Escherichia coli

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 occurs when the molecular center of mass has moved 0.3 to 0.5 L, where L is the chain contour length . A qualitative model is provided.

Arch Biochem Biophys, 1989 Jun, 271(2), 441 - 6
Expression, purification, and in vivo activity of atrial natriuretic factor prohormone produced in Escherichia coli; Gierse JK et al.; Atrial muscles of the heart are known to produce polypeptide hormones called atrial natriuretic factors (ANF) which have potent diuretic and hypotensive action . These hormones are synthesized as a larger protein precursor called pro atrial natriuretic factor or proANF which contains the biologically active ANF sequences at its C-terminus . Rat proANF (representing amino acids -1 to 128 of the coding sequence) was expressed in a soluble form in Escherichia coli . A simple purification procedure was developed which consists of boiling E . coli cell extracts in 1 M acetic acid and subjecting the supernatant to reversed-phase HPLC . The effect of intravenous administration of the purified recombinant proANF on mean arterial blood pressure was examined . The displacement dose-response curves obtained demonstrated that proANF exhibits similar, albeit less potent, physiological activity than ANF.

Am Rev Respir Dis, 1989 Jun, 139(6), 1356 - 60
Cell recruitment into lung wall and airways of conventional and pathogen-free guinea pigs after inhalation of endotoxin; Venaille T et al.; The cell kinetics of the acute inflammatory response to inhaled endotoxin (lipopolysaccharide, LPS) was studied in the lungs of conventional (CV) and pathogen-free (SPF) guinea pigs . Airway cells were obtained by bronchoalveolar lavage (BAL) . Lung wall cells were prepared via collagenase digestion of lung tissue slices . Acute exposure to LPS triggered the influx within 4 to 12 h of equivalent numbers (approximately 70 x 10(6)) of neutrophils into the lung walls of both CV and SPF guinea pigs . The recruited neutrophils then proceeded into the airways of CV animals, and by 48 h all recruited neutrophils were recoverable by BAL . In contrast, only one third of recruited neutrophils in the lungs of SPF animals moved from the lung wall into the airways . Analysis of neutrophil chemotactic factor (NCF) production identified lung wall cells as the major source of LPS-induced NCF activity in both groups and as virtually the sole source in SPF animals . The results emphasize the importance of studies on the precise lung tissue distribution of both recruited neutrophils, and endogenous NCF-producing cells, in elucidating the acute inflammatory response in the lungs.

J Bacteriol, 1989 Jun, 171(6), 3039 - 45
Use of lacZ fusions to measure in vivo expression of the first three genes of the Escherichia coli unc operon; Solomon KA et al.; We have constructed in-frame lacZ protein fusions to the first three genes of the Escherichia coli unc operon, which codes for the subunits of the proton-translocating ATPase . We have used these constructions to measure the relative in vivo expression of these genes . The second and third genes, uncB and uncE, which code for the a and c subunits of the F0 sector, were expressed at relative levels of approximately 1:10, although the measured expression of uncB depended upon how much of the gene was fused to lacZ . These rates compared favorably with the relative numbers of a and c subunits (a1:c10) in the purified F1F0 complex . The in vivo expression of uncI, the first gene of the operon, was very low, at best 10 to 20 times less than the expression of uncB.

J Immunol, 1989 Jun 1, 142(11), 3923 - 30
Carbohydrates on human Fc gamma receptors . Interdependence of the classical IgG and nonclassical lectin-binding sites on human Fc gamma RIII expressed on neutrophils; Kimberly RP et al.; To explore the molecular basis for the ability of aggregated IgG to block the phagocytosis by human polymorphonuclear leukocytes of Con A-opsonized E and of nonopsonized Escherichia coli with mannose-binding adhesins, we examined specific aspects of the glycoprotein structure of both the 40- to 43-kDa receptor for the Fc portion of IgG (Fc gamma RII) and the 50- to 78-kDa receptor for the Fc portion of IgG (Fc gamma RIIIPMN) from human polymorphonuclear leukocytes . Fc gamma RIIIPMN isolated by both mAb and ligand affinity chromatography, but not Fc gamma RII, binds Con A in Western blots . This binding is specifically inhibitable by alpha-methylmannoside . Digestion of Fc gamma RIIIPMN by recombinant endoglycosidase H, which is specific for high mannose-type (Con A-binding) oligosaccharides, alters the epitope recognized by mAb 3G8 in or near the IgG ligand-binding site of the receptor . Similarly, the ability of Fc gamma RIIIPMN to bind human IgG ligand is sensitive to endoglycosidase H digestion . Our data indicate that ligands other than the classical IgG opsonins can bind to human Fc gamma RIIIPMN per se through lectin-carbohydrate interactions . Furthermore, Fc gamma RIIIPMN contains a high mannose type oligosaccharide chain which contributes importantly to the integrity of the classical IgG ligand-binding site . Thus, specific glycosylations of the receptor are important for both classical and nonclassical engagement of Fc gamma RIII and may play a role in determining the properties of the ligand-binding site.

Biotechniques, 1989 Jun, 7(6), 576 - 9
A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for beta-galactosidase; Lim K et al.; Transfection efficiency of different cell types as well as promoter strength of cloned genes can be easily determined by direct assay of beta-galactosidase activity encoded from recombinant genes containing the E . coli beta-galactosidase gene . A substrate for beta-galactosidase, o-nitrophenyl-beta-D-galactopyranoside (ONPG), can be added to dishes containing the transfected cells, and the intensity of the colored enzyme product released from either the intact cell or cells lysed in the dishes can be determined . The results obtained by this assay are a reliable measure of transfection efficiency as well as promotor strength of the genes introduced into the cells . In addition, cells expressing the transfected gene can be identified and quantitated under a light microscope after incubation with X-gal . Thus, it is more convenient to use the E . coli beta-galactosidase gene than the chloramphenicol acetyltransferase gene as a reporter gene in the evaluation of DNA transfection.

Neuron, 1989 Jun, 2(6), 1615 - 24
The microtubule binding domain of tau protein; Lee G et al.; Tau protein is a microtubule-associated protein implicated in the spatial and temporal specification of microtubules and has been found in the neurofibrillary tangles of Alzheimer's disease . Determination of tau protein structure has revealed three 18 amino acid repeated sequences hypothesized to be tubulin binding sites . Using tau cDNA clones from human fetal brain, we employed E . coli expression systems to synthesize tau protein and fragments of tau protein in order to identify the microtubule binding site . A fragment containing the three repeated sequences binds microtubules, while the amino-terminal half of the protein does not bind . Fragments containing two or one repeat are also capable of binding, indicating that the basic tubulin interacting unit is one repeat.

J Gen Microbiol, 1989 Jun, 135 ( Pt 6), 1715 - 26
Functioning of the colicin A lysis protein is affected by Triton X-100, divalent cations and EDTA; Cavard D et al.; The colicin A lysis protein, Cal, is synthesized at the same time as colicin A by Escherichia coli harbouring plasmid pColA after induction by mitomycin C . Its function in the induced bacteria involves the release of colicin A, quasi-lysis, the death of the producing cells and the activation of the outer membrane phospholipase A . We have found that these various functions are affected differently by treatment of the induced cells with Triton X-100, divalent cations or EDTA . Triton X-100 and EDTA caused increased quasi-lysis and a higher level of mortality of the producing cells, but while Triton X-100 enhanced the release of colicin A, EDTA reduced it . Divalent cations protected the cells against both killing and quasi-lysis without greatly affecting colicin release . The effects of these agents were similar for both wild-type and phospholipase A mutants and depended only on the presence of a functional cal gene.

Agents Actions, 1989 Jun, 27(3-4), 477 - 80
Biochemical characterization of soluble phospholipase A2 from rheumatoid synovial fluid; Gonzalez-Buritica H et al.; Radiolabeled E . coli, Phosphatidylethanolamine (PE) and Phosphatidylcholine (PC), were used to characterize the phospholipase A2 (PLA2) activity in synovial fluid (SF) from rheumatoid arthritis (RA) patients . Cell-free fractions of SF contain a PLA2 enzyme that preferentially releases {14C}oleic acid from E . coli, requires calcium and is optimally active at neutral pH . Purified PE, but not PC is also readily degraded by the soluble enzyme . A cell-associated PLA2 present in sonicates of SF mononuclear cells and neutrophils preferentially releases {3H}AA from E . coli . These studies suggest the presence of at least two different enzymes with activity of PLA2 in rheumatoid SF.

Mol Gen Genet, 1989 Jun, 217(2-3), 511 - 9
High-level expression of the colicin A lysis protein; Cavard D et al.; Two plasmids that overproduce the colicin A lysis proteins, Cal, are described . Plasmid AT1 was constructed by a deletion in the colicin A operon, which placed the cal gene near a truncated caa gene in such a way that both gene products wer synthesized at high levels following induction . Plasmid CK4 was constructed by insertion of the cal gene downstream from the tac promoter of an expression vector . Overproduction of Cal was obtained after mitomycin C induction of pAT1 cells and after IPTG induction of pCK4 cells . The kinetics of Cal synthesis were examined with {35S} methionine and {2-3H} glycerol in lpp or lpp+ host strains . Each of the steps of the lipid modification and maturation pathway of Cal was demonstrated . The modified precursor form of overproduced Cal was not chased as efficiently as when it is produced in pColA cells . After treatment with globomycin, a significant amount of this modified precursor form accumulated and was degraded with time into smaller acylated proteins, but without release of the signal peptide . Release of cellular proteins and quasi-lysis were observed after about 1 hour of induction for cells containing either plasmid . In addition, in Cal-overproducing cells, the rate of quasi-lysis was increased but not its extent . In pldA cells, quasi-lysis was reduced but not abolished . Lethality of the Cal induction in the overproducing cells was in th same range as that in wild-type cells.

Mol Cell Biol, 1989 Jun, 9(6), 2360 - 9
Localization of a minimal binding domain and activation regions in yeast regulatory protein ADR1; Thukral SK et al.; ADR1 is a transcription factor required for activation of the glucose-repressible alcohol dehydrogenase 2 (ADH2) gene in Saccharomyces cerevisiae . ADR1 has two zinc finger domains between amino acids 102 and 159, and it binds to an upstream activation sequence (UAS1) in the ADH2 promoter . A functional dissection of ADR1 was performed by using a series of amino- and carboxy-terminal deletion mutants of ADR1, most of which were fused to the Escherichia coli beta-galactosidase . These deletion mutants were assayed for binding to UAS1 in vitro, for the ability to activate ADH2 transcription in vivo, and for level of expression . Deletion of ADR1 amino acids 150 to 172 and 76 to 98 eliminated DNA binding in vitro, which accounted for the loss of transcriptional activation in vivo . Results with the former deletion mutant indicated that both of the ADR1 zinc fingers are necessary for sequence-specific DNA binding . Results with the latter deletion mutant suggested that at least part of the sequence between amino acids 76 to 98, in addition to the two finger domains, is required for high-affinity DNA binding . The smallest fusion protein able to activate ADH2 transcription, containing ADR1 amino acids 76 to 172, was much less active in vivo than was the longest fusion protein containing amino acids 1 to 642 of ADR1 . In addition, multiple regions of the ADR1 polypeptide (including amino acids 40 to 76, 260 to 302, and 302 to 505), which are required for full activation of ADH2, were identified . An ADR1-beta-galactosidase fusion protein containing only the amino-terminal 16 amino acids of ADR1 was present at a much higher level than were larger fusion proteins, which suggested that the sequences within ADR1 influence the expression of the gene fusion.

Biochimie, 1989 Jun, 71(6), 729 - 39
Missense and nonsense suppressors can correct frameshift mutations; Tucker SD et al.; Missense and nonsense suppressor tRNAs, selected for their ability to read a new triplet codon, were observed to suppress one or more frameshift mutations in trpA of Escherichia coli . Two of the suppressible frameshift mutants, trpA8 and trpA46AspPR3, were cloned, sequenced, and found to be of the +1 type, resulting from the insertion of four nucleotides and one nucleotide, respectively . Twenty-two suppressor tRNAs were examined, 20 derived from one of the 3 glycine isoacceptor species, one from lysT, and one from trpT . The sequences of all but four of the mutant tRNAs are known, and two of those four were converted to suppressor tRNAs that were subsequently sequenced . Consideration of the coding specificities and anticodon sequences of the suppressor tRNAs does not suggest a unitary mechanism of frameshift suppression . Rather, the results indicate that different suppressors may shift frame according to different mechanisms . Examination of the suppression windows of the suppressible frameshift mutations indicates that some of the suppressors may work at cognate codons, either in the 0 frame or in the +1 frame, and others may act at noncognate codons (in either frame) by some as-yet-unspecified mechanism . Whatever the mechanisms, it is clear that some +1 frameshifting can occur at non-monotonous sequences . A striking example of a frameshifting missense suppressor is a mutant lysine tRNA that differs from wild-type lysine tRNA by only a single base in the amino acid acceptor stem, a C to U70 transition that results in a G.U base pair . It is suggested that when this mutant lysine tRNA reads its cognate codon, AAA, the presence of the G.U base pair sometimes leads either to a conformational change in the tRNA or to an altered interaction with some component of the translation machinery involved in translocation, resulting in a shift of reading frame . In general, the results indicate that translocation is not simply a function of anticodon loop size, that different frameshifting mechanisms may operate with different tRNAs, and that conformational features, some far removed from the anticodon region, are involved in maintaining fidelity in translocation.

Biochimie, 1989 Jun, 71(6), 721 - 8
Reversion of trpA nonsense mutations by deletion of the chain-termination codons; Tucker SD et al.; This paper describes a novel mechanism for reversion of nonsense mutations in the trpA gene of Escherichia coli . This mechanism, deletion of the nonsense codon, was discovered in the course of selecting for missense revertants of trpA(UGA211) and for catalytically active tryptophan synthetase alpha chain revertants of trpA(UAA234) and trpA(UAG234) . Each type of revertant trpA was cloned and its DNA sequence determined . trpA(UGA211) gave rise to two previously unidentified types of missense revertant . The first type was expected, namely trpA(CGA211), the result of a base substitution event . The other type, representing approximately 1% of the missense revertants, was unexpected on the basis of single base substitutions and an understanding of which amino acids are functional at alpha chain position 211 . It was found to be the result of a 21 base-pair deletion of a region containing codon 211 . The tryptophan-independent revertants of both position 234 nonsense mutants occurred at a frequency of approximately 2 per 10(9) viable cells . They were identical in that they both resulted from a 3 base-pair deletion, namely deletion of the chain-terminating codon at position 234 . One of them, however, also displayed an A instead of the normal G in the third position of codon 235 . The revertants were characterized according to growth in different media and tryptophan synthetase assays performed on crude extracts . These types of mutants should prove interesting and important for the elucidation of alpha chain structure-function relationships, for insight into the assembly and interaction of subunits in this model multienzyme complex, and for the study of mechanisms by which deletions can be generated.

Biochimie, 1989 Jun, 71(6), 711 - 20
Role of cysteine residues in tryptophanase for monovalent cation-induced activation; Tokushige M et al.; We cloned and sequenced the tryptophanase structural gene of Escherichia coli B/1t7-A strain . The results indicate that tryptophanase proteins of E . coli B/1t7-A and K-12 are identical . When cysteine residues in tryptophanase were chemically modified with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), the stabilizing effect of the active cations such as K+ and NH4+ was abolished . In consideration of our previous results that Cys-298 was selectively modified by SH reagents {Honda T . et al . (1986) J . Chromatogr . 371, 353-360}, Cys-298 seems to have a close relation to the expression of the effect of monovalent cations . Fluorescence decay measurement of the holoenzyme revealed that the fluorescence lifetime derived from the coenzyme, pyridoxal 5'-phosphate (PLP), was dependent on coexisting monovalent cations, whereas that of the tryptophyl residue was not, in either the apo- or the holoenzyme preparation . The results of the synchrotron small-angle X-ray scattering measurements showed that radii of gyration which reflect the size and shape of the enzyme were constant at around 38 A irrespective of the presence or absence of the K+ ion . These results suggest that the monovalent cations interact specifically with the PLP-binding site, and that the conformational change of enzyme protein caused by the monovalent-cation binding is limited to a small range . The above results are compatible with the possibility that Cys-298 is involved in the formation of "monovalent cation binding site" in the holoenzyme.

Biochem Int, 1989 Jun, 18(6), 1305 - 14
Calcium calmodulin in altered NaCl transport by heat-labile enterotoxin of Escherichia coli; Goyal J et al.; The mucosal-to-serosal and serosal-to-mucosal fluxes of Na+ and Cl- were carried out in control and experimental groups treated with different doses of heat-labile enterotoxin in the presence or absence of Ca2+-ionophore, Ca2+ channel blocker and calmodulin inhibitor . There was net secretion of Na+ and Cl- in 16 and 32 units of heat-labile enterotoxin treated groups in comparison to net absorption in control group, however, in animals treated with 8 units of heat-labile enterotoxin, no change in Na+ and Cl- fluxes was found when compared to control . Ca2+- ionophore increased net secretion of Na+ and Cl- in 16 and 32 units of heat-labile enterotoxin treated groups and also caused secretion in control group instead of net absorption . Ca2+ channel blocker and calmodulin inhibitor partially reversed the effect of heat-labile enterotoxin . The effect of Ca2+-ionophore was more pronounced in the control group while that of Ca2+ channel blocker and calmodulin inhibitor was more pronounced in 16 and 32 units of heat-labile enterotoxin treated groups . The findings suggest the involvement of Ca2+ and calmodulin in the action of heat-labile enterotoxin of Escherichia coli in mice.

Genes Dev, 1989 Jun, 3(6), 882 - 9
Regulated expression at high copy number allows production of a growth-inhibitory oncogene product in Drosophila Schneider cells; Johansen H et al.; The Drosophila metallothionein promoter (Mtn) was used to obtain efficient, regulated expression of foreign gene products inserted in high copy numbers into Drosophila melanogaster Schneider 2 cells . An expression unit comprised of a reporter gene {Escherichia coli galactokinase (galK)} fused to the Mtn promoter was stably introduced into Schneider 2 cells in up to several hundred copies per cell in a single transfection-selection event . This system contrasts dramatically with other eukaryotic systems that permit only a few copies of a gene to be stably inserted in a single transfection-selection event . The transfected Drosophila S2 cell lines expressed high levels of both galK mRNA and protein in response to metal induction . Most important, and in contrast to mammalian cells, expression remained fully regulated even at high copy number, with low basal expression maintained in the absence of inducer . This regulated system was used to obtain efficient expression in Drosophila cells of an otherwise lethal or growth-inhibitory gene product, the human H-ras oncogene . The ability to obtain regulated high-level expression of potentially lethal foreign proteins is unique to the Drosophila cell system.

Proc Natl Acad Sci U S A, 1989 Jun, 86(12), 4639 - 43
Theileria annulata sporozoite surface antigen expressed in Escherichia coli elicits neutralizing antibody; Williamson S et al.; Theileria annulata is an economically important protozoan parasite that threatens an estimated 250 million cattle with the disease tropical theileriosis . Development of a defined subunit vaccine is one means of trying to develop control measures against the disease . To this end we have characterized a surface antigen complex of the infective stage (sporozoite), by using a monoclonal antibody that neutralizes sporozoite infectivity in vitro . We have cloned the gene coding for this complex and have demonstrated that a fusion protein expressed from a fragment of this gene elicits strong neutralizing antibodies . Furthermore we provide data on the structure and expression of this gene . In particular we show that the region of the gene, expressed in one clone, codes for a protein segment relatively rich in proline residues . Also we demonstrate that expression of this gene appears to be stage specific, transcripts being present only in the sporoblast and sporozoite stages . The relevance of these findings to the production of a defined subunit vaccine is discussed.

Dev Biol, 1989 Jun, 133(2), 313 - 21
Tissue-specific expression of the 79B actin gene during Drosophila development; Courchesne-Smith CL et al.; We report temporal and tissue-specific patterns of expression of the 79B actin gene in Drosophila . The pattern of accumulation of 79B actin transcripts was determined and compared to the expression of a 79B actin promoter fused to the Escherichia coli beta-galactosidase reporter gene . This chimeric gene contained approximately 4 kb of 5' flanking DNA from the 79B actin gene . We found that beta-galactosidase activity in transformants was localized in the same tissues and possessed the same developmental timing as the transcripts of the 79B actin gene . Expression is limited to tubular-type muscles found predominantly in the thorax and leg, including the direct flight muscles, pleurosternal muscles, and muscles of the various leg segments . In addition, the 79B actin gene is expressed in muscles which support the head and abdomen, in the scutellar pulsatile organ, and in abdominal muscles which are present only in male flies.

Eur J Biochem, 1989 Jun 1, 182(1), 95 - 104
The influence of tryptophan on mobility of residues in the trp repressor of Escherichia coli; Lane AN; The relative mobility of residues in the trp repressor of Escherichia coli has been examined in the absence and presence of the corepressor L-tryptophan by one- and two-dimensional 1H NMR . A comparison of relative intensities of cross peaks in NOESY and COSY spectra allowed a rigid Tyr and a mobile Tyr residue, three mobile Ser residues and three mobile Lys residues to be detected . The two Tyr residues were assigned by selective nitration with tetranitromethane . The singly nitrated molecule (on Tyr7) binds the trp operator with an affinity close to that of the unmodified repressor . Measurements of the intraring cross-relaxation rate constant as a function of temperature for Tyr7 shows the presence of considerable internal motion on the subnanosecond time scale in the flexible N-terminal arm . The order parameter, S2, characterising the motion is 0.35, which increases to about 0.5 in the presence of Trp . Trp decreases both the amplitude of the motion and the rate of the motion . At least three of the six Ser residues of the trp repressor have greater mobility than expected for a rigid body, and two of the Ser residues are sensitive to the presence of Trp . The more mobile Ser residues are probably those on the N-terminal arm and the C-terminal sequence . These results complement the single-crystal X-ray diffraction studies for which the electron density of the first ten and last three amino acid residues is weak . The solution data are consistent with proposals that the flexible N-terminal arm of the trp repressor makes important contacts with the DNA.

Gastroenterology, 1989 Jun, 96(6), 1572 - 82
Evidence that rat Kupffer cells stimulate and inhibit hepatocyte protein synthesis in vitro by different mechanisms; West MA et al.; Kupffer cell control of hepatocyte protein synthesis may be an important mechanism involved in the regulation of normal liver function and may be one mechanism responsible for the alterations in liver function seen during sepsis . The present series of in vitro experiments compare the response to various inflammatory stimuli of hepatocytes cocultured with Kupffer cells with that of hepatocytes cultured alone . In the absence of inflammatory stimuli, Kupffer cells stimulated hepatocyte protein synthesis . Lipopolysaccharide or gentamicin-killed Escherichia coli triggered Kupffer cell-mediated inhibition of cocultured hepatocyte protein synthesis but had no effect on protein synthesis of hepatocytes cultured alone . Phorbol myristate acetate, muramyl dipeptide, and calcium ionophore had no effect on hepatocytes cultured alone but resulted in a loss of Kupffer cell-mediated stimulation of cocultured hepatocyte protein synthesis without inhibition . Addition of dexamethasone to cocultures prevented the Kupffer cell-mediated inhibition of hepatocyte protein synthesis triggered by lipopolysaccharide, but did not block Kupffer cell-mediated stimulation in the absence of lipopolysaccharide . The data suggest that Kupffer cells can stimulate and inhibit hepatocyte protein synthesis by independent mechanisms . Kupffer cells may be important regulators of hepatocellular function in health and disease.

J Cell Sci, 1989 Jun, 93 ( Pt 2), 227 - 32
Molecular cloning of the cDNA for ligatin; Jakoi ER et al.; We describe the first isolation and sequence of a partial cDNA clone encoding ligatin, a trafficking receptor for phosphoglycoproteins . The clone was isolated from a human U937 promonocyte lambda gt11 cDNA library using rabbit antiserum to rat ileal ligatin . RNA blot hybridization revealed that the intact receptor transcript in human cells is 2.4 kilobases (kb) . DNA sequencing together with expression of protein fusion products in Escherichia coli demonstrated that the cloned segment begins with a 1.2 kb open reading frame potentially encoding a 7.5 x 10(3) Mr section of the 10 x 10(3) Mr receptor followed by a 3' tail of 948 bases . The 225 bases of coding sequence correspond to the carboxyl region of ligatin and contain a potential acceptor site for asparagine-linked glycosylation . Neither a poly(A) sequence nor polyadenylation signal was found at the 3' end of the clone . In the 3' untranslated region there is a paired consensus sequence (TGAGnnnTTTTTCA) that is analogous to a conserved 12 base-pair sequence present in clusters in several growth-controlled mRNAs, including those for c-fos and beta-actin . The identity of this clone as ligatin was confirmed immunologically using antisera to an encoded fusion protein and three independent regions of its derived sequence . In addition, one of these antibodies produced a punctate immunofluorescence pattern within the cytosol of U937 cells in a similar fashion to anti-ligatin serum.

Agents Actions, 1989 Jun, 27(3-4), 274 - 6
Effects of prostaglandins and cAMP levels on monocyte IL-1 production; Kassis S et al.; The effects of prostaglandin E2 (PGE2), as well as other cAMP-elevating agents, on the in vitro production of interleukin-1 (IL-1) by human monocytes (HM) were examined . Exposure to E . coli lipopolysacharide (LPS) resulted in a dose- and time-dependent increase of IL-1 activity in monocytes culture supernatants . Maximal levels of secreted IL-1 in response to 10 ng LPS/ml were obtained at 18 h . PGE1, PGE2, cholera toxin (CT) and the phosphodiesterase inhibitor, isobutylmethylxanthin (IBMX), when added with LPS, resulted in a dose-dependent increase in cellular cAMP and in secreted IL-1 . Maximal levels of secreted IL-1 were 2.5-5.0-fold over LPS alone . When CT or PGE2 was added with IBMX a further increase was observed . These agents exhibited marginal effect on cell-associated IL-1 . Maximum cAMP levels was achieved at 10 min in response to either PGE1 or PGE2 and returned to near basal levels after 18 h . While PGE1 elevated cAMP to a larger extent than PGE2 (58- vs . 30-fold) the latter resulted in a higher levels of secreted IL-1 . Elevated cAMP persisted throughout the entire culture period in response to CT (4-6-fold) or IBMX (7-fold) . Thus, we conclude that in adherent HM, IL-1 production is potentiated and not inhibited by prostaglandins or agents that elevate cellular cAMP.

Anal Biochem, 1989 Jun, 179(2), 366 - 70
A modified primer extension procedure for specific detection of DNA-RNA hybrids on nylon membranes; Kainz P et al.; We have developed a modified primer extension procedure for specific detection of mRNA . Alkali-fragmented total cellular RNA or some RNA fraction is hybridized to single-stranded or double-stranded M13 DNA containing the insert of interest which is immobilized on nylon membranes . Hybridized RNA is then detected by incubation of membranes with Escherichia coli RNase H and DNA polymerase I . RNase H is used for nicking the RNA in the hybrids . The resulting 3'-OH groups can subsequently be used by DNA polymerase I to synthesize a labeled complementary strand . The method described is both relatively fast and sensitive and particularly useful for screening large numbers of DNA clones for their representation in RNA populations . Using total cellular RNA as hybridization probe and single-stranded M13 DNA as template as low as 0.25 ng of a specific mRNA was detected (2.5-fold background) when adding 1 microCi {3H}dCTP or 2.5 microCi {32P}d-CTP alternatively as radioactive precursor for the labeling reaction . The detection limit increased to 1 ng (2-fold background) with denatured replicative form double-stranded M13 DNA as template.

Mol Gen Genet, 1989 Jun, 217(2-3), 499 - 504
A precursor for a small stable RNA (10Sa RNA) of Escherichia coli; Subbarao MN et al.; Strains carrying plasmids that code for 10Sa RNA synthesize a larger molecule when the RNA processing enzyme RNase E is inactivated . The T1 fingerprint of 10Sa RNA and the larger molecule is very similar, but the latter contains additional oligonucleotides . We show that the larger RNA is converted to the smaller, mature RNA . The precursor molecule starts with an adenosine triphosphate and is therefore a primary transcript . RNase E is not the enzyme that processes p10Sa (precursor 10Sa) RNA into 10Sa RNA . The cell extract contains an activity that carries out this conversion . This activity requires the dication Mn2+.

Mol Gen Genet, 1989 Jun, 217(2-3), 411 - 8
Aminoglycoside suppression at UAG, UAA and UGA codons in Escherichia coli and human tissue culture cells; Martin R et al.; We have compared the suppression of nonsense mutations by aminoglycoside antibiotics in Escherichia coli and in human 293 cells . Six nonsense alleles of the chloramphenicol acetyl transferase (cat) gene, in the vector pRSVcat, were suppressed by growth in G418 and paromomycin . Readthrough at UAG, UAA and UGA codons was monitored with enzyme assays for chloramphenicol acetyl transferase (CAT), in stably transformed bacteria and during transient expression from the same plasmid in human 293 tissue culture cells . We have found significant differences in the degree of suppression amongst three UAG codons and two UAA codons in different mRNA contexts . However, the pattern of these effects are not the same in the two organisms . Our data suggest that context effects of nonsense suppression may operate under different rules in E . coli and human cells.

Microb Pathog, 1989 Jun, 6(6), 415 - 24
Recombinant interleukin-1 stimulates clearance of Escherichia coli K1 bacteraemia; Pelkonen S et al.; Enhancement of non-specific resistance to neonatal Escherichia coli K1 infection by interleukin-1 (IL-1) was analysed . Recombinant human IL-1 administered prophylactically to newborn LPS-non-responsive C3H/HeJ mice induced rapid clearance of E . coli 018:K1 bacteraemia . The effect was dose-dependent and was observed with mice treated immediately to 1 day before bacterial challenge, whereas treatment 2 days before challenge was ineffective . was ineffective . Clearance of intravenously injected radiolabelled 018:K1 E . coli suggested that IL-1 triggered defence mechanisms that contribute to bacterial sequestration and killing in the spleen and liver . Comparable increase in bacterial clearance occurred in naturally resistant LPS-responsive mice that had been subjected to transient E . coli K1 bacteraemia and showed increased resistance to reinfection . In the course of E . coli K1 bacteraemia a strong synthesis of acute phase reactants was observed in both susceptible and resistant mouse strains, which indicated that these proteins alone cannot confer natural resistance to E . coli K1 . IL-1 induced a very rapid synthesis of acute phase proteins . The clearance of K1 E . coli when still viable in IL-1-treated animals suggested that acute phase proteins are not likely to be major mediators of the IL-1-enhanced non-specific resistance.

Microb Pathog, 1989 Jun, 6(6), 403 - 14
Molecular cloning and expression of a T-cell stimulating membrane protein of Francisella tularensis; Sjostedt A et al.; The isolation and expression in Escherichia coli of a gene encoding a T-cell stimulating 17 kiloDalton (kDa) membrane protein of Francisella tularensis is described . A genomic library of DNA from the live vaccine strain LVS of F . tularensis was constructed in the E . coli expression vector phage lambda gt11 . The library was probed with antibodies directed against the 17 kDa protein . One recombinant phage was isolated, containing a 2.8 kilobase (kb) DNA insert . The insert was cleaved and a resulting 1.2 kb fragment was found to express the 17 kDa protein . The 1.2 kb fragment was inserted in the high copy number plasmid pUC18 and expressed in E . coli . Membrane preparations of these bacteria induced a response in T cells from F . tularensis-primed individuals but not in T cells from non-primed individuals . The cloned gene may become useful in studies on host interaction with F . tularensis and enable a precise identification of bacterial structures involved in the T-cell response.

DNA, 1989 Jun, 8(5), 369 - 75
A strategy to optimize translation initiation in recombinant mRNA: application to the Rop gene; Bottaro G et al.; Using the Escherichia coli Rop gene, we demonstrate a strategy that could be applied generally to optimize the initiation of translation of recombinant genes in E . coli . This involves cloning of the gene encoding the protein of interest in a suitable expression vector between an "efficient" ribosome binding site and the gene for the alpha-peptide of beta-galactosidase . By oligonucleotide-directed deletion mutagenesis, the two coding sequences are then fused in the correct frame . A second oligonucleotide is then used to place the initiator AUG (or GUG) at the correct distance from the Shine-Dalgarno sequence . In this step, however, we use an oligonucleotide that has a degenerate sequence . That is, on the basis of the "efficient" ribosome binding site sequence, we introduce random substitutions at various positions, both upstream and downstream from the initiator ATG, to obtain, after the mutagenesis experiment, a collection of random ribosome binding sites fused to the coding sequence . The development of blue colonies on indicator plates permits selection of clones in which an efficient ribosome binding site has been created for the specific gene of interest . We discuss the results obtained by applying the method to the Rop gene.

DNA, 1989 Jun, 8(5), 351 - 9
Genomic cloning and heterologous expression of human differentiation-stimulating factor; Lowe DG et al.; Differentiation-stimulating factor (D-factor) purified from mouse Ehrlich ascites cells was sequenced partially and found to be almost identical to leukemia inhibitory factor (LIF) from mouse Krebs II ascites cells . In comparison to LIF, D-factor had an additional amino-terminal serine residue . Using synthetic oligonucleotide probes designed from the murine D-factor sequence, we cloned the human gene encoding D-factor . A partial D-factor cDNA was cloned from COS-1 cells transfected with the human D-factor gene under the control of a heterologous promoter . We used this cDNA to construct a vector for direct expression of the protein in Escherichia coli . A mammalian cell expression vector was constructed using the signal sequence of interferon-alpha A linked to the D-factor cDNA . Both forms of recombinant human D-factor were active on the murine myeloid leukemia cell line M1 in a dose- and time-dependent manner for the inhibition of {3H}thymidine incorporation, and also induced phagocytosis, Fc receptor expression, and prostaglandin E2 synthesis by M1 cells.

Mol Microbiol, 1989 Jun, 3(6), 715 - 22
Transcription of the sulA-ompA region of Escherichia coli during the SOS response and the role of an antisense RNA molecule; Cole ST et al.; The transcriptional pattern of the 22 min region of the Escherichia coli chromosome containing the linked sulA and ompA genes, which encode an SOS-inducible inhibitor of cell division and a constitutively expressed, major outer membrane protein, respectively, has been re-examined . During normal growth, the sulA gene was repressed whereas the ompA gene produced a stable 1250 nucleotide transcript . Counter-transcription of sulA occurred from a promoter situated in the sulA-ompA intergenic region and the product of this transcriptional circuit, named isf, is a 353 nucleotide untranslated RNA . Since the isf RNA is complementary to the 3'-end of the sulA transcript, it could modulate sulA function by serving as an anti-messenger . On induction of the SOS-response, massive transcription of sulA took place, resulting in the 'silencing' of the isf gene, production of an abundant approximately 615 nucleotide sulA mRNA and a novel hybrid transcript of approximately 2100 nucleotides encoding both the SulA and OmpA proteins . Production of the latter RNA species, caused by transcription reading through the sulA terminator, the intergenic region and the coding sequences, was accompanied by a decrease in the abundance of the ompA mRNA as a result of promoter occlusion . However, the amount of OmpA protein produced was only slightly reduced.

Arch Surg, 1989 Jun, 124(6), 669 - 72
Thromboxane A2-receptor blockade and prostacyclin in porcine Escherichia coli shock; Svartholm E et al.; Septic shock was induced by intravenous infusion of live Escherichia coli in pigs to investigate the influences on central hemodynamic, coagulation, and fibrinolytic reactions by a thromboxane A2 (TxA2)-receptor blocker (BM 13.177; n = 6) and a prostacyclin analogue (iloprost or ZK 36,374; n = 7) . The early pulmonary vasoconstriction following E coli infusion was attenuated, but not abolished, by BM 13.177 . Only minor effects were seen after pretreatment with iloprost . Neither drug had any major effect on the coagulation and fibrinolytic activation . These results confirm that TxA2 is an important, but not the only, mediator of early pulmonary vascular response in porcine septicemia and that neither TxA2 nor prostacyclin is of major importance for the hemostatic reactions in this shock model.

J Immunol, 1989 Jun 1, 142(11), 3884 - 93
Generation and characterization of hamster monoclonal antibodies that neutralize murine tumor necrosis factors; Sheehan KC et al.; mAb to murine TNF (MuTNF) were produced after immunization of Armenian hamsters with purified, Escherichia coli-derived rMuTNF-alpha . Antibody produced from clone TN3-19.12, was purified and was found to inhibit 100% of the lytic activity of either recombinant or natural MuTNF-alpha at an antibody input of 25 ng/U . TN3-19.12 also inhibited all the lytic activity in culture supernatants from a variety of T cell sources, including activated T cell clones and T cell hybridomas (all of which expressed high levels of TNF-alpha and TNF-beta (lymphotoxin, LT) mRNA) . Western blot analysis was used to document the physical form(s) of MuTNF recognized by TN3-19.12 . Recombinant and macrophage-derived TNF displayed identical patterns of a single band with Mr 17 kDa . In contrast, T cell culture supernatants exhibited patterns consisting of two bands with Mr 17 and 24.7 kDa . The higher m.w . form was glycosylated based on its sensitivity to n-glycanase and displayed a m.w . consistent with that of TNF-beta (LT) . These data suggest that TN3-19.12 recognizes both MuTNF-alpha and MuTNF-beta (LT) . Monoclonal TN3-19.12 and polyvalent rabbit anti-rTNF were used to establish a MuTNF-specific ELISA capable of detecting picogram quantities of recombinant or natural TNF . This assay was used to detect TNF in the sera of mice challenged with a lethal dose of LPS . Peak TNF serum levels of 11 ng/ml were observed in these animals 90 min after i.p . LPS administration and then rapidly declined to near base line levels by 3 h . These values were confirmed by quantitating levels of TNF functional activity in the same samples . TN3-19.12 injected into mice subsequently treated with LPS prevented the detection of TNF in the circulation by either assay and protected mice from the lethal effects of endotoxin shock . Thus, TN3-19.12 effectively neutralizes endogenously produced TNF in vivo.

Gene, 1989 May 30, 78(2), 365 - 70
Functional expression of human glucose-6-phosphate dehydrogenase in Escherichia coli; Persico MG et al.; The complete coding sequence for human glucose-6-phosphate-dehydrogenase (G6PD) was inserted downstream from the tac promoter of a plasmid, pJF118EH, which also carries the lacIq repressor gene . When Escherichia coli strains (that are unable to grow on glucose due to the absence of functional zwf (G6PD-) and pgi genes) were transformed with this plasmid (pAC1), they were able to grow on glucose as sole carbon source . The rate of growth on glucose was faster in the presence of the inducer of the tac promoter, isopropyl-beta-D-thiogalactopyranoside (IPTG) . Extracts of the transformed cells contained a G6PD activity that was not detectable in the parental strains and that was inducible by IPTG . The G6PD activities from normal E . coli and from pAC1-transformed cells comigrated with human G6PD when subjected to electrophoresis on agarose gels . However, when denatured, the G6PD produced by pAC1 was, like the human enzyme, distinguishable from the E . coli-encoded enzyme on the basis of its immunoreactivity with antibody specific for human G6PD . Therefore, human G6PD can be expressed in E . coli and can function to complement the bacterial enzyme deficiency.

Gene, 1989 May 30, 78(2), 243 - 54
Human desmin-coding gene: complete nucleotide sequence, characterization and regulation of expression during myogenesis and development; Li ZL et al.; A recombinant clone encoding for the human desmin gene (des) has been isolated and characterized and its complete nucleotide sequence has been determined . The 8.4-kb gene has nine exons separated by introns ranging in size from 0.1-2.2 kb . Comparison of the human des gene with that of the hamster has shown that there is a full correspondence in position, size and sequence of the exons . There are eight introns in both the human and the hamster des genes . Although the nucleotide sequence of the introns reveals a large divergence, splice junction sequence signals are conserved . A particularly striking feature of the human des gene is the 1.2-kb repetitive sequence found in the introns . These sequences all belong to the human AluI family . When the 5'- and 3'-untranslated regions of the human vim and des genes were compared it was found that there was a 16-mer consensus element similar to that described by Quax et al . {Cell 43 (1985) 327-338} for the hamster and an 11-bp sequence with homology to the distal regulatory sequence of human and mouse alpha-cardiac actin-coding genes {Minty and Kedes, Mol . Cell . Biol . 6 (1986) 2125-2136} in the 5'-flanking region . The 3'-untranslated region of the human des gene was found to be conserved when compared to the hamster des gene . Only one species of desmin RNA of 2.2 kb was found in human striated and smooth muscle both in vivo and in vitro.

Biochemistry, 1989 May 30, 28(11), 4914 - 22
Energetics of complementary side-chain packing in a protein hydrophobic core; Kellis JT Jr et al.; The energetics of complementary packing of nonpolar side chains in the hydrophobic core of a protein were analyzed by protein engineering experiments . We have made the mutations Ile----Val, Ile----Ala, and Leu----Ala in a region of the small bacterial ribonuclease barnase where the major alpha-helix packs onto the central beta-sheet . The destabilization resulting from the creation of cavities was determined by measuring the decrease in free energy of folding from reversible denaturation induced by urea, guanidinium chloride, or heat . The different methods give consistent and reproducible results . The loss in free energy of folding for the mutant proteins is 1.0-1.6 kcal/mol per methylene group removed . This exceeds by severalfold the values obtained from model experiments of the partitioning of relevant side chains between aqueous and nonpolar solvents . Much of this discrepancy arises because two surfaces are buried when a protein folds--both the amino acid side chain in question and the portions of the protein into which it packs . These experiments directly demonstrate that the interior packing of a protein is crucial in stabilizing its three-dimensional structure: the conversion of leucine or isoleucine to alanine in the hydrophobic core loses half the net free energy of folding of barnase with a concomitant decrease in yield of the expressed recombinant protein.

Biochemistry, 1989 May 30, 28(11), 4607 - 15
An NMR study of the helix V-loop E region of the 5S RNA from Escherichia coli; Zhang P et al.; Experiments are described that complete the assignment of the imino proton NMR spectrum of the fragment 1 domain from the 5S RNA of Escherichia coli . Most of the new assignments fall in the helix V-loop E portion of the molecule (bases 70-78 and 98-106), the region most sensitive to the binding of ribosomal protein L25 . The spectroscopic data are incompatible with the standard, phylogenetically derived model for 5S RNA, which makes all the base pairs possible in loop E with the sequences aligned in parallel (C70-G106, C71-G105, etc.) {see Delihas et al . (1984) Prog . Nucleic Acid Res . Mol . Biol . 31, 161-190} . Furthermore, the alternative loop E model proposed for spinach chloroplast 5S RNA by Romby et al . {(1988) Biochemistry 27, 4721-4730} does not apply to the closely homologous 5S RNA from E . coli . The 5S RNAs from E . coli and spinach chloroplasts do not have the same secondary structures in solution despite their strong sequence homologies, and neither appears to conform to the standard model for 5S RNA in the loop E region.

Biochemistry, 1989 May 30, 28(11), 4601 - 7
Fluorescent oligonucleotides and deoxynucleotide triphosphates: preparation and their interaction with the large (Klenow) fragment of Escherichia coli DNA polymerase I; Allen DJ et al.; Fluorescent derivatives of short oligonucleotides of defined sequence were prepared by the incorporation of 5-(propylamino)uridine via current phosphoramidite chemistry, followed by derivatization of the propylamine function with mansyl chloride . These oligomers, annealed to complementary oligomers, yielded short duplex DNA fluorescently labeled at a specific base . The fluorescence emission from this labeled duplex increases upon binding to the Klenow fragment of DNA polymerase I (KF) at specific positions within the duplex DNA . By varying the position of the label within the duplex DNA and observing the emission, points of strong enzyme-DNA interactions were elucidated . A similar fluorescent derivative of a deoxynucleoside triphosphate (dNTP), 5-{{{{{{(5- sulfonaphthalenyl)amino}ethyl}amino}carbonyl}- methyl}thio}-2'-deoxyuridine 5'-triphosphate (AEDANS-S-dUTP), was synthesized, whose emission also was increased upon binding to KF . The change in emission intensities between unbound and bound substrates enabled the measurements of KDs for the DNA and dNTP derivative, which were found to be 0.15 nM and 2.9 microM, respectively . Stopped-flow measurements on these species yielded association and dissociation rates for each . Anisotropy measurements of the labeled base at various positions in the duplex yielded values that support the measurements made by observing the emission intensities.

Biochem Biophys Res Commun, 1989 May 30, 161(1), 236 - 41
Molecular cloning and amino acid sequence of rat kidney aminopeptidase M: a member of a super family of zinc-metallohydrolases; Malfroy B et al.; Using a polyclonal antibody, a partial cDNA clone for rat aminopeptidase M was identified in a lambda gt11 library from rat kidney . A synthetic oligonucleotide probe derived from the sequence of the insert was used to screen a randomly primed lambda gt10 library . This allowed the identification of several overlapping clones encoding the full sequence of the enzyme . The reading frame, 2898 base pairs in length, encodes a 966 amino acid polypeptide . A highly hydrophobic segment, 24 amino acids in length, located close to the aminoterminus, is proposed to serve as the membrane-spanning domain for this membrane-bound enzyme . The sequence includes nine potential N-linked glycosylation sites and one potential sulfation site . In addition, the rat aminopeptidase M sequence contains an eight amino acid consensus sequence believed to serve as the zinc binding domain in a family of zinc-metallohydrolases . Rat aminopeptidase M shows 77% similarity with the recently cloned human enzyme, as well as weaker but significant similarity with aminopeptidase N from E . coli (18%) and with human leukotriene A4 hydrolase (21%).

Biochemistry, 1989 May 30, 28(11), 4568 - 74
Crystal structure of a cAMP-independent form of catabolite gene activator protein with adenosine substituted in one of two cAMP-binding sites; Vaney MC et al.; Catabolite gene activator protein (CAP) in the presence of cAMP stimulates transcription from several operons in Escherichia coli . A cAMP-independent variant, in which Ala-144 is replaced by Thr (CAP91), is activated by analogues of cAMP, such as adenosine, which do not activate the wild-type CAP . In order to test the effect of adenosine on the structure, a crystal of CAP91 grown as a complex with cAMP was soaked in a solution of 10 mM adenosine, and X-ray diffraction data were measured to 3.5-A resolution . The difference Fourier map calculated with phases from the CAP91 structure showed significant negative density at the position of the phosphate of cAMP bound in one subunit of the CAP91 dimer . Adenosine was preferentially substituted for cAMP in the subunit in the "closed" conformation, while the cAMP-binding site of the "open" subunit was apparently still occupied by cAMP . The structure was refined by restrained least-squares methods to an R factor of 20.2% . Adenosine is not bound in exactly the same position as cAMP; instead, the 5'-OH of adenosine is in a new position that allows formation of two hydrogen bonds with Ser-83, replacing two of the three interactions of the phosphate of cAMP with Arg-82 and Ser-83.

Biochemistry, 1989 May 30, 28(11), 4531 - 5
Antisera specific for the alpha 1, alpha 2, alpha 3, and beta subunits of the Na,K-ATPase: differential expression of alpha and beta subunits in rat tissue membranes; Shyjan AW et al.; We have developed a panel of antibodies specific for the alpha 1, alpha 2, alpha 3, and beta subunits of the rat Na,K-ATPase . TrpE-alpha subunit isoform fusion proteins were used to generate three antisera, each of which reacted specifically with a distinct alpha subunit isotype . Western blot analysis of rat tissue microsomes revealed that alpha 1 subunits were expressed in all tissues while alpha 2 subunits were expressed in brain, heart, and lung . The alpha 3 subunit, a protein whose existence had been inferred from cDNA cloning, was expressed primarily in brain and copurified with ouabain-inhibitable Na,K-ATPase activity . An antiserum specific for the rat Na,K-ATPase beta subunit was generated from a TrpE-beta subunit fusion protein . Western blot analysis showed that beta subunits were present in kidney, brain, and heart . However, no beta subunits were detected in liver, lung, spleen, thymus, or lactating mammary gland . The distinct tissue distributions of alpha and beta subunits suggest that different members of the Na,K-ATPase family may have specialized functions.

Biochem Biophys Res Commun, 1989 May 30, 161(1), 229 - 35
A long-acting heat-stable enterotoxin analog of enterotoxigenic Escherichia coli with a single D-amino acid; Kubota H et al.; A heat-stable enterotoxin (STp) consisting of 18 amino acid residues including 6 half-cystine residues is produced by a porcine strain of enterotoxigenic Escherichia coli . Analogs of STp with replacements of single residues at each from positions 5 to 17 by the corresponding D-amino acid residue were synthesized by a solid-phase method . Of these analogs, {D-Cys5}-STp{5-17} showed the same biological properties as STp{5-17} . Moreover, its activity to cause fluid accumulation in suckling mouse lasts more than 24 hours, whereas the activity of STp{5-17} decreases after 6-10 hours . These results indicate that the action of the analog {D-Cys5}-STp{5-17} is strongly agonistic to that of the native ST.

Biochem Biophys Res Commun, 1989 May 30, 161(1), 197 - 203
Inositol lipid metabolism in vasopressin stimulated hepatocytes from rats infused with tumor necrosis factor; Spitzer JA et al.; We studied the effect of i.v . infusion of human recombinant tumor necrosis factor alpha (rHuTNF alpha, Cetus, 15 micrograms/100 g bw over 3 h) on vasopressin (VP)-stimulated 32P-inositol lipid turnover and the release of 3H-inositol phosphates in isolated rat hepatocytes . The early VP-induced decrease (within 30 s) in 32P-phosphatidylinositol 4-phosphate and 32P-phosphatidylinositol 4,5-bisphosphate labeling was significantly reduced (-40%) and at the same time the uptake of 32P into phosphatidic acid was 50% lower than in saline-infused (matched control) rats . Within 5 min of VP-stimulation, lower 32P phosphatidylinositol (-40%) and higher 32P-phosphatidic acid (+30%) labeling were observed in rHuTNF alpha-infused rats . Infusion of rHuTNF alpha also affected the VP-induced release of 3H-inositol phosphates . The accumulation of 3H-inositol-labeled water soluble products was decreased by 25% and 17% at 30 s and 10 min, respectively . These data show that rHuTNF alpha mimics early perturbations induced by Escherichia coli endotoxin infusion in VP-stimulated inositol lipid metabolism in rat hepatocytes.

Biochemistry, 1989 May 30, 28(11), 4749 - 54
Identification of lung major GTP-binding protein as Gi2 and its distribution in various rat tissues determined by immunoassay; Asano T et al.; Antisera were raised in rabbits against the 40-kDa alpha subunit of bovine lung GTP-binding protein, which were identified as the alpha subunit of Gi2 (Gi2 alpha) by the analysis of the partial amino acid sequence . Antibodies were purified with a Gi2 alpha-coupled Sepharose column and then were passed through a Gi1 alpha-coupled Sepharose column to remove antibodies reactive also with 41-kDa alpha . Purified antibodies reacted with Gi2 alpha, but not with Gi1 alpha, Gi3 alpha, or Go alpha in an immunoblot assay . A sensitive enzyme immunoassay method for the quantification of Gi2 alpha was developed by using these purified antibodies . The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli . The minimal detection limit of the assay was 1 fmol, or 40 pg . Samples from various tissues were solubilized with 2% sodium cholate and 1 M NaCl, and the concentrations of Gi2 alpha were determined . Gi2 alpha was detected in all the tissues examined in the rat . The highest concentration was found in platelets and leukocytes when the data were expressed as picomoles per milligram of protein . The spleen, lung, and cerebral cortex contained relatively high levels of Gi2 alpha . In the bovine brain, Gi2 alpha was distributed almost uniformly among the various regions . The concentrations of Gi2 alpha were constant in the rat brain throughout ontogenic development, in contrast with those of Go alpha which were markedly increased with age.

Biochem Biophys Res Commun, 1989 May 30, 161(1), 335 - 41
Characterization of a cysteine-free analog of recombinant human basic fibroblast growth factor; Arakawa T et al.; Using oligo site-directed mutagenesis, we have modified our synthetic gene for human basic fibroblast growth factor (bFGF) to replace all four cysteine codons with serine codons . The corresponding protein was expressed in Escherichia coli and purified from inclusion bodies by solubilization in urea followed by a series of column chromatographies and a folding step . The resulting protein, having no cysteine residues, is unable to form either intramolecular or intermolecular disulfide bonds . The secondary and tertiary structures of the purified analog, as determined by circular dichroism and fluorescence spectroscopy, were identical within experimental error to recombinant bovine and human bFGF with unaltered amino acid sequences . Reflecting the similar conformation, the analog protein exhibited mitogenic activity on NIH 3T3 cells which was indistinguishable from the natural sequence molecule.

Biochemistry, 1989 May 30, 28(11), 4717 - 24
Cryoelectron microscopy of Escherichia coli F1 adenosinetriphosphatase decorated with monoclonal antibodies to individual subunits of the complex; Gogol EP et al.; Monoclonal antibodies directed against epitopes on each of the five subunits (alpha, beta, gamma, delta, and epsilon) of the Escherichia coli F1 ATPase (ECF1) have been prepared and used to localize the subunits in the enzyme complex . Fab' fragments, prepared by pepsin digestion of the antibodies, were bound to ECF1 and visualized by cryoelectron microscopy of the unstained, frozen hydrated ECF1-Fab' complexes . Besides aiding in the identification of the ECF1 subunits, addition of Fab's to the specimen fortuitously offers additional advantages in this technique . ECF1 labeled with anti-alpha Fab' is uniformly oriented in the amorphous ice layer, in contrast to unlabeled ECF1, which exhibits a multitude of projection views when examined in ice . Almost all complexes display a triangular projection, which image averaging reveals to be a hexagonal view of ECF1 with Fab' fragments labeling every other peripheral subunit, confirming the alternating arrangement of alpha and beta subunits in the enzyme . A density in the interior of the structure is positioned asymmetrically, adjacent to an unlabeled peripheral mass, indicating that its primary linkage is to a beta rather than an alpha subunit . The composition of the asymmetric density was explored by examining the trypsin-treated ECF1, taking advantage of the unique orientation induced by the binding of anti-alpha Fab' . Trypsin treatment releases the delta and epsilon subunits and cleaves the gamma subunit; the internal density is reduced but not eliminated, showing the contribution of the gamma subunit to the residual structure, and suggesting that the loss of the delta and epsilon subunits, or a structural rearrangement of the gamma subunit, is responsible for its smaller size.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1989 May 30, 28(11), 4709 - 16
Molecular architecture of Escherichia coli F1 adenosinetriphosphatase; Gogol EP et al.; The structure of the E . coli F1 ATPase (ECF1) has been studied by a novel combination of two specimen preparation and image analysis techniques . The molecular outline of the ECF1 was determined by three-dimensional reconstruction of images of negatively stained two-dimensional crystals of ECF1 . Internal features were revealed by analysis of single particles of ECF1, preserved in their native state in a thin layer of amorphous ice, and examined by cryoelectron microscopy . Various projections of the unstained ECF1 were interpreted consistently with the three-dimensional structure in negative stain, yielding a more informative description of the enzyme than otherwise possible . Results show that the ECF1 is a roughly spherical complex approximately 90-100 A in diameter . Six elongated protein densities (the alpha and beta subunits, each approximately 90 A X approximately 30 A in size) comprise its hexagonally modulated periphery . At the center of the ECF1 is an aqueous cavity which extends nearly or entirely through the length of the complex . A compact protein density, located at one end of the hexagonal barrel and closely associated with one of the peripheral subunits, partially obstructs the central cavity.

Nucleic Acids Res, 1989 May 25, 17(10), 3655 - 61
Complementation of a Dictyostelium discoideum thymidylate synthase mutation with the mouse gene provides a new selectable marker for transformation; Chang AC et al.; A cDNA encoding mouse thymidylate synthase has been inserted 3' to the Dictyostelium discoideum actin 15 promoter in an E . coli-D.discoideum shuttle vector . When this construct was introduced into a D.discoideum thymidylate synthase mutant strain HPS400, stable transformants were obtained at high frequency . These transformants grew in standard axenic medium without requiring exogenous thymidine . This construct provides a second selectable marker for use in transformation of D.discoideum.

J Biol Chem, 1989 May 25, 264(15), 8978 - 84
Intracellular thionins of barley . A second group of leaf thionins closely related to but distinct from cell wall-bound thionins; Reimann-Philipp U et al.; Leaf thionins of barley have been identified as a novel class of cell wall proteins, toxic to plant pathogenic fungi, and possibly involved in the defense mechanism of plants (Bohlmann, H., Clausen, S., Behnke, S., Giese, H., Hiller, C., Reimann-Philipp, U., Schrader, G., Barkholt, V., and Apel, K., (1988) EMBO J . 7, 1559-1565) . In the present work a second subfraction of thionins has been detected within the leaf cell, mainly in the vacuole . Thionins of both groups are closely related to each other . They are toxic to phytopathogenic fungi as well as to plant protoplasts, they share similar amino acid sequences, and their synthesis in etiolated seedlings of barley is down-regulated by light . Despite these similarities each of the two subfractions of thionins could be clearly distinguished by its subcellular distribution . In ultrathin sections of embedded etiolated leaf material, cell wall thionins could be immunogold labeled specifically by an antiserum raised against a fusion protein of Escherichia coli beta-galactosidase and the 15,000 Mr precursor polypeptide of thionins . This antiserum did not react with intracellular thionins . Inversely, intracellular thionins were recognized specifically by an anti-serum raised against soluble leaf thionins . The possible function of intracellular thionins as part of a defense mechanism has been discussed.

Nucleic Acids Res, 1989 May 25, 17(10), 3757 - 62
Ribosomal protein L7/L12 has a helix-turn-helix motif similar to that found in DNA-binding regulatory proteins; Rice PA et al.; Inspection of the structure of the C-terminal domain of ribosomal protein L7/L12 (1) reveals a helix-turn-helix motif similar to the one found in many DNA-binding regulatory proteins (2-5) . The 19 alpha-carbon atoms of the L7/L12 alpha-helices superimpose on the DNA binding helices of CAP and cro with root-mean-square distances between corresponding alpha carbons of 1.45 and 1.55 A, respectively . These helices in L7/L12 are within a patch of highly conserved residues on the surface of L7/L12 whose role is as yet uncertain . We raise the possibility that they may constitute a binding site for nucleic acids, most probably RNA . Consistent with this hypothesis are calculations of the electrostatic charge potential surrounding the protein, which show a region of positive potential centered on the first of these helices.

Nucleic Acids Res, 1989 May 25, 17(10), 3673 - 88
In vitro transcription analysis of the role of flanking sequence on the DNA sequence specificity of adriamycin; Trist H et al.; An in vitro transcription assay has been used to define twelve high occupancy transcriptional blockage sites in 260 bp of heterogenous DNA on three promoter-containing DNA fragments . Transcription proceeded to immediately upstream of CpA sequences in nine of these sites, and this defines the most preferred intercalation site as CpA . In almost all cases, this sequence was flanked by T on the 5' end . The consensus sequence for the highest affinity Adriamycin site is therefore 5'-TCA, with some evidence for preference of AT base pairs flanking both ends of this trinucleotide {i.e., (t)TCA(a.t)(a.t)} . In contrast, lower occupancy CpA sites were flanked on the 5' end by a GC base pair, with a preference for up to three GC base pairs on the 3' end . The low affinity consensus sequence is therefore (G.C)CA(g.c)(g.c)(g.c).

J Biol Chem, 1989 May 25, 264(15), 8597 - 601
para-aminobenzoate synthesis from chorismate occurs in two steps; Nichols BP et al.; Escherichia coli p-aminobenzoate synthase is composed of two nonidentical subunits encoded by pabA and pabB and has been assumed to be the sole enzyme responsible for p-aminobenzoate biosynthesis from chorismate and glutamine . Plasmids were constructed that overproduce the p-aminobenzoate synthase subunits 250-500-fold . Partial purification of the subunits revealed that they form a diffusible intermediate that is subsequently converted to p-aminobenzoate by a second enzyme (Mr = 49,000) temporarily designated enzyme X.

J Biol Chem, 1989 May 25, 264(15), 8563 - 7
Transfer of phosphoryl group between two regulatory proteins involved in osmoregulatory expression of the ompF and ompC genes in Escherichia coli; Aiba H et al.; EnvZ is a cytoplasmic membrane protein which is involved in osmoregulatory expression of the ompF and ompC genes in Escherichia coli possibly by sensing the environmental osmotic signal . A truncated form of the EnvZ protein (EnvZ*), comprising 82% of EnvZ starting from the C terminus, was purified to homogeneity . The purified EnvZ* was autophosphorylated with ATP . The phosphoryl group on EnvZ* could then be rapidly transferred to OmpR, which is a positive regulator of the ompF and ompC genes and which was proposed to interact with EnvZ in the process of osmoregulation . In the presence of ATP, the phosphorylated OmpR was rapidly dephosphorylated . These results suggest that the transfer of the phosphoryl group between EnvZ and OmpR plays an important role in the signaling pathway in osmoregulation.

J Biol Chem, 1989 May 25, 264(15), 8509 - 19
The primary structure of rabbit liver mitochondrial serine hydroxymethyltransferase; Martini F et al.; The complete amino acid sequence of mitochondrial serine hydroxymethyltransferase from rabbit liver was determined . The sequence was obtained from analysis of peptides isolated from chymotryptic, cyanogen bromide, and limited acid cleavages of the protein . The enzyme consists of four identical subunits, each of 475 residues, i.e . 8 residues shorter than the subunit of the corresponding cytosolic isoenzyme . The sequences of the two rabbit proteins are easily aligned, provided a gap of 5 residues near the amino terminus and a gap of 3 residues near the carboxyl terminus are included in the mitochondrial sequence . The overall degree of identity between the two isoenzymes is 61.9%, whereas the structural identity of each eukaryotic isoenzyme with the corresponding Escherichia coli enzyme is about 40% . The rabbit isoenzymes are about 70 residues longer than the E . coli enzyme, with one-half of these residues accounted for by insertions in both isoenzymes near their carboxyl terminus . Predictions of secondary structure and calculations of hydropathy profiles are also presented, suggesting an even more extensive degree of identity in the three-dimensional folding of the three proteins, in accord with the known similarity of their catalytic properties . Evidence was obtained for the existence of additional molecular forms of the mitochondrial protein, differing in the absence of some amino acid residues at the amino terminus of the polypeptide chain.

J Biol Chem, 1989 May 25, 264(15), 8479 - 82
Zinc protects Escherichia coli against copper-mediated paraquat-induced damage; Korbashi P et al.; The essential mediatory role of copper and iron in paraquat-induced biological damage has been recently demonstrated . It was postulated that these transition metals undergo cyclic redox reactions and serve as centers for repeated production of hydroxyl radical, which are the ultimate deleterious agents . Additionally, we had presented evidence indicating efficient protection against paraquat toxicity by agents commonly employed (chelators, chemical scavengers, and protecting enzymes) . In this study we have used the Escherichia coli model in order to develop a new approach for protection against paraquat-induced metal-mediated cellular injury . It entails the administration of excess zinc (up to 50-fold over copper), which results in an inhibition of the toxic effect of paraquat . Lineweaver-Burk analysis demonstrates the competitive mode of this inhibition . The suggested mechanism involves either the direct displacement of copper by zinc or the formation of a ternary complex, (formula; see text) in which the binding of Cu(II) is weakened by the binding of Zn(II), interfering with the copper-mediated free radicals formation . Thus, use of redox-inactive metals, which possess high similarity of their ligand chemistry to that of iron and copper but are of relative low toxicity by themselves, should be considered for intervention in paraquat toxicity and in other metal-mediated free radical-induced injurious processes.

J Biol Chem, 1989 May 25, 264(15), 8686 - 91
Identification of amino acid residues modified by pyridoxal 5'-phosphate in Escherichia coli glutamine synthetase; Di Ianni CL et al.; Chemical modification studies with pyridoxal 5'-phosphate have indicated that lysine(s) appear to be at or near the active site of Escherichia coli glutamine synthetase (Colanduoni, J., and Villafranca, J . J . (1985) J . Biol . Chem . 260, 15042-15050; Whitley, E . J., Jr., and Ginsburg, A . (1978) J . Biol . Chem . 253, 7017-7025) . Enzyme samples were prepared that contained approximately 1, approximately 2, and approximately 3 pyridoxamine 5'-phosphate residues/50,000-Da monomer; the activity of each sample was 100, 25, and 14% of the activity of unmodified enzyme, respectively . Cyanogen bromide cleavage of each enzyme sample was performed, the peptides were separated by high performance liquid chromatography, and the peptides containing pyridoxamine 5'-phosphate were identified by their absorbance at 320 nm . These isolated peptides were analyzed for amino acid composition and sequenced . The N terminus of the protein (a serine residue) was modified by pyridoxal 5'-phosphate at a stoichiometry of approximately 1/50,000 Da and this modified enzyme had full catalytic activity . Beyond a stoichiometry of approximately 1, lysines 383 and 352 reacted with pyridoxal 5'-phosphate and each modification results in a partial loss of activity . When various combinations of substrates and substrate analogs (ADP/Pi or L-methionine-SR-sulfoximine phosphate/ADP) were used to protect the enzyme from modification, Lys-352 was protected from modification indicating that this residue is at the active site . Under all experimental conditions employed, Lys-47, which reacts with the ATP analog 5'-p-fluorosulfonylbenzoyl-adenosine does not react with pyridoxal 5'-phosphate.

Nucleic Acids Res, 1989 May 25, 17(10), 3865 - 74
Cloning of binding sequences for the Escherichia coli transcription activators, FNR and CRP: location of bases involved in discrimination between FNR and CRP; Bell AI et al.; Expression from the E.coli melR promoter (pmelR) is normally totally dependent on the transcription activator protein, CRP . We describe experiments with a genetically engineered DNA fragment carrying pmelR in which the wild type CRP binding site was replaced with synthetic oligonucleotides containing either FNR or CRP binding sequences . When the synthetic oligonucleotide contains the 22 bp consensus for FNR binding sites, expression from pmelR is dependent on FNR but not CRP . Single changes at either of two symmetrically-related positions create sites that are recognised by both FNR and CRP . Changes at both positions result in a site that is not recognised by FNR but which binds CRP tightly.

J Biol Chem, 1989 May 25, 264(15), 8447 - 50
Human apolipoprotein E . Receptor binding activity of truncated variants with carboxyl-terminal deletions; Lalazar A et al.; The amino-terminal thrombolytic fragment (residues 1-191) of human apolipoprotein (apo) E was previously shown to be fully active in binding to the low density lipoprotein receptor . In this study, truncated apoE variants with progressive deletions at the carboxyl terminus were produced in Escherichia coli by linker-insertion mutagenesis to define the minimum amino-terminal structure necessary for full receptor binding . These truncated forms of apoE, comprising residues 1-166, 1-170, 1-174, or 1-183, were combined with the phospholipid dimyristoylphosphatidylcholine and tested for their ability to bind to low density lipoprotein receptors on human fibroblasts . All of the truncated variants formed typical discoidal particles when combined with the phospholipid, and the particles could be isolated by density gradient ultracentrifugation . The 1-166 and 1-170 variants had very little receptor binding activity (1%), whereas the 1-183 variant had nearly full activity (85%) . The 1-174 variant had 19% activity . We conclude that the 171-183 region of apoE is important for receptor binding, either by contributing one or more residues essential for receptor binding or, more likely, by stabilizing or aligning the region known to be crucial for receptor binding, in the vicinity of residues 140-160.

Nucleic Acids Res, 1989 May 25, 17(10), 3927 - 49
Pseudorevertants of a lac promoter mutation reveal overlapping nascent promoters; Karls R et al.; Four pseudorevertants of a -10 region lacP mutation were isolated . Three of these mutations were found to activate nascent promoters . These mutations were: a -2 G/C----A/T change (-2A) promoting transcription at position +11, a +1 A/T----T/A change (+1T) promoting transcription initiation at position +13, and a +10 C/G----A/T change (+10A) promoting transcription initiation at a complex series of positions . The fourth mutation {a -12 T/A----A/T change (-12A)} promotes transcription initiation at -1 . The promoters activated by mutations -12A, -2A and +1T resembled the canonical sigma 70 promoter sequences . The +10A promoter activity is also dependent upon the sigma 70 holoenzyme but can not be readily assigned to a specific promoter sequence.

J Biol Chem, 1989 May 25, 264(15), 8746 - 52
Substrate binding in human immunodeficiency virus reverse transcriptase . An analysis of pyridoxal 5'-phosphate sensitivity and identification of lysine 263 in the substrate-binding domain; Basu A et al.; Human immunodeficiency virus reverse transcriptase (HIV-RT) exhibits a strong sensitivity to pyridoxal 5'-phosphate (PLP), a substrate-binding site directed reagent for DNA polymerases (Modak, M . J . (1976) Biochemistry 15, 3620-3626) . Treatment of HIV-RT with PLP followed by sodium borohydride reduction of the enzyme-PLP adduct results in irreversible inactivation of polymerase activity while RNase H activity associated with HIV-RT is minimally affected . Kinetic studies indicate that the PLP inhibition is complex . Yet one of the sites of PLP action appears to be involved in the process of dNTP binding as judged by (a) competitive mode of inhibition and (b) blockage of PLP into enzyme protein by the addition of substrate dNTP . Furthermore, this site is the only PLP reactive site which is accessible to borohydride reduction . Comparative tryptic peptide mapping of enzyme treated with PLP under a variety of conditions permitted the identification of a PLP reactive site containing peptide . Furthermore, reactivity of this site was also blocked by inclusion of substrate dNTP and appropriate template-primer . The amino acid composition and sequence analysis of this peptide showed that a lysine residue present at position 263 in the primary amino acid sequence of HIV-RT is the site of PLP reactivity . We therefore conclude that lysine 263 serves as an important part of the dNTP-binding domain in HIV-RT.

Practitioner, 1989 May 22, 233(1469), 784 - 6
Gastrointestinal infection; Gray J; Rotavirus and various types of E coli can cause life-threatening vomiting and diarrhoea in children . Food poisoning and traveller's diarrhoea cause problems for adults.

FEBS Lett, 1989 May 22, 249(1), 21 - 6
IS421, a new insertion sequence in Escherichia coli; Sato S et al.; The nucleotide sequence of a new insertion sequence (IS) in Escherichia coli, IS421, was determined . It is 1340 bp long and contains inverted repeats of 22 bp at its termini . It is flanked by 13 bp direct repeats apparently generated upon insertion . There are two ORFs longer than 200 bp in IS421 . One can encode a polypeptide of 371 amino acids (aa) and the other, which is on the other strand, can encode a polypeptide of 102 aa . The C-terminal part of the 371 aa polypeptide shows some homology to that of transposases encoded in some other known IS elements . The copy number of IS421 in chromosomal DNA was 4 for E . coli K-12 and B, and 5 for E . coli C, as determined by the Southern hybridization of restriction fragments.

J Mol Biol, 1989 May 20, 207(2), 335 - 53
Polymerase-specific differences in the DNA intermediates of frameshift mutagenesis . In vitro synthesis errors of Escherichia coli DNA polymerase I and its large fragment derivative; Papanicolaou C et al.; The sequences of more than 600 frameshift mutations produced as a consequence of in vitro DNA replication on an oligonucleotide-primed, single-stranded DNA template by the Escherichia coli polymerase I enzyme (PolI) or its large fragment derivative (PolLF) were compared . Four categories of mutants were found: (1) single-base deletions, (2) base substitutions, (3) multiple-base deletions and (4) complex frameshift mutations that change both the base sequence and the number of bases in a concerted mutational process . The template sequence 5'-Py-T-G-3', previously identified as a PolLF hotspot for single-base deletions opposite G, is also a hotspot for PolI . A PolI-specific warm spot for single-base deletions was identified . Among base substitutions, transitions were more frequent than transversions . Transversions were mediated by (template)G.G, (template)G.A, and (template)C.T mispairs . Multiple-base deletions were found only after PolI replication . Although each of these deletions can be explained by a misalignment mediated by directly repeated DNA sequences, deletion frequencies were often different for repeats of the same length . Both PolI and PolLF produced many complex frameshift mutants . The new sequences at the mutant sites are exactly complementary to nearby DNA sequences in the newly synthesized DNA strand . In each case, palindromic complementarity could mediate the misalignment needed to initiate the mutational process . The misaligned DNA synthesis accounts for the nucleotide changes at the mutant site and for homology that could direct realignment of the DNA onto the template . Most of the complex mutant sequences could be initiated by either intramolecular misalignments involving fold-back structures in newly synthesized DNA or by strand-switching during strand-displacement synthesis . The striking differences between the specificities of complex frameshift mutations and multiple-base deletions by PolI and PolLF identify the existence of polymerase-specific determinants that influence the frequency and specificity of misalignment-mediated frameshifts and deletions.

J Mol Biol, 1989 May 20, 207(2), 301 - 10
Consensus methods for finding and ranking DNA binding sites . Application to Escherichia coli promoters; O'Neill MC; There have been many different approaches employed to define the "consensus" sequence of various DNA binding sites and to use the definition obtained to locate and rank members of a given sequence family . The analysis presented here enlists two of these approaches, each in modified form, to develop a highly efficient search protocol for Escherichia coli promoters and to provide a relative ranking of these sites showing good agreement with in vitro measurements of promoter strength . Schneider et al . have applied Shannon's index of information content to evaluate the significance of each position within the consensus of a family of aligned sequences . In a formal sense, this index is only applicable to a group of sequences, providing at each position a negative entropy value between zero (random) and two bits (total conservation of a single base) for sequences in which all bases are equally represented . A method for evaluating how well an individual sequence conforms to the information content pattern of the consensus is described . A function is derived, by analogy to the information content of the sequence family, for application to individual sequences . Since this function is a measure of conformity, it can be used in a search protocol to identify new members of the family represented by the consensus . A protocol for locating E . coli promoters is presented . The Berg-von Hippel statistical-mechanical function is also tested in a similar application . While the information content function provides a superior search protocol, the Berg-von Hippel function, when scaled at each position by the information content, does well at ranking promoters according to their strength as measured in vitro.

J Mol Biol, 1989 May 20, 207(2), 365 - 77
Codon usage determines translation rate in Escherichia coli; Sorensen MA et al.; We wish to determine whether differences in translation rate are correlated with differences in codon usage or with differences in mRNA secondary structure . We therefore inserted a small DNA fragment in the lacZ gene either directly or flanked by a few frame-shifting bases, leaving the reading frame of the lacZ gene unchanged . The fragment was chosen to have "infrequent" codons in one reading frame and "common" codons in the other . The insert in these constructs does not seem to give mRNAs that are able to form extensive secondary structures . The translation time for these modified lacZ mRNAs was measured with a reproducibility better than plus or minus one second . We found that the mRNA with infrequent codons inserted has an approximately three-seconds longer translation time than the one with common codons . In another set of experiments we constructed two almost identical lacZ genes in which the lacZ mRNAs have the potential to generate stem structures with stabilities of about -75 kcal/mol . In this way we could investigate the influence of mRNA structure on translation rate . This type of modified gene was generated in two reading frames with either common or infrequent codons similar to our first experiments . We find that the yield of protein from these mRNAs is reduced, probably due to the action in vivo of an RNase . Nevertheless, the data do not indicate that there is any effect of mRNA secondary structure on translation rate . In contrast, our data persuade us that there is a difference in translation rate between infrequent codons and common codons that is of the order of sixfold.

Science, 1989 May 19, 244(4906), 783 - 90
RNA-protein interactions in 30S ribosomal subunits: folding and function of 16S rRNA; Stern S et al.; Chemical probing methods have been used to "footprint" 16S ribosomal RNA (rRNA) at each step during the in vitro assembly of twenty 30S subunit ribosomal proteins . These experiments yield information about the location of each protein relative to the structure of 16S rRNA and provide the basis for derivation of a detailed model for the three-dimensional folding of 16S rRNA . Several lines of evidence suggest that protein-dependent conformational changes in 16S rRNA play an important part in the cooperativity of ribosome assembly and in fine-tuning of the conformation and dynamics of 16S rRNA in the 30S subunit.

Biochim Biophys Acta, 1989 May 19, 981(1), 21 - 6
Sodium/proton antiport is required for growth of Escherichia coli at alkaline pH; McMorrow I et al.; Evidence is presented indicating that Escherichia coli requires the Na+/H+ antiporter and external sodium (or lithium) ion to grow at high pH . Cells were grown in plastic tubes containing medium with a very low Na+ content (5-15 microM) . Normal cells grew at pH 7 or 8 with or without added Na+, but at pH 8.5 external Na was required for growth . A mutant with low antiporter activity failed to grow at pH 8.5 with or without Na+ . On the other hand, another mutant with elevated antiporter activity grew at a higher pH than normal (pH 9) in the presence of added Na+ or Li+ . Amiloride, an inhibitor of the antiporter, prevented cells from growing at pH 8.5 (plus Na+), although it had no effect on growth in media of lower pH values.

Cell, 1989 May 19, 57(4), 585 - 97
Interaction of tRNA with 23S rRNA in the ribosomal A, P, and E sites; Moazed D et al.; Three sets of conserved nucleotides in 23 rRNA are protected from chemical probes by binding of tRNA to the ribosomal A, P, and E sites, respectively . They are located almost exclusively in domain V, primarily in or adjacent to the loop identified with the peptidyl transferase function . Some of these sites are also protected by antibiotics such as chloramphenicol, which could explain how these drugs interfere with protein synthesis . Certain tRNA-dependent protections are abolished when the 3'-terminal A or CA or 2',3'-linked acyl group is removed, providing direct evidence for the interaction of the conserved CCA terminus of tRNA with 23S rRNA . When the EF-Tu.GTP.aminoacyl-tRNA ternary complex is bound to the ribosome, no tRNA-dependent A site protections are detected in 23S rRNA until EF-Tu is released . Thus, EF-Tu prevents interaction of the 3' terminus of the incoming aminoacyl-tRNA with the peptidyl transferase region of the ribosome during anticodon selection, thereby permitting translational proofreading.

Cell, 1989 May 19, 57(4), 531 - 6
Unusual mRNA pseudoknot structure is recognized by a protein translational repressor; Tang CK et al.; Translation of ribosomal proteins in the alpha operon of E . coli is repressed by one of the encoded proteins, S4; it specifically recognizes an RNA fragment containing the translational initiation site for the first gene in the operon . RNA structure mapping experiments have suggested a pseudoknot structure for the S4 binding site: the loop of a hairpin is base paired to sequences downstream of the hairpin . Here, we systematically test this proposed structure by measuring S4 binding to an extensive set of site-directed mutations that create compensatory base pair changes in potential helices . The pseudoknot folding is confirmed, and two additional, unexpected interactions within the pseudoknot are also detected . The overall structure is an unusual "double pseudoknot" linking a hairpin upstream of the ribosome binding site with sequences 2-10 codons downstream of the initiation codon . Stabilization of this structure by S4 could account for translational repression.

Biochim Biophys Acta, 1989 May 19, 981(1), 8 - 14
Molecular basis of porin selectivity: membrane experiments with OmpC-PhoE and OmpF-PhoE hybrid proteins of Escherichia coli K-12; Benz R et al.; Lipid bilayer experiments were performed with one OmpF-PhoE and several OmpC-PhoE hybrid porins of Escherichia coli K-12 . All hybrid pores had approximately the same pore-forming activity, which indicated that the structure of the pores remained essentially unchanged by the genetic manipulation . This result was supported by single-channel experiments because all pores had similar single-channel conductances in potassium chloride . Measurements with other salts indicated a drastic change in the ionic selectivity when the fusion site in the ompC-phoE hybrid genes passed along the sequence of the porins from the N-terminal to the C-terminal end . Selectivity measurements using zero-current membrane potentials showed that the selectivity suddenly changed from anion to cation selectivity when a relatively short portion from the N-terminal end of PhoE was replaced by the corresponding part of OmpC . The replacement of increasing portions led to an increase in the cation selectivity until that of OmpC was reached . The change in the anion to cation selectivity is correlated with exchange of lysine-18 and serine-28 by aspartic acids . The anion selectivity of the phosphate starvation-inducible PhoE porin is closely related to the presence of several lysines spread along the primary sequence of the polypeptide chain.

Biochemistry, 1989 May 16, 28(10), 4455 - 61
Specific activation of open complex formation at an Escherichia coli promoter by oligo(N-methylpyrrolecarboxamide)s: effects of peptide length and identification of DNA target sites; Martello PA et al.; It has previously been shown that open complex formation at a promoter containing a block substitution of nonalternating A-T sequences in the spacer DNA separating the contacted -10 and -35 regions could be accelerated by distamycin . No stimulation was observed at a promoter with a substitution of alternating A-T base pairs in the same region or at the promoter with wild-type spacer . Here we compare the effect of distamycin {tris(N-methylpyrrolecarboxamide), formally a P3} with that of its extended homologues P4, P5, and P6 . It is found that the stimulatory potential of these synthetic oligopeptides which bind in the minor groove of DNA ranks in the order P4 greater than (distamycin, P5) greater than P6 . The interaction of these peptides with the three promoters was studied by monitoring the positions of the promoter DNA protected from MPE-Fe(II) cleavage in the presence of different concentrations of ligand . The results suggest that a higher affinity of oligopeptide for the spacer DNA than for the -10 and/or -35 region is a necessary, but not sufficient condition for stimulation . Different patterns of protected DNA regions are seen with each of the three promoters; with distamycin, P4, and P5, a unique arrangement of protected regions is observed for the variant containing nonalternating A-T base pairs in its spacer DNA . These data support the hypothesis that differences in the ways the minor-groove binders interact with each of the promoter variants account for the observed differential stimulation . We further postulate that it is a ligand-induced structural change in the nonalternating A-T DNA which is responsible for the activation of open complex formation at the promoter containing this substitution.

Biochemistry, 1989 May 16, 28(10), 4444 - 9
Purification and characterization of Escherichia coli endonuclease III from the cloned nth gene; Asahara H et al.; The gene which codes for endonuclease III of Escherichia coli has been sequenced . The nth gene was previously subcloned and defined as the gene which led to overproduction of endonuclease III when present on a multicopy plasmid and which created a deficiency in endonuclease III activity when mutated . The nth gene was sequenced and translated into a predicted polypeptide . The molecular weight (23,546), the amino-terminal amino acid sequence, and the amino acid composition of the polypeptide predicted from the nucleotide sequence are excellent agreement with those same properties determined for the purified protein . Thus, the nth gene is the structural gene for endonuclease III . Inspection of the nucleotide sequence reveals that there is an open reading frame immediately upstream of the nth gene, suggesting that it might be part of an operon . There is a region of dyad symmetry which could form a hairpin stem and loop structure if transcribed into RNA characteristic of a rho-dependent terminator downstream from the nth gene . The nth gene of Escherichia coli has been cloned onto a lambda PL expression vector which yields approximately 300-fold overproduction of endonuclease III . We have purified the enzyme to apparent homogeneity using two chromatographic steps . Our purification scheme allowed the preparation of 117 mg of protein from 190 g of E . coli with a 70% yield . The purified protein has both AP endonuclease activity and DNA N-glycosylase activity . The protein has a Stokes radius of 2.25 nm, a sedimentation coefficient of 2.65 S, a molecular weight of 26,300 in the native state and 27,300 in the denatured state, and a frictional ratio of 1.13.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1989 May 16, 28(10), 4382 - 7
Characterization of antibodies to dihydrothymine, a radiolysis product of DNA; Hubbard K et al.; Antibodies to dihydrothymine were elicited by immunizing rabbits with dihydrothymidine monophosphate conjugated by carbodiimide to BSA . By use of an ELISA assay, the antibodies produced were found to be specific for dihydrothymine . Hapten inhibition studies showed that dihydrothymidine monophosphate was 3 orders of magnitude more effective as an inhibitor than thymidine monophosphate and 4 orders of magnitude more effective than thymidine glycol monophosphate . With DNA containing dihydrothymine, antibody reactivity was observed at 20 fmol of dihydrothymine, which is approximately 0.1 dihydrothymine per 10,000 bases . Thus, the assay is very sensitive . The antibody reacted with denatured DNA containing dihydrothymine but not with native DNA containing this lesion . The antibody was used for measurement of in vivo incorporation of dihydrothymidine in wild-type Escherichia coli or mutants defective in their ability to remove dihydrothymine from DNA or in the de novo synthesis of thymidylate . Lastly, antibodies to dihydrothymine were use to quantitate the formation of dihydrothymine in DNA X-irradiated under N2 . Production of dihydrothymine in irradiated DNA correlated with the level of reducing species produced by X-rays, and dihydrothymine was produced preferentially in irradiated single-stranded or denatured DNA as compared to irradiated duplex DNA.

Biochemistry, 1989 May 16, 28(10), 4374 - 82
Inactivation of DNA polymerase I (Klenow fragment) by adenosine 2',3'-epoxide 5'-triphosphate: evidence for the formation of a tight-binding inhibitor; Catalano CE et al.; The suicidal inactivation of Escherichia coli DNA polymerase I by epoxy-ATP has been previously reported (Abboud et al., 1978) . We have examined in detail the mechanism of this inactivation utilizing a synthetic DNA template-primer of defined sequence . Epoxy-ATP inactivates the large fragment of DNA polymerase I (the Klenow fragment) in a time- and concentration-dependent manner (KI = 21 microM; kinact = 0.021 s-1) . Concomitant with inactivation is the incorporation of epoxy-AMP into the primer strand . The elongated DNA duplex directly inhibits the polymerase activity of the enzyme (no time dependence) and is resistant to degradation by the 3'----5' exonuclease and pyrophosphorylase activities of the enzyme . Inactivation of the enzyme results from slow (4 X 10(-4) s-1) dissociation of the intact epoxy-terminated template-primer from the enzyme and is thus characterized as a tight-binding inhibition . Surprisingly, while the polymerase activity of the enzyme is completely suppressed by epoxy-ATP, the 3'----5' exonuclease activity remains intact . The data presented demonstrate that even though the polymerase site is occupied with duplex DNA, the enzyme can bind a second DNA duplex and carry out exonucleolytic cleavage.

Biochemistry, 1989 May 16, 28(10), 4450 - 5
Endonuclease III is an iron-sulfur protein; Cunningham RP et al.; Elemental analyses, Mossbauer, and EPR data are reported to show that endonuclease III of Escherichia coli is an iron-sulfur protein . Mossbauer spectra of protein freshly prepared from E . coli grown on 57Fe-enriched medium demonstrate that the native enzyme contains a single 4Fe-4S cluster in the 2+ oxidation state, with a net spin of zero . Upon treatment with ferricyanide, a fraction (less than 25%) of the clusters is oxidized into a state which yields an EPR spectrum near g = 2.01 typical of a 3Fe-4S cluster . The magnetic field dependence of the linear electric field effect verifies this assignment . Electron spin echo modulation on the g = 2.01 form of the protein in deuterated solvent indicates the presence of exchangeable protons in the vicinity of the 3Fe-4S cluster . The data obtained show that the {4Fe-4S}2+ cluster of the native enzyme is resistant to either oxidation or reduction, although photoreduction elicited a g = 1.94 type EPR signal characteristic of a {4Fe-4S}1+ cluster . These studies show that endonuclease III is unique in being both a DNA repair enzyme and an iron-sulfur protein . The function of the 4Fe-4S cluster remains to be established.

Biochemistry, 1989 May 16, 28(10), 4187 - 93
Rapid isolation of OmpF porin-LPS complexes suitable for structure-function studies; Holzenburg A et al.; A gentle and rapid isolation procedure is described yielding fractions containing better than 95% pure OmpF porin of Escherichia coli BE with different amounts of bound lipopolysaccharide (LPS) . The procedure employs continuous free-flow electrophoresis (FFE) in the presence of detergent above its critical micelle concentration . Total yields of around 45% were typically obtained when porin-enriched membrane extracts were processed . By use of analytical ultracentrifugation a molecular mass of 114,000 and a sedimentation coefficient S20,w of 5.0 S were determined for porin trimers containing approximately 1 mol of tightly bound LPS . This porin readily formed 3D crystals suitable for high-resolution X-ray diffraction analysis . Three other porin-LPS isoforms isolated by FFE revealed molecular masses of 120,000, 124,000, and 151,000, suggesting that, in addition to the tightly bound LPS, 1, 2, and 8 mol of loosely bound LPS were present per mole of porin trimer . Each of the four different isoforms was suitable for reconstitution into highly ordered protein-lipid membrane arrays . The membrane crystals obtained with the 151-kDa isoform exhibited a unit cell polymorphism similar to that previously reported.

Biochemistry, 1989 May 16, 28(10), 4140 - 7
L-serine analogues form Schiff base and quinonoidal intermediates with Escherichia coli tryptophan synthase; Houben KF et al.; Substrate analogues of L-serine have been found that react with the alpha 2 beta 2 complex of Escherichia coli tryptophan synthase . Upon reaction with alpha 2 beta 2, the analogues glycine, L-histidine, L-alanine, and D-histidine form chemical intermediates derived from reaction with enzyme-bound pyridoxal 5'-phosphate with characteristic UV-visible spectral bands . The spectra of the products of the glycine, L-histidine, and L-alanine reactions with alpha 2 beta 2 contain contributions from the external aldimine, the quinonoid species, and other intermediates along the catalytic pathway . Just as previously reported for the reaction of L-serine with beta 2 {Goldberg, M . E., York, S., & Stryer, L . (1968) Biochemistry 7, 3662-3667}, the reactions of glycine, L-histidine, and L-alanine with the beta 2 form of tryptophan synthase yield spectra with no contributions from catalytic intermediates beyond the external aldimine . The kinetics of intermediate formation and comparisons of the time courses for the exchange of alpha-1H for solvent 2H catalyzed by alpha 2 beta 2 or beta 2 were found to be consistent with these assignments . Intermediates further along the tryptophan synthase catalytic pathway are stabilized to a greater degree in the alpha 2 beta 2 complex than in the beta 2 species alone . This observation strongly suggests that the association of alpha and beta subunits to form the native alpha 2 beta 2 species lowers the activation energies for the interconversion of the external aldimine with chemical species further along the catalytic path.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1989 May 16, 28(10), 4340 - 3
Organization of the F0 sector of Escherichia coli H+-ATPase: the polar loop region of subunit c extends from the cytoplasmic face of the membrane; Girvin ME et al.; The membrane-spanning F0 sector of the Escherichia coli H+-transporting ATP synthase (EC 3.6.1.34) contains multiple copies of subunit c, a 79 amino acid residue protein that is thought to insert in the membrane like a hairpin with two membrane traversing alpha-helices . The center of the protein is much more polar than the putative transmembrane alpha-helices and has been postulated to play a crucial role in coupling H+ translocation through F0 to ATP synthesis in the membrane extrinsic, F1 sector of the complex . However, the direction of insertion of subunit c in the membrane has not been established . We show here that the "polar loop" lies on the F1 binding side of the membrane . A peptide corresponding to Lys34----Ile46 of the polar loop was synthesized . Antisera were generated to the Lys34----Ile46 cognate peptide, and the polyclonal antipeptide IgG was shown to bind to a crude F0 fraction by using enzyme-linked immunosorbent assays . The antipeptide serum did not bind tightly enough to F0 to disrupt function . However, a polyclonal antiserum made to purified, whole subunit c was shown to block the binding of F1 to the F0 exposed in F1-stripped membranes . Incubation of the antisubunit c serum with the peptide reduced the inhibitory effect of the antiserum on the binding of F1 to F0 . The reversal of inhibition by the peptide was specific to the antisubunit c serum in that the peptide had no effect on inhibition of F1 binding to F0 by antiserum to subunit a of F0.(ABSTRACT TRUNCATED AT 250 WORDS)

Cancer Res, 1989 May 15, 49(10), 2735 - 42
Correlation between the induction of heat shock protein 70 and enhanced viral reactivation in mammalian cells treated with ultraviolet light and heat shock; Williams KJ et al.; Enhanced viral reactivation (EVR) is considered to be one manifestation of an inducible response to DNA damage in mammalian cells analogous to the SOS response in Escherichia coli . EVR is characterized by the increased survival of ultraviolet (UV)-irradiated virus in cells which have been pretreated with DNA-damaging agents or by another type of cellular stress, heat shock (HS) . In this study, we have analyzed the induction of nuclear proteins from Vero cells treated with either UV or HS, with the goal of identifying the protein(s) which mediate the EVR response . Results of 2-dimensional protein gel electrophoresis and fluorographic analysis of {35S}methionine-labeled nuclear proteins showed that UV-irradiation caused the increased synthesis of five proteins at 4-9 h after treatment . At 19-24 h, one of these proteins was still being synthesized at a higher level in UV-irradiated cells, and there were nine additional proteins whose syntheses were enhanced over control levels . In contrast, HS induced only one Mr 72,000 nuclear protein whose synthesis was maximal during the 4-9-h labeling period and corresponded to one of the proteins induced by UV at 19-24 h . Subsequent Western and Northern blot analyses have confirmed that this protein is a member of the heat shock protein (hsp) 70 family . Elevated nuclear levels of this protein correlated temporally with the maximum EVR response induced by each treatment (4 h after HS and 24 h after UV) . Since the kinetics of EVR is different following UV and HS and parallels the difference in the induction of nuclear levels of hsp70 following each treatment, the results suggest that hsp70 may be involved in mediating the EVR response . In addition, this protein may also play a role in the recovery of DNA synthesis in UV-irradiated cells.

Gene, 1989 May 15, 78(1), 37 - 46
Structure and expression in Escherichia coli K-12 of the L-asparaginase I-encoding ansA gene and its flanking regions; Jerlstrom PG et al.; Escherichia coli contains two L-asparaginase isozymes: a secreted high-affinity enzyme, L-asparaginase II (AnsII), and a low-affinity cytoplasmic enzyme, L-asparaginase I (AnsI), which is encoded by the ansA gene . The nucleotide sequence of ansA and flanking regions, comprising 2156 bp, has been determined . The ansA gene product has been identified and has a calculated Mr of 35,388; gel filtration of cell extracts indicates that the active form of the enzyme is a dimer . The deduced amino acid sequence of AnsI shows discernible similarity to AnsII in a region immediately adjacent to the proposed active-site peptide of asparaginase II as previously determined by substrate analogue binding experiments . A second open reading frame (ORF1), encoding a protein of Mr 23,336, is found 10 bp downstream from ansA; the ribosome-binding site of ORF1 overlaps the stop codon of ansA . Deletions within the 5' region of ansA abolish expression of ansA and also reduce expression of ORF1 . Together, these observations suggest that ansA and ORF1 constitute an operon . A palindromic sequence exists in the 3' region of ORF1 which may function as a bidirectional transcription terminator both for the ansA-ORF1 operon and a second, convergent, ORF.

Gene, 1989 May 15, 78(1), 195 - 9
Repression of damage-inducible (din) genes by the lexA3 mutation or by plasmid carrying the lexA gene; effect on pyrimidine dimer excision in UV-irradiated Escherichia coli; Masek F et al.; Dimer excision was followed in Escherichia coli K-12 AB1157 DM49 lexA3 mutant (whose repressor is not cleavable with RecA protease), and in E . coli K-12 AB2497{pGC3} carrying the cloned lexA gene . In either case din genes could not be efficiently derepressed . In such cells ultraviolet (UV) irradiation caused an extensive DNA degradation, which was not observed in cells with derepressed din genes . Even after a high UV dose (70 J/m2) dimers were being excised efficiently . However, progressive DNA degradation interfered with the precise detection of unexcised dimers . We conclude that induction of din genes is required for filling some of the gaps and for prevention of DNA degradation, but not for excision itself.

Gene, 1989 May 15, 78(1), 189 - 94
The structural basis of the high in vivo strength of the rRNA P2 promoter of Escherichia coli; Lukacsovich T et al.; Ribosomal RNA promoters of Escherichia coli are probably the strongest promoters in vivo and they can be used on plasmid vectors to express protein-coding sequences at a high rate . In fact, the P2 promoter of the rrnB gene is stronger (in vivo) than the tac promoter, which has a perfect consensus sequence . Conversion of the rrnB P2 promoter sequence to consensus significantly increases in vivo promoter strength . The removal of four nucleotides downstream of the -10 region also increases the strength of this promoter . On the other hand, shifting of the A + T-rich region upstream of this promoter by an 11-bp insertion drastically decreases in vivo activity . It is concluded that the two functionally important hexanucleotide sequences, -35 and -10, are necessary but not sufficient factors for the optimalization of in vivo promoter strength.

Anal Biochem, 1989 May 15, 179(1), 60 - 5
Synthesis of N7-ethyldeoxyguanosine 5'-triphosphate and placement of N7-ethylguanine in a specific site in a synthetic oligodeoxyribonucleotide; Farrance IK et al.; N7-Ethyldeoxyguanosine 5'-triphosphate (N7-Etd-GTP) was synthesized by direct ethylation of dGTP with diethyl sulfate and purified by TLC on cellulose plates at approximately 5% yield . N7-EtdGTP was identified by its uv spectra at pH 1, 7.4, and 13, by its absorbance maxima and minima, and by the lability of the glycosidic bond to acid- and heat-induced cleavage . At pH 7.4, spontaneous cleavage of the glycosidic bond proceeded with a half-life of greater than 48 h . An enzymatic method for placing an N7-ethylguanine in a specific site in DNA was developed using terminal deoxynucleotidyltransferase and the 3' to 5' exonuclease and 5' to 3' polymerase of the Klenow fragment of Escherichia coli DNA polymerase I . The method should be readily adaptable to other modified bases as long as the modification does not occur at a base-pairing site (e.g., 5-methylcytosine, N6-methyladenine, and others).

Anal Biochem, 1989 May 15, 179(1), 182 - 5
Assay for aliphatic amino acid decarboxylases by high-performance liquid chromatography; Kochhar S et al.; A sensitive and rapid assay for aliphatic amino acid decarboxylases based on separation of the product from the substrate by ion-pairing reversed-phase high-performance liquid chromatography and subsequent fluorometric detection has been developed . The resolution of substrates and products of seven amino acid decarboxylases, namely, arginine, aspartate, 2,6-diaminopimelate, histidine, glutamate, lysine, and ornithine decarboxylase, is complete within 15 to 35 min of isocratic elution . The limit of detection for the product is 40 pmol . The applicability of the procedure was assessed with glutamate decarboxylase . The formation of the product 4-aminobutyrate proved to be linear with time and protein concentration . The method allows the time course of the reaction to be followed in a single assay and works well with crude extracts of bacteria or tissues.

J Biol Chem, 1989 May 15, 264(14), 8026 - 32
Location of heme axial ligands in the cytochrome d terminal oxidase complex of Escherichia coli determined by site-directed mutagenesis; Fang H et al.; The cytochrome d terminal oxidase complex is one of two terminal oxidases which are components of the aerobic respiratory chain of Escherichia coli . This membrane-bound enzyme catalyzes the two-electron oxidation of ubiquinol and the four-electron reduction of oxygen to water . Enzyme turnover generates proton and voltage gradients across the bilayer . The oxidase is a heterodimer containing 2 mol of protoheme IX and 1 or 2 mol of heme d per mol of complex . To explain the functional properties of the enzyme, a simple model has been proposed in which it is speculated that the heme prosthetic groups define two separate active sites on opposite sides of the membrane at which the oxidation of quinol and the reduction of water, respectively, are catalyzed . This paper represents an initial effort to define the axial ligands of each of the three or four hemes within the amino acid sequence of the oxidase subunits . Each of the 10 histidine residues has been altered by site-directed mutagenesis with the expectation that histidine residues are likely candidates for heme ligands . Eight of the 10 histidine residues are not essential for enzyme activity, and 2 appear to function as heme axial ligands . Histidine 186 in subunit I is required for the cytochrome b558 component of the enzyme . This residue is likely to be located near the periplasmic surface of the membrane . Histidine 19, near the amino terminus of subunit I also appears to be a heme ligand . It is concluded that two of the four or five expected heme axial ligands have been tentatively identified, although further work is required to confirm these conclusions . A minimum of two additional axial ligands must be residues other than histidine.

Biochem Biophys Res Commun, 1989 May 15, 160(3), 1040 - 6
Expression of rat renal gamma-glutamyltransferase cDNA in Escherichia coli; Angele C et al.; To obtain the expression of rat kidney gamma-glutamyltransferase (GGT) cDNA in E . coli, plasmids containing the cDNA sequences coding for various parts of GGT were constructed . Transformation of E . coli cells by these hybrid vectors results in a production of unglycosylated recombinant proteins, immunologically recognized by specific antirat kidney GGT antibodies . Plasmid, expressing the complete coding sequence of GGT cDNA, allows the production of enzymatically active proteins localized in the periplasmic space, while the same sequence without the N-terminal hydrophobic region results in a production of cytoplasmic proteins . These recombinant proteins present a very basic isoelectric point (pI greater than 9) . These results suggest that the presence of the N-terminal region seems to be necessary to direct the expressed proteins enzymatically active in the periplasmic space.

J Biol Chem, 1989 May 15, 264(14), 7821 - 5
In an Escherichia coli coupled transcription-translation system, expression of the osmoregulated gene proU is stimulated at elevated potassium concentrations and by an extract from cells grown at high osmolality; Jovanovich SB et al.; We have studied the in vitro expression of the osmoregulated proU promoter in Escherichia coli coupled transcription-translation (S-30) extracts as a function of the osmolality of the culture medium used to grow the cells and of the salt concentration added to the extract . These variables represent novel extensions of the use of S-30 extracts to investigate gene regulatory phenomena in vitro . The concentrations of potassium acetate and of the physiologically relevant osmolyte potassium glutamate for maximal expression of the proU promoter are approximately 2-fold higher than the concentrations of these salts providing maximal expression of the lacUV5 promoter . The relative promoter activity of proU with respect to lacUV5 increases more than 30-fold with an increase in salt concentration from 50 to 300 mM . In comparative studies with S-30 extracts prepared from cells grown at high and low osmolalities, we find that at fixed salt concentrations expression of proU is increased 10-fold in the S-30 extract prepared from high osmolality-grown cells, whereas the expression of lacUV5 is increased less than 2-fold . Addition of anti-sigma 70 monoclonal antibodies or purified sigma 70 to the S-30 extract demonstrated that the major proU promoter(s) used in the S-30 extracts is sigma 70-dependent.

Arch Biochem Biophys, 1989 May 15, 271(1), 246 - 53
The recombinant spinach acyl-acyl carrier protein-I expressed in Escherichia coli is the 18:1 delta 11(cis) thioester; Guerra DJ et al.; A synthetic spinach acyl carrier protein-I (ACP-I) gene was cloned and expressed in the Escherichia coli beta-alanine auxotroph SJ16 (P . D . Beremand et al . (1987) Arch . Biochem . Biophys . 256, 90-100) . After characterization of the transformed cells and purification of the protein product it was evident that 50% of the recombinant spinach ACP-I was acylated during early log-phase growth (D . J . Guerra et al . (1988) J . Biol . Chem . 263, 4386-4391) . We have purified the recombinant acyl-acyl carrier protein-I to greater than 90% homogeneity and have made a fatty acid methyl ester of the delipidated and trypsin-treated preparation . We have found that the acyl moiety attached to recombinant spinach acyl carrier protein-I is 18:1 delta 11(cis) (cis-vaccenic acid) a major unsaturated end product of Escherichia coli de novo fatty acid synthesis . This result reflects previous work (D . S . Guerra et al . (1986) Plant Physiol . 82, 448-453) which suggested the acyl carrier protein-I structure has evolved from ancestral ACP structures to accommodate the eukaryotic pathway of lipid synthesis in higher plants . The accumulation of recombinant 18:1 delta 11(cis) acyl carrier protein-I in transformed E . coli SJ16 cells attests to the poor reactivity of this substrate to acyl transferase reactions and may help explain the lack of effect on pools of fatty acids found in vivo.

Gene, 1989 May 15, 78(1), 135 - 46
High-level synthesis of cowpea mosaic virus RNA polymerase and protease in Escherichia coli; Richards OC et al.; An expression system for the production of polymerase proteins of cowpea mosaic virus (CPMV) in Escherichia coli cells is described . High-level synthesis of proteins containing protease and polymerase moieties (110-kDa protein) and polymerase alone (87-kDa protein) were obtained from cells containing different plasmid constructions . Precursor and processed forms of CPMV proteins were detected by immunoblotting with antisera directed against 170-kDa precursor polyprotein and 24-kDa viral protease . Crude lysates and supernatant fractions of the lysates from E . coli cells harboring the various plasmid constructions were analysed for poly(A)-oligo(U) polymerase activity and found to be negative for CPMV activity under conditions where similar expression systems for the production of poliovirus RNA polymerase activity were positive . Thus, conditions for CPMV RNA replication may indeed be different from those for poliovirus even though the genomic organization of these viruses is similar.

J Biol Chem, 1989 May 15, 264(14), 8328 - 38
Mechanism of mRNA binding to bovine mitochondrial ribosomes; Denslow ND et al.; The binding of mRNA to bovine mitochondrial ribosomes was investigated using triplet codons, homopolymers and heteropolymers of various lengths, and human mitochondrial mRNAs . In the absence of initiation factors and initiator tRNA, mitochondrial ribosomes do not bind triplet codons (AUG and UUU) or homopolymers (oligo(U} shorter than about 10 nucleotides . The RNA binding domain on the 28 S mitoribosomal subunit spans approximately 80 nucleotides of the mRNA, judging from the size of the fragments of poly(U,G) and natural mRNAs protected from RNase T1 digestion by this subunit, but the major binding interaction with the ribosome appears to occur over a 30-nucleotide stretch . Human mitochondrial mRNAs coding for subunits II and III of cytochrome c oxidase and subunit 1 of the NADH-ubiquinone oxidoreductase (complex I) were used in studying in detail the binding of mRNA to the small subunit of bovine mitochondrial ribosomes . We have determined that these mRNAs have considerable secondary structure in their 5'-terminal regions and that the initiation codon of each mRNA is sequestered in a stem structure . Little mRNA was bound to ribosomes in a manner conferring protection of the 5' termini from RNase T1 digestion, under standard conditions supporting the binding of artificial templates, but such binding was greatly stimulated by the addition of a mitochondrial extract . Initiation factors and tRNAs from Escherichia coli were unable to stimulate the 5' terminus protected binding of these mRNA molecules, demonstrating a requirement for homologous factors . Our results strongly suggest that mitochondrial initiation factors are required for the proper recognition and melting of the secondary structure in the 5'-terminal region of mitochondrial mRNAs, as a prerequisite for initiation of protein synthesis in mammalian mitochondria.

J Biol Chem, 1989 May 15, 264(14), 8297 - 303
The molecular cloning of the gene encoding the Escherichia coli 75-kDa helicase and the determination of its nucleotide sequence and gentic map position; Wood ER et al.; A previously unreported DNA unwinding enzyme, referred to as the 75-kDa helicase, was recently purified from Escherichia coli cell extracts and biochemically characterized (Wood, E . R., and Matson, S . W . (1987) J . Biol . Chem . 262, 15269-15276) . In order to initiate the genetic analysis of the 75-kDa helicase, the gene encoding this enzyme was cloned . DNA sequencing confirmed the identity of the gene since the predicted amino acid sequence of the encoded polypeptide precisely matched the sequence of the first 27 NH2-terminal amino acid residues of the 75-kDa helicase as determined by peptide sequencing . The predicted amino acid sequence of the 75-kDa helicase is similar in several regions to the amino acid sequences of two other E . coli helicases, Rep protein and helicase II . The gene encoding the 75-kDa helicase was mapped to 22 min on the E . coli chromosome . We propose that this newly defined locus be referred to as helD, and, to avoid confusion with other E . coli helicases with a similar molecular size, we propose that the 75-kDa helicase be referred to as helicase IV.

J Biol Chem, 1989 May 15, 264(14), 8241 - 8
DNA methylation and the differential expression of C4 photosynthesis genes in mesophyll and bundle sheath cells of greening maize leaves; Ngernprasirtsiri J et al.; The methylation of nuclear and chloroplast DNAs has been examined in relation to the known differential expression of C4 photosynthesis genes in the bundle sheath and mesophyll cells of etiolated, greening, and fully green maize leaves . We have focused our research on phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RBUp2Case) which are coded by nuclear genes, and on the large subunit of RBUp2Case which is coded by a plastid gene . Reversed-phase high performance liquid chromatography revealed several kinds of methylated bases in DNAs of both photosynthetic cell types, with the largest amounts in fully green leaves . The occurrence of selective DNA methylation was investigated by employing an isoschizomeric pair of methyl-sensitive and -insensitive endonucleases followed by Southern hybridizations with specific DNA probes . Notably, there was an inverse correlation between the relative abundance of specific transcripts in a given cell type during greening and the methylation status of the corresponding nuclear or chloroplast gene . Furthermore, a heterologous in vitro transcription system using Escherichia coli RNA polymerase revealed that the plastid gene encoding the RBUp2Case large subunit in both cell types was active as a template in the unmethylated state, whereas it was inactive when methylated . Thus, the selective methylation of both chloroplast and nuclear DNA is likely one component of a multilevel control mechanism for the differential regulation of cell-specific C4 photosynthesis gene expression in greening maize leaves.

J Biol Chem, 1989 May 15, 264(14), 8135 - 40
Characterization and sequencing of the lac Y54-41 "uncoupled" mutant of the lactose permease; Brooker RJ et al.; The Escherichia coli strain carrying the lac Y54-41 gene encodes a mutant lactose permease which carries out normal downhill transport of galactosides but is defective in uphill accumulation . In this study, the mutant lac Y54-41 gene was cloned onto the multicopy vector pUR270 . As expected, the cloned gene was shown to express normal downhill transport activity but was markedly defective in the uphill transport of methyl-beta-D-thiogalactopyranoside . Direct measurements of H+ transport revealed that the mutant permease can transport H+ with methyl-beta-D-thiogalactopyranoside but at a significantly reduced capacity compared to the wild-type strain . However, under conditions where the mutant and wild-type strains both transport lactose at similar rates, no detectable H+ transport was observed in the mutant strain . The entire cloned lac Y54-41 gene was subjected to DNA sequencing, and a single base substitution was found which replaces glycine 262 in the protein with a cysteine residue . Inhibition experiments showed that the mutant permease is dramatically more sensitive to three different sulfhydryl reagents: N-ethylmaleimide, p-hydroxymericuribenzoate, and p-hydroxymercuriphenylsulfonic acid . However, the lactose analogue, thiodigalactoside, was only marginally effective at protecting against inhibition in the mutant strain . The results are consistent with the idea that the sulfhydryl reagents are inhibiting the mutant permease activity by reacting with cysteine 262.

J Biol Chem, 1989 May 15, 264(14), 8107 - 12
Structural and catalytic role of arginine 88 in Escherichia coli adenylate kinase as evidenced by chemical modification and site-directed mutagenesis; Reinstein J et al.; Phenylglyoxal inactivates Escherichia coli adenylate kinase by modifying a single arginine residue (Arg-88) . ATP, ADP, P1,P5-di(adenosine 5')-pentaphosphate, and to a lesser extent AMP protect the enzyme against inactivation by phenylglyoxal . Site-directed mutagenesis of Arg-88 to glycine yields a modified form of adenylate kinase (RG88 mutant) closely related structurally to the wild-type protein as indicated by Fourier transform infrared spectroscopy, differential scanning calorimetry, and limited proteolysis . However, this modified protein has only 1% of the maximum catalytic activity of the wild-type enzyme and 5- and 85-fold higher apparent Km values for ATP and AMP, respectively, than the parent adenylate kinase . Arg-88, which is a highly conserved residue in all known molecular forms of adenylate kinases (corresponding to Arg-97 in muscle cytosolic enzyme), should be located inside a big cleft of the molecule, close to the phosphate-binding loop . It possibly stabilizes the transferable gamma-phosphate group from ATP to AMP in the transition state.

J Biol Chem, 1989 May 15, 264(14), 8091 - 6
Mössbauer and EPR studies of the binuclear iron center in ribonucleotide reductase from Escherichia coli . A new iron-to-protein stoichiometry; Lynch JB et al.; 57Fe-enriched ribonucleotide reductase subunit B2 from Escherichia coli strain N6405/pSPS2 has been characterized by Mossbauer and EPR spectroscopy in its native diferric state and in a new differous form . The native protein exhibits two Mossbauer doublets in a 1:1 ratio with parameters that are in excellent agreement with those reported for the wild-type protein (Atkin, C . L., Thelander, L., Reichard, P., and Lang, G . (1983) J . Biol . Chem . 248, 7464-7472); in addition, our studies show the absence of adventitiously bound iron . The iron content in the present samples approached 4 per B2 subunit, and the tyrosyl radical content exceeded 1 per B2 subunit . The higher values are attributed to the use of a new epsilon 280 for the protein and more efficient methods for iron extraction . We thus propose that subunit B2 has two binuclear iron clusters, each associated with its own tyrosyl radical, in contradistinction from the prevailing model . Reduction of the native protein with dithionite or reconstitution of the apoprotein with Fe(II) afforded a protein complex with Mossbauer parameters, delta EQ = 3.13 mm/s and delta = 1.26 mm/s at 4.2 K, and a low field EPR signal associated with an integer spin system . These spectral properties resemble those of methane monooxygenase in its diferrous form . Upon exposure to O2, the reduced subunit B2 readily converts to the diferric state and yields active enzyme.

J Biol Chem, 1989 May 15, 264(14), 8082 - 90
Leading strand synthesis of R1 plasmid replication in vitro is primed by primase alone at a specific site downstream of oriR; Masai H et al.; By using an in vitro system for R1 plasmid replication dependent on a plasmid-encoded repA protein and host dnaA protein, 5' ends of the nascent leading strand were located at positions 1986-1992, some 380 base pair downstream of oriR . Analyses of early replication intermediates generated in vitro in the presence of dideoxy TTP also indicated that replication initiates about 400 base pair downstream of oriR and proceeds unidirectionally . When a 418-base single-stranded DNA from position 1778 to 2195, derived from the leading strand template, was cloned onto an M13 vector, the chimeric single-stranded phage could be replicated in vitro with only single-stranded DNA binding protein, primase (dnaG gene product), and DNA polymerase III holoenzyme . Furthermore, the priming occurred at a site identical to leading strand initiation . These results strongly suggest that the leading strand synthesis is primed by primase alone . The lagging strand synthesis is specifically terminated at position 1515 or 1516 within oriR, preventing further leftward fork movement . Based on these results, a scheme of R1 plasmid replication is presented.

J Biol Chem, 1989 May 15, 264(14), 8052 - 8
The effects of deletions in the central helix of calmodulin on enzyme activation and peptide binding; Persechini A et al.; Using site-directed mutagenesis we have expressed in Escherichia coli three engineered calmodulins (CaM) containing deletions in the solvent-exposed region of the central helix . These are CaM delta 84, Glu-84 removed; CaM delta 83-84, Glu-83 and Glu-84 removed; and CaM delta 81-84, Ser-81 through Glu-84 removed . The abilities of these proteins to activate skeletal muscle myosin light chain kinase, plant NAD kinase, and bovine brain calcineurin activities were determined, as were their abilities to bind a synthetic peptide based on the calmodulin-binding domain of skeletal muscle myosin light chain kinase . Similar results were obtained with all three deletion proteins . Vm values for enzymes activated by the deletion proteins are all within 10-20% of those values obtained with bacterial control calmodulin . Relative to bacterial control values, changes in Kact or Kd values associated with the deletions are all less than an order of magnitude: Kact values for NAD kinase and myosin light chain kinase are increased 5-7-fold, Kd values for binding of the synthetic peptide are increased 4-7-fold, and Kact values for calcineurin are increased only 1-3-fold . In assays of NAD kinase and myosin light chain kinase activation some differences between bovine calmodulin and bacterial control calmodulin were observed . With NAD kinase, Kact values for the bacterial control protein are increased 4-fold relative to values for bovine calmodulin, and Vm values are increased by 50%; with myosin light chain kinase, Kact values are increased 2-fold and Vm values are decreased 10-15% relative to those values obtained with bovine calmodulin . These differences between bacterial control and bovine calmodulins probably can be attributed to known differences in postranslational processing of calmodulin in bacterial and eucaryotic cells . No differences between bovine and control calmodulins were observed in assays of calcineurin activation or peptide binding . Our observations indicate that contacts with the deleted residues, Ser-81 through Glu-84, are not critical in the calmodulin-target complexes we have evaluated . Formation of these calmodulin-target complexes also does not appear to be greatly affected by the global alterations in the structure of calmodulin that are associated with the deletions . In models in which the central helix is maintained in the altered calmodulins, each deleted residue causes the two lobes of calmodulin to be twisted 100 degrees relative to one another and brought 1.5 A closer together.(ABSTRACT TRUNCATED AT 400 WORDS)

Gene, 1989 May 15, 78(1), 93 - 99
High level expression of genes cloned in phage lambda gt11; Stoker NG et al.; Plasmid cloning vectors have been constructed which allow genes originally cloned in lambda gt11 to be expressed at a high level in Escherichia coli . They are based on the pEMBL and pUC vectors, with the genes transcribed from the lac promoter . The EcoRI site in the vector has been altered to be in the same reading frame as the site used for cloning in lambda gt11 . Cloned proteins are expressed fused to a 2-kDa leader sequence containing a run of six Aparagine residues which considerably improves the stability of the recombinant proteins, but does not interfere with immunological assays . Using these vectors, the Mycobacterium leprae 18-kDa protein was expressed at 20 mg per litre of culture and constituted 15% of total cell protein.

Gene, 1989 May 15, 78(1), 9 - 17
Cloning and expression of the branching enzyme gene (glgB) from the cyanobacterium Synechococcus sp . PCC7942 in Escherichia coli; Kiel JA et al.; Using the glgB gene from Escherichia coli as a hybridization probe, the gene encoding the branching enzyme of the cyanobacterium Synechococcus sp . PCC7942 has been identified on a 3.9-kb PstI fragment which was cloned into plasmid pUC9 . Two types of plasmids have been isolated . Plasmid pKVN1 was expressing the Synechococcus sp . gene as was shown by complementation of the glgB mutation of E . coli KV832 . Plasmid pKVN2, which carried the same insert in the opposite orientation was unable to complement E . coli KV832, indicating that the promoter of the cloned gene was either absent or was not recognized in E . coli . Determination of branching activity in extracts of Synechococcus sp . and E . coli KV832{pKVN1} showed that the enzyme was optimally active at approximately 35 degrees C . No significant activity was present at temperatures higher than 55 degrees C, reflecting the mesophilic nature of the cloned enzyme . In a cell-free coupled transcription-translation system the cloned gene specified two proteins of 84 kDa and 72 kDa, respectively, which are probably translated independently from the same gene by initiation at two different start codons.

Biochem J, 1989 May 15, 260(1), 109 - 14
The catalytic consequences of experimental evolution . Transition-state structure during catalysis by the evolved beta-galactosidases of Escherichia coli (ebg enzymes) changed by a single mutational event; Li BF et al.; 1 . The first chemical step in the hydrolysis of galactosylpyridinium ions by the evolvant ebg enzyme is less sensitive to leaving-group acidity than in the case of the wild-type ebg enzyme, implying less glycone-aglycone-bond fission at the transition state . 2 . The first chemical step in the hydrolysis of aryl galactosides by ebg enzyme is probably less sensitive to leaving-group acidity than in the case of ebg enzyme, possibly as a consequence of resulting in more effective proton donation to the leaving aglycone . 3 . alpha-Deuterium kinetic isotope effects of 1.1(0) and beta-deuterium kinetic isotope effects of 1.0(0) were measured for the hydrolysis of galactosyl-enzyme intermediates derived from ebg and ebg enzymes: these effects are not compatible with reaction of the sugar ring through a 4C1-like conformation, or with an ionic glycosyl-enzyme intermediate . 4 . The variation with pH of steady-state kinetic parameters for hydrolysis of p-nitrophenyl galactoside by ebg and ebg enzymes and of 3-methylphenyl beta-galactoside, 3,4-dinitrophenyl beta-galactoside and beta-galactosyl-3-bromopyridinium ion by ebg enzyme was measured . The steep, non-classical, fall in activity against p-nitrophenyl galactoside at low pH observed with ebg and ebg enzymes is not observed with ebg enzymes.

Anal Biochem, 1989 May 15, 179(1), 202 - 5
Effect of stress conditions on Escherichia coli outer and inner membranes, separated by Sephacryl S-500 chromatography; Sanchez RA et al.; A chromatographic method for the separation of outer and inner membranes of Escherichia coli is described . This method is much faster than the generally used equilibrium centrifugation and has a greater versatility in the volumes that can be treated . Both techniques are used to examine the effects of different stress conditions on the E . coli membrane . The obtained results illustrate the equivalence of both methods.

J Biol Chem, 1989 May 15, 264(14), 8039 - 45
Lipoamide dehydrogenase from Escherichia coli . Steady-state kinetics of the physiological reaction; Sahlman L et al.; Lipoamide dehydrogenase from Escherichia coli operates qualitatively by the same mechanism as the enzyme from pig heart . It has been suggested that quantitative differences between the two, in particular the marked inhibition of the bacterial enzyme by its product NADH, are related to the fact that the E . coli enzyme lacks the phosphorylation/dephosphorylation control present in the mammalian enzyme (Wilkinson, K . D., and Williams, C . H., Jr . (1981) J . Biol . Chem . 256, 2307-2314) . Because of the inhibition by NADH, the kinetics of the E . coli enzyme have not been studied previously in the physiological direction with the natural substrate, dihydrolipoamide . We have now measured the steady-state kinetics of the oxidation of dihydrolipoamide by NAD+ using the stopped-flow technique to follow only the early time course . The pH dependence of kcat revealed an apparent pKa value of 6.7, reflecting ionization(s) of the enzyme-substrate complex . The pH dependence of kcat/Km gave an apparent pKa of 7.4 reflecting ionization(s) of the free 2-electron-reduced enzyme . The inhibition pattern for NADH was mixed, consistent with the fact that NADH is both a product inhibitor and inhibits by reducing a fraction of the enzyme to the catalytically inactive 4-electron-reduced state . There is a modest pH-dependent positive cooperativity in the saturation curve for NAD+ decreasing with increasing pH . Spectral changes in the 530 and 446 nm bands of the 2-electron-reduced enzyme, associated with the titration of the nascent thiols and the base, showed tentative pKa values of 6.4 and 7.1, respectively, in a pH jump experiment . The properties of the wild type E . coli enzyme can now be compared with those of several site-directed mutants.

Gene, 1989 May 15, 78(1), 59 - 72
The role of bases upstream of the Shine-Dalgarno region and in the coding sequence in the control of gene expression in Escherichia coli: translation and stability of mRNAs in vivo; Schauder B et al.; A range of translational initiation regions (TIR) was created by combining synthetic DNA fragments derived from the atpB-atpE intercistronic sequence of Escherichia coli with the cDNA sequence encoding mature human interleukin 2 (IL-2), the E . coli fnr gene, or an fnr::lacZ gene fusion . Both the overall rates of gene expression and the relative concentrations and stabilities of the corresponding mRNA species were estimated in strains bearing the constructs on plasmids . These measurements served as the basis for analyses of the relationship between the structure of the TIR and the true rates of translation that it promotes . The constructs involving the IL-2 cDNA were predicted to allow much less stable secondary structure within the TIR than those involving the N-terminal region of the fnr gene . Thus by combining one set of upstream sequences with two different types of N-terminal coding sequence, it was possible to distinguish between the respective influences of primary and secondary structure upon initiation . The data indicate that in the presence of a given Shine-Dalgarno (SD)/start codon combination, the decisive factor for translational initiation efficiency is the stability of base pairing involving, or in the vicinity of, this region . The sequences contributing to this secondary structure can be many bases upstream of the SD region and/or downstream of the start codon . There was no indication that the specific base sequence upstream of the SD region could, other than to the extent that it contributed to the local secondary structure, significantly influence the efficiency of translational initiation.

Science, 1989 May 12, 244(4905), 700 - 2
Complementary DNA coding click beetle luciferases can elicit bioluminescence of different colors; Wood KV et al.; Eleven complementary DNA (cDNA) clones were generated from messenger RNA isolated from abdominal light organs of the bioluminescent click beetle, Pyrophorus plagiophthalamus . When expressed in Escherichia coli, these clones can elicit bioluminescence that is readily visible . The clones code for luciferases of four types, distinguished by the colors of bioluminescence they catalyze: green (546 nanometers), yellow-green (560 nanometers), yellow (578 nanometers), and orange (593 nanometers) . The amino acid sequences of the different luciferases are 95 to 99 percent identical with each other, but are only 48 percent identical with the sequence of firefly luciferase (Photinus pyralis) . Because of the different colors, these clones may be useful in experiments in which multiple reporter genes are needed.

Nucleic Acids Res, 1989 May 11, 17(9), 3469 - 78
Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants; Woodcock DM et al.; Many strains of E . coli K12 restrict DNA containing cytosine methylation such as that present in plant and animal genomes . Such restriction can severely inhibit the efficiency of cloning genomic DNAs . We have quantitatively evaluated a total of 39 E . coli strains for their tolerance to cytosine methylation in phage and plasmid cloning systems . Quantitative estimations of relative tolerance to methylation for these strains are presented, together with the evaluation of the most promising strains in practical recombinant cloning situations . Host strains are recommended for different recombinant cloning requirements . These data also provide a rational basis for future construction of 'ideal' hosts combining optimal methylation tolerance with additional advantageous mutations.

FEBS Lett, 1989 May 8, 248(1-2), 83 - 6
The effect of nonreceptor adsorption on the lethal action of colicin E1; Malkhosyan SR et al.; The survivability of Escherichia coli K12s cells has been studied after treatment with 125I-labeled colicin E1 . It has been shown that for low amounts of adsorbed colicin the survivability follows single-hit kinetics . When the number of colicin molecules adsorbed exceeds approx . 50 per cell, deviation from single-hit kinetics occurs towards higher survivability . Colicin E1 adsorbed nonreceptorwise by the cell's surface has been shown to inhibit the lethal action of colicin E1 molecules adsorbed at specific receptors . This fact has been used in accounting for the elevated survivability of cells at high colicin doses . The functional significance of the phenomenon is discussed.

Cell, 1989 May 5, 57(3), 413 - 22
Early and late periodic patterns of even skipped expression are controlled by distinct regulatory elements that respond to different spatial cues; Goto T et al.; We have identified the regulatory sequences required for the periodic expression of the Drosophila pair rule gene even skipped (eve) . We find that the gradually changing pattern of periodic eve expression during early embryogenesis is directed by two distinct regulatory programs . Initially, eve expression in individual stripes is established by different regulatory elements, each of which responds to nonperiodic spatial cues provided, at least in part, by the gap genes . Later, coordinate expression of eve in all seven stripes is directed by a single regulatory region that responds to periodic cues provided by primary pair rule genes, including eve itself . As a consequence of this two-step regulatory program, eve functions both in the establishment of the periodic pattern of gene expression and in the subsequent specification of parasegmental boundaries.

J Biol Chem, 1989 May 5, 264(13), 7584 - 9
Structural analysis of the FMN binding domain of NADPH-cytochrome P-450 oxidoreductase by site-directed mutagenesis; Shen AL et al.; Comparison of the amino acid sequence of rat liver NADPH-cytochrome P-450 oxidoreductase with that of flavoproteins of known three-dimensional structure suggested that residues Tyr-140 and Tyr-178 are involved in binding of FMN to the protein . To test this hypothesis, NADPH-cytochrome P-450 oxidoreductase was expressed in Escherichia coli using the expression-secretion vector pIN-III-ompA3, and site-directed mutagenesis was employed to selectively alter these residues and demonstrate that they are major determinants of the FMN-binding site . Bacterial expression produced a membrane-bound 80-kDa protein containing 1 mol each of FMN and FAD per mol of enzyme, which reduced cytochrome c at a rate of 51.5 mumol/min/mg of protein and had absorption spectra and kinetic properties very similar to those of the rat liver enzyme . Replacement of Tyr-178 with aspartate abolished FMN binding and cytochrome c reductase activity . Incubation with FMN increased catalytic activity to a maximum of 8.6 mumol/min/mg of protein . Replacement of Tyr-140 with aspartate did not eliminate FMN binding, but reduced cytochrome c reductase activity about 5-fold, suggesting that FMN may be bound in a conformation which does not permit efficient electron transfer . Substitution of phenylalanine at either position 140 or 178 had no effect on FMN content or catalytic activity . The FAD level in the Asp-178 mutant was also decreased, suggesting that FAD binding is dependent upon FMN; FAD incorporation may occur co-translationally and require prior formation of an intact FMN domain.

J Mol Biol, 1989 May 5, 207(1), 53 - 60
SOS processing of unique oxidative DNA damages in Escherichia coli; Laspia MF et al.; phi X174 replicative form (RF) I transfecting DNA containing thymine glycols (5,6-dihydroxy-5,6-dihydrothymine), urea glycosides or apurinic (AP) sites was used to study SOS processing of unique DNA damages in Escherichia coli . All three lesions can be found in DNA damaged by chemical oxidants or radiation and are representative of several common structural modifications of DNA bases . When phi X DNA containing thymine glycols was transfected into host cells that were ultraviolet-irradiated to induce the SOS response, a substantial increase in survival was observed compared to transfection into uninduced hosts . Studies with mutants demonstrated that both the activated form of RecA and UmuDC proteins were required for this reactivation . In contrast, no increase in survival was observed when DNA containing urea glycosides or AP sites was transfected into ultraviolet-induced hosts . These data suggest that SOS-induced reactivation does not reflect a generalized repair system for all replication-blocking, lethal lesions but rather that the efficiency of reactivation is damage dependent . Further, we found that a significant fraction of potentially lethal thymine glycols could be ultraviolet-reactivated in an umuC lexA recA-independent manner, suggesting the existence of an as yet uncharacterized damage-inducible SOS-independent mode of thymine glycol repair.

J Mol Biol, 1989 May 5, 207(1), 269 - 88
Negative co-operativity in Escherichia coli single strand binding protein-oligonucleotide interactions . II . Salt, temperature and oligonucleotide length effects; Bujalowski W et al.; We have examined the salt and temperature dependences of the equilibrium binding of the Escherichia coli single strand binding (SSB) tetramer to a series of oligodeoxythymidylates, dT(pT)N-1, with N = 16, 28, 35, 56 and 70 . Absolute binding isotherms were obtained, based on the quenching of the intrinsic protein fluorescence upon formation of the complexes . The shorter oligonucleotides, with N = 16, 28 and 35, bind to multiple sites on the SSB tetramer and negative co-operativity is observed among these binding sites . We have quantitatively analyzed these isotherms, using a statistical thermodynamic ("square") model to obtain the intrinsic binding constant KN, and the negative co-operativity constant, sigma N . For all oligonucleotides, we find that KN decreases significantly with increasing concentration of monovalent salt, indicating a large electrostatic component to the free energy of the interaction (e.g . delta log KN/delta log {NaBr} = -2.7, -4.6 and -7.1 for N = 16, 35 and 70, respectively), with contributions from both cations and anions . For oligonucleotides that span two or more subunits, there is a significant unfavorable contribution to the binding free energy for each intersubunit crossing, with an accompanying uptake of anions . Therefore, the extent of anion uptake increases as the number of intersubunit crossings increase . There is a strong temperature dependence for the intrinsic binding of dT(pT)15, such that delta Ho = -26(+/- 3) kcal/mol dT(pT)15 . Negative co-operativity exists under all solution conditions tested, i.e . sigma N less than 1, and this is independent of anion concentration and type . However, the negative co-operativity constant does decrease with decreasing concentration of cation . The dependence of sigma 16 on Na+ concentration indicates that an average of one sodium ion is taken up as a result of the negative co-operativity between two dT(pT)15 binding sites . These data and the lack of a temperature dependence for sigma 16 suggest that the molecular basis for the negative co-operativity is predominantly electrostatic and may be due to the repulsion of regions of single-stranded DNA that are required to bind in close proximity on an individual SSB tetramer.

J Mol Biol, 1989 May 5, 207(1), 249 - 68
Negative co-operativity in Escherichia coli single strand binding protein-oligonucleotide interactions . I . Evidence and a quantitative model; Bujalowski W et al.; The interaction of the Escherichia coli single strand binding (SSB) protein with single-stranded DNA is complex, since a number of different binding modes have been observed, with different DNA site sizes and binding properties and the transitions among these binding modes are strongly influenced by solution conditions in vitro . Recent experiments have suggested the existence of negative co-operativity among the multiple DNA binding sites within individual SSB tetramers . In order to probe this negative co-operativity, we have examined the binding of a series of oligonucleotides of varying length, using the quenching of the intrinsic SSB protein fluorescence to monitor binding . The stoichiometries for saturation of the SSB tetramer are 4, 2, 2, 1 and 1, for the oligonucleotides, dT(pT)N-1, with N = 16, 28, 35, 56 and 70, respectively, indicating that one molecule of either dT(pT)27 or dT(pT)34 interacts with two SSB subunits, whereas one molecule of dT(pT)15 interacts with only a single subunit . Saturation of the SSB tetramer with dT(pT)15, dT(pT)34, dT(pT)69 or poly(dT) results in 85 to 90% quenching of the SSB fluorescence, whereas saturation with dT(pT)27 or dT(pT)55 results in only 80% and 72% quenching, respectively . Therefore, a single-stranded DNA of at least 64 nucleotides is required to wrap around an SSB tetramer fully and interact with all four subunits . A quenching of 50(+/- 2)% is observed upon filling only half of the subunits with either one molecule of dT(pT)34 or two molecules of dT(pT)15, which agrees with the quenching and site size observed in the (SSB)35 polynucleotide binding mode . Direct binding measurements indicate that the binding of dT(pT)27 to its second site is influenced by the oligonucleotide that occupies the first binding site (either dT(pT)27 or dT(pT)34), providing proof for the existence of a true negative co-operativity . This negative co-operativity is observed also for the binding of the shorter oligonucleotide, dT(pT)15 . A statistical thermodynamic ("square") model gives an excellent description of the binding of all oligonucleotides possessing multiple sites on the SSB tetramer, based on only two interaction constants, the intrinsic binding constant, KN, and the negative co-operativity parameter, sigma N . These data indicate that the binding sites (subunits) on the unliganded SSB tetramer are all equivalent, but that a non-equivalence between dimers of subunits within the tetramer is induced upon binding ssDNA.

J Biol Chem, 1989 May 5, 264(13), 7590 - 5
The structural stability of a protein is an important determinant of its proteolytic susceptibility in Escherichia coli; Parsell DA et al.; To investigate the relationship between the degradation rate of a protein in Escherichia coli and its thermal stability in vitro, we constructed a set of variants of the N-terminal domain of lambda repressor with a wide range of melting temperatures . Pulse-chase experiments showed that, within this set, the proteins that are most thermally stable have the longest intracellular half-lives and vice versa . Moreover, second-site mutations which act directly or indirectly to increase the thermodynamic stability of the native N-terminal domain were found to suppress the intracellular degradation of one of the unstable mutants . These data suggest that thermal stability is, indeed, a key determinant of the proteolytic susceptibility of this protein in the cell . It is not the sole determinant, however, as sequences at the extreme C terminus of the N-terminal domain can influence proteolytic sensitivity without affecting the stability of the native structure . We propose that the thermal stability of the N-terminal domain of lambda repressor is an important determinant of its proteolytic sensitivity because degradation proceeds primarily from the unfolded form and that sequence determinants within the unfolded chain influence whether the unfolded protein will be a good substrate for proteolytic enzymes.

J Biol Chem, 1989 May 5, 264(13), 7425 - 30
Discrimination between nucleotide effector responses of aspartate transcarbamoylase due to a single site substitution in the allosteric binding site; Corder TS et al.; The substitution of alanine for lysine at position 56 of the regulatory polypeptide of aspartate transcarbamoylase affected both homotropic and heterotropic characteristics . In the absence of effectors, the ALAr56-substituted holoenzyme lost the homotropic cooperativity observed for aspartate in the wild-type holoenzyme . Under conditions of allosteric inhibition in the presence of 2mM CTP, the cooperative character of ATCase was restored, and the Hill coefficient increased from 1.0 to 1.7 . In contrast to the native enzyme, the altered enzyme did not respond to ATP; however, ATP could still bind to the enzyme as demonstrated by its direct competition with CTP . Furthermore, the recently observed CTP-UTP synergism of the wild-type enzyme was not detectable . The site-directed mutant enzyme could not be activated by low levels of the bisubstrate analogue, N-(phosphonacetyl)-L-aspartate, and the rate of association of pHMB with the cysteine residues located at the interface of the catalytic and regulatory chains was slightly altered . These characteristics suggested that the mutant holoenzyme assumed a relaxed (or abnormal T state) conformation . Thus, this single substitution differentially affected the heterotropic responses to the various allosteric effectors of ATCase and eliminated the homotropic characteristics in response to aspartate in the absence of CTP.

J Biol Chem, 1989 May 5, 264(13), 7291 - 301
The allosteric three-site model for the ribosomal elongation cycle . Analysis with a heteropolymeric mRNA; Gnirke A et al.; Ribosomal tRNA binding studies and functional tests were performed at 6 mM Mg2+ using the mRNA analogue C17AUGA4C17 which contains three unique codons in its central region . The following results were obtained . 1) The relative binding affinities of 20 different deacylated tRNAs to nonprogrammed 70 S ribosomes were assessed and were found to vary substantially . 2) When added as the first tRNA, fMet-tRNA and deacylated tRNAs (but not N-acetylated aminoacyl-tRNAs) can bind to internal codons of the mRNA and are therefore suitable for setting the reading frame via codon-anticodon interaction in the peptidyl-tRNA site (P site) . 3) After prefilling the P site with deacylated tRNA, the exit site for deacylated tRNA (E site) can be quantitatively occupied only if the cognate codon is present at that site . 4) The translocation of peptidyl-tRNA from the aminoacyl-tRNA site (A site) to the P site is not accompanied by a release of deacylated tRNA . The codon sequence excludes a release and rebinding of deacylated tRNA to the newly exposed A site . Rather, the deacylated tRNA is cotranslocated from the P to the E site where it remains stably bound . 5) After one round of elongation, addition of an A site ligand triggers the dissociation of deacylated tRNA from the E site . Conversely, E site occupation reduces the affinity of the A site for N-acetylated aminoacyl-tRNA . Thus, A and E sites are allosterically linked via negative cooperativity . The results support the allosteric three-site model as an appropriate description of the ribosomal elongation cycle.

J Mol Biol, 1989 May 5, 207(1), 151 - 62
Adenylate kinases from thermosensitive Escherichia coli strains; Haase GH et al.; The adk genes from several thermosensitive (ts) mutants of Escherichia coli were cloned and sequenced . The mutations responsible for the thermolability of the gene product, the enzyme adenylate kinase, were established . From five independently isolated strains analysed, two contain a CCG to TCG transition changing proline 87 to serine (P87S), another two have a TCT to TTT transition that mutates serine 129 to phenylalanine (S129F), and the last one was found not to contain a mutation in the adk gene . Overproducing strains were constructed that contain ts genes in the genome as well as in the plasmids . These strains grow at high temperature, although much slower than wild-type . Most probably, the high rate of synthesis of adenylate kinase compensates for the destruction of the thermolabile protein by the elevated temperature . Mutated proteins were purified . The P87S but not the S129F mutation was found to cause thermosensitivity of the adenylate kinase reaction . Revertants of thermosensitivity were isolated and the nature of the mutation was determined by the RNase digestion method of RNA-DNA hybrids and by DNA sequencing . The revertants of the P87S mutation regained the wild-type sequence, whereas the revertants of the S129F strain retained the original mutation in the adenylate kinase gene . These results are discussed in the light of the three-dimensional structure of the enzyme and the possible role of adenylate kinase in phospholipid synthesis.

J Biol Chem, 1989 May 5, 264(13), 7715 - 9
Increasing gene expression in yeast by fusion to ubiquitin; Ecker DJ et al.; Heterologous gene expression in yeast can be increased up to several hundred-fold by expressing a foreign gene as a fusion to the ubiquitin gene . An endogenous yeast endoprotease (Ub-Xase) removes the ubiquitin from the fusion product to produce the authentic protein . The utility of this technique has been demonstrated by expression of three different proteins in yeast as both unfused and ubiquitin-fused forms: 1) the alpha subunit of the mammalian stimulating G-protein of the adenylate cyclase complex (Gs alpha); 2) a soluble fragment of the T cell receptor protein (sCD4); and 3) the protease domain of human urokinase (UKP) . The sequence specificity of the Ub-Xase was demonstrated by mutagenesis of the carboxyl-terminal glycine of ubiquitin to an alanine, which inhibited ubiquitin removal in vivo . Processing of the ubiquitin-Gs alpha fusion protein (ub-Gs alpha) in vivo resulted in Gs alpha which could be reconstituted in mammalian membrane preparations and had the same specific activity as the authentic Gs alpha expressed in yeast . The yeast Ub-Xase has also been shown to work in vitro by the processing of a ub-sCD4 fusion protein synthesized in Escherichia coli . This technology should greatly enhance the utility of yeast for heterologous protein production.

J Biol Chem, 1989 May 5, 264(13), 7390 - 4
Galactoside-dependent proton transport by mutants of the Escherichia coli lactose carrier . Replacement of histidine 322 by tyrosine or phenylalanine; King SC et al.; Mutations have been introduced into the Escherichia coli lac Y gene by oligonucleotide-directed mutagenesis such that the lactose carrier contains either tyrosine or phenylalanine in place of histidine 322 . These mutants did not carry out active accumulation of lactose, melibiose, or methyl-beta-D-galactopyranoside, but facilitated diffusion was still catalyzed . Galactoside-dependent H+ transport, measured with the pH electrode, was retained in both mutants . We conclude that although histidine 322 is important for energy transduction, neither an electronegative atom nor a dissociable proton is essential for proton cotransport with lactose or melibiose.

J Biol Chem, 1989 May 5, 264(13), 7384 - 9
2-Acylglycerolphosphoethanolamine acyltransferase/acyl-acyl carrier protein synthetase is a membrane-associated acyl carrier protein binding protein; Cooper CL et al.; Two enzymatic activities, 2-acylglycerolphosphoethanolamine (2-acyl-GPE) acyltransferase and acyl-acyl carrier protein (acyl-ACP) synthetase, were solubilized and purified from Escherichia coli membranes . Electrophoretic analysis of the final product of the purification procedure revealed a single protein species with an apparent molecular mass of 27 kilodaltons . The ratio of acyltransferase to synthetase activities remained the same throughout the purification scheme indicating that both activities were catalyzed by the same enzyme . 2-Acyl-GPE acyltransferase exhibited an apparent ACP Km of 64 nM under standard assay conditions that increased to 10 microM when the assay was conducted in the presence of 0.4 M LiCl . Acyl-ACP synthetase activity was not detected in the absence of 0.4 M LiCl, and the apparent ACP Km for this reaction was 16 microM . Direct evidence that ACP was a subunit of the acyltransferase/synthetase was obtained by the adsorption of both catalytic activities to an ACP-Sepharose affinity column and by the binding of {3H}ACP to the purified enzyme preparation . The apparent Km for acyl-ACP was 13 microM, and the rate of acyl transfer from this acyl donor was enhanced by the addition of 0.4 M LiCl indicating that the exchange of enzyme-bound ACP for acyl-ACP was a determinant factor in the rate of phosphatidylethanolamine formation from acyl-ACP . These data indicate that the 2-acyl-GPE acyltransferase and acyl-ACP synthetase reactions are catalyzed by the same membrane protein that possesses a high affinity binding site for soluble ACP.

J Biol Chem, 1989 May 5, 264(13), 7338 - 44
dnaA protein regulates transcriptions of the rpoH gene of Escherichia coli; Wang QP et al.; The rpoH (htpR) gene of Escherichia coli encodes a sigma factor which confers upon RNA polymerase the ability to recognize the promoters for genes responsive to the phenomenon termed the heat shock response . dnaA protein, a sequence-specific DNA binding protein, is required for initiation of chromosomal replication by binding to sites within the chromosomal origin . dnaA protein also autoregulates its expression by binding to a site in the dnaA promoter region . Two copies of the dnaA protein recognition sequence are present within the rpoH promoter region . Using filter binding assays, dnaA protein was observed to bind specifically to DNA fragments containing the rpoH promoter region with greater affinity than its binding to the dnaA promoter region . By contrast, reduced binding to a DNA fragment containing the lacUV5 promoter was observed . DNase I footprint analysis indicated that dnaA protein protected specific sites within the rpoH promoter region . The binding of dnaA protein to the rpoH promoter region resulted in transcriptional repression from two of the three promoters of the rpoH gene in vitro . Elevated levels of dnaA protein repressed transcription from these two rpoH promoters in vivo . These results indicate that dnaA protein regulates rpoH transcription to influence the expression of genes under rpoH control.

J Biol Chem, 1989 May 5, 264(13), 7135 - 40
Spectroscopic and quantitative analysis of the oxygenated and peroxy states of the purified cytochrome d complex of Escherichia coli; Lorence RM et al.; Oxygenated and peroxy states of the cytochrome d complex of Escherichia coli have been proposed as intermediates in the reaction mechanism of this ubiquinol oxidase . In this report, several stable states of the purified enzyme were examined spectroscopically at room temperature . As purified, the cytochrome d complex exists in an oxygenated state characterized by an absorbance band at 650 nm . Removal of oxygen results in loss of absorbance at this wavelength, which is restored upon the return of oxygen . The presence of one oxygen molecule in the oxygenated state was quantified by measuring oxygen released when excess hydrogen peroxide was added to the oxygenated state by passage of argon generates a "partially reduced" state with an absorbance peak at 628 nm, apparently due to reduced cytochrome d . Addition of equimolar hydrogen peroxide to the fully oxidized state produces the peroxy state . This peroxy state is also formed upon addition of excess hydrogen peroxide to the oxygenated state via a stable intermediate termed "peroxy intermediate." It is likely that 1) the oxygenated state consists of one molecule of oxygen bound to reduced heme d, and 2) there are at least two stable states that have bound peroxide at room temperature, the peroxy state and a newly discovered peroxy intermediate.

N Engl J Med, 1989 May 4, 320(18), 1165 - 72
Promotion and subsequent inhibition of plasminogen activation after administration of intravenous endotoxin to normal subjects; Suffredini AF et al.; To evaluate the effect of endotoxin on the fibrinolytic response, we administered Escherichia coli endotoxin (4 ng per kilogram of body weight) intravenously to 19 healthy volunteers and measured fibrinolytic proteins, protease inhibitors, neutrophil elastase, and von Willebrand factor in serial blood samples obtained over 24 hours . One hour after endotoxin administration, the level of tissue plasminogen activator (t-PA) antigen rose from 10 to 23 ng per milliliter, peaking at 52 ng per milliliter at three hours . The level of alpha 2-plasmin inhibitor-plasmin complexes increased sevenfold, peaking at three hours . Plasminogen-activator inhibitor-1 activity rose more slowly, from 7 U per milliliter to a maximum of 49 U per milliliter at five hours . The concentrations of neutrophil elastase and von Willebrand antigen were unchanged at one hour, increased approximately threefold by 3 hours, and remained elevated at 24 hours . None of these measures changed in a control group (n = 5) given intravenous saline instead of endotoxin . We studied t-PA functional activity in four subjects . The level of activity rose rapidly, from 1.2 ng per milliliter at base line to 8.3 ng per milliliter at one hour and 13.9 ng per milliliter at two hours; it was undetectable at three hours . This increase in plasminogen activator activity was abolished in vitro by incubation of t-PA with an antiserum specific for human t-PA, suggesting that t-PA may be directly responsible for plasmin generation in the response to endotoxin . We conclude from this study of healthy subjects that endotoxin activates the fibrinolytic system, beginning with release of t-PA in the blood within one hour . The early activation of plasmin by endotoxin may prevent thrombosis, and the increase in fibrinolysis is then offset by the release of plasminogen activator inhibitor.

Biochemistry, 1989 May 2, 28(9), 4105 - 8
15N-labeled 5S RNA . Identification of uridine base pairs in Escherichia coli 5S RNA by 1H-15N multiple quantum NMR; Davis DR et al.; Escherichia coli 5S RNA labeled with 15N at N3 of the uridines was isolated from the S phi-187 uracil auxotroph grown on a minimal medium supplemented with {3-15N}uracil . 1H-15N multiple quantum filtered and 2D chemical shift correlated spectra gave resonances for the uridine imino 1H-15N units whose protons were exchanging slowly with solvent . Peaks with 1H/15N shifts at 11.6/154.8, 11.7/155.0, 11.8/155.5, 12.1/155.0, and 12.2/155.0 ppm were assigned to GU interactions . Two labile high-field AU resonances at 12.6/156.8 and 12.8/157.3 ppm typical of AU pairs in a shielded environment at the end of a helix were seen . Intense AU signals were also found at 13.4/158.5 and 13.6/159.2 ppm where 1H-15N units in normal Watson-Crick pairs resonate . 1H resonances at 10.6 and 13.8 ppm were too weak, presumably because of exchange with water, to give peaks in chemical shift correlated spectra . 1H chemical shifts suggest that the resonance at 13.8 ppm represents a labile AU pair, while the resonance at 10.6 ppm is typical of a tertiary interaction between U and a tightly bound water or a phosphate residue . The NMR data are consistent with proposed secondary structures for 5S RNA.

Biochemistry, 1989 May 2, 28(9), 4099 - 105
Comparative cross-linking study on the 50S ribosomal subunit from Escherichia coli; Walleczek J et al.; We have carried out an extensive protein-protein cross-linking study on the 50S ribosomal subunit of Escherichia coli using four different cross-linking reagents of varying length and specificity . For the unambiguous identification of the members of the cross-linked protein complexes, immunoblotting techniques using antisera specific for each individual ribosomal protein have been used, and for each cross-link, the cross-linking yield has been determined . With the smallest cross-linking reagent diepoxybutane (4 A), four cross-links have been identified, namely, L3-L19, L10-L11, L13-L21, and L14-L19 . With the sulfhydryl-specific cross-linking reagent o-phenylenedimaleimide (5.2 A) and p-phenylenedimaleimide (12 A), the cross-links L2-L9, L3-L13, L3-L19, L9-L28, L13-L20, L14-L19, L16-L27, L17-L32, and L20-L21 were formed; in addition, the cross-link L23-L29 was exclusively found with the shorter o-phenylenedimaleimide . The cross-links obtained with dithiobis(succinimidyl propionate) (12 A) were L1-L33, L2-L9, L2-L9-L28, L3-L19, L9-L28, L13-L21, L14-L19, L16-L27, L17-L32, L19-L25, L20-L21, and L23-L34 . The good agreement of the cross-links obtained with the different cross-linking reagents used in this study demonstrates the reliability of our cross-linking approach . Incorporation of our cross-linking results into the three-dimensional model of the 50S ribosomal subunit derived from immunoelectron microscopy yields the locations for 29 of the 33 proteins within the larger ribosomal subunit.

Biochemistry, 1989 May 2, 28(9), 3875 - 9
NMR assignments for the amino-terminal residues of trp repressor and their role in DNA binding; Arrowsmith CH et al.; The trp repressor of Escherichia coli specifically binds to operator DNAs in three operons involved in tryptophan metabolism . The NMR spectra of repressor and a chymotryptic fragment lacking the six amino-terminal residues are compared . Two-dimensional J-correlated spectra of the two forms of the protein are superimposable except for cross-peaks that are associated with the N-terminal region . The chemical shifts and relaxation behavior of the N-terminal resonances suggest mobile "arms" . Spin-echo experiments on a ternary complex of repressor with L-tryptophan and operator DNA indicate that the termini are also disordered in the complex, although removal of the arms reduces the DNA binding energy . Relaxation measurements on the armless protein show increased mobility for several residues, probably due to helix fraying in the newly exposed N-terminal region . DNA binding by the armless protein does not reduce the mobility of these residues . Thus, it appears that the arms serve to stabilize the N-terminal helix but that this structural role does not explain their contribution to the DNA binding energy . These results suggest that the promiscuous DNA binding by the arms seen in the X-ray crystal structure is found in solution as well.

Biochemistry, 1989 May 2, 28(9), 3908 - 11
Modulation of mitomycin cross-linking by DNA bending in the Escherichia coli CAP protein-DNA complex; Cera C et al.; We have examined the comparative reactivity of mitomycin cross-linking sites in DNA molecules either free in solution or complexed with Escherichia coli CAP protein . Sites in the region to which the protein is bound show strongly variable cross-linking by the drug . The reactivity of a CpG site located where the minor groove is narrowed by bending toward the protein was decreased by about 4-fold, compared to free DNA . The reactivity of a site placed so that the minor groove is widened by the bend was reduced by about 25%, and the reactivity of a (CpG)3 sequence facing primarily away from the protein was reduced 25-fold by CAP binding . These results support the view that local DNA structure plays a critical role in determining the efficiency of cross-linking.

Biochemistry, 1989 May 2, 28(9), 3868 - 75
Recognition of beta beta'-substituted and alpha beta,alpha'beta'-disubstituted phosphonate analogues of bis(5'-adenosyl) tetraphosphate by the bis(5'-nucleosidyl)-tetraphosphate pyrophosphohydrolases from Artemia embryos and Escherichia coli; McLennan AG et al.; A total of 13 phosphonate analogues of bis(5'-adenosyl) tetraphosphate (AppppA) have been tested as substrates and inhibitors of the asymmetrically cleaving bis(5'-nucleosidyl) tetraphosphatase (NppppNase) from Artemia and the symmetrically cleaving NppppNase from Escherichia coli . With the Artemia enzyme, the substrate efficiency of beta beta'-substituted compounds decreased with decreasing substituent electronegativity (O greater than CF2 greater than CHF greater than CCl2 greater than CHCl greater than CH2) such that AppCF2ppA and AppCH2ppA were hydrolyzed at 70% and 2.5% of the rate of AppppA, respectively . These compounds were competitive inhibitors of this enzyme with Ki values that generally also decreased with electronegativity from 12 microM for AppCF2ppA to 0.4 microM for AppCH2ppA (Km for AppppA = 33 microM) . AppCH = CHppA and AppCH2CH2ppA were neither effective substrates nor inhibitors of the Artemia enzyme . Alpha beta,alpha'beta'-Disubstituted analogues were generally less effective inhibitors with Ki values ranging from 23 microM (ApCH2ppCH2pA) to greater than 1.5 mM (ApCH2CH2ppCH2CH2pA) . However, they displayed a low and unexpected rate of symmetrical cleavage by the Artemia enzyme: e.g., ApCHFppCHFpA yielded ApCHFp at 3% of the rate of AppppA breakdown . Both sets of analogues were also competitive inhibitors of the E . coli NppppNase with Ki values ranging from 7 microM (AppCH2ppA) to 250 microM (ApCH2CH2ppCH2CH2pA) (Km for AppppA = 28 microM) . The only alpha beta,alpha'beta'-disubstituted analogue to be hydrolyzed by the E . coli enzyme was ApCF2ppCF2pA at 0.2% of the rate of AppppA; however, several of the beta beta'-substituted compounds showed a limited degree of asymmetrical cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1989 May 2, 28(9), 4047 - 54
Effect of residue 65 substitutions on thermal stability of tissue plasminogen activator kringle-2 domain; Kelley RF et al.; We have used differential scanning calorimetry to measure the effect of replacements of valine 65 on thermal stability of the isolated kringle-2 domain of tissue plasminogen activator (t-PA) . The role of this site in stability was examined because a human t-PA variant having this valine (residue 245 in t-PA numbering) replaced with a methionine has been described {Johnston, M.D., & Berger, H . (1987) U.K . Patent Application GB 2176702A} . Mutants of kringle-2 having valine 65 replaced with Met, Leu, Ile, Thr, Ala, or Ser were constructed by using site-directed mutagenesis in conjunction with a restricted site selection strategy . Isolated kringle-2 domains were expressed in Escherichia coli and purified as previously described for the wild-type domain {Cleary, S., Mulkerrin, M.G., & Kelley, R.F . (1989) Biochemistry 28, 1884-1891} . None of these substitutions results in a significant perturbation of the native conformation of kringle-2 as judged by far-UV circular dichroism and equilibrium dialysis measurements of L-lysine affinity . A two-state analysis of the heat capacity profile observed for heating a solution of wild-type (w-t) kringle-2 containing 100 mM citrate, pH 4.5, provides values of 64.3 +/- 0.8 degrees C for Tg (melting temperature), 81 +/- 5 kcal/mol for delta H g, and 1.2 +/- 0.9 kcal/(mol-deg) for delta C p . Thermal denaturation of w-t kringle-2 is reversible in the pH range 3-6 as indicated by the observation of similar heat capacity profiles for consecutive heating cycles and also recovery of spectroscopic and lysine binding properties upon cooling the heat-denatured protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1989 May 2, 28(9), 3738 - 43
Isolation and preliminary characterization of wild-type OmpC porin dimers from Escherichia coli K-12; Rocque WJ et al.; Escherichia coli K-12 strain PLB3255 contains a mutation in the ompF gene that results in a 15 amino acid deletion in the porin protein . The mutation (dex) appears to increase the OmpF channel size, allowing the PLB3255 cells to grow on maltodextrins in the absence of a functional maltoporin . Porin isolated from strain PLB3255, which contains a wild-type ompC gene, was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and shown to contain 50-60% trimer aggregates and 35-40% of a 50-kDa "dimer" . When the porin isolate was heated to 100 degrees C and separated on SDS gels containing 8 M urea, both the trimer and the "dimer" were recovered in a single band at 36 kDa that corresponded in mobility to wild-type OmpC porin . An analysis of the temperature stability of the isolate showed that the OmpC "dimer" was less stable and denatured at 66 degrees C compared to 81 degrees C for the trimer . To separate these aggregates, the unheated porin was suspended in 30% SDS, applied to a Sephadex G-200 gel filtration column, and eluted with 0.5% sodium deoxycholate . Two peaks were recovered containing separated trimers and "dimers" . Circular dichroism spectra of isolated dimers and trimers indicated similar amounts of beta-structure . The isolated dimers and trimers were reconstituted into artificial membranes . Electrical conductance across planar bilayer lipid membranes and liposome swelling assays showed that the two isolates had similar channel-forming activity . Four other ompF deletion mutants of the same phenotype were also shown to produce 50-kDa OmpC porin "dimers".(ABSTRACT TRUNCATED AT 250 WORDS)

Sci China B, 1989 May, 32(5), 534 - 42
Limited tryptic digestion of leucyl-tRNA synthetase and characterization of its active fragment; Miao F et al.; Leucyl-tRNA synthetase (LeuRS, EC 6.1.1.4) from E . coli underwent limited proteolysis by trypsin which cut off 6K peptide and converted the intact LeuRS into a 96K fragment . The truncated enzyme retained the PPi exchange activity with the same kinetic parameters as those of native LeuRS but lost the tRNALeu charging, binding and other tRNALeu-related activities . N-terminus analysis showed that the 6K peptide was located at the C-terminus of Leu-RS . This small part played a crucial role in tRNALeu binding . Our results suggest that the two activities, PPi exchange and tRNA charging are independent of each other and correspond to different structural regions of LeuRS . The C-terminal region might be the tRNALeu binding site of LeuRS.

Res Immunol, 1989 May, 140(4), 355 - 76
Polypeptide-antibody binding mechanism: conformational adaptation investigated by equilibrium and kinetic analysis; Friguet B et al.; The mechanism of polypeptide-antibody binding was analysed by kinetic and equilibrium studies to find out whether or not the binding of an antibody to a large protein or a polypeptide antigen behaves as a one-step reaction or involves conformational adaptation . Three monoclonal antibodies recognizing 3 distinct epitopes on the C-terminal domain (F2) of Escherichia coli tryptophan synthase beta 2 subunit were used . The dissociation equilibrium constant (KD), the association rate constant (kon) and the dissociation rate constant (koff) of these antibodies for the native beta 2 subunit, its C-terminal fragment (F2) and different polypeptides obtained by chemical cleavage of the F2 fragment were measured . It was found that for some polypeptide-antibody complexes, binding could not be described as a one-step association-dissociation reaction, thus indicating the existence of conformational adaptation upon antibody-antigen complex formation . It was also shown that differences in affinities of a given antibody for its epitope carried by different polypeptides were mainly due to differences in the dissociation rate constant (koff) and not in the association rate constant (kon) . Moreover, the immunoreactivity of various polypeptides obtained by cleavage of the F2 fragment enabled us to localize the 3 epitopes on the beta chain in light of the 3-dimensional structure of tryptophan synthase described recently by Hyde et al . (1988).

DNA, 1989 May, 8(4), 233 - 43
Characterization of the hsp70 multigene family of Caenorhabditis elegans; Heschl MF et al.; Our laboratory has been characterizing the hsp70 multigene family from the nematode Caenorhabditis elegans as the first step to the genetic characterization of the heat shock response in a relatively simple multicellular eukaryote . Two gene members, hsp-1 and hsp-2ps have already been characterized (Snutch et al., 1988; Heschl and Baillie, 1989) . The third gene member, hsp-3, is expressed constitutively and is non-heat inducible; its mRNA is most abundant at the L1 larval stage . The hsp-3 protein (hsp70C) shares a high degree of identity with the rat grp78 protein and has a long, hydrophobic leader sequence . The carboxyl terminus of hsp70C has the putative ER-retention signal, KDEL . The fourth gene member, hsp-6 is expressed constitutively and moderately heat inducible . A partial hsp-6 protein (hsp70F) sequence shares a higher degree of identity with the Escherichia coli dnaK protein than with eukaryotic hsp70 proteins . The predicted amino-terminal half of the hsp70F polypeptide also contains a long, amphiphilic leader sequence similar to mitochondrial import leader sequences . These two genes encode proteins that potentially cross intracellular membranes . We compared the 5'-flanking DNA from the C . elegans hsp-3 gene to fragment containing enhancer activity from the rat grp78 gene regulatory region (Lin et al., 1986) . A 23-nucleotide sequence was conserved between the two promoter regions . This sequence shares approximately 80% identity between these two evolutionary distant organisms . A comparison to other hsp70 genes did not reveal any conservation of this 23-nucleotide sequence . We propose that this sequence may be involved in a unique aspect of the regulation of the C . elegans' grp78-like gene and the rat grp78 gene.

Mol Cell Endocrinol, 1989 May, 63(1-2), 15 - 22
Generation of polyclonal antibodies against purified rat whey acidic proteins and the synthesis of a tracer fusion protein suitable for use in radioimmunoassays; Krozowski Z; Rodent milk consists mainly of caseins and whey proteins . A major component of the latter group is the whey acidic proteins (WAP) the gene for which has been cloned recently and shown to contain several potential glucocorticoid receptor binding sites . Studies on the regulation of this gene by glucocorticoids would be greatly enhanced by the availability of a radioimmunoassay for WAP . Rat milk was obtained from lactating Sprague-Dawley rats and the WAP purified to homogeneity by ammonium sulphate fractionation, gel filtration and ion exchange chromatography . Rabbit polyclonal antibodies were raised against the purified WAP and used to probe a Western blot of whey proteins . The major band recognized by the antibody corresponded in molecular weight to purified WAP . Problems associated with radiolabelling the tyrosine-free WAP molecule necessitated the fusion of a tyrosine containing protein with the rat milk protein . A rat WAP cDNA clone was ligated to the glutathione transferase gene, the fusion protein expressed in Escherichia coli and purified by a one-step procedure on a glutathione affinity column . Purified WAP readily displaced the radiolabelled recombinant tracer in a radioimmunoassay.

Am J Vet Res, 1989 May, 50(5), 778 - 81
Phagocytic and nitroblue tetrazolium reductive properties of mature and immature neutrophils and eosinophils from blood and bone marrow from cows; Silva ID et al.; Functional capabilities of morphologically mature (segmented) and immature granulocytes (neutrophils and eosinophils) from bone marrow from cows were studied and compared with similar activities of segmented granulocytes from blood . Phagocytosis of Escherichia coli and postphagocytic oxidative metabolic stimulation, measured by nitroblue tetrazolium (NBT) reduction, were evaluated simultaneously . Phagocytosis was observed readily in segmented neutrophils, neutrophilic bands, and metamyelocytes and rarely in myelocytes . Phagocytosis was not seen in promyelocytes and myeloblasts . Neutrophilic bands and metamyelocytes were phagocytically less active than were segmented neutrophils . Washed segmented bone marrow neutrophils possessed phagocytic activity similar to that of blood neutrophils, whereas the activity of unwashed segmented bone marrow neutrophils was markedly less than that of blood neutrophils . Reduction of NBT was observed only in blood segmented neutrophils and bone marrow segmented neutrophils; the magnitude of NBT reduction was significantly (P = less than 0.005) less in bone marrow neutrophils than in blood neutrophils . Eosinophils were phagocytically less competent than were neutrophils . The NBT reduction was observed only in eosinophils from blood, but not in eosinophils from bone marrow.

J Bacteriol, 1989 May, 171(5), 2735 - 9
Identification and sequence of the basic replication region of a broad-host-range plasmid isolated from Thiobacillus ferrooxidans; Dorrington RA et al.; The minimum region required for replication of the broad-host-range Thiobacillus ferrooxidans plasmid pTF-FC2 in Escherichia coli was shown to be contained on a 2.05-kilobase fragment of DNA . A 184-base-pair fragment that was required in cis for plasmid replication was identified . This region was also involved in plasmid incompatibility . Nucleotide sequencing of this region revealed three perfectly conserved 22-base-pair tandemly repeated sequences . A comparison of this region with the equivalent region of the broad-host-range plasmid R1162 showed that the repeated sequences had 60% nucleotide homology . The 106-base-pair region immediately adjacent to the repeated sequences was 75% homologous . These plasmids were compatible.

J Clin Lab Immunol, 1989 May, 29(1), 17 - 23
Response of humans to gamma-irradiated reference Escherichia coli endotoxin; Elin RJ et al.; This study assesses the effect of gamma-irradiation of endotoxin given intravenously to healthy human volunteers . The national reference standard endotoxin derived from E . coli was placed in aqueous medium in sterile-sealed ampoules and divided into four groups . One group received endotoxin with no radiation while the other three received endotoxin with gamma-irradiation at doses of 0.18, 0.36, or 1.08 Mrad . These doses of radiation cause characteristic alterations to the endotoxin molecule, primarily to the O-polysaccharide moiety . Each of the four different preparations of endotoxin was given intravenously to four volunteers at a concentration of 4 ng/kg . The responses for clinical symptoms, cortisol, and growth hormone were significantly and progressively reduced by increasing the irradiation to the endotoxin . Most strikingly, no clinical symptoms were noted with the endotoxin exposed to the highest dose of radiation (1.08 Mrad) . Fever, vital signs, white blood cell count, and differential exhibited no statistically significant differences among the groups, but the kinetics of change were altered by increasing doses of gamma-irradiation . Irradiated endotoxin was significantly more effective in decreasing the platelet count than untreated endotoxin . The fever index correlated significantly with maximum temperature, change in temperature, white blood cell count index, mature neutrophil count index, and the cortisol index . Thus, there is dissociation of biological activities for endotoxin in humans due to molecular changes primarily in the O-polysaccharide moiety from exposure to gamma-irradiation.

Clin Endocrinol (Oxf), 1989 May, 30(5), 531 - 8
Antigenicity and efficacy of authentic sequence recombinant human growth hormone (somatropin): first-year experience in the United Kingdom; Buzi F et al.; Twenty-one children were treated for GH deficiency with authentic sequence biosynthetic human GH (somatropin), 60 micrograms/kg body weight, subcutaneously, three times weekly for 1 year or longer . The magnitude of growth response and rise in serum insulin-like growth factor I levels were similar to those expected from experience with pituitary GH and somatrem . Three patients developed serum antibodies to GH with a binding capacity greater than 0.02 mg/l, but in only one patient was the GH binding capacity greater than 1.0 mg/l and he showed no attenuation of growth response . Escherichia coli polypeptide antibodies did not rise significantly and no clinically important side-effects occurred . Somatropin is safe, effective and of low immunogenicity in the treatment of GH deficiency.

Mikrobiologiia, 1989 May-Jun, 58(3), 467 - 70
{The effect of growth rate and culturing conditions on the stability of plasmid pCJ55 and on the level of expression of a large fragment of DNA-polymerase I, cloned in Escherichia coli}; Kriukova EG et al.; Plasmid pCJ55 with a cloned gene for the large fragment of Escherichia coli DNA polymerase I is stable in the population of a recombinant strain under the conditions of batch and continuous cultivation at different dilution rates in the presence of ampicillin . The level of Klenow fragment expression is determined by at least two factors: the stability of the recombinant strain and its specific growth rate . The maximal activity of the Klenow fragment was found after thermoinduction of the culture growing at a rate of mu = 0.6 h-1 in a synthetic medium with bactopeptone and glucose as a carbon source.

Nippon Shokakibyo Gakkai Zasshi, 1989 May, 86(5), 1089 - 95
{An experimental study on the severe type of alcoholic liver disease--a pathogenetic role of potentiated activation of complement system by endotoxin after chronic ethanol consumption}; Arai M et al.; Endotoxemia frequently appears in severe type of alcoholic liver disease . However, we have little knowledge how endotoxin influences the progression of alcoholic liver injury . Thus, to study the causal mechanism for the progression to the severe type of alcoholic liver diseases, endotoxin (0.2 mg/100 g BW, E . Coli O26:B6) was intravenously injected in chronic ethanol-fed rats and controls, and then, rats were sacrificed after 16 hours of endotoxin treatment . The elevation of serum GOT and GPT activities induced by endotoxin was significantly higher in chronic ethanol-fed rats than controls, and these biochemical changes were well correlated with the grade of necrosis of liver histology . Furthermore, in chronic ethanol-fed rats, endotoxin treatment tremendously increased blood BUN and creatinine levels and produced the degeneration of renal tubuli with neutrophil infiltration into glomerulus . These experimental findings are very similar to the severe type of alcoholic liver disease . On the other hand, endotoxin significantly decreased serum values of CH50 in chronic ethanol-fed rats, but not in controls . Such alterations of CH50 induced by endotoxin were well correlated with the several parameters indicating the injury of liver and kidney . Therefore, the present study may indicate that chronic ethanol ingestion aggravates endotoxin-induced organ injury, and that the activation of complement system may associate with such progression of organ injury.

Plasmid, 1989 May, 21(3), 238 - 41
Single-stranded circular DNA generated from broad host range plasmid R1162 and its derivatives; Kok M et al.; Extraction of R1162 plasmid DNA with the alkaline lysis method yields considerable amounts of single-stranded circular plasmid DNA . Destabilization of plasmid DNA is stimulated by the R1162 mob region in cis . The formation of single-stranded circular DNA is initiated at a specific site on the plasmid, presumably the origin of transfer (oriT).

Tsitol Genet, 1989 May-Jun, 23(3), 16 - 20
{Ultrastructural changes in alveolar macrophages in rats under the effect of Escherichia coli endotoxin}; Kharlanova NG et al.; Electron-microscopic investigation has shown that alveolar macrophages phagocyte surfactant in the initial period of the endotoxin shock, necrotized cells--in the intermediate period, fibrin--at the stage of late endotoxemia.

Mol Biol (Mosk), 1989 May-Jun, 23(3), 889 - 98
{Cloning and expression of the hemagglutinin gene of influenza virus subtype H1 in Escherichia coli}; Petrenko VA et al.; The full-length copy of the hemagglutinin gene of influenza virus was inserted into M13 phage DNA . The DNA sequence coding for the hydrophobic prepeptide was removed from the gene by oligonucleotide-directed mutagenesis . The possibilities of expression of the full-length and mutant genes in E . coli were investigated . The beta-galactosidase-hemagglutinin fusion proteins were isolated . The fusion proteins exhibited specific binding to antiviral antibodies . This binding could be competitively inhibited by excess of viral hemagglutinin, demonstrating that these fusion proteins contained antigenic determinants of hemagglutinin.

Mol Gen Genet, 1989 May, 217(1), 162 - 7
In vitro and in vivo studies on the mitochondrial import of CBS1, a translational activator of cytochrome b in yeast; Korte A et al.; Translation of mitochondrial cytochrome b mRNA in yeast is activated by the product of the nuclear gene CBS1 . CBS1 encodes a 27 kDa precursor protein, which is cleaved to a 24 kDa mature protein during the import into isolated mitochondria . The sequences required for mitochondrial import reside in the amino-terminal end of the CBS1 precursor . Deletion of the 76 amino-terminal amino acids renders the protein incompetent for mitochondrial import in vitro and non-functional in vivo . When present on a high copy number plasmid and under the control of a strong yeast promoter, biological function can be restored by this truncated derivative . This observation indicates that the CBS1 protein devoid of mitochondrial targeting sequences can enter mitochondria in vivo, possibly due to a bypass of the mitochondrial import system.

Mol Gen Genet, 1989 May, 217(1), 13 - 9
Ribonucleotide reductase: a determinant of 5-bromodeoxyuridine mutagenesis in phage T4; Sargent RG et al.; Mutagenesis by 5-bromodeoxyuridine (BrdUrd) can result from base-pairing errors either during replication of a BrdUrd-containing template or at the nucleotide incorporation step . Replication errors give rise predominantly to AT-to-GC transitions, while incorporation errors, in which 5-bromo-dUTP competes with dCTP at a template guanine site, should give rise to GC-to-AT transitions . The latter pathway should be sensitive to deoxyribonucleoside triphosphate (dNTP) pool fluctuations . Since dNTP pools are regulated through allosteric control of ribonucleotide reductase, the control of this enzyme should be a determinant of BrdUrd mutagenesis--if mutagenesis results largely from incorporation errors . Since T4 phage-encoded ribonucleotide reductase is insensitive to feedback inhibition, we established conditions under which phage DNA replication is dependent upon ribonucleotide reductase of the host, Escherichia coli . We examined BrdUrd mutagenesis of rII mutants known to revert to wild type either by AT-to-GC or GC-to-AT transition pathways . While both reversion pathways were stimulated under all conditions analyzed, the AT-to-GC pathway was stimulated more when the E . coli reductase was functioning, while the GC-to-AT pathway was more specifically enhanced when the T4 reductase was active . These results confirm that ribonucleotide reductase is a determinant of BrdUrd mutagenesis, but our observations, plus experiments showing that BrdUrd has relatively small effects upon dNTP pool sizes, indicate that the relationship between deoxyribonucleotide metabolism and BrdUrd mutagenesis is more complex than anticipated.

Mol Gen Genet, 1989 May, 217(1), 1 - 5
The secY-rpmJ region of the spc ribosomal protein operon in Escherichia coli: structural alterations affecting secY expression; Ueguchi C et al.; A unique feature of the spc ribosomal protein operon is that its region distal to the promoter contains a gene (secY) for an integral membrane protein, followed by an open reading frame termed X which has recently been proposed to encode a new ribosomal protein (protein B) . We now show that the open reading frame X indeed directs the synthesis of a protein with electrophoretic mobilities similar to the B protein, and this supports the proposal that X may be more appropriately called rpmJ . Insertion of a plasmid sequence into the secY-rpmJ boundary of the chromosome caused a reduced expression of secY probably by destabilizing the secY part of the message . The results of complementation experiments suggested that a normal level of expression of rpmJ is not required for growth or protein secretion.

Microb Pathog, 1989 May, 6(5), 381 - 90
Shiga-like toxin converting phage of enterohemorrhagic Escherichia coli strain 933; O'Brien AD et al.; Production of Shiga-like toxin (SLT) by enterohemorrhagic Escherichia coli (EHEC) is controlled by phage conversion, and specific phages carry either the SLT-I or SLT-II operon . EHEC strain 933 produces both SLT-I and SLT-II . Previous studies demonstrated that the vast majority of phages recovered from strain 993 have hexagonal heads with short tails and encode SLT-II . However, conflicting results were obtained concerning the properties of SLT-I converting phages from strain 933 . The present study reexamined the recovery of phages from 933 by various methods and characterized the restriction fragments from strain 933 DNA that hybridized with radiolabeled DNA from the SLT-I converting phage 933J, which has an elongated head with a long tail, and the SLT-II converting phage 933W . In the present study, only SLT-II converting phages like 933W were recovered from strain 933 . A set of restriction fragments that hybridized with DNA from phage 933J but not 933W was present both in wild type strain 933 and in the variant 933D, which produces only SLT-I and was shown here to be cured of phage 933W . The sizes of the restriction fragments in strain 933 that were homologous with phage 933J differed, however, from those of phage 933J . These data indicate that the phage we isolated and named 933J probably did not originate from strain 933 as we originally reported . The present evidence demonstrates that strain 933 contains both the SLT-II converting phage 933W and other sequences of DNA homologous with phage 933J that probably represent a defective SLT-I converting phage.

Microb Pathog, 1989 May, 6(5), 351 - 9
Isolation and characterization of the non-fimbrial adhesin NFA-4 from uropathogenic Escherichia coli O7:K98:H6; Hoschutzky H et al.; The non-fimbrial adhesin NFA-4 from uropathogenic Escherichia coli O7:K98:H6 mediates the agglutination of human red cells (RBC), notably of blood group MM . The adhesin can be separated from the bacteria by heat extraction and was purified to homogeneity by ammonium sulphate precipitation and anion exchange chromatography in the presence of 8 M urea . NFA-4 consists of non-covalently linked 28 kDa subunits which tend to form aggregates of an apparent molecular weight in excess of 10(6) Da . The first 23 amino-terminal amino acids were sequenced, and no homology of this region was found with that of the blood group M specific non-fimbrial adhesin of an unrelated uropathogenic E . coli . It has, however, an about 70% homology to the corresponding region of the K88 antigen from animal-pathogenic enterotoxic E . coli . Both polyclonal and monoclonal antibodies against NFA-4 were prepared . One of the monoclonal antibodies strongly inhibits the hemagglutinating activity of both whole bacteria and purified NFA-4.

Kidney Int, 1989 May, 35(5), 1133 - 7
Ascending pyelonephritis in young rats retards kidney growth; Hannerz L et al.; Several radiological studies have suggested that pyelonephritis in infancy and childhood may result in kidney growth retardation without renal scarring . In the present study, we induced ascending pyelonephritis in 20-day-old rats with intravesical infusion of E . coli . Four days after infusion, E . coli was cultured from all renal cortex . The rats were either left untreated (PNu) or were treated with trimethoprim-sulfa (PNt) . The rats were investigated one month after infection and compared with an age-matched control group (C) . Seventy-nine percent of the PNu rats had recovered spontaneously from infection . Body weight was the same in all groups . In PNu rats, kidney weight (KW), kidney area (KA) and glomerular filtration rate (GFR) were significantly decreased . KW, KA and GFR were similar in PNt and C rats . The numbers of filtering nephrons were not reduced by the infection . The total cortical DNA content (index of cell number) was significantly lower in PNu (5.30 +/- 0.32 mg) and PNt (6.62 +/- 0.44 mg) than in C rats (8.48 +/- 0.49 mg) . The cortical DNA content was significantly lower in PNu than in PNt rats . The cortical protein/DNA ratio was significantly higher in PNu rats than in C rats . The protein/DNA ratio was similar in PNt and PNu rats . The increase in protein/DNA ratio was interpreted as a sign of cell hypertrophy . The inflammatory process as such did not increase the protein/DNA ratio . The kidneys were also examined for structural lesions . Signs of scarring, inflammation and cell necrosis were almost absent in all groups.(ABSTRACT TRUNCATED AT 250 WORDS)

EMBO J, 1989 May, 8(5), 1623 - 7
xerB, an Escherichia coli gene required for plasmid ColE1 site-specific recombination, is identical to pepA, encoding aminopeptidase A, a protein with substantial similarity to bovine lens leucine aminopeptidase; Stirling CJ et al.; The heritable stability of ColE1 is dependent on a site-specific recombination system which acts to resolve plasmid multimers into monomers . This plasmid stabilizing recombination system requires the presence in cis of the ColE1 cer region, plus at least two trans-acting factors encoded by the xerA and xerB genes of Escherichia coli . The xerB gene has been cloned and sequenced and found to encode a polypeptide with a calculated mol . wt of 55.3 kd . The predicted amino acid sequence of this protein exhibits striking similarity to that of bovine lens leucine aminopeptidase (53 kd) . The biological significance of this similarity is corroborated by genetic and biochemical evidence which suggests that xerB is identical to the E.coli and S.typhimurium pepA genes that encode aminopeptidase A.

EMBO J, 1989 May, 8(5), 1479 - 84
Nuclear transport kinetics depend on phosphorylation-site-containing sequences flanking the karyophilic signal of the Simian virus 40 T-antigen; Rihs HP et al.; Selective nuclear protein transport was analyzed in single living cells . Hybrid proteins consisting of short stretches of the Simian virus 40 T-antigen and of the almost complete beta-galactosidase moiety were generated by molecular genetic methods and injected into the cytoplasm of rodent hepatoma cells . A histochemical assay showed that all proteins containing the karyophilic signal of the T-antigen (residues 126/127-132) were equally well accumulated by the nucleus within 15 h after injection . Microfluorimetric measurements of nuclear transport kinetics, however, revealed large differences . Proteins containing the karyophilic signal without flanking sequences were taken up by the nucleus on a time scale of hours . The same held for a protein containing T-antigen residues 127-147 . However, a protein containing T-antigen residues 111-135 was accumulated by the nucleus with a half-time of 8-10 min reaching an equilibrium nucleocytoplasmic concentration ratio of greater than or equal to 15 . Photobleaching measurements showed that, independently of subcellular localization, the mobility of all proteins was quite large . Thus, our measurements revealed a striking effect of T-antigen residues 111-125 on the kinetics of nuclear transport . Residues 111-125 do not seem to contain a second karyophilic signal . Conspicuously, however, they comprise a cluster of phosphorylation sites.

Electrophoresis, 1989 May-Jun, 10(5-6), 366 - 76
Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay; Fried MG; Native gel electrophoresis (mobility shift) assays may be used to obtain quantitative information about the site distribution, equilibria and kinetics of protein-DNA interactions . These applications depend on the ability of the electrophoretic system to resolve the reaction components, and on their stabilities during the separation process . Factors which affect the lifetimes and mobilities of protein-DNA complexes during electrophoresis include reaction and electrophoresis buffer composition, pH, and ionic strength; the presence of low molecular weight effectors and enzymatic substrates; the nature and concentration of the gel matrix; the temperature; the molecular weights of protein and DNA; the stoichiometric ratios of their complexes; and the possibility of conformational and configurational isomerization of reaction components . We discuss how these factors influence the acquisition of quantitative data from electrophoretic patterns and band intensities, and present formulas for the estimation of equilibrium constants and rate constants for prototypical DNA-protein interactions.

DNA, 1989 May, 8(4), 271 - 8
Tilapia growth hormone: molecular cloning of cDNA and expression in Escherichia coli; Rentier-Delrue F et al.; A cDNA library was prepared from poly(A)+RNA extracted from tilapia Oreochromis niloticus anterior pituitaries . The recombinant clones carrying the cDNA sequence of tilapia growth hormone (tiGH) were selected using a fragment of the trout growth hormone (tGH) cDNA as hybridization probe . The nucleotide sequence of the full-length tiGH cDNA was determined . This cDNA encodes a protein of 204 amino acids, including the putative signal peptide of 17 amino acids . Mature tiGH cDNA was inserted in an Escherichia coli expression vector which led to the production of tiGH protein with a yield estimated to be 20% of the total bacterial proteins.

Rev Infect Dis, 1989 May-Jun, 11 Suppl 3, S579 - 85
Antisporozoite vaccine for malaria: experimental basis and current status; Nussenzweig RS et al.; A major sporozoite surface antigen, the circumsporozoite protein, has been identified in all four malaria parasites affecting humans and in numerous species causing malaria in rodents and simians . The corresponding genes have been cloned and sequenced, and considerable similarities are apparent . An extensive central region of these proteins consists of tandemly repeated sequences of four to 16 amino acids . The sporozoite protein of Plasmodium falciparum has 37-41 repeats of four amino acids: NANP (asparagine-alanine-asparagine-proline) . Most sera from people in endemic areas that react with sporozoites also recognize the dodecamer (NANP)3 . Conjugated to a carrier, (NANP)3 is an excellent immunogen for rabbits and mice . NANP has recently served as the basis for two experimental malaria vaccines tested in volunteers . One of these vaccines, (NANP)32 tet32, was genetically engineered in Escherichia coli; the other consisted of the synthetic peptide (NANP)3 conjugated to tetanus toxoid . Most peptide-immunized volunteers developed antipeptide/sporozoite antibodies; however, there was no booster effect, and only one of three individuals was completely protected . For optimal protection, future vaccines must not only contain the B cell epitope but also induce T helper cells and cytotoxic T cells producing interferon-gamma, which has been shown to inhibit the development of liver-stage parasites.

Mol Microbiol, 1989 May, 3(5), 679 - 87
Topology and function of the integral membrane protein conferring immunity to colicin A; Geli V et al.; The topology of the integral membrane protein Cai (colicin A immunity protein), which is required to protect producing cells from the pore-forming colicin A, was analysed using fusions to alkaline phosphatase . The properties of these fusion proteins support the model for Cai topology previously proposed on theoretical grounds . The protein was found to contain four transmembrane sequences and its N- and C-terminal regions were found to be directed towards the cytoplasm . Oligonucleotide-directed mutagenesis and sequence comparisons between Cai, Cbi (colicin B immunity protein), and Cni (colicin N immunity protein) were carried out to determine the functional regions of Cai . The possible roles of the various regions of Cai in its protective function and in its topological organization are discussed.

Mol Microbiol, 1989 May, 3(5), 645 - 52
Detection and identification of FaeC as a minor component of K88 fibrillae of Escherichia coli; Oudega B et al.; A tribrid gene containing ompF, faeC, and lacZ sequences was constructed by subcloning a large central segment of the K88ab gene encoding the fibrillar subunit-like protein FaeC into the open reading frame expression vector pORF2 . The resulting tribrid protein was isolated and used to raise antibodies against the FaeC protein . These antibodies were then used for the detection and subcellular localization of the FaeC protein in Escherichia coli harbouring the K88ab-encoding plasmid pFM205 or mutant derivatives . Immunoblotting of subcellular fractions and of purified fibrillae, and agglutination experiments using whole cells revealed that the FaeC protein is present in the periplasm and as a minor component in the K88ab fibrillae . FaeC was also detected in purified K88ac and K88ad fibrillae . Immunoelectron microscopy confirmed the presence of FaeC in K88ab fibrillae, particularly at the tips of the longer fibrillae.

Environ Health Perspect, 1989 May, 81, 77 - 9
Mutagenic metabolites of benzene detected in the Microscreen assay; Rossman RG et al.; The reactive metobolite responsible for benzene hematotoxicity and carcinogenicity is unknown . It can be hypothesized that the ultimate carcinogen derived from benzene metabolism might also act as a mutagen . This laboratory has recently developed a new assay that can detect mutagens of all types, using a single strain of bacteria, E . coli WP2s (lambda), as a target . Different genetic end points can be monitored in the same exposed population of bacteria . When a number of known metabolites of benzene were assayed, only trans,trans-muconic acid gave a strong positive response . Mutations were induced at two genetic loci (Trp+ revertants and T5 resistance) . The mutagenic activity was greatly increased when a rat liver metabolizing system was added . We speculate that trans,trans-muconic acid is metabolized to a diepoxide, which may be the ultimate mutagen and possibly the ultimate carcinogen.

Appl Environ Microbiol, 1989 May, 55(5), 1298 - 300
Survival of chlorine-injured enterotoxigenic Escherichia coli in an in vitro water system; Walsh SM et al.; Survival of chlorine-injured and noninjured subpopulations of enterotoxigenic Escherichia coli was compared in KH2PO4-buffered water and chlorine-neutralized tap water . Injured cells were no less persistent than noninjured cells and did not exhibit limited survival as a consequence of chlorine injury . At high inoculum densities, some injured cells were able to repair, apparently owing to the accumulation of materials arising from the chlorination procedure.

Mol Endocrinol, 1989 May, 3(5), 822 - 31
Bioactive recombinant methionyl bovine prolactin: structure-function studies using site-specific mutagenesis; Luck DN et al.; A method has been developed for the extraction from transformed Escherichia coli cells of methionyl bovine PRL (met-bPRL) in a relatively pure form . While the extracted met-bPRL was as reactive as the native hormone with respect to polyclonal anti-bPRL antibodies, its bioactivity, as measured by the Nb2 lactogen in vitro bioassay, was relatively low . The bioactivity of the met-bPRL could be increased to the same order as that of the native hormone by treatment with a mixture of oxidized and reduced thioredoxin . A number of variant met-bPRLs containing specific amino acid changes have been generated by site-specific mutagenesis . The changes involved the substitution (or deletion) of some of the conserved amino acids in bPRL by the different amino acids present at the corresponding positions in the related, but nonlactogenic bovine GH . Nine mutants containing single amino acid changes had bio- and immunoactivities of the same order as those of met-bPRL . One mutant, which incorporated two of the single amino acid changes (serine 62 to threonine and threonine 65 to alanine), had immunoactivity approximating that of met-bPRL but much lower bioactivity (45%) . A further mutant, generated by the deletion of tyrosine 28, had essentially no bioactivity although it could not be distinguished immunologically from met-bPRL or bPRL . The findings are discussed in the light of the putative three-dimensional PRL structure and current hypotheses which seek to relate specific regions of PRL to lactogenic activity . It appears that the first putative alpha-helix of bPRL is important for the binding and mitogenic activity of the hormone.

J Biochem (Tokyo), 1989 May, 105(5), 813 - 7
Properties of electroporation-mediated DNA transfer in Escherichia coli; Taketo A; Efficient and reproducible DNA-transfection was attained in E . coli, by electroporation . The yield of the transfectants was affected by pretreatment of the recipient cells as well as by the composition of the electroporation medium . Using a single pulse procedure, relationships among the electrical parameters, the transfection efficiency, and the cellular viability were investigated in 10 mM Tris-HCl buffer (pH 7.5) containing 5% sucrose . Certain sodium salts (e.g., citrate, phosphate, and sulfate) were promotive, whereas Mg2+, DEAE-dextran, and polyvinylpyrrolidone were inhibitory to the transfection . Heterologous nucleic acids (native DNA, denatured DNA, and tRNA) exerted only a marginal effect on transfection with a viral replicative-form DNA . The efficiency of DNA transfer was affected by culture conditions, and bacteria grown at a higher temperature were more competent . The electroporation system was more efficient than an improved CaCl2 method, not only in transfection with viral single- and double-stranded DNAs, but also in transformation with plasmid DNAs.

J Biochem (Tokyo), 1989 May, 105(5), 671 - 2
Crystallization and preliminary X-ray characterization of branched-chain amino acid aminotransferase from Escherichia coli; Kamitori S et al.; The branched-chain amino acid aminotransferase of Escherichia coli was crystallized in two crystal systems, monoclinic and tetragonal, from polyethylene glycol and ammonium sulfate solutions, pH 7.0, respectively . The crystals were of good quality, with diffractions extending beyond 2.8 A . The space group and unit cell dimensions of the monoclinic system crystals were determined from precession photographs to be C2, and a = 93.9, b = 143.6, c = 143.9 A and beta = 134.3 degrees . For the tetragonal system crystals, the possible space group P422 or P4122, and cell dimensions of a = b = 101 A and c = 249 A were determined . Three identical subunits exist per an asymmetric unit in both types of crystals.

Mol Cell Biol, 1989 May, 9(5), 2050 - 7
The two positively acting regulatory proteins PHO2 and PHO4 physically interact with PHO5 upstream activation regions; Vogel K et al.; The repressible acid phosphatase gene PHO5 of Saccharomyces cerevisiae requires the two positively acting regulatory proteins PHO2 and PHO4 for expression . pho2 or pho4 mutants are not able to derepress the PHO5 gene under low-Pi conditions . Here we show that both PHO2 and PHO4 bind specifically to the PHO5 promoter in vitro . Gel retardation assays using promoter deletions revealed two regions involved in PHO4 binding . Further characterization by DNase I footprinting showed two protected areas, one located at -347 to -373 (relative to the ATG initiator codon) (UASp1) and the other located at -239 to -262 (UASp2) . Exonuclease III footprint experiments revealed stops at -349 and -368 (UASp1) as well as at -245 and -260 (UASp2) . Gel retardation assays with the PHO2 protein revealed a binding region that lay between the two PHO4-binding sites . DNase I footprint analysis suggested a PHO2-binding site covering the region between -277 and -296.

Mol Cell Biol, 1989 May, 9(5), 1987 - 95
Synthesis of an enzymatically active FLP recombinase in vitro: search for a DNA-binding domain; Amin AA et al.; We have used an in vitro transcription and translation system to synthesize an enzymatically active FLP protein . The FLP mRNA synthesized in vitro by SP6 polymerase is translated efficiently in a rabbit reticulocyte lysate to produce enzymatically active FLP . Using this system, we assessed the effect of deletions and tetrapeptide insertions on the ability of the respective variant proteins synthesized in vitro to bind to the FLP recognition target site and to carry out excisive recombination . Deletions of as few as six amino acids from either the carboxy- or amino-terminal region of FLP resulted in loss of binding activity . Likewise, insertions at amino acid positions 79, 203, and 286 abolished DNA-binding activity . On the other hand, a protein with an insertion at amino acid 364 retained significant DNA-binding activity but had no detectable recombination activity . Also, an insertion at amino acid 115 had no measurable effect on DNA binding, but recombination was reduced by 95% . In addition, an insertion at amino acid 411 had no effect on DNA binding and recombination . On the basis of these results, we conclude that this approach fails to define a discrete DNA-binding domain . The possible reasons for this result are discussed.

J Clin Microbiol, 1989 May, 27(5), 983 - 8
Immunochemical identification and biological characterization of cytotoxic necrotizing factor from Escherichia coli; De Rycke J et al.; The aim of this study was to identify Escherichia coli cytotoxic necrotizing factor (CNF) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and to investigate the possible dissociation of CNF from hemolysin (Hly), which is often produced by CNF-producing strains . CNF was purified from cell lysates of a CNF-producing strain by using ammonium sulfate precipitation, ion-exchange chromatography, gel filtration, and preparative nondenaturing PAGE . All eluates from successive longitudinal slices of a preparative polyacrylamide gel were tested for cytoxicity and analyzed by SDS-PAGE; CNF activity was quantitatively correlated with a protein of 115 kilodaltons (kDa) . This procedure increased both cytotoxicity and lethal activity (about 300-fold) . We then compared SDS-PAGE protein patterns of enriched lysates from eight field and mutant E . coli strains producing both CNF and Hly, Hly alone, or neither; the 115-kDa band was present solely in CNF-producing strains, irrespective of Hly production . A neutralizing antiserum was produced against unpurified CNF from strain BM2-1 and then extensively adsorbed with cells and extracts of a CNF-defective mutant from BM2-1 . The adsorbed antiserum possessed antitoxin activity and neutralized both lethal and necrotic effects of cell lysates from all the CNF-producing strains tested . In an immunoblot of enriched extract from BM2-1, the adsorbed antiserum recognized, besides the 115-kDa protein, another protein of 59 kDa, which was present in the CNF-defective mutant from BM2-1 and was not associated with cytotoxicity . We can conclude from these findings that CNF is a protein of 115 kDa associated with both cytotoxicity and in vivo toxicity, distinct from Hly, and present in all presumed CNF-producing strains tested.

FEMS Microbiol Lett, 1989 May, 50(1-2), 177 - 80
Vero cytotoxin production and HeLa cell adherence of enteropathogenic Escherichia coli of infantile diarrhoea in Iran; Katouli M et al.; A total of 70 enteropathogenic Escherichia coli (EPEC) strains belonging to 11 serogroups, isolated from infantile diarrhoea in Tehran, Iran, were tested for the production of verocytotoxin (VT), enterotoxin, and also for their adherence to HeLa cells . In total 55 (78.5%) strains were either VT (32 strains) or enterotoxin (23 strains) producers, and of these 8 strains produced both VT and enterotoxins . 57 (81.4%) strains showed either Localized (LA) or Diffuse adherence (DA) or both types of adhesion (LA/DA) on HeLa cells, with strains showing LA/DA in the same preparations being dominant (32 strains), followed by those showing LA (14 strains) and DA (11 strains) . Among adherent EPEC, 26 (37.1%) strains belonging to the serogroups 020, 086, 0119, 0125, 0126, 0127 and 0128 also produced VT . These findings suggest that production of VT and enterotoxin is an important factor in the pathogenesis of EPEC diarrhoea in Iran and that the combination of adherence and production of toxins is a common feature of EPEC strains which cause diarrhoea in this country.

FEMS Microbiol Lett, 1989 May, 50(1-2), 153 - 6
Molecular cloning of mycobacterial promoters in Escherichia coli; Sirakova TD et al.; Mycobacterium bovis BCG chromosomal DNA, digested with EcoR1 and HindIII, was used to construct a promoter library in Escherichia coli using the promoter probe plasmid pKO-1 . DNA inserts of various sizes showed promoter activity judged by the level of galactokinase (galK) whose synthesis they activate (between 50 and 850 galactokinase units) . No correlation between the length of the DNA insert and the level of the galactokinase was found suggesting that the multicopy pool of the promoters does not influence the level of the transcription of the galK gene.

Fiziol Zh, 1989 May-Jun, 35(3), 43 - 9
{Disorders of cardio- and hemodynamics in endotoxic shock}; Kvochina LI et al.; Relative significance of cardiac and vessel components as well as the role of arachidonic acid metabolites in the development of endotoxic shock have been investigated in two series of experiments on the mongrel dogs . It is determined that endotoxin exerts no direct negative inotropic influence on the myocardium: blood pool in peripheral capacitance vessels plays a main role in the development of the first phase of the endotoxic shock (the first 30 min), that is a result of prostacyclin influence on these vessels, while in the subsequent phase it is a result of the bloodflow disturbance in the myocardium or arachidonic acid metabolites influence on the myocardium . Administration of endotoxin to the bloodflow significantly increased concentration of prostanoids; thromboxane A2 and prostacyclin in it . Indomethacin, inhibitor of prostaglandin synthesis, prevents development of the endotoxic shock.

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