells showed that AD 143 at the same concentrations did not have any effect . Influx of {3H}thymidine or {3H}uridine was studied by centrifugation of the cells through phthalate oil mixture, and it was found that the influx of the labeled nucleosides was decreased after treatment of the cells with AD 143 . An increase in the membrane fluidity was observed after treatment of the cells with AD 143, as revealed by electron paramagnetic resonance spectroscopy studies . These observations suggest that the decreased incorporation of {3H}thymidine and {3H}uridine into acid-precipitable material that we observed earlier in the AD 143-treated cells may in part be the result of the AD 143-induced alteration of cell membrane activities, which in turn causes an inhibition of nucleoside uptake. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1989 Jun, 11(3), 170 - 4 {Expression of gene encoding HBsAg in yeast cell with its alpha-factor promoter}; Lin HN; Construction of an E . coli-yeast shuttle plasmid was reported with yeast alpha-factor as promoter of synthesis and secretion of mature foreign proteins by yeast cells (Saccharomyces cerevisiae) . The HBsAg gene was successfully recombined into the plasmid with its frame precisely adjusted, thus resulting in extracellular secretion of HBsAg protein by the yeast cells. Mol Gen Genet, 1989 Jun, 217(2-3), 533 - 5 Restriction analysis of lambda EMBL3 background recombinants: occurrence of lambda phages carrying a head to tail oriented left arm DNA sequence; Troster H et al.; Eight representative recombinant background clones of lambda EMBL3 were analysed using KpnI, BamHI, SalI, EcoRI and HindIII digestion . We found that lambda EMBL3 carries its own left arm in the BamHI cloning site . In the way, recombinant molecules were found to be generated which can grow on Escherichia coli strain NM539 . In all cases analysed, the left arm DNA was inserted in a head to tail orientation . Seven clones carried a restored BamHI site at the cos site-BamHI site connection . In the region where the inserted left arm and the right arm were ligated, BamHI cloning produces a large palindromic sequence consisting of two polylinkers . This BamHI site was incompletely cleaved in all cases analysed . We assume that a part of the lambda DNA molecule in this region shows a cruciform structure prohibiting recognition or cleavage of this site by restriction endonuclease BamHI. EMBO J, 1989 Jun, 8(6), 1827 - 31 Nuclear protein p68 is an RNA-dependent ATPase; Iggo RD et al.; The human nuclear antigen p68 cross reacts with a monoclonal antibody to SV40 large-T antigen . Its deduced amino acid sequence contains short motifs which place it in a large superfamily of proteins of known or putative helicase activity . Recently, a p68 subfamily (DEAD box proteins) which share more extensive regions of homology has been identified in mouse, Drosophila, Saccharomyces cerevisiae and Escherichia coli . These proteins are involved in translation, ribosome assembly, mitochondrial splicing, spermatogenesis and embryogenesis . We show here that immunopurified human p68 has RNA dependent ATPase activity . In addition, we show that the protein undergoes dramatic changes in cellular location during the cell cycle. Mol Microbiol, 1989 Jun, 3(6), 757 - 66 The Escherichia coli enterobactin biosynthesis gene, entD: nucleotide sequence and membrane localization of its protein product; Armstrong SK et al.; The nucleotide sequence of the Escherichia coli enterobactin biosynthesis gene entD has been determined . entD specifies a predicted 23579 Dalton protein containing several helical regions, a transmembrane segment and one positively charged domain . The EntD polypeptide was overexpressed and identified in electrophoretic gels as a membrane protein . Although results of conventional membrane fractionation techniques were inconclusive, protease accessibility studies provided evidence that EntD domains are exposed on the inner leaflet of the cytoplasmic membrane . The presence of repetitive extragenic palindromic (REP) sequences within the fepA-entD intercistronic region was confirmed . Lack of a canonical promoter and an iron control region 5' to entD, along with RNA hybridization data, suggest that an iron-regulated transcript contains both fepA and entD. Hybridoma, 1989 Jun, 8(3), 277 - 91 Development of a monoclonal antibody specific for the gamma chain of the T-cell antigen receptor using an open reading frame expression vector; Nanno M et al.; To develop an anti-framework monoclonal antibody (mab) specific for the gamma (gamma)-chain of the T-cell antigen receptor (TCR), we expressed a part of the constant region of the gamma-chain (C gamma 2 gene segment) in E . coli using the pWR590 vector . This plasmid contains the E . coli lac promoter, operator, a truncated beta-galactosidase (beta-gal) gene (coding for the first 590 of the 1,007 amino acids of the beta-gal) and a polylinker region (at the 3' end of the beta-gal) containing nine restriction sites . These can be cleaved by any one of eight common restriction enzymes, permitting the introduction of the DNA fragment of interest . We employed the pT gamma 1 gamma-chain cDNA probe, which like the vast majority of the gamma-chain specific probes is aberrant and contains an in-frame stop codon at the junction of V and J regions . Computer analysis of the pT gamma 1 sequence revealed several MaeIII restriction sites that could result in a number of fragments . One of these fragments consisted of 245 base pairs (nucleotides 404-648) and contained most of the CI exon of the C gamma 2 . Successful insertion of this fragment to the pWR590 vector was confirmed using restriction enzyme analysis . The C gamma insert was 12% of the construct . Expression of the pWR590-HpT gamma 1 recombinant plasmid in E . coli followed by SDS-PAGE analysis revealed a hybrid protein with a molecular weight of 85 kd which constituted at least 25% of the total E . coli insoluble protein . In contrast, cells transformed with the control pWR590 vector without insert expressed a 78 kd polypeptide chain . We developed several mabs against the pWR590-HpT gamma 1 hybrid protein by fusing spleen lymphocytes from BALB/c mice immunized with the pWR590-HpT gamma 1 protein, with cells of the NS1 mouse myeloma cell line . Screening of the mabs was carried out by ELISA against the pWR590-HpT gamma 1 hybrid protein and the control pWR590 beta-gal protein (beta-gal 590), derived by expressing in E . coli the pWR590 vector without gamma-chain insert . Two groups of mabs were obtained, those reacting with the pWR590-HpT gamma 1 hybrid protein only and those reacting with both the hybrid and the control beta-gal 590 proteins . The specificity of these mabs was further studied by Western blotting with similar results.(ABSTRACT TRUNCATED AT 400 WORDS) Mutat Res, 1989 Jun, 223(2), 183 - 9 SOS Chromotest study concerning some appreciation criteria of humic substances' genotoxic potency; Pommery J et al.; The genotoxicity of 5 compounds: 2 fulvic acids, a trade humic acid, a synthetic humic material (SHM), and 2,5-dihydroxybenzoic acid, was assessed after chlorination, by means of the SOS Chromotest for tester strain E . coli PQ 37 without metabolic activation . Chlorination was carried out for humic material concentration of 0.5 mg total organic carbon per liter, and chlorine concentrations in the range of 0.1-2.0 chlorine equivalents per mole of carbon . Among all the considered criteria that can account for potent toxicity: chemical degradation determined by the UV absorption decrease, chlorine consumption, average molecular weight, only the polymerization index (O.D . 665 nm/O.D . 465 nm) can be related to the genotoxicity of humic samples . This latter criterion appears a possible predictor of genotoxic potency, revealed subsequent to the aqueous chlorination of humic materials . Looking at the various genotoxic activities of the tested compounds, SHM can be considered a better model of natural humic materials than the trade humic acid. Biopolymers, 1989 Jun, 28(6), 1043 - 58 The acceleration of linear DNA during pulsed-field gel electrophoresis; Holzwarth G et al.; The velocity and orientation of T4 and lambda DNA have been measured for the first 20 s during pulsed-field gel electrophoresis in order to clarify the DNA motions that occur . For a square pulse with field strength E = 10 V/cm, the velocity of lambda DNA increases gradually to 10.5 microns/s in 1.0 s, declines to 8.6 microns/s, and then rises to a plateau value of 9.3 microns/s after 4 s . T4 DNA behaves similarly, but more slowly . Parallel measurements of fluorescence-detected linear dichroism show that the DNA becomes substantially aligned with its chain axis parallel to the electrophoretic field E after the pulse is applied . The alignment also shows an overshoot, an undershoot, and a plateau comparable to those seen for velocity . When the field strength increases, both the velocity and the alignment reach their peaks more quickly . For all field strengths and both molecular weights, the velocity peak occ