Escherichia coli

Biochemistry, 1992 Oct 13, 31(40), 9717 - 24
Differential scanning calorimetry of thermal unfolding of the methionine repressor protein (MetJ) from Escherichia coli; Johnson CM et al.; The thermal stability of the methionine repressor protein from Escherichia coli (MetJ) has been examined over a wide range of pH (pH 3.5-10) and ionic strength conditions using differential scanning calorimetry . Under reducing conditions, the transitions are fully reversible, and thermograms are characteristic of the cooperative unfolding of a globular protein with a molecular weight corresponding to the MetJ dimer, indicating that no dissociation of this dimeric protein occurs before unfolding of the polypeptide chains under most conditions . In the absence of reducing agent, repeated scans in the calorimeter show only partial reversibility, though the thermodynamic parameters derived from the first scans are comparable to those obtained under fully reversible conditions . The protein is maximally stable (Tm 58.5 degrees C) at about pH 6, close to the estimated isoelectric point, and stability is enhanced by increasing ionic strength in the range I = 0.01-0.4 M . The average calorimetric transition enthalpy (delta Hm) for the dimer is 505 +/- 28 kJ mol-1 under physiological conditions (pH 7, I = 0.125, Tm = 53.2 degrees C) and shows a small temperature dependence which is consistent with an apparent denaturational heat capacity change (delta Cp) of about +8.9 kJ K-1 mol-1 . The effects of both pH and ionic strength on the transition temperature and free energy of MetJ unfolding are inconsistent with any single amino acid contribution and are more likely the result of more general electrostatic interactions, possibly including significant contributions from electrostatic repulsion between the like-charged monomers which can be modeled by a Debye-Huckel screened potential.

Biochemistry, 1992 Oct 13, 31(40), 9526 - 32
The C-terminal domain of Escherichia coli ribosomal protein L7/L12 can occupy a location near the factor-binding domain of the 50S subunit as shown by cross-linking with N-{4-(p-azidosalicylamido)butyl}-3-(2'-pyridyldithio)propionamide; Zecherle GN et al.; All large ribosomal subunits contain two dimers composed of small acidic proteins that are involved in binding elongation factors during protein synthesis . The ribosomal location of the C-terminal globular domain of the Escherichia coli ribosomal acidic protein L7/L12 has been determined by protein cross-linking with a new heterobifunctional, reversible, photoactivatable reagent, N-{4-(p-azidosalicylamido)-butyl}-3-(2'-pyridyldithio)propionamide . Properties of this reagent are described . It was first radiolabeled with 125I and then attached through the formation of a disulfide bond to a unique cysteine of L7/L12, introduced by site-directed mutagenesis at residue 89 . Intact 50S ribosomal subunits were reconstituted from L7/L12-depleted cores and the radiolabeled L7/L12Cys89 . Irradiation of the reconstituted subunits resulted in photo-cross-linking between residue 89 and other ribosomal components . Reductive cleavage of the disulfide cross-link resulted in transfer of the 125I label from L7/L12Cys89 to the other cross-linked components . Two radiolabeled proteins were identified, L11 and L10 . The location of both of these proteins is well established to be at the base of the L7/L12 stalk near the binding sites for the N-terminal domain of both L7/L12 dimers, and for elongation factors . The result indicates that L7/L12 can have a bent conformation bringing the C-terminal domain of at least one of the L7/L12 dimers at or near the factor-binding domain . The cross-linking method with radiolabeled N-{4-(p-azidosalicylamido)butyl}-3-(2'-pyridyldithio)propionamide should be applicable for studies of other multicomponent complexes that can be reconstituted.

Biochim Biophys Acta, 1992 Oct 13, 1180(1), 65 - 72
Expression of wild-type and mutant medium-chain acyl-CoA dehydrogenase (MCAD) cDNA in eucaryotic cells; Jensen TG et al.; An effective EBV-based expression system for eucaryotic cells has been developed and used for the study of the mitochondrial enzyme medium-chain acyl-CoA dehydrogenase (MCAD) . 1325 bp of PCR-generated MCAD cDNA, containing the entire coding region, was placed between the SV40 early promoter and polyadenylation signals in the EBV-based vector . Both wild-type MCAD cDNA and cDNA containing the prevalent disease-causing mutation A to G at position 985 of the MCAD cDNA were tested . In transfected COS-7 cells, the steady state amount of mutant MCAD protein was consistently lower than the amount of wild-type human enzyme . The enzyme activity in extracts from cells harbouring the wild-type MCAD cDNA was dramatically higher than in the controls (harbouring the vector without the MCAD gene) while only a slightly higher activity was measured with the mutant MCAD . The mutant MCAD present behaves like wild-type MCAD with respect to solubility, subcellular location, mature protein size and tetrameric structure . In immunoblot comparisons, the MCAD protein was present in normal fibroblasts, but essentially undetectable in patient fibroblasts homozygous for the prevalent mutation . We suggest that the MCAD protein carrying this mutation has an impaired ability to form correct tetramers, leading to instability and subsequent degradation of the enzyme . This finding is discussed in relation to the results from expression of human MCAD in Escherichia coli, where preliminary results show that production of mutant MCAD leads to the formation of aggregates.

Biochemistry, 1992 Oct 13, 31(40), 9813 - 22
Structure and function of alternative proton-relay mutants of dihydrofolate reductase; David CL et al.; Using site-specific mutagenesis, we have constructed two mutants of Escherichia coli dihydrofolate reductase (ecDHFR) to investigate further the function of a weakly acidic side chain at position 27 in substrate protonation: Asp27-->Glu (D27E) and Asp27-->Cys (D27C) . The crystal structure of D27E ecDHFR in a binary complex with methotrexate shows that the side-chain oxygen atoms of Glu27 are in almost precisely the same location as those of Asp27 in the wild-type enzyme . Kinetic evidence indicates that Glu27 can indeed function efficiently in the proton relay to dihydrofolate . Even though vertebrate DHFRs all have a glutamic acid at the structurally equivalent position, the kinetic properties of Glu27 ecDHFR more closely resemble those of wild-type bacterial DHFRs than of vertebrate DHFRs . The D27C mutation produced an enzyme still capable of relaying a proton to dihydrofolate, but with the intrinsic pKa in its pH-activity profiles shifted upward to values characteristic of the more basic thiolate group . The crystal structure of the binary complex with methotrexate reveals two unexpected features: (1) the Cys27 sulfhydryl group does not point toward the pteridine-binding site, but the side chain of this residue is instead rotated 120 degrees to interact with a tyrosine side chain projecting from a neighboring beta-strand; (2) a bound ethanol molecule occupies a cavity adjacent to methotrexate . Ethanol is a component of the crystallization medium.

Biochemistry, 1992 Oct 13, 31(40), 9647 - 56
Transcription activation by cAMP receptor protein (CRP) at the Escherichia coli gal P1 promoter . Crucial role for the spacing between the CRP binding site and the -10 region; Lavigne M et al.; The cAMP-CRP complex activates the initiation of transcription at the Escherichia coli gal P1 promoter, and the activation efficiency is highly sensitive to the location of the complex on this promoter region . Moving the CRP binding site by one base pair toward the start of transcription significantly decreases the extent of activation in vivo and actually turns the cAMP-CRP complex into an inhibitor in in vitro experiments . A structural analysis of open complexes formed on the two promoter fragments at 37 degrees C has revealed three elements crucial for an optimal activation process: a strong upstream anchorage of RNA polymerase, a cooperative binding of CRP and RNA polymerase, and an accurate orientation of the two promoter regions located upstream and downstream of the CRP binding site . Furthermore, structural analysis of polymerase promoter complexes at lower temperatures suggests that RNA polymerase initially recognizes the upstream region of the gal P1 promoter and subsequently interacts with sequences from the -10 to +20 region to yield the final open complex structure . The involvement of CRP in these sequential events has been examined.

Biochemistry, 1992 Oct 13, 31(40), 9642 - 6
Energy coupling in DNA gyrase: a thermodynamic limit to the extent of DNA supercoiling; Cullis PM et al.; ATP alpha S (Rp) has been shown to support the supercoiling of plasmid pBR322 catalyzed by Escherichia coli DNA gyrase at comparable rates to the natural substrate ATP and is able to promote the introduction of one more superhelical turn than ATP . The difference in free energy change between consecutive rounds of supercoiling in gyrase-mediated reactions is calculated to be 2.6 kJ mol-1 . The difference in free energy of hydrolysis of ATP and ATP alpha S (Rp) has been determined from the difference in the equilibrium constants for the phosphorylation of arginine established by arginine kinase . This equilibrium constant has been found to be displaced by a factor of about 1.5, corresponding to a greater free energy of hydrolysis of ATP alpha S (Rp) compared to ATP of approximately 1 kJ mol-1 . This difference in free energy can be tentatively ascribed to a relative destabilization of the MgATP alpha S (Rp) complex with respect to MgATP . Assuming that the stoichiometry of the coupled reactions requires two ATPs hydrolyzed per round of supercoiling, ATP alpha S (Rp) should be capable of providing an additional ca . 2 kJ mol-1 of free energy for DNA supercoiling, which is in good agreement with estimates for the additional free energy required to achieve a further round of supercoiling . These results provide direct evidence to support the proposal that the extent of DNA supercoiling by DNA gyrase is limited by the free energy of hydrolysis of the nucleotide.

Gene, 1992 Oct 12, 120(1), 85 - 8
Construction of lambda gt103, a derivative of phage lambda gt10 that has unique EcoRI, NotI, SacI and SpeI sites and retains positive selection for recombinants; Lee J et al.; A phage vector, lambda gt103, that has unique EcoRI, NotI, SacI and SpeI sites within the imm434 cI repressor gene, was constructed by PCR-aided site-directed mutagenesis of lambda gt10 {Huynh et al., DNA Cloning Techniques: A Practical Approach, 1985, pp . 49-78} . This vector allows directional cloning and retains positive selection for recombinants on Escherichia coli C600hfl strains (since only phages with disrupted cI genes plate on this host) . Libraries made with this phage vector can be efficiently screened for clones in which a part of the insert is homologous to probe DNAs derived from a plasmid-based library, without cross-hybridization.

Gene, 1992 Oct 12, 120(1), 11 - 6
Role of the histone-like proteins OsmZ and HU in homologous recombination; Dri AM et al.; The HU protein of Escherichia coli has been implicated in various site-specific recombination reactions . Moreover, recent data suggest that HU may also participate in homologous recombination . In particular, it has been shown that P1 transduction is inhibited in the absence of HU {Kano and Imamoto, Gene 89 (1990) 133-137} . In contrast, we found that transductional recombination and conjugational recombination were almost normal in hupA hupB mutants . However, it appeared that the recombination proficiency of hupA hupB mutant bacteria was reduced tenfold in an intrachromosomal recombination assay . Moreover, we found that intrachromosomal recombination was reduced tenfold in a gyrB226 strain and by more than 100-fold in an osmZ205 strain . The gyrB226 mutation affects the DNA gyrase activity, while mutations in osmZ are highly pleiotropic, affecting the expression of a variety of genes and increasing the frequency of site-specific inversion events . Since it has been shown that the hupA hupB mutations, like the gyrB226 mutation, decrease the level of DNA supercoiling, whereas the osmZ205 mutation increases the level of DNA supercoiling, it appears that the histone-like proteins HU and OsmZ may play a key role in intrachromosomal recombination by affecting the DNA topology.

Gene, 1992 Oct 12, 120(1), 1 - 9
Mutational analysis of the Escherichia coli serB promoter region reveals transcriptional linkage to a downstream gene; Neuwald AF et al.; Genes encoding proteins with unrelated functions can be cotranscribed, and this may be used by cells to coordinate different metabolic pathways during growth . We describe a gene, designated sms, which is downstream from the serine biosynthetic gene serB in Escherichia coli but does not appear to be involved in amino acid (aa) biosynthesis . The sms gene is 1380 bp long . The Sms product migrates at 55 kDa on sodium dodecyl sulfate(SDS)-polyacrylamide gels and has a M(r) of 49472 (460 aa residues) calculated from the nucleotide sequence . The deduced Sms aa sequence shares regions of similarity with two ATP-dependent proteases, Lon and RecA, and contains two motifs: a C-x(2)-C-x(n)-C-x(2)-C motif, which is found in some nucleic acid binding proteins, and an ATP/GTP binding site motif . Insertional inactivation of sms led to increased sensitivity to the alkylating agent methylmethane sulfonate, but not to a requirement for serine or other metabolites . Several promoter mutations were isolated and characterized, which suggest that serB has a typical promoter recognized by sigma 70 . After the serB coding sequence there is a 48-bp region with no obvious promoter sequence preceding the sms translation start codon . Analyses using sms'-lacZ fusions cloned downstream from wild-type and mutant serB promoters showed that sms is cotranscribed with serB.

Vet Microbiol, 1992 Oct, 32(3-4), 351 - 62
Molecular cloning and characterization of Mycobacterium paratuberculosis promoters in Escherichia coli; Thomas TJ et al.; DNA fragments from Mycobacterium paratuberculosis were cloned in the promoter probe plasmid pKO1 . Of 957 recombinant DNA clones, 24 induced synthesis of galactokinase (the reporter gene) when these plasmids were transformed into an Escherichia coli strain deficient for the enzyme . A DNA insert from one putative promoter-containing plasmid, designated pAG5, was sequenced and shown to contain, a characteristic RNA polymerase binding site, a probable ribosomal binding site and a putative open reading frame.

Circ Shock, 1992 Oct, 38(2), 75 - 84
Efficacy of monoclonal antibody against tumor necrosis factor alpha in an endotoxemic baboon model; Emerson TE Jr et al.; Tumor necrosis factor alpha (TNF) has been described as a primary mediator of the pathophysiology associated with bacterial sepsis/endotoxemia . We tested the efficacy and possible mechanisms of protection of a monoclonal antibody against TNF (TNF Mab) in a low mortality (29%), endotoxemic baboon model . A number of parameters were monitored at days 0, 1, 2 and 5-7 after challenge with 2 mg E . coli endotoxin/kg . TNF Mab pretreatment (15 mg/kg) prevented the increase in detectable serum TNF and the early perturbations in cardiovascular function which occurred in the control group . Early metabolic dysfunction was delayed in the TNF MAb group and was attenuated thereafter . Dysfunction of the kidney, liver, and coagulation systems and the increased IL-6 levels were significantly attenuated in the TNF MAb group; neutrophil activation was not affected by TNF MAb . No deaths occurred in the TNF MAb group . These results support the hypothesis that TNF plays a central role in the pathophysiology of endotoxic shock.

J Natl Med Assoc, 1992 Oct, 84(10), 850 - 2
Appendicitis in children: a continuing clinical challenge; Marrero RR Jr et al.; This article discusses the findings of a study of pre-adolescent children to determine if the mode of presentation of appendicitis had changed over the past 10 years, if the incidence of perforations decreased with age, and if diagnosis related groups (DRGs) impacted the length of hospital stay . The charts of 42 children under the age of 12 years who were discharged from two inner-city hospitals with a diagnosis of acute appendicitis from 1980 to 1989 were reviewed . There were 20 blacks and 22 whites, 26 males and 16 females with an average age of 7.31 years (range: 2 to 11 years) . Over 95% of patients presented with right lower quadrant pain, 78% with guarding, 80% with a positive psoas sign, 93% with a positive Rovsing's sign, and 65% with rectal tenderness . Over 85% of patients had a history of nausea, vomiting, and anorexia . The mean duration of pain was 52.8 hours and the mean temperature was 99.6 degrees F . The mean white blood cell count was 18,176 +/- 4682 for whites versus 14,615 +/- 5459 for blacks . At surgery 15/42 (36%) of patients had a perforation, 11 of whom had positive wound cultures . Escherichia coli was recovered in all 11 of these patients . The average duration of pain in the perforated group was 50.9 hours, and the average age was 7 years . Eleven of these patients had normal bowel sounds on admission . Only 31% of the total cohort had a fecalith identified by pathology . The average postoperative length of stay was 6.5 +/- 2.5 days before the initiation of DRGs and 7.5 +/- 3 days afterward.

Crit Care Med, 1992 Oct, 20(10), 1448 - 53
Pluronic F 127 liquid sensitizes mice to low doses of Escherichia coli lipopolysaccharide; Pickett WC et al.; BACKGROUND AND METHODS: In murine models of endotoxemia, large amounts of lipopolysaccharide have to be administered to induce mortality . If mice are pretreated with D-galactosamine, the amount of lipopolysaccharide required to induce mortality is significantly lowered . Pluronic F 127 liquid is a relatively non-toxic copolymer that exhibits reverse gelation properties . Thus, it is a liquid at cold temperature and a gel at body temperature . The present studies were performed to ascertain whether the reverse gelation properties of Pluronic F 127 liquid could be used in devising a model of septic shock where a sustained delivery of lipopolysaccharide occurred . In evaluating this model, dose-response studies were conducted with lipopolysaccharide when a) it was administered intraperitoneally in saline or in Pluronic F 127 liquid, and b) it was administered intravenously to mice that had been pretreated with saline or Pluronic F 127 liquid . Mortality was followed for up to 72 hrs . RESULTS: Various doses of Escherichia coli lipopolysaccharide dissolved in saline or in Pluronic F 127 liquid were administered intraperitoneally to mice . The lethal dose of lipopolysaccharide required to kill 50% of the mice (LD50) administered in Pluronic F 127 liquid was approximately ten- to 15-fold less than the values obtained for lipopolysaccharide administered in saline . This decrease in the LD50 of lipopolysaccharide was also observed if the mice were treated intraperitoneally with Pluronic F 127 liquid and challenged 6 hrs later with iv lipopolysaccharide . The concentrations of tumor necrosis factor and interleukin-6 in the plasma were significantly higher when a low dose of lipopolysaccharide was administered to mice that had been pretreated with Pluronic F 127 liquid . While there was no effect on the liver enzymes, Pluronic F 127 liquid caused an increase in the plasma triglycerides . CONCLUSIONS: The data reported in this paper indicate that the LD50 of lipopolysaccharide is significantly decreased if it is administered in Pluronic F 127 liquid or administered to mice that have been pretreated with the Pluronic F 127 liquid . Thus, Pluronic F 127 liquid appears to sensitize mice to low levels of lipopolysaccharide . Unlike the D-galactosamine model, lipopolysaccharide can be administered as late as 6 hrs after treatment with Pluronic F 127 liquid . While the mechanisms by which Pluronic F 127 liquid sensitizes mice is not known, plasma triglycerides were increased in mice treated with this agent, suggesting that tissues responsible for the synthesis and/or degradation of triglycerides play a role in this sensitization process.

Nucleic Acids Res, 1992 Oct 11, 20(19), 5215 - 21
Yeast RNC1 encodes a chimeric protein, RhoNUC, with a human rho motif and deoxyribonuclease activity; Chow TY et al.; The yeast Saccharomyces cerevisiae contains an endoexonuclease yNucR that has been implicated in both recombination and repair . We describe the isolation and characterization of the corresponding gene . Within the predicted N-terminal half of the protein there is extensive homology (approximately 50%) with human rho genes, which are related to the ras oncogene, particularly in the proposed GTP-binding region . The C-terminal region, which is related to the Escherichia coli recC protein, presumably encodes the endoexonuclease activity . The yNucR may thus represent a new class of GTP-binding proteins . Because of the chimeric nature of the polypeptide, this protein is renamed RhoNUC (rather than the original yNucR) and the gene is RNC1 for Rho-associated-NuClease . Over expression of the gene leads to altered cell growth and nuclear morphology . We propose that the gene plays an important role in cell development as well as DNA repair/recombination.

Nucleic Acids Res, 1992 Oct 11, 20(19), 5035 - 9
Specificities of three tight-binding Lac repressors; Kolkhof P; Tight binding mutants of Lac repressor exhibit complex repression phenomena . In this work, in vivo Lac operator binding of three such mutants of E . coli Lac repressor (X86: ser 61-leu, l12: pro 3-tyr and the double mutant l12X86: pro 3-tyr, ser 61-leu) was analyzed . Repression of beta-galactosidase synthesis controlled by ideal lac operator and its 27 symmetric operator variants containing each possible base-pair at each single half-operator position in the presence of the tight-binding Lac repressor mutants was determined . The average increase of repression with all operator variants was about 3 fold with the X86 mutant . It was about 4 fold with the l12 mutant and about 2 fold with the double mutant l12X86 as compared to wildtype Lac repressor . The X86 mutant showed the same increase of affinity to all operator variants, whereas the l12 and l12X86 mutants exhibited lower repression with some variants than with most others . These results suggest that the X86 mutant has gained no additional specificity . In contrast the l12 mutant and the l12X86 mutant exhibit a relaxed specificity for certain base pairs in positions 1 and 3 of lac operator . This suggests that the extreme N-terminus of Lac repressor may interact with the inner base-pairs in the minor groove.

Nucleic Acids Res, 1992 Oct 11, 20(19), 5159 - 66
Chemical synthesis of a biologically active natural tRNA with its minor bases; Gasparutto D et al.; The complete chemical synthesis of an E . coli tRNA(Ala) with its specific minor nucleosides, dihydrouridine, ribothymidine and pseudouridine, is reported . The method makes use of protected 2'-O-tertiobutyldimethylsilyl-ribonucleoside-3'-O-(2-cyanoethyl-N- ethyl-N- methyl)phosphoramidites . The exocyclic amino functions of the bases were protected by the phenoxyacetyl group for purines and acetyl for cytosine . The assembling has been performed on a silica support with coupling yield better than 98% within 2 min of condensation . Triethylamine tris-hydrofluoride allowed a clean and complete deprotection of the tBDMS groups . The synthetic tRNA(Ala) has been transcribed into cDNA by reverse transcriptase and sequenced . With E . coli alanyl-tRNA synthetase the alanyl acceptance activity and kcat/Km were 672 pmol/A260 and 6 x 10(4)M-1s-1, respectively.

Nucleic Acids Res, 1992 Oct 11, 20(19), 5119 - 25
A comparison of the fidelity of copying 5-methylcytosine and cytosine at a defined DNA template site; Shen JC et al.; 5-Methylcytosine has been postulated to be an endogenous mutagen in procaryotes and eucaryotes leading to base substitution hot spots, C-->T transitions, resulting from spontaneous deamination of mC to T . The possibility remains, however, that a second mechanism involving mispairing of mC with A might also contribute to base substitution mutagenesis via G-->A transitions . Stimulation of the G-->A mutational pathway could involve preferential misincorporation of dAMP opposite template mC compared to C . To investigate this possibility, we synthesized a sequence containing mC at a defined template location . We compared the fidelity of copying mC versus C and the efficiency of extending mismatched base pairs at the mC position using three DNA polymerases, AMV reverse transcriptase, Drosophila DNA polymerase alpha, and mutant Escherichia coli Klenow fragment containing no proofreading exonuclease activity . Significant differences in misinsertion and mismatch extension efficiencies were observed only for the case of AMV reverse transcriptase . AMV reverse transcriptase was observed to incorporate dAMP 4 to 5-fold more efficiently opposite mC than C . Favored extension of a 5-MeC.A over C.A mispair was also observed with a difference of about 3-fold . In contrast to AMV reverse transcriptase, Klenow fragment showed no significant difference when copying either mC or C sites or when extending mispairs involving mC and C . Incorporation of dAMP opposite either C or mC was barely detectable using pol alpha, although pol alpha has been observed to form A.C mismatches in other sequences . While we cannot completely exclude the possibility that dAMP might be incorporated opposite mC in preference to C, our results suggest that contributions of the G-->A pathway to mC mutagenic hot spots are likely to be minor, lending additional support to the model invoking deamination of mC.

Nucleic Acids Res, 1992 Oct 11, 20(19), 5115 - 8
Characterization of the double stranded RNA dependent RNase activity associated with recombinant reverse transcriptases; Ben-Artzi H et al.; An in situ gel assay was applied to the study of double stranded RNA dependent RNase activity associated with reverse transcriptase (RT) of HIV-1 and murine leukemia virus . Polyacrylamide gels containing {32P} RNA/RNA substrate were used for electrophoresis of proteins under denaturing conditions . The proteins were renatured and in situ enzymatic degradation of 32P-RNA/RNA was followed . E . coli RNaseIII, but not E . coli RNaseH, was active in this in situ gel assay, indicating specificity of the assay to RNA/RNA dependent nucleases . Analysis of purified preparations of HIV-1 RT p66/p51 expressed in E . coli demonstrated an RNA/RNA dependent RNase activity comigrating with the large subunit (p66) of the enzyme . In addition, this activity of the RT was often accompanied by a contaminating RNA/RNA dependent RNase, with a molecular weight approximately 30,000 dalton identical to that of E . coli RNaseIII . As the p51 small subunit of HIV-1 RT and a mutant of RT p66/p51, at Glutamic acid #478, did not exhibit RNA/RNA dependent RNase activity, at least part of the active site of the RNA/RNA dependent RNase activity appeared to reside at the carboxy end of the molecule . As these RT proteins are also deficient of RNaseH, our results suggest overlapping or identical catalytic sites for degradation of the substrates RNA/DNA and RNA/RNA.

J Mol Biol, 1992 Oct 5, 227(3), 961 - 70
Large ATP synthase operon of the red alga Antithamnion sp . resembles the corresponding operon in cyanobacteria; Kostrzewa M et al.; The large plastid ATP synthase operon of the multicellular red alga Antithamnion sp . was cloned and the sequence of six ATPase genes determined . The operon resembles more the one from cyanobacteria than the ATP synthase operon of the chloroplast genome . The gene order is atpI, H, G, F, D and A, coding for the ATPase subunits a, c, b', b, delta and alpha, respectively . In green plants, the genes atpG and atpD are located in the nucleus . Unlike the situation in three published cyanobacterial ATP synthase operons, atpC, coding for the gamma subunit, is not a part of the rhodoplast operon . A single 4.5 kb transcript was detected with atpG, F, D and A gene probes that could span the whole operon, but no transcript could be detected with atpI and atpH probes . The end of an open reading frame preceding the atp genes shows remarkable homology to elongation factor TS from Escherichia coli . Behind the ATPase cluster, two open reading frames were detected that are not homologous to any known chloroplast gene . One of them may code for a transport protein of unknown specificity . Gene arrangement and sequence comparisons support the hypothesis of a polyphyletic origin of rhodoplasts and chloroplasts.

J Mol Biol, 1992 Oct 5, 227(3), 597 - 601
Mutation in the specificity polypeptide of the type I restriction endonuclease R.EcoK that affects subunit assembly; Zinkevich V et al.; We describe the isolation and characterization of a temperature-sensitive mutation within the hsdS gene of the type I restriction and modification system EcoK . This mutation appears to affect the ability of the HsdR subunit to interact with the HsdS subunit when forming an active endonuclease . We discuss the possibility that this mutant, together with another mutation described previously, may define a discontinuous domain, involved in protein-protein interactions, within the HsdS polypeptide.

J Biol Chem, 1992 Oct 5, 267(28), 20429 - 34
The heat-shock protein ClpB in Escherichia coli is a protein-activated ATPase; Woo KM et al.; The clpB gene in Escherichia coli encodes a heat-shock protein that is a close homolog of the clpA gene product . The latter is the ATPase subunit of the multimeric ATP-dependent protease Ti (Clp) in E . coli, which also contains the 21-kDa proteolytic subunit (ClpP) . The clpB gene product has been purified to near homogeneity by DEAE-Sepharose and heparin-agarose column chromatographies . The purified ClpB consists of a major 93-kDa protein and a minor 79-kDa polypeptide as analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate . Upon gel filtration on a Superose-6 column, it behaves as a 350-kDa protein . Thus, ClpB appears to be a tetrameric complex of the 93-kDa subunit . The purified ClpB has ATPase activity which is stimulated 5-10-fold by casein . It is also activated by insulin, but not by other proteins, including globin and denatured bovine serum albumin . ClpB cleaves adenosine 5'-(alpha,beta-methylene)-triphosphate as rapidly as ATP, but not adenosine 5'-(beta,gamma-methylene)-triphosphate . GTP, CTP, and UTP are hydrolyzed 15-25% as well as ATP . ADP strongly inhibits ATP hydrolysis with a Ki of 34 microM . ClpB has a Km for ATP of 1.1 mM, and casein increases its Vmax for ATP without affecting its Km . A Mg2+ concentration of 3 mM is necessary for half-maximal ATP hydrolysis . Mn2+ supports ATPase activity as well as Mg2+, and Ca2+ has about 20% their activity . Anti-ClpB antiserum does not cross-react with ClpA nor does anti-ClpA antiserum react with ClpB . In addition, ClpB cannot replace ClpA in supporting the casein-degrading activity of ClpP . Thus, ClpB is distinct from ClpA in its structural and biochemical properties despite the similarities in their sequences.

J Biol Chem, 1992 Oct 5, 267(28), 20416 - 21
Enzymatic instability of NADH-cytochrome b5 reductase as a cause of hereditary methemoglobinemia type I (red cell type); Shirabe K et al.; Nucleotide substitutions in the gene for NADH-cytochrome b5 reductase were identified in three independent probands of hereditary methemoglobinemia type I . Patients in Kagoshima and Okinawa in Japan were shown to possess the same base change, from guanine to adenine at codon 57, which results in amino acid substitution from Arg to Gln . This nucleotide change was the same as formerly found in a patient in Toyoake, Japan (Katsube, T., Sakamoto, N., Kobayashi, Y., Seki, R., Hirano, M., Tanishima, K., Tomoda, A., Takazakura, E., Yubisui, T., Takeshita, M., Sakaki, Y., and Fukumaki, Y . (1991) Am . J . Hum . Genet . 48, 799-808) . A type I patient in Italy was shown to have a base change from guanine to adenine at codon 105 which causes substitution from Val to Met . To characterize the enzymes of type I patients, Arg-57----Gln and Val-105----Met mutant enzymes were overexpressed in Escherichia coli and purified to homogeneity . kcat/Km values (NADH) of these two enzymes were 25% in Arg-57----Gln and 14.5% in Val-105----Met compared with that of the wild type enzyme, while the value of type II (generalized, severe form of the disease) mutant enzyme was 3% of the normal value (Yubisui, T., Shirabe, K., Takeshita, M., Kobayashi, Y., Fukumaki, Y., Sakaki, Y., and Takano, T . (1991) J . Biol . Chem . 266, 66-70) . The type I mutant enzymes were less heat-stable and more susceptible to proteinase treatment than the wild type . From these results we conclude that restriction of enzyme deficiency to red cells in hereditary methemoglobinemia type I may be generally derived from instability and increased proteolytic susceptibility of variant NADH-cytochrome b5 reductases due to a point mutation.

J Biol Chem, 1992 Oct 5, 267(28), 20331 - 8
Mutational analysis of the consensus nucleotide binding sequences in the rat liver mitochondrial ATP synthase beta-subunit; Thomas PJ et al.; The coupling step in the biosynthesis of ATP in biological systems is generally believed to involve an energy-requiring release of ATP bound to the beta-subunit of the ATP synthase complex . A molecular description of the ATP binding site on the beta-subunit is, therefore, critical to understanding the mechanism of coupling in the enzyme . Previously, we reported that a purified, bacterially expressed rat liver beta-subunit binds adenine nucleotides tightly and specifically (Garboczi, D . N., Hullihen, J . H., and Pedersen, P . L . (1988) J . Biol . Chem . 263, 15694-15698) . In order to assess the contribution of various regions of the isolated beta-subunit to the ATP binding site we have systematically deleted four different regions: the N-terminal region, the Walker A consensus region, the Walker B consensus region (Walker, J . E., Saraste, M., Runswick, M . J., and Gay, N . (1982) EMBO J . 1, 945-951), and a "C" region, which, like the A and B regions, bears homology to adenylate kinase . Plasmids directing the expression of double deletions of A and B regions, and B and C regions were also constructed . In addition, 2 residues outside of these regions, His-177 and Tyr-345, which have been predicted to play a central role in nucleotide binding, were mutated . Rabbit antisera to synthetic peptides of the A and C regions verified the identity of the bacterially expressed mutant proteins . Seven of the eight mutant proteins overexpressed in Escherichia coli were resistant to E . coli proteases in the preparative stages, as predicted for compact folded proteins . Furthermore, circular dichroism spectropolarimetry revealed no profound structural alterations in the purified mutant proteins . Relative to the overexpressed full-length beta-subunit, the mutant lacking the A consensus region suffered a 30-fold loss of affinity for ATP and a loss of specificity for 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) over 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate . The mutant proteins lacking either the N-terminal region or the B region exhibited nucleotide binding properties similar to the full-length beta-subunit, whereas the mutant protein lacking the C region suffered an order of magnitude reduction in affinity for ATP . The affinity of the A and B region double deletion was indistinguishable from the A region deletion in regard to TNP-ATP binding, while the double deletion mutant lacking the B and C regions was not stably expressed in the E . coli SE6004.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1992 Oct 5, 267(28), 20277 - 81
MAK3 encodes an N-acetyltransferase whose modification of the L-A gag NH2 terminus is necessary for virus particle assembly; Tercero JC et al.; The MAK3 gene is necessary for propagation of the L-A double-stranded RNA virus of Saccharomyces cerevisiae . MAK3 encodes a protein with substantial homology to the Escherichia coli rimI N-acetyltransferase that acetylates the NH2 terminus of ribosomal protein S18, and shares consensus sequences with a group of N-acetyltransferases . The NH2 terminus of the viral major coat protein encoded by L-A is normally blocked, but we find that it is unblocked in a mak3-1 mutant . L-A virus-encoded proteins produced from a cDNA clone of L-A can encapsidate the L-A (+)-strands in a wild-type host, but not in a mak3-1 mutant strain . The amount of major coat protein found in the particle fraction is reduced greater than 100-fold, and the amount in the total cell extract is reduced 5-10-fold . A modified beta-galactosidase, having as its NH2-terminal the NH2-terminal 13 residues of the L-A-encoded major coat protein, is blocked in a wild-type host, but not in a mak3-1 host . We propose that MAK3 encodes an N-acetyltransferase whose modification of the L-A major coat protein NH2 terminus is essential for viral assembly, and that unassembled coat protein is unstable.

J Biol Chem, 1992 Oct 5, 267(28), 20175 - 80
Genetic dissection of the transcription cycle . A mutant RNA polymerase that cannot hold onto a promoter; Martin E et al.; Deletion of 10 amino acids from a conserved motif in the beta subunit of Escherichia coli RNA polymerase (RNAP) leads to an interrupted transcription cycle and lethal phenotype . RNAP carrying the mutant subunit retains catalytic function and specificity of promoter recognition but is unable to efficiently hold onto DNA in the binary complex, resulting in a diminished initiation frequency . However, inefficient initiation by the mutant enzyme leads to processive and stable ternary elongating complex . Thus, the mutation dissects the traits of promoter selectivity, binary complex stability, and ternary complex processivity reflecting compartmentalization of function within the RNAP molecule.

J Biol Chem, 1992 Oct 5, 267(28), 20011 - 6
Expression of mature bovine H-protein of the glycine cleavage system in Escherichia coli and in vitro lipoylation of the apoform; Fujiwara K et al.; H-protein, a component of the glycine cleavage system with lipoic acid as a prosthetic group, was expressed in Escherichia coli using a T7 RNA polymerase plasmid expression system . After induction with 25 microM isopropyl-beta-D-thiogalactopyranoside, bacteria harboring the recombinant plasmid expressed mature bovine H-protein as a soluble form at a level of about 10% of the total bacterial protein . Little of the H-protein was lipoylated in E . coli cultured without added lipoate, but when the cells were cultured in medium supplemented with 30 microM lipoate, about 10% of the recombinant protein expressed was the correctly lipoylated active form, 10% was an inactive aberrantly modified form, presumably with an octanoyl group, and the remaining 80% was the unlipoylated apoform . Each of the three forms was purified to homogeneity and shown to have the same NH2-terminal amino acid sequence as that of native bovine H-protein . The specific activity of the lipoylated form of H-protein expressed was consistent with that of H-protein purified from bovine liver . The purified recombinant apo-H-protein was lipoylated and consequently activated in vitro with lipoyl-AMP as a lipoyl donor by lipoyltransferase purified 150-fold from bovine liver mitochondria . The lipoylation was dependent on lipoyl-AMP, apo-H-protein, and lipoyltransferase . The partially purified lipoyltransferase had no lipoate-activating activity . These results provide the first evidence that in mammals two consecutive reactions are required for the attachment of lipoic acid to the acceptor protein: the activation of lipoic acid to lipoyl-AMP catalyzed by lipoate-activating enzyme and the transfer of the lipoyl group to an N epsilon-amino group of a lysine residue to apoprotein by lipoyl-AMP:N epsilon-lysine lipoyltransferase.

J Biol Chem, 1992 Oct 5, 267(28), 19866 - 71
Site-directed mutagenesis of glutathione S-transferase YaYa . Important roles of tyrosine 9 and aspartic acid 101 in catalysis; Wang RW et al.; The roles of tyrosine 9 and aspartic acid 101 in the catalytic mechanism of rat glutathione S-transferase YaYa were studied by site-directed mutagenesis . Replacement of tyrosine 9 with phenylalanine (Y9F), threonine (Y9T), histidine (Y9H), or valine (Y9V) resulted in mutant enzymes with less than 5% catalytic activity of the wild type enzymes . Kinetic studies with purified Y9F and Y9T mutants demonstrated poor catalytic efficiencies which were largely due to a drastic decrease in kcat . The estimated pK alpha values of the sulfhydryl group of glutathione bound to Y9F and Y9T mutant enzymes were 8.5 to 8.7, similar to the chemical reaction, in contrast to the estimated pK alpha value of 6.7 to 6.8 for the glutathione enzyme complex of wild type glutathione S-transferase . These results indicate that tyrosine 9 is directly responsible for the lowering of the pKa of the sulfhydryl group of glutathione, presumably due to the stabilization of the thiolate anion through hydrogen bonding with the hydroxyl group of tyrosine . To examine the role of aspartic acid in the binding of glutathione to YaYa, 4 conserved aspartic acid residues at positions 61, 93, 101, and 157 were changed to glutamic acid and asparagine . All mutant enzymes retained either full or partial activity except D157N, which was virtually inactive . Kinetic studies with four mutant enzymes (D93E, D93N, D101E, and D101N) indicate that only D101N exhibited a 5-fold increase in Km toward glutathione . Also, the binding of this mutant to the affinity column was greatly reduced . These results demonstrate that aspartic acid 101 plays an important role in glutathione interaction to YaYa . The role of aspartic acid 157 in catalysis remains to be determined.

J Biol Chem, 1992 Oct 5, 267(28), 19813 - 8
DNA melting within stable closed complexes at the Escherichia coli rrnB P1 promoter; Ohlsen KL et al.; Several transcription complexes are shown to form on the E . coli ribosomal rrnB P1 promoter in vitro . These include two closed complexes that are sensitive to heparin attack, and one open complex . The closed complexes are unusual in that they are both highly specific and stable, properties associated with the atypical DNA sequence of this promoter . The effector ppGpp does not prevent closed complex formation but does reduce the level of open complexes that form.

FEBS Lett, 1992 Oct 5, 310(3), 246 - 8
Accumulation of protoporphyrin IX in light-sensitive mutants of Escherichia coli; Miyamoto K et al.; The accumulation of protoporphyrin IX (Proto IX) in light-sensitive mutants of Escherichia coli was detected by spectrofluorimetry . Fluorescence emission and excitation spectra were recorded from extracts of bacterial cells . Proto IX clearly accumulated in cells with mutations in the visA (hemH) gene but not in the wild-type strain CA274 or in visA mutants that had been rendered light-resistant by introduction of the wild-type visA+ gene . Accumulation of Proto IX was also not observed in cells with a mutation in the visB gene . These results confirm the hypothesis that the sensitivity of the visA mutants to light is caused by the abnormal accumulation of Proto IX, a substrate of ferrochelatase, as the result of a genetic defect in the gene for ferrochelatase.

J Mol Biol, 1992 Oct 5, 227(3), 621 - 34
Domains of the Escherichia coli threonyl-tRNA synthetase translational operator and their relation to threonine tRNA isoacceptors; Brunel C et al.; The expression of the gene for threonyl-tRNA synthetase (thrS) is negatively autoregulated at the translational level in Escherichia coli . The synthetase binds to a region of the thrS leader mRNA upstream from the ribosomal binding site inhibiting subsequent translation . The leader mRNA consists of four structural domains . The present work shows that mutations in these four domains affect expression and/or regulation in different ways . Domain 1, the 3' end of the leader, contains the ribosomal binding site, which appears not to be essential for synthetase binding . Mutations in this domain probably affect regulation by changing the competition between the ribosome and the synthetase for binding to the leader . Domain 2, 3' from the ribosomal binding site, is a stem and loop with structural similarities to the tRNA(Thr) anticodon arm . In tRNAs the anticodon loop is seven nucleotides long, mutations that increase or decrease the length of the anticodon-like loop of domain 2 from seven nucleotides abolish control . The nucleotides in the second and third positions of the anticodon-like sequence are essential for recognition and the nucleotide in the wobble position is not, again like tRNA(Thr) . The effect of mutations in domain 3 indicate that it acts as an articulation between domains 2 and 4 . Domain 4 is a stable arm that has similarities to the acceptor arm of tRNA(Thr) and is shown to be necessary for regulation . Based on this mutational analysis and previous footprinting experiments, it appears that domains 2 and 4, those analogous to tRNA(Thr), are involved in binding the synthetase which inhibits translation probably by interfering with ribosome loading at the nearby translation initiation site.

J Biol Chem, 1992 Oct 5, 267(28), 19963 - 70
Role of the A protein-binding sites in the in vitro transposition of mu DNA . A complex circuit of interactions involving the mu ends and the transpositional enhancer; Allison RG et al.; To investigate the role of the A protein-binding sites at the Mu ends in the DNA strand transfer reaction, we constructed mutant mini-Mu molecules in which these sites were deleted (L3 or R3) or substituted (L2 or R2) to conserve the spacing arrangements at the adjacent sites . The single site mutants are poor substrates for phosphodiester bond hydrolysis at the Mu ends in Type 1 reactions in the absence of Escherichia coli integration host factor (IHF) . Addition of IHF to the reaction stimulates Type 1 cleavage more than 10 times for the delta-R3, delta-L3, S-L2 mutants and more than five times in the case of the S-R2 mutant under alternate conditions . The site of IHF stimulation resides within the transpositional enhancer which implicates the end-binding sites L2, L3, R2, and R3 in interactions with the enhancer . At least two of the L2, L3, and R3 sites are required for proficient reaction in the presence of IHF . By combining the single site mutants with O1 or O2 partially deleted enhancer elements, we have tentatively localized some of the interactions to each side of the functional enhancer revealing a complex circuit of end-enhancer interactions . The R3 site is suggested to be involved in interactions only with O2 and the L3 site only with O1 . The data also suggest the possibility that L2 and R2 may be involved in interactions with both O1 and O2 . Finally, our working model predicts that the L3-O1 and R3-O2 interactions may be required contacts for discriminating between the Mu left and right ends in transpososome formation.

FEBS Lett, 1992 Oct 5, 310(3), 240 - 5
Isolation of a cDNA fragment coding for Chlamydomonas reinhardtii ferredoxin and expression of the recombinant protein in Escherichia coli; Rogers WJ et al.; A cDNA clone coding for mature C . reinhardtii ferredoxin has been isolated from a cDNA library using PCR and two oligonucleotide primers based on the N- and C-termini of the protein's amino acid sequence . The nucleotidic sequence of the PCR fragment (299 bp) agreed well with the amino acid sequence since a single conservative substitution (Thr-7 to Ser) could be deduced . The PCR fragment was inserted into the expression vector pTrc 99A, using the incorporated NcoI and BamHI restriction sites and the construction used to transform E . coli (DH5 alpha F') . After subsequent large scale expression and purification of the recombinant protein, biochemical and biophysical analysis have indicated that the product isolated from E . coli is homologous to native ferredoxin isolated from green algae.

Mol Immunol, 1992 Oct, 29(10), 1237 - 47
Production of stable anti-digoxin Fv in Escherichia coli; Anthony J et al.; We have created a bacterial expression-export system and have used it to express (14 mg l-1) the variable region fragment (Fv) of an anti-digoxin antibody (26-10) in Escherichia coli . The expression-export plasmid contains a T7 promoter and the E . coli signal sequences ompA {Movva et al., J . biol . Chem . 255, 27-29 (1980)} and phoA {Inouye et al., J . Bacteriol . 149, 434-439 (1982)} fused to heavy chain (VH) and light chain (VL) variable region sequences to generate an artificial cistron . The 26-10 Fv protein made using this system was soluble, unlike many other expression systems which produce insoluble proteins in the form of inclusion bodies . The 26-10 VH and VL proteins were cleaved at their mature N-termini and exported into the bacterial periplasm where they could be easily extracted and affinity purified on ouabain-Sepharose . 26-10 Fv bound to digoxin with similar affinity and specificity as the whole 26-10 antibody (Ka for Fv, 1.3 x 10(9) M-1, Ka for IgG, 7 x 10(9) M-1) . 26-10 Fv appears to be remarkably stable in comparison with other Fv fragments . The half-life for chain dissociation of 26-10 Fv was 48 hr compared to the reported 1.5 hr half-life of McPC603 Fv . We present the proton NMR spectra of the 26-10 Fv as preliminary evidence that this expression-export system can be used to facilitate the analysis of the solution structure of 26-10 Fv by NMR.

J Infect Dis, 1992 Oct, 166(4), 803 - 11
Lipopolysaccharide heterogeneity in Escherichia coli J5 variants: analysis by flow cytometry; Evans ME et al.; A lipopolysaccharide O-side chain-producing phenotypic variant was isolated from a uridine 5'-diphosphogalactose 4-epimeraseless Escherichia coli J5 rough mutant strain by fluorescence-activated cell sorting with a monoclonal antibody (MAb) specific for the O-side chain of E . coli O111:B4 smooth parent lipopolysaccharide . The variant (J5-2) was recognized by both core- and O-side chain-specific MAbs, while the "original" rough mutant (J5-1) and smooth parent strains stained only with core- and O-side chain-specific MAb, respectively . J5-2 produced complete and incomplete (Rc chemotype) core and O polysaccharide in the presence of galactose . Three other E . coli J5 variants were either phenotypically similar to J5-1 (J5-UK) or distinct from J5-1 and J5-2 (J5-A, -B) . The latter phenotype had a lower-molecular-weight core, compared with J5-1 and J5-2, and distinct MAb specificities . Various J5 phenotypes also differed in galactokinase levels, the ability to use galactose, and susceptibility to core-specific MAb binding on solid media . The J5-2 strain showed reciprocal changes in O-side chain and core expression during log-phase growth . E . coli J5 thus undergoes spontaneous alterations in lipopolysaccharide phenotype.

J Cell Biol, 1992 Oct, 119(1), 191 - 202
Homophilic and heterophilic binding activities of Nr-CAM, a nervous system cell adhesion molecule; Mauro VP et al.; Nr-CAM is a membrane glycoprotein that is expressed on neurons . It is structurally related to members of the N-CAM superfamily of neural cell adhesion molecules having six immunoglobulin-like domains and five fibronectin type III repeats in the extracellular region . We have found that the aggregation of chick brain cells was inhibited by anti-Nr-CAM Fab' fragments, indicating that Nr-CAM can act as a cell adhesion molecule . To clarify the mode of action of Nr-CAM, a mouse fibroblast cell line L-M(TK-) (or L cells) was transfected with a DNA expression construct encoding an entire chicken Nr-CAM cDNA sequence . After transfection, L cells expressed Nr-CAM on their surface and aggregated . Aggregation was specifically inhibited by anti-Nr-CAM Fab' fragments . To check the specificity of this aggregation, a fusion protein (FGTNr) consisting of glutathione S-transferase linked to the six immunoglobulin domains and the first fibronectin type III repeat of Nr-CAM was expressed in Escherichia coli . Addition of FGTNr to the transfected cells blocked their aggregation . Further analysis using a combination of cell aggregation assays, binding of cells to FGTNr-coated substrates, aggregation of FGTNr-coated Covaspheres and binding of FGTNr-coated Covaspheres to FGTNr-coated substrates revealed that Nr-CAM mediates two types of cell interactions: a homophilic, divalent cation-independent binding, and a heterophilic, divalent cation-dependent binding . Homophilic binding was demonstrated between transfected L cells, between chick embryo brain cells and FGTNr, and between Covaspheres to which FGTNr was covalently attached . Heterophilic binding was shown to occur between transfected and untransfected L cells, and between FGTNr and primary chick embryo fibroblasts; in all cases, it was dependent on the presence of either calcium or magnesium . Primary chick embryo glia or a human glial cell line did not bind to FGTNr-coated substrates . The results indicate that Nr-CAM is a cell adhesion molecule of the nervous system that can bind by two distinct mechanisms, a homophilic mechanism that can mediate interactions between neurons and a heterophilic mechanism that can mediate binding between neurons and other cells such as fibroblasts.

Plant Mol Biol, 1992 Oct, 20(1), 123 - 31
Transfection of heteroduplexes containing uracil.guanine or thymine.guanine mispairs into plant cells; Inamdar NM et al.; We have compared the fate of U.G mispairs or analogous T.G mispairs in DNA heteroduplexes transfected into tobacco protoplasts . The heteroduplex DNA consisted of tomato golden mosaic virus DNA sequences in the Escherichia coli vectors pUC118 or pUC119 . After transfection, the mismatched U residues were lost with an efficiency of greater than 95%, probably as a result of the uracil-DNA glycosylase pathway for excision of U residues in any sequence context . In contrast to the preferential removal of the mispaired U residues, biased removal of T residues from analogous heteroduplexes was not seen in the transfected plant cells . Also, we investigated the effect of extensively methylating one strand of the heteroduplex DNA used for transfection . Surprisingly, such methylation resulted in highly biased loss of the mismatched base from the 5-methylcytosine-rich strand of T.G-containing heteroduplexes.

J Biochem Biophys Methods, 1992 Oct, 25(2-3), 95 - 112
Statistical testing of equality of two break-points in experimental data; Shanubhogue A et al.; Two examples in quantitative biology are examined to emphasize the need for two-phase regression models: the osmotic behaviour of cells and the non-linear temperature kinetics of membrane-bound enzyme systems . Existing statistical techniques are inadequate to test the equality of break-points of two data sets for specific reasons . We suggest here a pragmatic solution by way of a computer programme useful in applying two-phase regression models to such data sets wherein a decision needs to be made whether the critical transition differs or not.

Avian Dis, 1992 Oct-Dec, 36(4), 1012 - 4
Failure of the Congo red dye uptake test to discriminate between virulent and avirulent avian Escherichia coli; Spears KR et al.; Twenty avian Escherichia coli isolates from normal and diseased chickens were compared by use of three virulence tests . These tests included the uptake of Congo red dye, an embryo lethality test, and a quantitative microtiter complement resistance test . A direct correlation was seen between the results of the complement resistance test and the embryo lethality test . The results of the Congo red test did not correlate with the two other tests.

Pathol Biol (Paris), 1992 Oct, 40(8), 807 - 12
Lethal and non-lethal course of endotoxic shock is determined by interactions between tumor necrosis factor, platelet activating factor and eicosanoids; Mozes T et al.; Continuous lipopolysaccharide (LPS) infusion in pigs induced death in approximately half of the animals and a prolonged state of shock (up to 3 hours of experimental observation period, i.e., two hours after discontinuation of LPS infusion) in the surviving animals . Lethal-induced shock was marked by huge release of Tumor Necrosis Factor (TNF) into the blood, whereas eicosanoid and Platelet Activating Factor (PAF) levels remained unchanged . In pigs surviving LPS-infusion but still remaining in a state of shock, transient increases in PAF and thromboxane levels were observed, whereas prostacyclin and leukotrienes values remained above normal levels up to the end of the observation period . It is concluded that different types of mediators play a role in LPS-induced lethal shock as compared to non-lethal prolonged state of shock.

Wei Sheng Wu Xue Bao, 1992 Oct, 32(5), 364 - 9
{Detection of Mycobacterium tuberculosis in sputum specimens of pulmonary tuberculosis by DNA amplification}; Zhuang Y et al.; The PCR was used to detect M . tuberculosis DNA sequences in uncultured clinical specimens . Two oligonucleotide primers with 20 bp each amplified target template DNA of M . tuberculosis . Amplified DNA product was 245 bp which was identified by agarose gel electrophoresis . The sensitivity of detection of M . tuberculosis genomic DNA and bacteria suspension by PCR was lpg and 13 viable bacteria cell/ml, respectively . In specificity experiments, only M . tuberculosis, M . bovis and BCG were positive by PCR, but all other 14 Mycobacterium tested, including streptomyces lividans and E . coli plasmid PUC19 were negative . The sensitivity of detection of M . tuberculosis by PCR was determined by comparing the fast-acid staining and culture on total 54 sputum specimens of pulmonary tuberculosis and 12 nontuberculosis lung disease . The positive rate of PCR in pulmonary tuberculosis were 37.0%, culture method showed only 14.8%, fast-acid staining were 16.7% . Nontuberculosis lung disease were negative . The results show that DNA amplification is a superior method with high degree of sensitivity and specificity for rapid diagnosis of pulmonary tuberculosis.

Mol Microbiol, 1992 Oct, 6(20), 2961 - 73
Sequence alterations affecting F plasmid transfer gene expression: a conjugation system dependent on transcription by the RNA polymerase of phage T7; Maneewannakul K et al.; We constructed derivatives of the Escherichia coli conjugative plasmid F that carry altered sequences in place of the major transfer operon promoter, PY . Replacement of PY with a promoter-deficient sequence resulted in a transfer-deficient, F-pilus-specific phage-resistant plasmid (pOX38-tra701) that could still express TraJ and TraT; TraY, F-pilin, TraD, and TraI were not detectable on Western blots . On a second plasmid (pOX38-tra715) we replaced PY with a phage T7 late promoter sequence . In hosts carrying a lacUV5-promoter-regulated T7 RNA polymerase gene, all transfer-associated properties of pOX38-tra715 could be regulated with IPTG . After induction, pOX38-tra715 transferred at the wild-type frequency, expressed normal numbers of F-pili and conferred sensitivity to pilus-specific phages . No adverse effects on cell viability were apparent, and additional mutations could easily be crossed onto pOX38-tra715 . A traJ deletion (pOX38-tra716) had no effect on the IPTG-induced transfer phenotype . Insertion of cam into trbC, resulted in a mutant (pOX38-tra715trbC33) which, after induction, exhibited the same phenotype associated with other trbC mutants; it could also be complemented by expression of trbC in trans . With pOX38-tra715 or its derivatives, we were able to label specifically the products of tra genes located throughout the long tra operon, by using rifampicin . This feature can be used to investigate transfer protein interactions and to follow changes in these proteins that are associated with conjugal mating events.

Mol Microbiol, 1992 Oct, 6(20), 2951 - 9
The TraM protein of the conjugative plasmid F binds to the origin of transfer of the F and ColE1 plasmids; Di Laurenzio L et al.; The gene encoding the TraM protein of the conjugative plasmid F was cloned, overexpressed and the gene product was purified . The TraM protein was found in the cytoplasm of cells carrying the F plasmid with a smaller amount in the inner membrane . DNase I footprinting experiments showed that the purified protein protects three regions in the F oriT locus with different affinity for the upper and lower strands of DNA . A 15-nucleotide motif was identified within the protected regions that represented the DNA-binding site . The TraM protein was also found to bind to a sequence in the oriT region of the non-conjugative plasmid ColE1 that resembles the three binding sites in the F oriT region.

J Gen Microbiol, 1992 Oct, 138 ( Pt 10), 2167 - 71
Identification of hydrophobic proteins FepD and FepG of the Escherichia coli ferrienterobactin permease; Chenault SS et al.; In Escherichia coli, iron assimilation by means of the siderophore enterobactin requires two hydrophobic cytoplasmic membrane proteins, FepD and FepG, which are essential components of a binding-protein-dependent transport system . Such components are typically difficult to detect . Here we report observation of the fepD and fepG gene products in polyacrylamide gels; they appeared as diffuse bands at positions consistent with smaller sizes than those predicted by sequence analysis . Translational coupling was suggested by the lack of a detectable product from the fepG message in the absence of translation of the upstream fepD message . The orientation of FepD/FepG in the membrane was predicted based on their similarities in sequence and hydrophobicity to FhuB.

J Gen Microbiol, 1992 Oct, 138 ( Pt 10), 2007 - 14
Two mechanisms for growth inhibition by elevated transport of sugar phosphates in Escherichia coli; Kadner RJ et al.; The Escherichia coli uhp T gene encodes an active transport system for sugar phosphates . When the uhp T gene was carried on a multicopy plasmid, amplified levels of transport activity occurred, and growth of these strains was inhibited upon the addition of various sugar phosphates . Two different mechanisms for this growth inhibition were distinguished . Exposure to glucose-6-phosphate, fructose-6-phosphate or mannose-6-phosphate, which enter directly into the glycolytic pathway, resulted in cessation of growth and substantial loss of viability . Cell killing was correlated with the production of the toxic metabolite, methylglyoxal . In contrast, addition of 2-deoxyglucose-6-phosphate, galactose-6-phosphate, glucosamine-6-phosphate or arabinose-5-phosphate, which do not directly enter the glycolytic pathway, resulted in growth inhibition without engendering methylglyoxal production or cell death . Inhibition of growth could result from excessive accumulation of organophosphates in the cell or depletion of inorganic phosphate pools as a result of the sugar-P/Pi exchange process catalysed by UhpT . The phosphate-dependent uptake of glycerol-3-phosphate by the GlpT antiporter was strongly inhibited under conditions of elevated sugar-phosphate transport . There are thus two separate toxic effects of elevated sugar-phosphate transport, one of which was lethal and related to increased flux through glycolysis . It is likely that the control of uhpT transcription by catabolite repression exists to limit the level of UhpT transport activity and thereby prevent the toxic events that result from elevated uptake of its substrates.

Rev Clin Esp, 1992 Oct, 191(5), 264 - 6
{Spinal epidural abscess . 8 years' experience}; de la Fuente Aguado J et al.; We present four cases of spinal epidural abscess diagnosed in Clinica Puerta de Hierro between 1982 and 1990 . In three cases the localization was thoracic and in one it was lumbar . Fever and vertebral pain were the more constant clinical symptoms . Lumbar punction showed findings in Cerebro-Spinal Fluid compatible with a parameningeal inflammation focus in the three cases it was performed . Diagnosis was established with myelography or Computerized Axial Tomography . Treatment in two cases was laminectomy and systemic antibiotics: and only antibiotics in the other two cases . Evolution was favorable in the patients who underwent surgery, but the patients treated conservatively had a fatal outcome.

Domest Anim Endocrinol, 1992 Oct, 9(4), 273 - 83
Partial prostaglandin-mediated mechanism controlling the release of cortisol in plasma after intravenous administration of endotoxins; Massart-Leen AM et al.; In a first series of experiments we studied the influence of E . coli endotoxins or lipopolysaccharides (LPS) administered either intravenously (i.v.) or intramammarily (i.mam.) to lactating goats on plasma cortisol and rectal temperature (RT) . Differences in the magnitude of the cortisol release and shape of the fever curve were observed . In both models maximal pyrexia and fever index (FI) were comparable . In a second series of experiments the influence of LPS on the plasma cortisol and RT was studied after i.v . injection of increasing doses of LPS:low (25 ng/kg), moderate (200 ng/kg) and relatively high (500 ng/kg) . Although the cortisol response was dose dependent, the effect was not correlated with FI . The administration of flurbiprofen, a non steroidal antiinflammatory drug (NSAID), resulted in a complete inhibition of fever at all LPS doses and the cortisol release after administration of low doses LPS . This indicates a prostaglandin mediation . With moderate and high doses LPS the cortisol release was only partially inhibited and delayed suggesting a non prostaglandin mediated mechanism . In a third series of experiments the influence of flurbiprofen on fever and cortisol release was studied after i.mam . LPS administration . The observed increase of plasma cortisol and RT were completely abolished after flurbiprofen treatment . It is concluded that: 1) the increase of plasma cortisol after LPS administration in lactating goats is not related to hyperthermia per se, 2) the control of fever and cortisol release may, to some extent, differ according to the LPS dose and method of administration, 3) the cortisol release observed after moderate and high doses of LPS is probably controlled by two phenomena . The first being induced by cyclo-oxygenase metabolites, the second by intermediary mediators other than prostaglandins or by LPS itself . 4) Although an eight-fold higher dose of LPS was given i.mam., a cortisol release comparable to the lowest dose of LPS (25 ng/kg) was observed . These differences in cortisol release can be ascribed to 1) a detoxification of LPS at the level of the mammary gland or 2) a slower resorption of LPS from the mammary gland.

Cell Struct Funct, 1992 Oct, 17(5), 293 - 300
Effects of fibronectin-type V collagen recombinant fusion protein on cell adhesion and cell proliferation; Hatai M et al.; An expression vector pTF7520-Col-V-In, which encodes a fusion protein of the cell-binding domain of fibronectin (C277) and the insulin- and heparin-binding domain of the alpha 1 chain of human type V collagen, was constructed . E . coli transfected with this plasmid synthesized a 50-kDa fusion protein . This fusion protein, C277-V, was purified from the crude extract by a single step heparin HPLC . Similar amounts of insulin bound to purified C277-V and to the alpha 1 chain of type V collagen as judged by the binding of peroxidase-conjugated insulin . Cell-adhesive activity of C277-V was lower than that of the original fibronectin fragment C274, but similar numbers of cells adhered to both protein substrates when the culture dishes were coated with 1 mM of each protein . Insulin bound to the C277-V substratum stimulated the growth of mouse mammary tumor MTD cells in serum-free culture medium.

J Biomol Struct Dyn, 1992 Oct, 10(2), 317 - 31
A structural model for sequence-specific proflavin-DNA interactions during in vitro frameshift mutagenesis; Berman HM et al.; Molecular models describing intermediates that may lead to proflavin-induced 1 bp deletions during in vitro polymerization by E . coli DNA polymerase I Klenow fragment are proposed . The models provide structural explanations for the fact that the induced frameshifts always occur opposite template bases that are adjacent to 5' pyrimidines and are based on the underlying hypothesis that the deletions arise because the polymerase passes by a template base without copying it . Because the most frequent mutations are opposite Pu in the template sequence 5' Py Pu 3', a single-strand loop-out model was constructed for this sequence and proflavin was added, using structures found in crystalline oligonucleotides and their complexes with proflavin . The model seeks to rationalize the roles of the 5' pyrimidine and proflavin in facilitating the bypass . Four potential roles for proflavin in mutagenesis are described: 1) stacking on the looped-out base; 2) stacking on the base pair immediately preceding the site of mutation; 3) hydrogen bonding with the 5' pyrimidine; 4) hydrogen bonding with the phosphate backbone . These models point to the possibility that a number of proflavin-DNA interactions may be involved . In contrast, modeling does not suggest a role for classically intercalated proflavin in frameshift mutagenesis arising during in vitro DNA polymerization.

J Biomol Struct Dyn, 1992 Oct, 10(2), 311 - 6
Binding of DNA to large fragment of DNA polymerase I: identification of strong and weak electrostatic forces and their biological implications; Yadav PN et al.; Examination of the electrostatic potential of a modeled complex, consisting of the Klenow fragment of E . coli DNA polymerase I and DNA template-primer, suggested the presence of two distinct interacting regions . The one displaying a strong electropositive potential field is generated by side chains of basic amino acid pairs and is directed towards the major groove site in DNA . The second electrostatic potential field around DNA is somewhat weaker and appears to be exerted by a pair of vicinal side chains of acidic and basic amino acids . The distribution of charges in this manner appears well suited for the binding of enzyme to the template-primer required in the enzymatic synthesis of DNA.

Res Commun Chem Pathol Pharmacol, 1992 Oct, 78(1), 57 - 68
Cardiovascular actions of NG-methyl-L-arginine are abolished in a canine shock model using high-dose endotoxin; Klabunde RE et al.; This study was designed to test the hypothesis that endotoxin-induced hypotension is caused, in part, by increased endothelial synthesis of nitric oxide and that inhibition of nitric oxide synthesis with NG-monomethyl-L-arginine (NMA) would reverse the cardiovascular actions of endotoxin . A high dose of endotoxin (1.5 mg/kg, i.v.) was administered by rapid intravenous infusion to pentobarbital-anesthetized dogs . Endotoxin caused rapid and profound reductions in cardiac output and mean arterial pressure; systemic vascular resistance, however, was unaltered except for a transient increase . Coronary and mesenteric blood flows were reduced . NMA (30 mg/kg, i.v.) given 60 min after endotoxin administration, had no significant effect on cardiac function or systemic vascular function except for a transient increase in cardiac output and decrease in systemic vascular resistance . This same dose of NMA given to dogs not receiving endotoxin caused systemic vasoconstriction, increased arterial pressure and decreased cardiac output . These results suggest that increased nitric oxide production is not a primary factor causing endotoxin-induced hypotension and that nitric oxide does not modulate vascular tone following administration of high doses of endotoxin.

Med Hypotheses, 1992 Oct, 39(2), 127 - 9
Sudden infant death syndrome (SIDS) and the immune response; Reid GM; Vitamin E pretreatment significantly prevented E . coli-induced Disseminated Intravascular Coagulation (DIC) in rats (1) . DIC, a reduction in fibrinogen and a falling platelet count and diffuse haemorrhage are part of the clinical features of Haemorrhagic Shock Encephalopathy Syndrome (HSES), recognised as a disease entity in the 1980s (2) . At the SIDS Conference 1974 Reisinger described the effect of Escherichia coli (E . coli) endotoxin on the rabbit (3) . An early effect was a reduction in fibrinogen and a falling platelet count, resulting in the release of relatively large amounts of the neuro-transmitter serotonin, stored in platelets (3, 4) . Fibrinogen inhibited the release of serotonin from platelets (24) . Serotonin is released from platelets during platelet aggregation (14) . Platelet aggregation is inhibited by vitamin E (1) . Serotonin is a neuro-transmitter associated with deep sleep, respiratory movements and cardiovascular collapse (3) . Death at a later stage involved vascular permeability, edema and haemorrhage . After fibrin-platelet clots had formed DIC was present in lungs, kidneys and other organs (3) . Medical researchers in Australia linked almost half of SIDS victims with a poisonous strain of intestinal E . coli bacteria (5) . Dietary selenium in the intestinal villous tip is considered a daily modulator of cytochrome P450-dependent metabolism of drugs and toxins absorbed by intestinal mucosa (6) . Villous atrophy occurs in HSES (2).

Protein Expr Purif, 1992 Oct, 3(5), 386 - 94
High-level expression and simplified purification of recombinant ricin A chain; Li BY et al.; Ricin toxin is a glycoprotein which catalytically inactivates eukaryotic ribosomes by depurination of a single adenosine residue from the 28S ribosomal RNA . The enzymatic activity is present in the A chain of the toxin molecule, whereas the B chain contains two binding sites for galactose . Since it is highly potent in inhibiting protein synthesis, the A chain is used to prepare cytotoxic conjugates effective against tumor cells . Such chimeric proteins are highly selective and have a wide range of clinical applications . Extensive preclinical studies on these conjugates require large amounts of purified A chain . Native ricin A chain is heterogeneous, since plants produce a number of isoforms of ricin toxin . Purified, native preparations often contain two types of ricin A chain which differ in the extent of glycosylation . By cloning and expressing the gene of A chain, one could obtain homogeneous toxin molecules devoid of carbohydrates . In addition, structural changes in the toxin polypeptide could be introduced by in vitro mutagenesis, which can improve the pharmacological properties and antitumor activity . Earlier methods of expression strategies using Escherichia coli have yielded only moderate levels of expression . In the present study, the coding region of ricin A chain was cloned into pET3b, a high-level expression vector under the control of the T7 promoter . Recombinant ricin A chain produced by this construct has an additional 14 amino acid residues at the NH2 terminus . Subsequently, a NdeI site was created at the 5' end of the gene by oligonucleotide-directed mutagenesis . The modified fragment was then introduced into pET3b vector to produce toxin polypeptide identical to the native sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Expr Purif, 1992 Oct, 3(5), 374 - 9
Expression in Escherichia coli: purification and properties of the recombinant human general transcription factor rTFIIB; Moncollin V et al.; The human class II transcription factor TFIIB (rTFIIB) was overexpressed in Escherichia coli using a T7 RNA polymerase expression system and further purified to apparent homogeneity . The purified rTFIIB is identical to the endogenous factor according to the following criteria: molecular weight, microsequencing and mass spectra studies, ability to recognize the stable preinitiation complex formed between TFIID and the adenovirus 2 major late TATA box as demonstrated by gel shift as well as by DNase I footprinting assays, and transcription activity.

Curr Opin Genet Dev, 1992 Oct, 2(5), 792 - 8
What is the minimum number of dedicated functions required for a basic cell cycle?
Donachie WD.
The genome of Escherichia coli has a coding capacity for about 4500 proteins but only a small number of these appear to be specific for the periodic events (initiation of DNA replication, chromosome partitioning and cell division) that punctuate the cell-duplication cycle: furthermore, many of these cell cycle dedicated functions are dispensible under certain conditions, although their presence undoubtedly increases the fitness of the organism to survive in a competitive environment . A simplified but effective cell replication cycle can probably operate with only a few cycle-dedicated proteins, in addition to those required for cell growth itself.

Biochem Int, 1992 Oct, 28(2), 255 - 63
Improved production and purification of interferon-alpha-4a using a T7 RNA polymerase expression system; Waine GJ et al.; An improved method for the synthesis and purification of human interferon-alpha-4a is presented . Interferon-alpha-4a was prepared using a T7 RNA polymerase expression system, where it was expressed from the vector pET-3a . Biologically active interferon-alpha-4a was isolated from the E . coli cells and purified using protamine sulphate precipitation, anion-exchange chromatography and affinity chromatography utilising a monoclonal antibody specific for human interferons-alpha.

Anal Biochem, 1992 Oct, 206(1), 43 - 9
Trans-diamminedichlorplatinum (II)-modified probes for detection of picogram quantities of DNA; Kiseleva VI et al.; A novel simple nonradioactive method for detection of specific nucleotide sequences has been developed . This method consists of the hybridization of a target DNA with a DNA probe modified with trans-diamminedichlorplatinum(II) (trans-DDP) followed by detection of DNA/DNA hybrids with affinity-isolated anti-DNA-trans-DDP antibodies and poly-horseradish peroxidase-protein A conjugate . Major advantages of this approach are the low cost and the extreme simplicity of the labeling procedure, which involves only mixing of the reagents . The sensitivity of the proposed technique is sufficient to detect 0.8 pg of DNA in Southern blot hybridization and 25 fg in dot hybridization and permits colony screening.

ASAIO J, 1992 Oct-Dec, 38(4), 815 - 9
Effect of endotoxin clearance using whole body rinse-out on some blood-gas and metabolic parameters in rats; Voynikov TI et al.; Conscious and unrestrained male Wistar rats were infused over a 10 min period with 60 mg/kg Escherichia coli endotoxin (LD100) and observed for 6 hr . Hypotension, metabolic acidosis, hyperlactemia, hypoglycemia, and elevated oxygen extraction from blood developed in the rats . Another group of rats was subjected to blood substitution with FC-43 emulsion (artificial blood) 30 min after endotoxin application until hematocrit dropped to 3%, after which 6-9 ml of donor blood was exchange transfused with the emulsion . This method resulted in 98% endotoxin clearance, improved blood alkaline reserves, preserved blood glucose, and normalized arterial mixed venous oxygen gradient and O2 extraction from the blood (which implies preserved blood circulation status) . Side effects included hypotension, a transitory rise in blood lactate level, and a fall in arterial oxygen content due to the lower hemoglobin (erythrocyte) level.

J Appl Physiol, 1992 Oct, 73(4), 1517 - 22
Acute heat stress protects rats against endotoxin shock; Ryan AJ et al.; The purpose of this study was to determine 1) whether prior (24-h) heat stress could render rats cross-resistant to the lethal activity of bacterial lipopolysaccharide (LPS) and 2) whether this acquired state of resistance is associated with endotoxemia during the heat stress event . Four groups (n = 7/group) of rats were examined: 1) saline treated, 2) LPS treated, 3) heat stressed and saline treated, and 4) heat stressed and LPS treated . Saline or LPS (Escherichia coli, serotype 0111:B4, 20 mg/kg body wt) was given intravenously 24 h after exposure to heat (ambient temperature 47-50 degrees C, relative humidity 30%) for heat-stressed rats and at the same time of day for nonheated rats; survival was monitored for 48 h . Thermal responses were similar (P > 0.05); values for maximum core temperature (Tc) and time above Tc of 40 degrees C were 42.7 +/- 0.1 and 42.6 +/- 0.1 degrees C (SE) and 44.0 +/- 2.1 and 47.9 +/- 3.7 (SE) min for the heat-stressed saline-treated and heat-stressed LPS-treated rats, respectively . Administration of LPS to nonheated rats resulted in 71.4% (5 of 7 rats) lethality . In contrast, all (7 of 7) rats subjected to a single nonlethal heat stress event 24 h before LPS treatment survived (P < 0.05) . Endotoxin was not detected in arterial plasma immediately after heat stress in rats (n = 6) exposed to a Tc of 42.9 +/- 0.1 degrees C . These findings demonstrate that acute heat stress can protect rats from the lethal activity of LPS.

Biochem Int, 1992 Oct, 28(1), 121 - 7
Action of a nuclease from rat nuclei on UV-irradiated DNA; Hibino Y et al.; An Mg(2+)-dependent nuclease was highly purified from rat-liver nuclei . The nuclease activity was enhanced in ultraviolet light (UV)-irradiated dsDNA, but not in unirradiated dsDNA . Irrespective of UV irradiation, ssDNA was readily cleaved by this enzyme . UV-irradiated plasmid DNA was incubated with this enzyme and subjected to the template primer activity assay with a Klenow polymerase . With increasing incubation time, the activity was enhanced in the circular-relaxed and linear forms, but not in the superhelical form . These results implied that this enzyme excises UV-damaged sites in dsDNA to form single strand gaps for repair synthesis.

Appl Environ Microbiol, 1992 Oct, 58(10), 3399 - 403
Cloning and expression of the aspartate carbamoyltransferase gene from Treponema denticola; Ishihara K et al.; Treponema denticola seems to play a central role in the etiology of human periodontal disease . We have cloned an antigenic protein-coding sequence from T . denticola ATCC 33520 . The protein-coding region was found to be a 3-kbp HindIII-HindIII fragment . The open reading frame consists of 1,426 bp and codes for a protein with an M(r) of 54,919 . The deduced amino acid sequence showed 33.8% homology with that of the aspartate carbamoyltransferase of Escherichia coli . The gene products showed aspartate carbamoyltransferase activity.

Vet Immunol Immunopathol, 1992 Oct, 34(1-2), 159 - 72
Immunological parameters in meat-type chicken lines divergently selected by antibody response to Escherichia coli vaccination; Heller ED et al.; Our group has established two lines of meat-type chickens divergently selected for early (HC line) and late (LC line) antibody responsiveness at 10 days of age to immunization with inactivated pathogenic Escherichia coli bacteria . The question addressed in the study presented here is whether this selection has changed other immunological responses, increasing the overall 'early' immunocompetence . Broilers of the third and fourth generations (S3 and S4) of the selected lines (HC and LC) and a control, unselected line (CT) were vaccinated at 10 days of age with E . coli vaccine, Newcastle virus vaccine (NDV), sheep erythrocytes (SRBC) or bovine serum albumin (BSA) . Line-HC chicks exhibited higher antibody titers to E . coli, NDV and SRBC than CT or LC chicks . At 20 days of age HC chicks demonstrated a higher total protein and a higher beta- and gamma-globulin levels in their serum . At 21 days of age, HC chicks cleared carbon particles faster than LC chicks . Peripheral blood lymphocytes (PBL) from HC chicks vaccinated with E . coli vaccine, proliferated in vitro more actively in the presence of the stimulating antigen than the PBL of LC chicks . Peripheral blood lymphocytes (PBL) obtained from HC-line chicks exhibited a higher proliferative response to concanavalin A (Con A)-, phytohemagglutinin (PHA)- or pokeweed mitogen (PWM)-stimulation than LC PBL . These results demonstrate that the selection for high or low antibody response to E . coli at a young age resulted also in a significant change in the response of other parameters of the immune system . The high response to E . coli was found to be associated with a high antibody response to other antigens (NDV and SRBC), increased phagocytic activity and increased proliferative response to antigen or mitogens . The selection most probably affected early immunocompetence.

Mol Biochem Parasitol, 1992 Oct, 55(1-2), 95 - 104
The translation initiation site of recombinant Trypanosoma brucei ornithine decarboxylase varies with different promoters; Kuntz DA et al.; Expression of the Trypanosoma brucei ornithine decarboxylase (ODC) gene in Escherichia coli behind the lambda phage PR promoter led to the production of a recombinant enzyme having the same subunit molecular weight as the native enzyme {4} . However, when the same gene is expressed behind the tac promoter or the phoA promoter, the ODCs produced by the transformed E . coli have subunit molecular weights approximately 2 kDa higher than that of the native enzyme . Amino terminal sequencing of the recombinant proteins indicates that the ODC synthesized under control of the lambda PR promoter actually starts at the second methionine (Met23) of the open reading frame, whereas those produced in the latter two cases begin at the first methionine (Met1) . Analysis of the 5'-end of T . brucei ODC mRNA supports the conclusion that translation initiates at Met23 . We postulate that, for the lambda PR promoter, translation initiates at Met23 instead of Met1 because of the formation of a stable secondary structure in the region of the Met1 and the presence of a good E . coli consensus translation initiation site upstream of Met23 . We have constructed a new plasmid using the pho A promoter to express recombinant T . brucei ODC starting at Met23 in large quantities.

Mol Biochem Parasitol, 1992 Oct, 55(1-2), 155 - 65
Identification and expression of a Fasciola hepatica gene encoding a gut antigen protein bearing repetitive sequences; Marin MS et al.; A Fasciola hepatica cDNA clone of about 2 kb was isolated from an expression library by immunological screening using blood serum from an experimentally infected calf . The cDNA clone hybridised to a RNA of about 3 kb in a Northern blot experiment . The nucleotide sequence of the cDNA revealed the presence of an open reading frame of 1636 bp which encoded 24 tandemly arranged 20-amino acid-long repeats, followed by 65 non-repeated residues preceding the stop codon . This antigen was expressed in Escherichia coli as beta-galactosidase fusion proteins which were used for the production of specific antibodies . Immunofluorescence studies using specific antifusion sera revealed that the antigen was specifically expressed in the parasite intestine epithelial cells . Due to its early appearance it might be possible to design diagnostic assays based on this repeated antigen for identification of recently infected animals.

Mol Gen Genet, 1992 Oct, 235(1), 55 - 63
The region essential for efficient autonomous replication of pSa in Escherichia coli; Okumura MS et al.; Comparative analyses were made between plasmid pSa17, a deletion derivative of pSa that is capable of replicating efficiently in Escherichia coli and plasmid pSa3, a derivative that is defective for replication . By comparing the restriction maps of these two derivatives, the regions essential for replication and for stable maintenance of the plasmid were determined . A 2.5 kb DNA segment bearing the origin of DNA replication of pSa17 was sequenced . A 36 kDa RepA protein was encoded in the region essential for replication . Downstream of the RepA coding region was a characteristic sequence including six 17 bp direct repeats, the possible binding sites of RepA protein, followed by AT-rich and GC-rich sequences . Furthermore, an 8 bp incomplete copy of the 17 bp repeat was found in the promoter region of the repA gene . Based on the hypothesis that RepA protein binds to this partial sequence as well as to intact 17 bp sequences, an autoregulatory system for the synthesis of RepA protein may be operative . Another open reading frame (ORF) was found in the region required for the stability of the plasmid . The putative protein encoded in this ORF showed significant homology to several site-specific recombination proteins . A possible role of this putative protein in stable maintenance of the plasmid is discussed.

Mol Gen Genet, 1992 Oct, 235(1), 22 - 32
Characterization of a chromosomally encoded, non-PTS metabolic pathway for sucrose utilization in Escherichia coli EC3132; Bockmann J et al.; A wild-type isolate, EC3132, of Escherichia coli, that is able to grow on sucrose was isolated and its csc genes (mnemonic for chromosomally coded sucrose genes) transferred to strains of E . coli K12 . EC3132 and all sucrose-positive exconjugants and transductants invariably showed a D-serine deaminase (Dsd)-negative phenotype . The csc locus maps adjacent to dsdA, the structural gene for the D-serine deaminase, and contains an inducible regulon, controlled by a sucrose-specific repressor CscR, together with structural genes for a sucrose hydrolase (invertase) CscA, for a D-fructokinase CscK, and for a transport system CscB . Based on DNA sequencing studies, this last codes for a hydrophobic protein of 415 amino acids . CscB is closely related to the beta-galactoside transport system LacY (31.2% identical residues) and a raffinose transport system RafB (32.3% identical residues) of the enteric bacteria, both of the proton symport type . A two-dimensional model common to the three transport proteins, which is based on the integrated consensus sequence, will be discussed.

Mol Gen Genet, 1992 Oct, 235(1), 131 - 9
FinOP repression of the F plasmid involves extension of the half-life of FinP antisense RNA by FinO; Lee SH et al.; The transfer operon of the F plasmid is positively regulated by the traJ gene product, expression of which, in turn, is regulated by both an antisense RNA, FinP, and the FinO protein (the FinOP system) . A finP- F plasmid, pSFL20, was constructed by site-directed mutagenesis and was found to produce wild-type levels of pili encoded by the transfer operon . Transcription of the traJ gene was decreased by a factor of 3-5 fold in the presence of FinOP with no accumulation of a stable RNA duplex between the FinP RNA and the portion of the traJ mRNA which is complementary to finP . Stabilization of FinP RNA by FinO occurs in the absence of traJ transcripts, suggesting that FinO may interact directly with FinP to prevent its degradation.

Mol Gen Genet, 1992 Oct, 235(1), 1 - 10
Purification and properties of the RuvA and RuvB proteins of Escherichia coli; Tsaneva IR et al.; The RuvA and RuvB proteins of Escherichia coli play important roles in the post-replicational repair of damaged DNA, genetic recombination and cell division . In this paper, we describe the construction of over expression vectors for RuvA and RuvB and detail simple purification schemes for each protein . The purified 22 kDa RuvA polypeptide forms a tetrameric protein (M(r) ca . 100,000) as observed by gel filtration . The tetramer is stabilised by strong disulphide bridges that resist denaturation during SDS-PAGE (in the absence of boiling and beta-mercaptoethanol) . In contrast, purified RuvB polypeptides (37 kDa) weakly associate to form a dimeric protein (M(r) ca . 85,000) . At low protein concentrations, the RuvB dimer dissociates into monomers . The multimeric forms of each protein may be covalently linked by the bifunctional cross-linking reagent dimethyl suberimidate . Addition of purified RuvA and RuvB to a RecA-mediated recombination reaction was found to stimulate the rate of strand exchange leading to the rapid formation of heteroduplex DNA.

Mol Microbiol, 1992 Oct, 6(19), 2887 - 95
Cloning of mycobacterial histidine synthesis genes by complementation of a Mycobacterium smegmatis auxotroph; Hinshelwood S et al.; Histidine-requiring auxotrophs of Mycobacterium smegmatis were isolated following N-methyl-N'-nitro-N-nitrosoguanidine treatment . One of these mutants, his5, was transformed with an M . smegmatis shuttle cosmid library, and complementing clones were isolated at a frequency of approximately 1% . A 2.3 kb fragment was subcloned and sequenced, and found to contain the start of an operon including the hisD gene and part of the hisC gene . No hisG gene was detected upstream of hisD, suggesting that the regulation of histidine biosynthesis in mycobacteria may differ from that of Escherichia coli . The strategy used here will allow the molecular genetics of complex mycobacterial-specific biosynthetic pathways involved in the virulence of pathogenic species to be studied.

Mol Microbiol, 1992 Oct, 6(19), 2805 - 13
Transcriptional control, translation and function of the products of the five open reading frames of the Escherichia coli nir operon; Harborne NR et al.; Five open reading frames designated nirB, nirD, nirE, nirC and cysG have been identified from the DNA sequence of the Escherichia coli nir operon . Complementation experiments established that the NirB, NirD and CysG polypeptides are essential and sufficient for NADH-dependent nitrite reductase activity (EC 1.6.6.4) . A series of plasmids has been constructed in which each of the open reading frames has been fused in-phase with the beta-galactosidase gene, lacZ . Rates of beta-galactosidase synthesis during growth in different media revealed that nirB, -D, -E and -C are transcribed from the FNR-dependent promoter, p-nirB, located just upstream of the nirB gene: expression is co-ordinately repressed by oxygen and induced during anaerobic growth . Although the nirB, -D and -C open reading frames are translated into protein, no translation of nirE mRNA was detected . The cysG gene product is expressed from both p-nirB and a second, FNR-independent promoter, p-cysG, located within the nirC gene . No NADH-dependent nitrite reductase activity was detected in extracts from bacteria lacking either NirB or NirD, but a mixture of the two was as active as an extract from wild-type bacteria . Reconstitution of enzyme activity in vitro required stoichiometric quantities of NirB and NirD and was rapid and independent of the temperature during mixing . NirD remained associated with NirB during the initial stages of purification of the active enzyme, suggesting that NirD is a second structural subunit of the enzyme.

Mol Microbiol, 1992 Oct, 6(19), 2777 - 84
Frameshifting in the expression of the Escherichia coli trpR gene; Benhar I et al.; The trpR gene of Escherichia coli carries an open reading frame that encodes the trp repressor, 108 amino acids long . Here we show that translation of an additional (+1) reading frame of trpR occurs both in vivo and in vitro . This results in the synthesis of a stable +1 frame polypeptide . Using site-specific mutagenesis, immunological techniques and amino acid sequencing we have found that the N-terminus of the +1 frame product and that of the known 0 frame product are identical but that their C-termini differ . Our results are discussed in relation to the role of natural frameshifting as a regulatory mechanism of gene expression in general, and with respect to tryptophan biosynthesis in particular.

Mol Microbiol, 1992 Oct, 6(19), 2755 - 9
Biological roles of the Escherichia coli RuvA, RuvB and RuvC proteins revealed; West SC et al.; In Escherichia coli, the ruvA, ruvB and ruvC gene products are required for genetic recombination and the recombinational repair of DNA damage . New studies suggest that these three proteins function late in recombination and process Holliday junctions made by RecA protein-mediated strand exchange . In vitro, RuvA protein binds a Holliday junction with high affinity and, together with RuvB (an ATPase), promotes ATP-dependent branch migration of the junction leading to the formation of heteroduplex DNA . The third protein, RuvC, which acts independently of RuvA and RuvB, resolves recombination intermediates by specific endonucleolytic cleavage of the Holliday junction.

J Bioenerg Biomembr, 1992 Oct, 24(5), 479 - 84
Catalytic sites of Escherichia coli F1-ATPase; Senior AE; The catalytic site of Escherichia coli F1-ATPase is reviewed in terms of structure and function . Structural prediction, biochemical analyses, and mutagenesis experiments suggest that the catalytic site is formed primarily by residues 137-335 of beta-subunit . Subdomains of the site involved in phosphate-bond cleavage/synthesis and adenine-ring binding are discussed . Ambiguities inherent in steady-state catalytic measurements due to catalytic site cooperativity are discussed, and the advantages of pre-steady-state ("unisite") techniques are emphasized . The emergence of a single high-affinity catalytic site occurs as a result of F1-oligomer assembly . Measurements of unisite catalysis rate and equilibrium constants, and their modulation by varied pH, dimethylsulfoxide, and mutations, are described and conclusions regarding the nature of the high-affinity catalytic site and mechanism of catalysis are presented.

J Bioenerg Biomembr, 1992 Oct, 24(5), 463 - 7
A glycine-rich sequence in the catalytic site of F-type ATPase; Futai M et al.; Affinity labeling and genetic studies on the glycine-rich sequence of the beta subunit of E . coli F-type ATPase are discussed . A model of the structure of the enzyme near the gamma phosphate moiety is proposed.

J Bioenerg Biomembr, 1992 Oct, 24(5), 453 - 61
A model for the catalytic site of F1-ATPase based on analogies to nucleotide-binding domains of known structure; Duncan TM et al.; An updated topological model is constructed for the catalytic nucleotide-binding site of the F1-ATPase . The model is based on analogies to the known structures of the MgATP site on adenylate kinase and the guanine nucleotide sites on elongation factor Tu (Ef-Tu) and the ras p21 protein . Recent studies of these known nucleotide-binding domains have revealed several common functional features and similar alignment of nucleotide in their binding folds, and these are used as a framework for evaluating results of affinity labeling and mutagenesis studies of the beta subunit of F1 . Several potentially important residues on beta are noted that have not yet been studied by mutagenesis or affinity labeling.

J Bioenerg Biomembr, 1992 Oct, 24(5), 435 - 9
Structure of the Escherichia coli ATP synthase and role of the gamma and epsilon subunits in coupling catalytic site and proton channeling functions; Capaldi RA et al.; The structure of the Escherichia coli ATP synthase has been studied by electron microscopy and a model developed in which the alpha and beta subunits of the F1 part are arranged hexagonally (in top view) alternating with one another and surrounding a central cavity of around 35 A at its widest point . The alpha and beta subunits are interdigitated in side view for around 60 A of the 90 A length of the molecule . The F1 narrows and has three-fold symmetry at the end furthest from the F0 part . The F1 is linked to F0 by a stalk approximately 45 A long and 25-30 A in diameter . The F0 part is mostly buried in the lipid bilayer . The gamma subunit provides a domain that extends into the central cavity of the F1 part . The gamma and epsilon subunits are in a different conformation when ATP + Mg2+ are present in catalytic sites than when ATP + EDTA are present . This is consistent with these two small subunits switching conformations as a function of whether or not phosphate is bound to the enzyme at the position of the gamma phosphate of ATP . We suggest that this switching is the key to the coupling of catalytic site events with proton translocation in the F0 part of the complex.

Genomics, 1992 Oct, 14(2), 474 - 80
The human galactose-1-phosphate uridyltransferase gene; Leslie ND et al.; Classical galactosemia is an inborn error of metabolism caused by a deficiency of galactose-1-phosphate uridyltransferase (GALT) . Standard treatment with dietary galactose restriction will reverse the potentially lethal symptoms of the disease that are manifest in the newborn period . However, the long-term prognosis for these patients is variable . As a first step toward investigating the molecular basis for phenotypic variation in galactosemia, we have cloned and sequenced the entire gene for human galactose-1-phosphate uridyltransferase . This gene is organized into 11 exons spanning 4 kb . In exons 6, 9, and a portion of 10, there is a high degree of amino acid sequence conservation among Escherichia coli, yeast, mouse, and human . We have identified a number of nucleotide changes in the GALT genes of galactosemic patients that alter conserved amino acids . The most common of these is an A to G transition at nucleotide position 1470, converting a glutamine to an arginine at amino acid codon position 188 (Q188R).(ABSTRACT TRUNCATED AT 250 WORDS)

Genomics, 1992 Oct, 14(2), 249 - 55
Detection of single DNA base mutations with mismatch repair enzymes; Lu AL et al.; A novel method for identifying DNA point mutations has been developed by using mismatch repair enzymes . The high specificity of the Escherichia coli MutY protein has permitted the development of a reliable and sensitive method for the detection and characterization of point mutations in the human genome . The MutY protein is involved in a repair pathway that can convert A/G or A/C mismatches to C/G or G/C basepairs, respectively . A/G or A/C mismatches formed by hybridization between two amplified genomic DNA samples or between specific DNA probes and target DNA are nicked at the mispaired adenine strand by MutY protein . As little as 1% of the mutant sequence can be detected by the mismatch repair enzyme cleavage (MREC) method in a mixture of normal and mutated DNAs (e.g., mutant cells are only present in 1% of the normal cell background) . By using different probes, the assay also can determine the nucleotide sequence of the mutation . We have applied this method to detect single-base substitutions in human oncogenes.

Genetics, 1992 Oct, 132(2), 295 - 302
DNA inversions between short inverted repeats in Escherichia coli; Schofield MA et al.; Using site-specific mutagenesis in vitro, we have constructed Escherichia coli strains that allow the detection of the inversion of an 800-bp segment in the lac region . The invertible segment is bounded by inverted repeats of either 12 or 23 bp . Inversions occurring at these inverted repeats will restore the Lac+ phenotype . Inversions can be detected at both short homologies at frequencies ranging from 0.5 x 10(-8) to 1 x 10(-7) . These events, which have been verified by DNA sequence analysis, are reduced up to 1000-fold in strains deficient for either RecA, RecB or RecC . They are not reduced in strains deficient in the RecF, J pathway . These results show that the RecB,C,D system can mediate rearrangements at short sequence repeats, and probably plays a major role in cellular rearrangements.

FEMS Microbiol Lett, 1992 Oct 1, 76(1-2), 41 - 4
In vivo recombination and the production of hybrid genes; Calogero S et al.; In vivo recombination between homologous genes is increasingly being favoured as a means of generating proteins with altered and novel specificities . The typical procedure requires the cloning of two related genes on a single replicative plasmid of Escherichia coli and the selection or screening of recombinants . Up to now the recombination process between the cloned genes was generally thought to involve the recA function and the availability of free ends in the DNA molecule to be recombined . Our results show that neither is necessary . Recombinants are obtained by simply growing the bacteria that host the plasmid carrying the two cloned genes.

Chirurg, 1992 Oct, 63(10), 832 - 6
{Necrotizing fasciitis . Case report and review of the literature}; Wolters U et al.; The necrotizing fasciitis is an infection with no or only a small trauma . It is a rapidly progressing process especially with necrosis and edema of subcutaneous fat and adjacent fascia underlined by systemic toxicity but spearing of skin and muscle by the initial process . Late recognition and lack of immediate aggressive surgical treatment contribute to the distressingly high mortality . Early and complete debridement offers the best chance for cure.

Comput Appl Biosci, 1992 Oct, 8(5), 511 - 20
Dynamic programming algorithms for restriction map comparison; Huang X et al.; For most sequence comparison problems there is a corresponding map comparison algorithm . While map data may appear to be incompatible with dynamic programming, we show in this paper that the rigor and efficiency of dynamic programming algorithms carry over to the map comparison algorithms . We present algorithms for restriction map comparison that deal with two types of map errors: (i) closely spaced sites for different enzymes can be ordered incorrectly, and (ii) closely spaced sites for the same enzyme can be mapped as a single site . The new algorithms are a natural extension of a previous map comparison model . Dynamic programming algorithms for computing optimal global and local alignments under the new model are described . The new algorithms take about the same order of time as previous map comparison algorithms . Programs implementing some of the new algorithms are used to find similar regions within the Escherichia coli restriction map of Kohara et al.

Comput Appl Biosci, 1992 Oct, 8(5), 433 - 41
First and second moment of counts of words in random texts generated by Markov chains; Kleffe J et al.; An exact expression for the variance of random frequency that a given word has in text generated by a Markov chain is presented . The result is applied to periodic Markov chains, which describe the protein-coding DNA sequences better than simple Markov chains . A new solution to the problem of word overlap is proposed . It was found that the expected frequency and overlapping properties determine most of the variance . The expectation and variance of counts for triplets are compared with experimental counts in Escherichia coli coding sequences.

Biophys Chem, 1992 Oct, 44(3), 163 - 73
Time-resolved intrinsic fluorescence of Enzyme I . The monomer/dimer transition; Chauvin F et al.; Enzyme I of the bacterial phosphotransferase system can exist in a monomer/dimer equilibrium which may have functional significance . Each monomer contains two tryptophan residues . It is demonstrated that the decay of both the monomer and the dimer can be described by a biexponential . The decay times depend on the temperature and at 6 degrees C the decay times are tau 1 = 0.4 ns and tau 2 = 3.2 ns for the monomer and tau 3 = 3.2 ns and tau 4 = 7.2 ns for the dimer form of the enzyme . The changes in the fluorescence decay parameters can be utilized to measure the equilibrium constant for the monomer/dimer transition.

Biophys J, 1992 Oct, 63(4), 1026 - 31
Study of mechanisms of electric field-induced DNA transfection . IV . Effects of DNA topology on cell uptake and transfection efficiency; Xie TD et al.; Electric parameters and solvent conditions are known to influence the efficiency of DNA transfection of cells by a pulsed electric field (PEF) . A previous study (Neumann, E., M . Schaefer-Ridder, Y . Wang, and P . H . Hofschneider . 1982 . EMBO (Eur . Mol . Biol . Organ.) J . 1:841-845) has indicated that DNA topology is also an important determinant . We report an investigation of the PEF induced uptake, stability, and expression of three different topological isomers, circular supercoiled (scDNA), circular relaxed (crDNA), and linearized (lnDNA) forms of the plasmid pBR322, by Escherichia coli strain JM105 . Monomeric pBR322 prepared by the electroelution from an agarose gel was in the supercoiled form . Treatment of the scDNA with wheat germ topoisomerase I removed the superhelicity and the DNA assumed the relaxed circular form . Treatment of scDNA by a restriction endonuclease, EcoRI or Hind III, linearized the DNA . The MgCl2-dependent bindings of all three forms of DNA to the cell surface were indistinguishable . So was the PEF induced cell uptake . In contrast, the transfection efficiency (TE) for the scDNA and the crDNA were high (approximately 2 x 10(8) micrograms-1 DNA at neutral pH), whereas that for the lnDNA was approximately five orders of magnitude lower (less than 1 x 10(3) micrograms-1 DNA) . Analysis by agarose gel electrophoresis indicated that the PEF loaded lnDNA was degraded by the host cell within 3 h . However, the loaded scDNA and the crDNA were stable and expressed in the cytoplasm . We conclude that first, the PEF induced DNA entry into E . coli did not depend on the topology of the DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Reprod Dev, 1992 Oct, 33(2), 149 - 59
Unexpected position-dependent expression of H-2 and beta 2-microglobulin/lacZ transgenes; Cohen-Tannoudji M et al.; In order to study sequences involved in the developmentally regulated and tissue-specific expression of the class I Major Histocompatibility Complex (MHC) genes, we have constructed several H-2/lacZ transgenic lines in which the 5' regulatory sequences of the H-2Kb gene are linked to the Escherichia coli beta-galactosidase (lacZ) gene . In five H-2/lacZ lines, the pattern of lacZ expression, detected histochemically varied greatly from line to line . None of the H-2/lacZ transgenes were transcribed in cells normally expressing a high level of endogenous H-2 molecules, although these H-2 regulatory sequences have been shown to be sufficient to drive tissue-specific expression of other reporter genes . Interestingly, when constructs containing 5' beta 2-microglobulin (beta 2m) regulatory sequences linked to lacZ were used to derive transgenic lines, similar results were obtained . A survey of lacZ labeling in H-2/lacZ and beta 2m/lacZ transgenic mice strongly suggests that these transgenes are very sensitive to position effect, lacZ expression being controlled by endogenous chromosomal regulatory elements specific for each insertion site . Here we describe the complex pattern of lacZ expression in the different transgenic lines during development; we discuss the unusual properties of these transgenes and underline their potential use for developmental studies and characterization of genomic sequences involved in spatiotemporal gene expression.

Mol Reprod Dev, 1992 Oct, 33(2), 131 - 40
Cyclin B in fish oocytes: its cDNA and amino acid sequences, appearance during maturation, and induction of p34cdc2 activation; Hirai T et al.; Under the influence of maturation-inducing hormone (MIH) secreted from follicle cells, oocyte maturation is finally triggered by maturation-promoting factor (MPF), which consists of a homolog of the cdc2+ gene product of fission yeast (p34cdc2) and cyclin B . Two species of cyclin B clones were isolated from a cDNA library constructed from mature goldfish oocytes . Sequence comparisons revealed that these two clones are highly homologous (95%) and were found to be similar to Xenopus cyclin B1 . Using monoclonal antibodies against Escherichia coli-produced goldfish cyclin B and the PSTAIR sequence of p34cdc2, we examined the levels of cyclin B and p34cdc2 proteins during goldfish oocyte maturation induced in vitro by 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17 alpha, 20 beta-DP), a natural MIH in fish . Protein p34cdc2 was found in immature oocyte extracts and did not remarkably change during oocyte maturation . Cyclin B was not detected in immature oocyte extracts and appeared when oocytes underwent germinal vesicle breakdown . Cyclin B that appeared during oocyte maturation was labelled with {35S}methionine, indicating its de novo synthesis . Introduction of E . coli-produced cyclin B into immature oocyte extracts induced p34cdc2 (MPF) activation . Although the possibility that immature goldfish oocytes contain an insoluble cyclin B is not completely excluded, these results strongly suggest that 17 alpha, 20 beta-DP induces oocytes to synthesize cyclin B, which in turn activates preexisting p34cdc2, forming active MPF.

Biol Chem Hoppe Seyler, 1992 Oct, 373(10), 1067 - 73
Dihydropteridine reductase from Escherichia coli exhibits dihydrofolate reductase activity; Vasudevan SG et al.; E . coli Dihydropteridine reductase, known to have a pterin-independent oxidoreductase activity with potassium ferricyanide as electron donor, has now been shown to possess also dihydrofolate reductase activity . The kinetic parameters for dihydrofolate reductase activity have been determined . The ratio of the three activities, dihydropteridine reductase, dihydrofolate reductase and pterin-independent oxidoreductase activity is 1.0, 0.05 and 4.3, respectively . The enzyme, a flavoprotein which is unstable in the presence of dithiothreitol, was shown to be a monomer with a molecular mass of 25.7 kDa . The apparent lack of discrimination between hydride transfer from the pyridine nucleotide to N5 of the pterin in the dihydropteridine reductase reaction and C6 of folate in the dihydrofolate reaction suggested that the FAD prosthetic group may be involved in the hydride transfers . The flavoprotein inhibitor N,N- dimethylpropargylamine inhibited the dihydropteridine reductase and oxidoreductase reactions differently and did not affect the dihydrofolate reductase activity however.

Biochem J, 1992 Oct 1, 287 ( Pt 1), 21 - 9
Biosynthesis of heparin . The D-glucuronosyl- and N-acetyl-D-glucosaminyltransferase reactions and their relation to polymer modification; Lidholt K et al.; Oligosaccharides with the general structure {GlcA-GlcNAc}n-GlcA-aMan (aMan is 2,5-anhydro-D-mannose), derived from the Escherichia coli K5 capsular polysaccharide, were found to serve as monosaccharide acceptors for a GlcNAc-transferase, solubilized from a mouse mastocytoma microsomal fraction and implicated in heparin biosynthesis . Digestion of these oligosaccharides with beta-D-glucuronidase yielded acceptors for the GlcA-transferase that acts in concert with the GlcNAc-transferase . Assays based on the oligosaccharide acceptors showed broad pH optima for the two enzymes, centred around pH 6.5 for the GlcNAc-transferase and around pH 7.0 for the GlcA-transferase . The GlcNAc-transferase showed an absolute requirement for Mn2+, whereas the GlcA-transferase was stimulated by Ca2+ and Mg2+ but not by Mn2+ . The GlcNAc acceptor ability of the {GlcA-GlcNAc}n-GlcA-aMan oligosaccharides increased with increasing chain length, as reflected by the apparent Km, which was 60 microM for a hexasaccharide but 6 microM for a hexadecasaccharide . By contrast, the Km for {GlcNAc-GlcA}n-aMan oligosaccharides in the GlcA-transferase reaction was higher, approximately 0.5 mM, and unaffected by acceptor size . After chemical modification of the oligosaccharides to obtain mixed N-substituents (N-unsubstituted, N-acetylated or N-sulphated GlcN residues), GlcNAc transfer was found to be virtually independent of the N-substituent pattern of the acceptor sequence . The GlcA-transferase, on the other hand, showed marked preference for an acceptor with a non-reducing-terminal GlcNAc-GlcA-GlcNSO3- sequence, which would thus have a lower Km for the enzyme than the corresponding fully N-acetylated structure . These results, along with our previous finding that chain elongation in a mastocytoma microsomal system is strongly promoted by concomitant N-sulphation of the nascent chain {Lidholt, Kjellen & Lindahl (1989) Biochem . J . 261, 999-1007}, raise the possibility that the glycosyltransferases and the N-deacetylase/N-sulphotransferase act in concert during chain elongation, assembled into an enzyme complex.

Am Rev Respir Dis, 1992 Oct, 146(4), 997 - 1002
Effect of sulfidopeptide leukotriene receptor antagonists on endotoxin-induced pulmonary dysfunction in awake sheep; Miller RF et al.; We studied the effects of two structurally unrelated sulfidopeptide leukotriene receptor antagonists on endotoxin-induced pulmonary dysfunction in chronically instrumented unanesthetized sheep . The agents employed were L-660,711 (MK-571) (Merck-Frosst, Canada) and SK&F 104,353 (Smith Kline and French, King of Prussia, PA) . The efficacy and specificity of the agents were verified in sheep by administering boluses of exogenous leukotrienes (LTB4, LTC4, LTD4, and LTE4) in doses as great as 100 micrograms while monitoring lung mechanics and vascular pressures . The antagonists blocked the changes in lung mechanics and pulmonary hemodynamics induced by the sulfidopeptide leukotrienes (LTC4, LTD4, and LTE4) while having no effect on the animals' responses to LTB4 . The endotoxin studies were performed by administering endotoxin alone (Escherichia coli endotoxin 0.75 microgram/kg) or endotoxin after pretreatment with one of the sulfidopeptide leukotriene receptor antagonists . In control studies, each animal received a continuous infusion of one of the receptor antagonists for a duration identical to that of the endotoxin studies . Neither L-660,711 nor SK&F 104,353 significantly altered the endotoxin-induced changes in pulmonary hemodynamics, lung mechanics, lung fluid and solute exchange, oxygenation, or leukopenia . Peak lung lymph thromboxane B2 levels were significantly lower in sheep pretreated with L-660,711 . When the antagonists were given alone, no effects were seen . We conclude that (1) sulfidopeptide leukotrienes do not measurably contribute to endotoxin-induced pulmonary dysfunction in chronically instrumented sheep; (2) sulfidopeptide leukotrienes may contribute to thromboxane release after endotoxin.

Anesthesiology, 1992 Oct, 77(4), 742 - 9
Effects of glucose infusion on cerebral cortical glucose and lactate concentrations during endotoxemia in rats; Sawa T et al.; Hypoglycemia has been observed in several species before death from endotoxemia . Although several studies have emphasized the importance of maintaining brain glucose at normal concentration during endotoxemia, the effect of glucose infusion on cerebral glucose metabolism has not been studied . Accordingly, the effects of glucose infusion on interstitial glucose and lactate concentrations in the cerebral cortex during endotoxemia were studied in 22 Wistar rats . Cerebral glucose and lactate were measured at 30-min intervals for 4 h using microdialysis . Animals were divided into four groups: 1) saline-infused control (n = 5); 2) saline-infused endotoxemia (n = 7); 3) glucose-infused control (n = 5); and 4) glucose-infused endotoxemia (n = 5) . In groups 2 and 4, endotoxemia was induced by intravenous injection with E . coli lipopolysaccharide B (5 mg.100 g-1) . One hour after endotoxin administration, saline or 50% glucose was infused at a flow rate of 0.5 ml.100 g-1.h-1 for 3 h . Endotoxin induced a significant increase (P less than 0.05) in blood glucose in the saline-infused rats, which survived for 4 h (n = 5), from 91.6 +/- 15.4 mg.100 ml-1 at baseline to 136.3 +/- 23.3 mg.100 ml-1 (149%) at 1 h, followed by a gradual decrease (to 63% of the basal concentration at 4 h) . Similar changes were observed in brain glucose (14.9 +/- 1.9 mg.100 ml-1 baseline, 175% of baseline at 2 h, and 57% of baseline at 4 h).(ABSTRACT TRUNCATED AT 250 WORDS)

Proc Natl Acad Sci U S A, 1992 Oct 1, 89(19), 9331 - 4
Release of N2,3-ethenoguanine from chloroacetaldehyde-treated DNA by Escherichia coli 3-methyladenine DNA glycosylase II; Matijasevic Z et al.; The human carcinogen vinyl chloride is metabolized in the liver to reactive intermediates which form N2,3-ethenoguanine in DNA . N2,3-Ethenoguanine is known to cause G----A transitions during DNA replication in Escherichia coli, and its formation may be a carcinogenic event in higher organisms . To investigate the repair of N2,3-ethenoguanine, we have prepared an N2,3-etheno{14C}guanine-containing DNA substrate by nick-translating DNA with {14C}dGTP and modifying the product with chloroacetaldehyde . E . coli 3-methyladenine DNA glycosylase II, purified from cells which carry the plasmid pYN1000, releases N2,3-ethenoguanine from chloroacetaldehyde-modified DNA in a protein- and time-dependent manner . This finding widens the known substrate specificity of glycosylase II to include a modified base which may be associated with the carcinogenic process . Similar enzymatic activity in eukaryotic cell might protect them from exposure to metabolites of vinyl chloride.

Proc Natl Acad Sci U S A, 1992 Oct 1, 89(19), 9262 - 6
Striking effects of coupling mutations in the acceptor stem on recognition of tRNAs by Escherichia coli Met-tRNA synthetase and Met-tRNA transformylase; Lee CP et al.; We measured kinetic parameters in vitro and directly analyzed aminoacylation and formylation levels in vivo to study recognition of Escherichia coli initiator tRNA mutants by E . coli Met-tRNA synthetase and Met-tRNA transformylase . We show that, in addition to the anticodon sequence, mutations in the "discriminator" base A73 also affect aminoacylation . An A73----U change has a small effect, but a change to G73 or C73 significantly lowers Vmax/Kappm for in vitro aminoacylation and leads to appreciable accumulation of uncharged tRNA in vivo . Significantly, coupling of the G73 mutation with G72, a neighboring-base mutation, results in a tRNA essentially uncharged in vivo . Coupling of C73 and U73 mutations with G72 does not have such an effect . Elements crucial for Met-tRNA transformylase recognition of tRNAs are located at the end of the acceptor stem . These elements include a weak base pair or a mismatch between nucleotides (nt) 1 and 72 and base pairs 2.71 and 3.70 . The natures of nt 1 and 72 are less important than the fact that they do not form a strong Watson-Crick base pair . Interestingly, the negative effect of a C.G base pair between nt 1 and 72 is suppressed by mutation of the neighboring nucleotide A73 to either C73 or U73 . The presence of C73 or U73 could destabilize the C1.G72 base pair at the end of an RNA helix . Thus, in some tRNAs, the discriminator base could affect stability of the base pair between nt 1 and 72 and thereby the structure of tRNA at the end of the acceptor stem.

Proc Natl Acad Sci U S A, 1992 Oct 1, 89(19), 9257 - 61
Escherichia coli biotin holoenzyme synthetase/bio repressor crystal structure delineates the biotin- and DNA-binding domains; Wilson KP et al.; The three-dimensional structure of BirA, the repressor of the Escherichia coli biotin biosynthetic operon, has been determined by x-ray crystallography and refined to a crystallographic residual of 19.0% at 2.3-A resolution . BirA is a sequence-specific DNA-binding protein that also catalyzes the formation of biotinyl-5'-adenylate from biotin and ATP and transfers the biotin moiety to other proteins . The level of biotin biosynthetic enzymes in the cell is controlled by the amount of biotinyl-5'-adenylate, which is the BirA corepressor . The structure provides an example of a transcription factor that is also an enzyme . The structure of BirA is highly asymmetric and consists of three domains . The N-terminal domain is mostly alpha-helical, contains a helix-turn-helix DNA-binding motif, and is loosely connected to the remainder of the molecule . The central domain consists of a seven-stranded mixed beta-sheet with alpha-helices covering one face . The other side of the sheet is largely solvent-exposed and contains the active site . The C-terminal domain comprises a six-stranded, antiparallel beta-sheet sandwich . The location of biotin binding is consistent with mutations that affect enzymatic activity . A nearby loop has a sequence that has been associated with phosphate binding in other proteins . It is inferred that ATP binds in this region, adjacent to the biotin . It is proposed that the binding of corepressor to monomeric BirA may promote DNA binding by facilitating the formation of a multimeric BirA-corepressor-DNA complex . The structural details of this complex remain an open question, however.

Proc Natl Acad Sci U S A, 1992 Oct 1, 89(19), 9186 - 90
Interactions between natural polyamines and tRNA: an 15N NMR analysis; Frydman L et al.; 15N NMR spectroscopy was used to explore the interactions between natural polyamines and Escherichia coli tRNA . It was found that when tRNA is added to solutions of 15N-labeled spermine or spermidine, there is a considerable decrease in the relative heights of the -NH(2+)--resonances with respect to the signals arising from the -NH3+ groups . The presence of tRNA was also found to reduce the longitudinal relaxation times T1 of the nitrogens, mainly those of the -NH(2+)- groups . The longitudinal relaxation times of the nitrogens were used to characterize the temperature dependence of the binding, and they allowed us to calculate the activation energies that determine the correlation times of amino groups in the presence of tRNA . Both the thermodynamic and the relaxation results indicate that (i) spermine binds more strongly to tRNA than spermidine does and (ii) within each of these molecules the -NH(2+)- groups bind more strongly to tRNA than the more electropositive -NH3+ moieties . This specificity suggests that the interaction between polyamines and tRNA cannot be described exclusively in terms of electrostatic forces and that other interactions (most likely, hydrogen bonding) are very important for establishing the polyamine-tRNA link . Some of the factors that may conspire against the binding of -NH3+ groups to tRNA are briefly discussed.

Proc Natl Acad Sci U S A, 1992 Oct 1, 89(19), 9089 - 93
Precursor structure, expression, and tissue distribution of human guanylin; de Sauvage FJ et al.; Heat-stable enterotoxins (STa) are small, cysteine-rich peptides secreted by Escherichia coli that are able to induce diarrhea through the stimulation of an intestine-specific receptor-guanylyl cyclase known as STaR . A 15-amino acid peptide, guanylin, was recently purified from rat jejunum and proposed to be a potential endogenous activator of this receptor . We describe here the cloning and characterization of human and mouse cDNAs encoding precursor proteins of 115 and 116 amino acids, respectively, having guanylin present at their C termini . Expression of the human cDNA in mammalian cells leads to the secretion of proguanylin, an inactive 94-amino acid protein . Guanylin generated by either trypsin or acid treatment of proguanylin was purified and found to bind to, and activate, STaR . Northern blot and in situ hybridization show high-level expression of guanylin mRNA restricted to the intestine, with localization to Paneth cells at the base of the small intestinal crypts . These results demonstrate that guanylin is an endogenous activator of STaR.

Proc Natl Acad Sci U S A, 1992 Oct 1, 89(19), 8923 - 7
Monomers and dimers of the RepA protein in plasmid pSC101 replication: domains in RepA; Manen D et al.; The replication of plasmid pSC101 requires the plasmid-encoded protein RepA . This protein has a double role: it binds to three directly repeated sequences in the pSC101 origin and promotes replication of the plasmid; it binds to two inversely repeated sequences in its promoter region and regulates its own transcription . A series of RepA protein derivatives carrying deletions of the C-terminal region were assayed for specific binding . We found that the last third of the protein is not needed for binding to the various specific sites . Truncated proteins that still bind can also form heterodimers with a wild-type protein . Analysis of band retardation assays conducted with wild-type and truncated proteins indicates that RepA binds to directly repeated sequences as a monomer and to inversely repeated sequences as a dimer.

Proteins, 1992 Oct, 14(2), 168 - 77
Functional roles of amino acid residues involved in forming the alpha-helix-turn-alpha-helix operator DNA binding motif of Tet repressor from Tn10; Baumeister R et al.; The Tn10 derived Tet repressor contains an amino acid segment with high homology to the alpha-helix-turn-alpha-helix motif (HTH) of other DNA binding proteins . The five most conserved amino acids in HTH are probably involved in structural formation of the motif . Their functional role was probed by saturation mutagenesis yielding 95 single amino acid replacement mutants of Tet repressor . Their binding efficiencies to tet operator were quantitatively determined in vivo . All functional mutants contain amino acid substitutions consistent with their proposed role in a HTH . In particular, only the two smallest amino acids (serine, glycine) can substitute a conserved alanine in the proposed first alpha-helix without loss of activity . The last position of the first alpha-helix, the second position in the turn, and the fourth position in the second alpha-helix require mostly hydrophobic residues . The proposed C-terminus of the first alpha-helix is supported by a more active asparagine compared to glutamine replacement mutant of the wt leucine residue . The turn is located close to the protein surface as indicated by functional lysine and arginine replacements for valine . A glycine residue at the first position in the turn can be replaced by any amino acid yielding mutants with at least residual tet operator affinity . A structural model of the HTH of Tet repressor is presented.

Pediatrics, 1992 Oct, 90(4), 616 - 21
Risk factors for the central nervous system manifestations of gastroenteritis-associated hemolytic-uremic syndrome; Cimolai N et al.; Hemolytic-uremic syndrome is usually a consequence of enteric verotoxigenic Escherichia coli infection, and a prevailing hypothesis contends that systemically absorbed verotoxins are responsible for the multiple organ involvement . In an attempt to determine whether the central nervous system (CNS) manifestations could occur owing to factors that reflect a toxin insult, the authors studied the association of clinical and laboratory variables with the development of neurological disease . Ninety-one patients with hemolytic-uremic syndrome from 1982 through 1990 were included . Twenty-seven (18 female, 9 male) had a CNS disorder; 17 of these had seizures and there were two deaths . Multivariate analyses led to the following observations: female gender (odds ratio {OR} 8.50; 95% confidence interval {CI} 2.08 to 50.0), prolonged use of an antimotility pharmacological agent (OR 8.50; 95% CI 1.69 to 42.81), and an increased hemoglobin level (OR 1.11; 95% CI 1.05 to 1.17) were associated with an increased risk for developing a neurological manifestation . Prior administration of a blood product was associated with a decreased risk (OR 0.12; 95% CI 0.02 to 0.52) . The findings suggest that other mechanisms for CNS disease may exist in addition to direct toxin insult.

Otolaryngol Clin North Am, 1992 Oct, 25(5), 1011 - 6
Strategies for constructing a guinea pig organ of Corti cDNA library and its potential use; Wilcox ER; Mutations of genes common to several tissues or organs can lead to cellular damage, which may result in hearing impairment as part of a syndromic disorder . Mutations of genes that are unique to the organ of Corti would have a high probability of causing nonsyndromic hearing impairment . It is expected that such genes are involved in auditory transduction as well as in maintaining specific hair cell and supporting cell functions in the organ of Corti . Cloning and describing genes involved with nonsyndromic hearing impairment thus require the construction of a guinea pig cDNA library of the organ of Corti.

Nature, 1992 Oct 1, 359(6394), 387 - 93
Crystal structure of the met repressor-operator complex at 2.8 A resolution reveals DNA recognition by beta-strands; Somers WS et al.; The crystal structure of the met repressor-operator complex shows two dimeric repressor molecules bound to adjacent sites 8 base pairs apart on an 18-base-pair DNA fragment . Sequence specificity is achieved by insertion of double-stranded antiparallel protein beta-ribbons into the major groove of B-form DNA, with direct hydrogen-bonding between amino-acid side chains and the base pairs . The repressor also recognizes sequence-dependent distortion or flexibility of the operator phosphate backbone, conferring specificity even for inaccessible base pairs.

Mol Cell Biol, 1992 Oct, 12(10), 4271 - 8
Regulated expression of a mammalian nonsense suppressor tRNA gene in vivo and in vitro using the lac operator/repressor system; Syroid DE et al.; We have exploited the Escherichia coli lac operator/repressor system as a means to regulate the expression of a mammalian tRNA gene in vivo and in vitro . An oligonucleotide containing a lac operator (lacO) site was cloned immediately upstream of a human serine amber suppressor (Su+) tRNA gene . Insertion of a single lac repressor binding site at position -1 or -32 relative to the coding region had no effect on the amount of functional tRNA made in vivo, as measured by suppression of a nonsense mutation in the E . coli chloramphenicol acetyltransferase gene following cotransfection of mammalian cells . Inclusion of a plasmid expressing the lac repressor in the transfections resulted in 75 to 98% inhibition of suppression activity of lac operator-linked tRNA genes but had no effect on expression of the wild-type gene . Inhibition could be quantitatively relieved with the allosteric inducer isopropylthio-beta-D-galactoside (IPTG) . Similarly, transcription in vitro of lac operator-linked tRNA genes in HeLa cell extracts was repressed in the presence of lac repressor, and this inhibition was reversible with IPTG . These results demonstrate that the bacterial lac operator/repressor system can be used to reversibly control the expression of mammalian genes that are transcribed by RNA polymerase III.

Leuk Res, 1992 Oct, 16(10), 1025 - 9
Prolongation by differentiation-stimulating factor/leukemia inhibitory factor of the survival time of mice implanted with mouse myeloid leukemia cells; Yamamoto-Yamaguchi Y et al.; Mouse myeloid leukemic M1 cells can be induced to differentiate into macrophages by differentiation-stimulating factor (D-factor)/leukemia inhibitory factor (LIF) . We examined the effect of D-factor on the survival times of syngeneic mice implanted with two different clones (T-22 and R-4) of M1 cells . D-factor induced differentiation and suppressed DNA synthesis of sensitive T-22 cells but not resistant R-4 cells in vitro . For in vivo experiments, we used recombinant mouse D-factor (rmD-factor) produced in mammalian cells, which is glycosylated and is more stable in vitro and in vivo than unglycosylated rmD-factor produced in Escherichia coli . Treatment with rmD-factor prolonged the survival times of mice implanted with T-22 cells but not R-4 cells.

J Trop Med Hyg, 1992 Oct, 95(5), 316 - 21
Enterotoxigenic drug resistant plasmids in animal isolates of Escherichia coli and their zoonotic importance; Singh M et al.; A study was undertaken to determine how widely drug resistant enterotoxigenic plasmids are distributed among the animal isolates of Escherichia coli, and their potential for exchange . Thirty-one strains of E . coli isolated from metritis, septicaemia and diarrhoea of animals were tested for the production of enterotoxins . Thirteen strains were enterotoxin producers . Five produced heat-stable enterotoxins (ST), four heat-labile (LT) and four strains produced both (LT/ST) types of enterotoxins . Most of the enterotoxin producing E . coli strains were isolated from diarrhoeal sources, followed by septicaemia and metritis . Of the 13 enterotoxigenic E . coli, nine were resistant to various antibiotics which transferred their drug resistance partially or completely into an E . coli K-12 recipient . Only one strain of serogroup 032, isolated from equine metritis, transferred ampicillin, oxytetracycline and doxycycline resistance and heat-labile enterotoxin biosynthesis determinants en bloc . Plasmid DNA analysis of the exconjugants showed the presence of a 41.8 MDa conjugative plasmid . The presence of such autotransferable enterotoxin biosynthesis replicons in their unusual ecological niches such as metritis and septicaemia suggests the ubiquitous nature of enterotoxin genes which perhaps were selected and spread due to the presence of genes for drug resistance . The potential of cross-infection of human beings from animals and vice versa via such plasmids is discussed.

J Mol Evol, 1992 Oct, 35(4), 318 - 28
The nucleotide sequence of the entire ribosomal DNA operon and the structure of the large subunit rRNA of Giardia muris; van Keulen H et al.; The total nucleotide sequence of the rDNA of Giardia muris, an intestinal protozoan parasite of rodents, has been determined . The repeat unit is 7668 basepairs (bp) in size and consists of a spacer of 3314 bp, a small-subunit rRNA (SSU-rRNA) gene of 1429, and a large-subunit rRNA (LSU-rRNA) gene of 2698 bp . The spacer contains long direct repeats and is heterogeneous in size . The LSU-rRNA of G . muris was compared to that of the human intestinal parasite Giardia duodenalis, to the bird parasite Giardia ardeae, and to that of Escherichia coli . The LSU-rRNA has a size comparable to the 23S rRNA of E . coli but shows structural features typical for eukaryotes . Some variable regions are typically small and account for the overall smaller size of this rRNA . The structure of the G . muris LSU-rRNA is similar to that of the other Giardia rRNA, but each rRNA has characteristic features residing in a number of variable regions.

J Nucl Med, 1992 Oct, 33(10), 1810 - 5
Localization of infection using streptavidin and biotin: an alternative to nonspecific polyclonal immunoglobulin; Rusckowski M et al.; Since favorable images of infection are obtained with radio-labeled nonspecific IgG, streptavidin has been considered as an alternative protein in this investigation . The advantage of streptavidin is that once localized it may be targeted with radiolabeled biotin . Studies were conducted in a mouse model with an Escherichia coli infection in one thigh . Indium-111-labeled streptavidin showed equivalent localization to the infection as that obtained with 111In-labeled polyclonal nonspecific IgG, however blood levels with streptavidin were lower at all time points; consequently, target-to-blood ratios were improved . Pretargeting with unlabeled streptavidin followed 3 hr later with 111In-labeled biotin showed equivalent localization in the target and reduced activity in all organs sampled . As such, infected thigh-to-normal thigh ratios were improved 3-fold for pretargeting versus either labeled IgG or streptavidin . Improvements in infected thigh-to-liver and blood ratios were greater than 8-fold . Only in the case of kidneys was the ratio unimproved . In conclusion, we have shown that by preadministration of unlabeled streptavidin followed by labeled biotin, infectious lesions in a mouse model may be imaged earlier with lower background levels relative to the administration of labeled nonspecific IgG.

J Exp Med, 1992 Oct 1, 176(4), 1007 - 13
Identification of a motif for HLA-DR1 binding peptides using M13 display libraries; Hammer J et al.; Oligonucleotides encoding peptides known to bind to HLA-DR1 molecules have been inserted into the gene III of filamentous M13 phages . DR1 molecules purified from human lymphoblastoid cell lines could specifically bind to these peptide sequences expressed on the phage surface . A M13 phage peptide library was next constructed and screened with DR1 molecules . After four rounds of selection, more than 80% of the phages were able to bind to DR1 . Competition experiments with both isolated phages and corresponding synthetic peptides showed that the binding was specific . Sequence analysis of the peptide encoding region of 60 phages binding to DR1 molecules and comparison with phages of the original library revealed two potential anchor positions . The first was an aromatic residue (Tyr, Phe, or Trp) at the NH2 terminus of the peptide sequences, and the second was located three residues downstream and consisted of Met or Leu . In addition, the negatively charged amino acids Asp and Glu were mostly excluded from the DR1 binding sequences, and the small amino acid residues Gly and Ala were enriched at position 6 . As for DR1, this approach should enable one to easily determine the binding motifs of other MHC class II alleles and isotypes . Furthermore, it could have interesting applications in the design of major histocompatibility complex-specific antagonists.

J Lab Clin Med, 1992 Oct, 120(4), 633 - 8
Cirrhosis impairs serum bacteriostasis for Escherichia coli; Lister PD et al.; To study the effects of cirrhosis on serum inhibition of Escherichia coli, cirrhosis with ascites was induced in male Sprague-Dawley rats by intragastric administration of carbon tetrachloride . Heat-inactivated (56 degrees C for 30 minutes) serum from cirrhotic rats (CRS) or that from control rats (NRS) was inoculated with 1 x 10(5) colony-forming units per milliliter (CFU/ml) of E . coli, and growth was measured after 24 hours . The mean growth of E . coli in CRS was significantly higher than growth in NRS: 3.5 +/- 5.4 x 10(8) CFU/ml versus 1.2 +/- 2.0 x 10(6) CFU/ml, respectively (p < 0.01) . Fifty-four percent of CRS samples (22/41) completely lacked bacteriostatic activity . These CRS samples were categorized as growth-supporting (G+CRS) because their growth exceeded the mean + 2 SD of NRS (5.2 x 10(6) CFU/ml) . Serum bacteriostasis could be restored to G+CRS by adding purified rat apotransferrin (1 mg/ml), suggesting the presence of excess iron in G+CRS . However, serum iron concentration (SI) and total iron binding capacity (TIBC) were virtually the same in G+CRS (SI = 120 +/- 22 micrograms/dl; TIBC = 351 +/- 45 micrograms/dl) as in growth-inhibitory CRS (SI = 131 +/- 16 micrograms/dl; TIBC = 347 +/- 46 micrograms/dl) but were significantly less than NRS (SI = 208 +/- 29 micrograms/dl; TIBC = 533 +/- 57 micrograms/dl), p < 0.01 . The percent transferrin saturation was similar in all groups: 34% +/- 6%; 38% +/- 5% and 39% +/- 9%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

J Bacteriol, 1992 Oct, 174(20), 6682 - 4
The presence of only one of five exoribonucleases is sufficient to support the growth of Escherichia coli; Kelly KO et al.; Escherichia coli contains multiple exoribonucleases . Strains lacking the exoribonucleases RNase II, D, BN, T, and PH are inviable . The introduction of a chromosomal, wild-type copy of the gene for any one of these enzymes is sufficient to allow cell growth, with the enzymes being in the following order of effectiveness: RNase T > RNase PH > RNase D > RNase II > RNase BN . The data indicate that these five exoribonucleases functionally overlap in vivo and that any one of them can take over the functions of all the others, although with various efficiencies.

J Bacteriol, 1992 Oct, 174(20), 6608 - 16
Structural characterization of ordered arrays of sn-glycerol-3-phosphate acyltransferase from Escherichia coli; Wilkison WO et al.; Overproduction of the sn-glycerol-3-phosphate acyltransferase in Escherichia coli leads to incorporation of this integral membrane protein into ordered tubular arrays within the cell . Freeze-fracture-etch shadowing was performed on suspensions of partially purified tubules and whole bacteria . This procedure revealed the presence of ridges and grooves defining a set of long-pitch left-handed helical ridges . The long-pitch helices represented chains of acyltransferase dimers . Tubules observed within the cell were often closely packed, with an apparent alignment of grooves and ridges in adjacent tubules . Fracture planes passing through the tubules indicated the presence of a bilayer structure, with some portion of the enzyme being associated with the membrane . The major portion of the enzyme extended from the hydrophilic surface, forming a large globular structure that, in favorable views, displayed a central cavity facing the cytoplasm . Computer analysis of shadowed tubules revealed that the left-handed helices were six stranded, with a pitch of 1,050 A (105.0 nm) and a spacing of 75 A (7.5 nm) between acyltransferase dimers along the chains . Analysis of the predicted secondary structure failed to reveal obvious transmembrane segments, suggesting that very little of the protein was inserted into the bilayer.

J Bacteriol, 1992 Oct, 174(20), 6600 - 7
Purification and properties of an Arthrobacter oxydans P52 carbamate hydrolase specific for the herbicide phenmedipham and nucleotide sequence of the corresponding gene; Pohlenz HD et al.; Arthrobacter oxydans P52 isolated from soil samples was found to degrade the phenylcarbamate herbicides phenmedipham and desmedipham cometabolically by hydrolyzing their central carbamate linkages . The phenylcarbamate hydrolase (phenmedipham hydrolase) responsible for the degradative reaction was purified to homogeneity . The enzyme was shown to be a monomer with a molecular weight of 55,000 . A 41-kb wild-type plasmid (pHP52) was identified in A . oxydans P52, but not in a derivative of this strain that had spontaneously lost the ability to hydrolyze phenylcarbamates, indicating that the gene for phenylcarbamate degradation (pcd) is plasmid encoded . Determination of two partial amino acid sequences allowed the localization of the coding sequence of the pcd gene on a 3.3-kb PstI restriction fragment within pHP52 DNA by hybridization with synthetic oligonucleotides . The phenylcarbamate hydrolase was functionally expressed in Escherichia coli under control of the lacZ promoter after the 3.3-kb PstI fragment was subcloned into the vector pUC19 . A stretch of 1,864 bases within the cloned Pst fragment was sequenced . Sequence analysis revealed an open reading frame of 1,479 bases containing the amino acid partial sequences determined for the purified enzyme . Sequence comparisons revealed significant homology between the pcd gene product and the amino acid sequences of esterases of eukaryotic origin . Subsequently, it was demonstrated that the esterase substrate p-nitrophenylbutyrate is hydrolyzed by phenmedipham hydrolase.

J Bacteriol, 1992 Oct, 174(20), 6432 - 7
An alpha-helical hydrophobic hairpin as a specific determinant in protein-protein interaction occurring in Escherichia coli colicin A and B immunity systems; Geli V et al.; A collection of chimeric pore-forming domains between colicins A and B was constructed to investigate the specific determinants responsible for recognition by the corresponding immunity proteins . The fusion sites in the hybrid proteins were positioned according to the three-dimensional structure of the soluble form of the colicin A pore-forming domain . The hydrophobic hairpin of colicin pore-forming domains, buried in the core of the soluble structure, was the main determinant recognized by the integral immunity proteins . The immunity protein function may require helix-helix recognition within the lipid bilayer.

J Bacteriol, 1992 Oct, 174(20), 6350 - 8
Identification of minor fimbrial subunits involved in biosynthesis of K88 fimbriae; Bakker D et al.; The nucleotide sequences of the genes faeF, faeH, faeI, and faeJ encoding K88 minor fimbrial subunits were determined . Analysis of the primary structure of the gene products revealed that all four proteins are synthesized with an amino-terminal signal sequence . The molecular masses of the mature FaeF, FaeH, FaeI, and FaeJ proteins were calculated to be 15,161, 25,461, 24,804, and 25,093 Da, respectively . FaeH, FaeI, and FaeJ showed significant homology with FaeG, the major fimbrial subunit of K88 fimbriae . Mutations in the respective genes were constructed . Analysis of the mutants showed that the minor fimbrial subunits FaeF and FaeH play an essential role in the biogenesis but not in the adhesive properties of the K88 fimbriae . Mutations in faeI or faeJ had no significant effect on K88 production or adhesive capacity . Specific antisera against FaeF and FaeH were raised by immunization with hybrid Cro-LacZ-FaeF and Cro-LacZ-FaeH proteins . Immunoblotting and immunoelectron microscopy revealed that FaeF and FaeH are located in or along the K88 fimbrial structure.

J Bacteriol, 1992 Oct, 174(19), 6314 - 6
Inhibition of cell division initiation by an imbalance in the ratio of FtsA to FtsZ; Dewar SJ et al.; Elevated levels of FtsA protein block cell division at a very early stage, similar to that caused by inhibition of the action of FtsZ . In contrast, overexpression of FtsA and FtsZ together does not block division . A specific ratio of FtsA to FtsZ protein, therefore, is required for cell division.

J Bacteriol, 1992 Oct, 174(19), 6311 - 3
DNA polymerase I modulates inducible stable DNA replication in Escherichia coli; Ruscitti T et al.; Mutants of Escherichia coli lacking RNase HI activity and cells induced for the SOS response express modes of DNA replication independent of protein synthesis, called constitutive and induced stable DNA replication, respectively . We report here that mutants deleted for the polA gene express induced stable DNA replication at approximately 25-fold the rate of wild-type cells, whereas constitutive stable DNA replication is not enhanced.

J Bacteriol, 1992 Oct, 174(19), 6264 - 9
New mutations in and around the L2 disordered loop of the RecA protein modulate recombination and/or coprotease activity; Larminat F et al.; The RecA protein plays a key role in Escherichia coli recombination and DNA repair . We have created new recA mutants with mutations in the vicinity of the recA430 mutation (Gly-204----Ser) which is known to affect RecA coprotease activity . Mutants carrying recA659 or recA611, located 3 and 7 amino acids downstream of residue 204, respectively, lose all RecA activities, while the mutant carrying recA616, which is located at 12 amino acids from this residue, keeps the coprotease activity but is unable to promote recombination . Complementation experiments show that both mutations recA611 and recA659 are dominant over the wild-type or recA430 allele while recA616 seems to be recessive to recA+ and dominant over recA430 . It is suggested that these mutations are located in RecA domains which direct conformational modifications.

J Bacteriol, 1992 Oct, 174(19), 6256 - 63
DnaK, DnaJ, and GrpE are required for flagellum synthesis in Escherichia coli; Shi W et al.; The DnaK, DnaJ, and GrpE heat shock proteins are required for motility of Escherichia coli . Cells deleted for dnaK or dnaJ, or with some mutations in the dnaK or grpE gene, are nonmotile, lack flagella, exhibit a 10- to 20-fold decrease in the rate of synthesis of flagellin, and show reduced rates of transcription of both the flhD master operon (encoding FlhD and FlhC) and the fliA operon (encoding sigma F) . Genetic studies suggest that DnaK and DnaJ define a regulatory pathway affecting flhD and fliA synthesis that is independent of cyclic AMP-catabolite gene activator protein or the chemotaxis system.

J Bacteriol, 1992 Oct, 174(19), 6247 - 55
A chemotactic signaling surface on CheY defined by suppressors of flagellar switch mutations; Roman SJ et al.; CheY is the response regulator protein that interacts with the flagellar switch apparatus to modulate flagellar rotation during chemotactic signaling . CheY can be phosphorylated and dephosphorylated in vitro, and evidence indicates that CheY-P is the activated form that induces clockwise flagellar rotation, resulting in a tumble in the cell's swimming pattern . The flagellar switch apparatus is a complex macromolecular structure composed of at least three gene products, FliG, FliM, and FliN . Genetic analysis of Escherichia coli has identified fliG and fliM as genes in which mutations occur that allele specifically suppress cheY mutations, indicating interactions among these gene products . We have generated a class of cheY mutations selected for dominant suppression of fliG mutations . Interestingly, these cheY mutations dominantly suppressed both fliG and fliM mutations; this is consistent with the idea that the CheY protein interacts with both switch gene products during signaling . Biochemical characterization of wild-type and suppressor CheY proteins did not reveal altered phosphorylation properties or evidence for phosphorylation-dependent CheY multimerization . These data indicate that suppressor CheY proteins are specifically altered in the ability to transduce chemotactic signals to the switch at some point subsequent to phosphorylation . Physical mapping of suppressor amino acid substitutions on the crystal structure of CheY revealed a high degree of spatial clustering, suggesting that this region of CheY is a signaling surface that transduces chemotactic signals to the switch.

J Bacteriol, 1992 Oct, 174(19), 6207 - 14
Structural characterization and corepressor binding of the Escherichia coli purine repressor; Choi KY et al.; The Escherichia coli purine repressor, PurR, binds to a 16-bp operator sequence and coregulates the genes for de novo synthesis of purine and pyrimidine nucleotides, formation of a one-carbon unit for biosynthesis, and deamination of cytosine . We have characterized the purified repressor . Chemical cross-linking indicates that PurR is dimeric . Each subunit has an N-terminal domain of 52 amino acids for DNA binding and a C-terminal 289-residue domain for corepressor binding . Each domain was isolated after cleavage by trypsin . Sites for dimer formation are present within the corepressor binding domain . The corepressors hypoxanthine and guanine bind cooperatively to distinct sites in each subunit . Competition experiments indicate that binding of one purine abolishes cooperativity and decreases the affinity and the binding of the second corepressor . Binding of each corepressor results in a conformation change in the corepressor binding domain that was detected by intrinsic fluorescence of three tryptophan residues . These experiments characterize PurR as a complex allosteric regulatory protein.

J Bacteriol, 1992 Oct, 174(19), 6145 - 51
The proper ratio of FtsZ to FtsA is required for cell division to occur in Escherichia coli; Dai K et al.; Interactions among cell division genes in Escherichia coli were investigated by examining the effect on cell division of increasing the expression of the ftsZ, ftsA, or ftsQ genes . We determined that cell division was quite sensitive to the levels of FtsZ and FtsA but much less so to FtsQ . Inhibition of cell division due to an increase in FtsZ could be suppressed by an increase in FtsA . Inhibition of cell division due to increased FtsA could be suppressed by an increase in FtsZ . In addition, although wild-type strains were relatively insensitive to overexpression of ftsQ, we observed that cell division was sensitized to ftsQ overexpression in ftsI, ftsA, and ftsZ mutants . Among these, the ftsI mutant was the most sensitive . These results suggest that these gene products may interact and that the proper ratio of FtsZ to FtsA is critical for cell division to occur.

J Bacteriol, 1992 Oct, 174(19), 6054 - 60
Two-stage control of an oxidative stress regulon: the Escherichia coli SoxR protein triggers redox-inducible expression of the soxS regulatory gene; Nunoshiba T et al.; Escherichia coli responds to the redox stress imposed by superoxide-generating agents such as paraquat by activating the synthesis of as many as 80 polypeptides . Expression of a key group of these inducible proteins is controlled at the transcriptional level by the soxRS locus (the soxRS regulon) . A two-stage control system was hypothesized for soxRS, in which an intracellular redox signal would trigger the SoxR protein as a transcriptional activator of the soxS gene and the resulting increased levels of SoxS protein would activate transcription of the various soxRS regulon genes (B . Demple and C.F . Amabile Cuevas, Cell 67:837-839, 1990) . We have constructed operon fusions of the E . coli lac genes to the soxS promoter to monitor soxS transcription . Expression from the soxS promoter is strongly inducible by paraquat in a manner strictly dependent on a functional soxR gene . Several other superoxide-generating agents also trigger soxR(+)-dependent soxS expression, and the inductions by paraquat and phenazine methosulfate were dependent on the presence of oxygen . Numerous other oxidative stress agents (H2O2, gamma rays, heat shock, etc.) failed to induce soxS, while aerobic growth of superoxide dismutase-deficient bacteria triggered soxR-dependent soxS expression . These results indicate a specific redox signal for soxS induction . A direct role for SoxR protein in the activation of the soxS gene is indicated by band-shift and DNase I footprinting experiments that demonstrate specific binding of the SoxR protein in cell extracts to the soxS promoter . The mode of SoxR binding to DNA appears to be similar to that of its homolog MerR in that the SoxR footprint spans the -10 to -35 region of the soxS promoter.

Infect Immun, 1992 Oct, 60(10), 4328 - 34
Influence of Actinobacillus pleuropneumoniae serotype 2 and its cytolysins on porcine neutrophil chemiluminescence; Dom P et al.; The effects of Actinobacillus pleuropneumoniae serotype 2 and its metabolites on the oxidative activity of porcine neutrophils were studied by using a chemiluminescence technique . Viable A . pleuropneumoniae stimulated the production of oxygen radicals by neutrophils . After having reached a peak value, the oxidative activity decreased until a total inhibition of the oxidative activity of the neutrophils was achieved . All effects were neutralized with homologous convalescent-phase pig sera which had been adsorbed by heat-inactivated A . pleuropneumoniae . Inactivated bacteria and bacteria in the presence of chloramphenicol each had no influence on the oxidative activity of neutrophils . In contrast, a heat-labile factor in A . pleuropneumoniae culture supernatants stimulated and inhibited the oxidative activity of the neutrophils in a dose-dependent manner . Undiluted and low dilutions of culture supernatants were toxic for the phagocytes, while high dilutions stimulated the oxygen radical production of the neutrophils . These effects were neutralized with homologous convalescent-phase pig sera . In order to investigate whether the heat-labile factors in the culture supernatant could be cytolysins, we repeated the experiments with cytolysin II and cytolysin III produced by recombinant Escherichia coli . It was demonstrated that stimulation and inhibition could be reproduced by both cytolysins . In conclusion, the observations made in this study showed that A . pleuropneumoniae secretes heat-labile metabolites that stimulate neutrophil-oxidative metabolism at relatively low concentrations and kill the neutrophils at higher concentrations . Cytolysins may be responsible, at least in part, for these effects.

Infect Immun, 1992 Oct, 60(10), 4154 - 67
Experimental verocytotoxemia in rabbits; Richardson SE et al.; The clinicopathologic effects of intravenously administered purified verocytotoxin 1 (VT1; Shiga-like toxin 1) in 2-kg male rabbits was studied . The 50% lethal dose was 0.2 micrograms of protein per kg of body weight (2 x 10(4) 50% cytotoxic doses per kg) . The clinical features included nonbloody diarrhea and a progressive flaccid paresis, usually culminating in death . The histopathology was characterized by edema and hemorrhage in the mucosa and submucosa of the cecum and edema, hemorrhage, and neuronal necrosis in the brain and gray matter of the spinal cord . Thrombotic microangiopathy, the characteristic histopathologic renal lesion in the hemolytic-uremic syndrome, was also found to be the underlying lesion in verocytotoxemic rabbits . To determine the specific distribution of VT1 in rabbit tissues, purified 125I-labelled VT1 was administered intravenously to 20 rabbits (both immunologically naive and VT1-immune rabbits) . The highest specific uptake of 125I-VT1 was in the spinal cord, brain, cecum, colon, and small bowel in unimmunized animals but in the liver, spleen, and lungs in immune animals . Immunofluorescent staining of cecal and spinal cord tissues after intravenous administration of VT1 showed evidence of specific vascular endothelial cell binding of the toxin . The striking correlation of the central nervous system and gastrointestinal localization of 125I-VT1 with the sites of known histopathology is consistent with direct toxin-mediated injury to these tissues, initiated by the specific binding of VT1 to the vascular endothelium . We conclude that the vascular damage induced by VT1 in affected rabbit tissues is similar to that seen in the kidneys and other tissues in patients with verocytotoxin-producing Escherichia coli-associated hemolytic-uremic syndrome . This suggests that although the rabbit model fails to replicate human hemolytic-uremic syndrome, it is useful for studying the pathogenesis of the vascular lesions in verocytotoxin-producing E . coli-associated diseases.

Infect Immun, 1992 Oct, 60(10), 4040 - 50
Identification of tumor necrosis factor as a transcriptional regulator of the phosphoenolpyruvate carboxykinase gene following endotoxin treatment of mice; Hill MR et al.; The decreased synthesis of hepatic phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme of gluconeogenesis, that occurs during endotoxemia was shown previously in rats to occur at the transcriptional level . In the current study, the exogenous administration of human recombinant tumor necrosis factor (TNF), a proximal mediator of endotoxic shock, reduced the PEPCK transcription rate, mRNAPEPCK levels, and PEPCK enzyme activity in a time- and dose-dependent manner in CD-1 mice . Comparable amounts of circulating TNF were measured in mice 2 h after injection of human recombinant TNF (10(5) U) or a 50% lethal dose of Escherichia coli endotoxin (20 mg/kg) . Direct action of TNF to decrease the PEPCK transcription rate was confirmed in vitro with H-4-II-E Reuber hepatoma cells, in which a dose-dependent inhibition of PEPCK transcription was observed with 1 to 100 U of TNF per ml . A role for TNF-elicited changes in PEPCK gene expression during endotoxemia was confirmed by the protective effect of rabbit polyclonal antibodies to recombinant murine TNF . C57BL/6 mice passively immunized with anti-TNF 4 h prior to endotoxin challenge exhibited normal PEPCK enzyme activity . Neutralization of circulating TNF with anti-TNF failed, however, to prevent the hypoglycemia commonly observed during endotoxemia, suggesting the participation of other mediators . Anti-TNF treatment reduced circulating interleukins 1 and 6 at 3 and 6 h after endotoxin treatment, respectively . These results suggest that during endotoxemia, the development of hypoglycemia is multifaceted and that several cytokines are most likely involved . The findings from the Reuber hepatoma cell model afford an opportunity in future work to map putative cytokine response elements in the PEPCK promoter responsible for perturbed hormonal regulation of the gene during endotoxemia.

Epidemiol Infect, 1992 Oct, 109(2), 283 - 9
Virulence factors of enteric Escherichia coli in young Aboriginal children in north-west Australia; Gunzburg ST et al.; Enterotoxigenic Escherichia coli (ETEC) were the most frequently identified enteric pathogens associated with diarrhoea in 0-5 year old Aboriginal children in tropical north-west Australia with an incidence similar to those from other tropical regions . Heat-stable toxin-producing (ST+) strains were associated with diarrhoea throughout the year but heat-labile toxin-producing (LT+) strains were more important in the monsoonal summer season . ST+ strains were commonest in children with diarrhoea between 6 and 18 months of age while LT+ strains were associated with diarrhoea in children aged 18-24 months . Vero-toxigenic E . coli (VTEC) which produced VT1, but not VT2, and enteroadherent (EAF+) E . coli were significant causes of diarrhoea, mainly in children below 18 months but without a seasonal pattern.

Eur J Immunol, 1992 Oct, 22(10), 2609 - 15
Analysis of human/mouse interleukin-6 hybrid proteins: both amino and carboxy termini of human interleukin-6 are required for in vitro receptor binding; Fiorillo MT et al.; The multifunctional cytokine interleukin-6 (IL-6) is a single polypeptide chain consisting of 184 amino acids in man and 187 amino acids in mouse . Despite the relatively high degree of sequence similarity of these two molecules (about 57%), the biological activity in mouse and human IL-6 shows species specificity . Starting with this observation, we constructed interspecies hybrids with the goal of defining which segments of the human IL-6 molecule are involved in human receptor binding . In this manner we generated multiple amino acid substitution mutants which do not contain insertions or deletions as compared with the parental proteins, and which, therefore, should not show dramatic changes in folding . Using two biological assays on cells of human and mouse origin and a recently developed in vitro binding assay to recombinant soluble human IL-6 receptor, we obtained results which indicate that both the amino and carboxy termini are necessary and sufficient for efficient binding, but that the carboxy terminus plays the dominant role in receptor recognition.

Eur J Biochem, 1992 Oct 1, 209(1), 431 - 6
Fast kinetics studies of Escherichia coli electrotransformation; Eynard N et al.; Direct gene transfer is achieved in Escherichia coli by use of square wave electric pulsing . As observed by video monitoring, the field pulse causes bacteria to orientate parallel to the field lines . Rapid kinetic turbidity changes indicate that this process happens quickly . In these circumstances, and in pulsing conditions prone to inducing transformation, only caps are affected by the field . Considerable cytoplasmic ion leakage occurs during the pulse, affecting the interfacial ionic concentration . The pulsing-buffer osmolarity has to be close to that used with protoplasts . Contact between the plasmid and the bacteria can be very short before the pulse but must be present during the pulse . The plasmid remains accessible to externally added DNases up to 5 days after the pulse, suggesting that the transfer step is slow . Electric-field-mediated transfer can be described in two steps: the anchoring process during the pulse, followed by the crossing of the membrane.

Eur J Biochem, 1992 Oct 1, 209(1), 399 - 407
Characterization and molecular cloning of a flavoprotein catalyzing the synthesis of phytofluene and zeta-carotene in Capsicum chromoplasts; Hugueney P et al.; In plants, zeta-carotene is the first visible carotenoid formed in the biosynthetic pathway through the following two-step desaturation reaction: phytoene-->phytofluene--> zeta-carotene . Using Capsicum annuum chromoplast membranes and the reconstitution system previously described {Camara, B., Bardat, F . & Moneger, R . (1982) Eur . J . Biochem . 127, 255-258}, we have attempted to purify the desaturase(s) catalyzing these reactions . The two activities were coincidental during all the purification procedures . Only a single polypeptide with 56 +/- 2 kDa was detected by SDS/PAGE of all active fractions . The enzyme contained protein-bound FAD . Antibodies raised against the purified polypeptide selectively precipitated the phytoene and the phytofluene desaturase activities, thus demonstrating that the enzyme is a bifunctional flavoprotein . The antibodies were used to isolate a full-length cDNA clone from which was deduced the primary structure of the desaturase which contains a characteristic dinucleotide-binding site . Overexpression of the cDNA in Escherichia coli allowed the production of a recombinant desaturase which had all the properties of the chromoplast desaturase . The phytoene/phytofluene desaturase mRNA levels were extremely low in green fruits and increased slightly before detectable carotenoid synthesis and remained constant throughout ripening . However, the desaturase activity and protein levels were found to increase significantly during the chloroplast to chromoplast transition in C . annuum fruits.

Eur J Biochem, 1992 Oct 1, 209(1), 391 - 7
Purification and biochemical characterization of the EcaI DNA methyltransferase; Szilak L et al.; The EcaI GGTNACC-specific DNA-adenine modification methyltransferase has been purified to apparent homogeneity . The active form of the DNA methyltransferase is a single polypeptide . The enzyme has a pH optimum at pH 8.0 and a temperature optimum at 25 degrees C . EcaI DNA methyltransferase transfers one methyl group to the adenine of the recognition site in a single binding event . The Km was 170 nM for DNA and 1.8 microM for the methyl donor S-adenosylmethionine . Methylated DNA is a competitive inhibitor with respect to DNA (Ki = 3.5 nM) . The other product of the DNA-methylation reaction, S-adenosylhomocysteine was found to be a competitive inhibitor with respect to S-adenosylmethionine (Ki = 2.7 microM) . The S-adenosylmethionine analog sinefungin was shown to be a very strong inhibitor (Ki = 3.5 nM) of the DNA methyltransferase reaction.

EMBO J, 1992 Oct, 11(10), 3767 - 75
Mutations in T7 RNA polymerase that support the proposal for a common polymerase active site structure; Bonner G et al.; In order to test the proposal that most nucleotide polymerases share a common active site structure and folding topology, we have generated 22 mutations of residues within motifs A, B and C of T7 RNA polymerase (RNAP) . Characterization of these T7 RNAP mutants showed the following: (i) most of the mutations resulted in moderate to drastic reductions in T7 RNAP transcriptional activity supporting the idea that motifs A, B and C identify part of the polymerase active site; (ii) the degree of conservation of an amino acid within these motifs correlated with the degree to which mutation of that amino acid reduced transcriptional activity, supporting the predictive ability of this alignment in identifying the most functionally critical residues; (iii) a comparison of DNAP I and T7 RNAP mutants revealed similarities (as well as differences) between corresponding mutant phenotypes; (iv) the Klenow fragment structure is shown to provide a reasonable basis for interpretation of the differential effects of mutating different amino acids within motifs A, B and C in T7 RNAP . These observations support the proposal that these polymerase active sites have similar three-dimensional structures.

EMBO J, 1992 Oct, 11(10), 3759 - 66
Coding from a distance: dissection of the mRNA determinants required for the incorporation of selenocysteine into protein; Heider J et al.; Incorporation of selenocysteine into proteins is directed by specifically 'programmed' UGA codons . The determinants for recognition of the selenocysteine codon have been investigated by analysing the effect of mutations in fdhF, the gene for formate dehydrogenase H of Escherichia coli, on selenocysteine incorporation . It was found that selenocysteine was also encoded when the UGA codon was replaced by UAA and UAG, provided a proper codon-anticodon interaction was possible with tRNA(Sec) . This indicates that none of the three termination codons can function as efficient translational stop signals in that particular mRNA position . The discrimination of the selenocysteine 'sense' codon from a regular stop codon has previously been shown to be dependent on an RNA secondary structure immediately 3' of the UGA codon in the fdhF mRNA . It is demonstrated here that the correct folding of this structure as well as the existence of primary sequence elements located within the loop portion at an appropriate distance to the UGA codon are absolutely required . A recognition sequence can be defined which mediates specific translation of a particular codon inside an mRNA with selenocysteine and a model is proposed in which translation factor SELB interacts with this recognition sequence, thus forming a quaternary complex at the mRNA together with GTP and selenocysteyl-tRNA(Sec).

EMBO J, 1992 Oct, 11(10), 3629 - 34
DNA transcription and repressor binding affect deletion formation in Escherichia coli plasmids; Vilette D et al.; Chimeric plasmids containing phage M13 and plasmid pBR322 sequences undergo deletions in Escherichia coli with a high frequency . In all plasmids one deletion endpoint is the M13 replication origin nick site . We examined the effects of transcription on the position of the other deletion end-point, by inserting in the plasmids an inducible promoter followed by a transcription terminator . Transcription dramatically affected deletions in an orientation-dependent way, such that greater than 95% of end-points were localized downstream from the inserted promoter when it faced the major plasmid transcripts . The end-points were not constrained to the transcribed region and were not affected by the orientation of pBR322 DNA replication . We propose that deletion events occur preferentially in a plasmid domain which is rendered positively supercoiled by convergent transcription . We also show that interaction of LacI repressor with the cognate operator generates a localized deletion hot spot . This hot spot is dependent on pBR322 replication, and therefore probably acts by arresting progression of DNA replication.

Chest, 1992 Oct, 102(4), 1095 - 8
Reduction of alveolar-capillary diffusion after inhalation of endotoxin in normal subjects; Herbert A et al.; Normal subjects were exposed to an aerosol of Escherichia coli endotoxin . Carbon monoxide diffusion (Dco), spirometry, blood neutrophils, white blood cells, and platelets were determined at various times thereafter . A significant decrease in Dco and an increase in blood neutrophils was found, with a maximum effect 4 to 8 h after exposure . Exposure to distilled water caused a tendency for Dco to decrease and a significant increase in blood neutrophils . No effect on spirometry or body temperature was detected . It is suggested that the changes observed represent an inflammation at the alveolar level that appears at dose levels of endotoxin below those which cause bronchoconstriction and fever.

Am J Respir Cell Mol Biol, 1992 Oct, 7(4), 455 - 61
Endotoxin induces the expression of macrophage inflammatory protein 1 alpha mRNA by rat alveolar and bone marrow-derived macrophages; Christman JW et al.; Macrophage inflammatory protein 1 alpha (MIP-1 alpha) is a newly described cytokine that is present in large amounts in the culture supernatant of an endotoxin-stimulated murine macrophage-like cell line (RAW 264.7) . There is increasing information that suggests that this cytokine mediates acute neutrophilic inflammation, although the mechanism of mediation is unknown . Data examining the production and regulation of MIP-1 alpha by primary rat macrophages are lacking, and MIP-1 alpha has not been studied previously in an animal model of endotoxin-induced neutrophilic alveolitis . In this study, we performed Northern analysis of steady-state rat MIP-1 alpha mRNA using an oligonucleotide probe complementary to amino acids 4-13 of murine MIP-1 alpha . Our data demonstrate that rat alveolar and bone marrow-derived macrophages can be induced by in vitro endotoxin treatment to express a 1.1-kb MIP-1 alpha mRNA . Expression of the mRNA could be elicited by treatment with 0.1 to 10.0 micrograms/ml of endotoxin in vitro with peak steady-state levels detectable up to 9 h after adding endotoxin to the media . Alveolar macrophages recovered by whole lung lavage from endotoxin-treated rats expressed increased amounts of the mRNA homologous to MIP-1 alpha mRNA when treated in vitro with endotoxin . We also found that rat neutrophils could be induced by endotoxin in vitro to express the MIP-1 alpha mRNA . We were able to identify MIP-1 alpha in culture supernatant from endotoxin-stimulated rat alveolar and bone marrow-derived macrophages by immunoprecipitation with a specific goat anti-murine MIP-1 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Virology, 1992 Oct, 190(2), 606 - 15
Dugbe Nairovirus M RNA: nucleotide sequence and coding strategy; Marriott AC et al.; The coding assignments of the medium-sized (M) RNA segment of the Dugbe (DUG) virus (Nairovirus, Bunyaviridae) were investigated . The complete nucleotide sequence of 4888 nucleotides (nt) contained one long open reading frame in the viral complementary RNA, extending from an AUG start codon at nt 48-50 to a stop codon at nt 4701-4703 (numbered from the 5' terminus of vcRNA) . Comparison of the terminal sequences with the ends of the DUG S segment revealed sequence identity between the first nine nucleotides of both segments . No sequence homologies were found with the M segments of other members of the Bunyaviridae, or with their polypeptide products . Expression of portions of the DUG M open reading frame in Escherichia coli demonstrated the carboxyl terminal region of the M open reading frame codes for the G1 structural glycoprotein, which is the target for neutralising antibodies . Confirmation of this assignment was obtained by sequencing the amino terminus of the G1 protein . Two nonstructural glycoproteins which share epitopes with G1 were identified in virus-infected cells, one of which (85 kDa) is processed over a period of several hours to produce G1 . The G2 coding region was located upstream of the G1 sequence . The region between the carboxyl terminus of G2 and the 5' end of the long open reading frame apparently encodes a nonstructural protein of about 70 kDa, which is a precursor of the G2 protein.

Mol Biol Cell, 1992 Oct, 3(10), 1095 - 105
Escherichia coli ribonucleotide reductase expression is cell cycle regulated; Sun L et al.; The expression of the genes encoding ribonucleotide reductase in Escherichia coli was investigated in cultures synchronized by obtaining the smallest cells in a population after sucrose gradient centrifugation . Specific activity of ribonucleotide reductase and DNA initiation were found to increase in parallel, periodically as a function of the cell cycle . The expression of nrd was also determined in cells synchronized by periodic repeated doubling in a phosphate limited medium . Antibodies directed against the B2 subunit of ribonucleotide reductase were raised in a rabbit and purified . Immunoprecipitation of the B2 subunit and RNA-DNA dot blot hybridization assays were developed and employed to determine the expression of ribonucleotide reductase translational and transcriptional products during the cell cycle . Both of nrd-mRNA and B2 subunit expression were found to increase each generation at approximately the same time DNA synthesis was initiated and then to decrease back to the basal level shortly after DNA initiation . These results provided evidence of cell cycle dependent regulation of ribonucleotide reductase in E . coli . When the upstream regulatory region of nrd was fused to a promoterless lacZ gene on a single copy plasmid, lac-mRNA and beta-galactosidase were found to be synthesized in parallel to nrd expression from the chromosomal operon . When nrd sequences surrounding the promoter were removed from this construct, lac-mRNA and beta-galactosidase synthesis were no longer cell cycle regulated.

Proc Natl Acad Sci U S A, 1992 Oct 1, 89(19), 9354 - 8
A bifunctional enzyme (delta 1-pyrroline-5-carboxylate synthetase) catalyzes the first two steps in proline biosynthesis in plants; Hu CA et al.; Many plants synthesize and accumulate proline in response to osmotic stress . Despite the importance of this pathway, however, the exact metabolic route and enzymes involved in the synthesis of proline in plants have not been unequivocally identified . We report here the isolation of a mothbean (Vigna aconitifolia) cDNA clone encoding a bifunctional enzyme, delta 1-pyrroline-5-carboxylate synthetase (P5CS), with both gamma-glutamyl kinase and glutamic-gamma-semialdehyde dehydrogenase activities that catalyzes the first two steps in proline biosynthesis . The two enzymatic domains of P5CS correspond to the ProB and ProA proteins of Escherichia coli and contain a leucine zipper in each domain, which may facilitate inter- or intramolecular interaction of this protein . The Vigna P5CS enzyme activity is feedback regulated by proline but is less sensitive to end-product inhibition than is the E . coli gamma-glutamyl kinase . The P5CS gene is expressed at high levels in Vigna leaves and is inducible in roots subjected to salt stress, suggesting that P5CS plays a key role in proline biosynthesis, leading to osmoregulation in plants.

Proc Natl Acad Sci U S A, 1992 Oct 1, 89(19), 8899 - 902
GreA protein: a transcription elongation factor from Escherichia coli; Borukhov S et al.; A protein identified as the 158-amino acid product of the greA gene was isolated from Escherichia coli . When added to a halted ternary transcription complex, the GreA protein induced cleavage and removal of the 3' proximal dinucleotide from the nascent RNA . The new 3' terminus generated by the cleavage could be extended into longer transcripts . GreA-mediated cleavage of a transcript appears to permit a ternary complex to resume transcription from a state of indefinite elongation arrest induced by a specific DNA site . The GreA protein tended to interact with RNA polymerase during purification and recycled between RNA polymerase molecules in the course of the in vitro cleavage reaction . Similar biochemical activities have been reported in eukaryotic RNA polymerases, indicating that transcript cleavage and restart of elongation may be a general transcriptional mechanism.

Mutat Res, 1992 Oct, 269(2), 285 - 99
The distribution of UV damage in the lacI gene of Escherichia coli: correlation with mutation spectrum; Sage E et al.; We have determined the UV (254 nm) damage distribution in the transcribed and non-transcribed strands of the i-d region of the Escherichia coli lacI gene . The locations of replication blocking lesions were revealed as termination sites of T7 DNA polymerase and/or T4 DNA polymerase 3'-5' exonuclease . Termination products, i.e . both cyclobutane pyrimidine dimers and 6-4 photoproducts, were resolved and analysed on an automated DNA sequencer . These two major photoproducts are not randomly distributed along the gene, but occur in clusters, in longer runs of pyrimidines . We also have compared the UV damage distribution with the previously reported UV-induced base substitutions in the same region . Mutational hotspots, in both repair-deficient and repair-proficient strains of E . coli, are all located in stretches of pyrimidines, and thus correlate well with the distribution of photolesions . One mutational hotspot in the wild-type strain may reflect the high frequency of closely opposed lesions . To explain the other mutational hotspots, we propose that the repair of UV lesions is impaired due to the local conformation of the DNA, which might deviate from the B-form . This hypothesis is supported by the excess of mutational hotspots in pyrimidine runs in the Uvr+ strain compared to Uvr- . Runs of pyrimidines thus represent both damage- and mutation-prone sequences following UV treatment.

J Bacteriol, 1992 Oct, 174(20), 6471 - 8
Spiroplasma citri UGG and UGA tryptophan codons: sequence of the two tryptophanyl-tRNAs and organization of the corresponding genes; Citti C et al.; From the total tRNAs of Spiroplasma citri, we isolated and purified two tRNA(Trp) species by using chromatography on an RPC-5 column followed by denaturing polyacrylamide gel electrophoresis . The sequence of the two tRNAs, as well as the sequences of the corresponding genes, were determined . One of the two tRNA(Trp) species has a CCA anticodon and is able to pair with the universal UGG tryptophan codon, while the second has a U*CA (U* is a modified uridine) anticodon and is able to pair with UGA but also with UGG in accordance with the "U:N wobble" rule . Thus, in S . citri, UGA is not a stop codon but codes for tryptophan . The two tRNA(Trp) genes, together with a third tRNA gene, tRNA(Ser) (CGA), belong to a single transcription unit . The nucleotide sequences of the two tRNA(Trp) species show 82.9% similarity . The two spiroplasmal tRNA(Trp) species can be aminoacylated by using an aminoacyl-tRNA synthetase fraction from S . citri . In contrast, the enzyme fraction from Escherichia coli aminoacylates tRNA(Trp) (CCA) but not tRNA(Trp) (U*CA).

Genes Dev, 1992 Oct, 6(10), 2010 - 9
Escherichia coli single-stranded DNA-binding protein is a supercoiled template-dependent transcriptional activator of N4 virion RNA polymerase; Markiewicz P et al.; Coliphage N4 is a double-stranded DNA virus that requires the sequential activity of three different RNA polymerases during infection . The N4 virion RNA polymerase, which is carried in the virion and is injected with the DNA at the start of infection, is responsible for the synthesis of N4 early RNAs . In vitro, the virion RNA polymerase can transcribe double-stranded N4 DNA accurately and efficiently but only when the DNA is denatured . We have shown previously that the activity of DNA gyrase is required for in vivo early N4 transcription . We report here that Escherichia coli single-stranded DNA-binding protein (SSB) is also required for N4 early transcription . In vitro, linear or relaxed templates cannot be activated by SSB; however, supercoiled template and SSB allow the virion polymerase to recognize its promoters on duplex DNA and activate transcription . The effects of supercoiling are limited to transcript initiation and are not required for transcript elongation . The activation is specific for SSB; no other single-stranded DNA-binding proteins can substitute . Therefore, SSB is one of a small number of proteins that function to stimulate both replication and transcription . The basis for the specificity of SSB, the mechanism of transcriptional activation by SSB and template supercoiling, and their role in the N4 transcriptional program during development are discussed.

EMBO J, 1992 Oct, 11(10), 3521 - 31
A mammalian dual specificity protein kinase, Nek1, is related to the NIMA cell cycle regulator and highly expressed in meiotic germ cells; Letwin K et al.; Screening of mouse cDNA expression libraries with antibodies to phosphotyrosine resulted in repeated isolation of cDNAs that encode a novel mammalian protein kinase of 774 amino acids, termed Nek1 . Nek1 contains an N-terminal protein kinase domain which is most similar (42% identity) to the catalytic domain of NIMA, a protein kinase which controls initiation of mitosis in Aspergillus nidulans . In addition, both Nek1 and NIMA have a long, basic C-terminal extension, and are therefore similar in overall structure . Despite its identification with anti-phosphotyrosine antibodies, Nek1 contains sequence motifs characteristic of protein serine/threonine kinases . The Nek1 kinase domain, when expressed in bacteria, phosphorylated exogenous substrates primarily on serine/threonine, but also on tyrosine, indicating that Nek1 is a dual specificity kinase with the capacity to phosphorylate all three hydroxyamino acids . Like NIMA, Nek1 preferentially phosphorylated beta-casein in vitro . In situ RNA analysis of nek1 expression in mouse gonads revealed a high level of expression in both male and female germ cells, with a distribution consistent with a role in meiosis . These results suggest that Nek1 is a mammalian relative of the fungal NIMA cell cycle regulator.

Clin Exp Immunol, 1992 Oct, 90(1), 93 - 8
Antigen-specific T cell recognition of affinity-purified and recombinant thyroid peroxidase in autoimmune thyroid disease; Ewins DL et al.; The T cell proliferative responses of peripheral blood lymphocytes from 20 patients with autoimmune thyroid disease (AITD) and 20 healthy controls were analysed to immunoaffinity-purified thyroid peroxidase (TPO) and recombinant antigen preparations generated in Escherichia coli as glutathione-s-transferase fusion proteins . The epitope specificity of the T cell response was investigated using a selection of eight discrete recombinant fragments encompassing the whole of the extracellular region of the TPO molecule . Significant differences in the proliferative responses between patients and controls were observed to the full length, affinity-purified TPO molecule (P less than 0.002) as well as to the recombinant fragments R1c (residues 145-250) (P less than 0.001) and R2b (residues 457-589) (P less than 0.001) suggesting the presence of at least two distinct T cell determinants on this autoantigen . One of these T cell epitopes, localized within the region R1c, has not previously been identified by studies with synthetic peptides.

Cancer Res, 1992 Oct 1, 52(19), 5213 - 8
Flow cytometric detection of drugs altering the DNA methylation pattern; Biard DS et al.; We have developed a model system for assessing the demethylating potential of external agents . Disruption in the DNA methylation pattern was evaluated at the translational level of the Escherichia coli beta-galactosidase coding gene (lacZ) . We have constructed a clonal cell line (A4/4 cells) derived from the adenovirus-transformed human embryonic kidney 293 strain . The A4/4 cells contain the E . coli lacZ gene under the control of the mouse metallothionein 1 promoter which is down-regulated by a natural DNA methylation pattern . Furthermore, the lacZ transcription is also regulated by the E . coli lac operator/repressor system and by mouse metallothionein 1 metal responsiveness offering a wide range in lacZ expression . In this system, the beta-galactosidase activity was only recovered in the presence of a demethylating agent such as 5-azacytidine . The demethylating potential of 5-azacytidine, 5-aza 2'-deoxycytidine and sodium butyrate was rapidly assessed by a flow cytometric method using fluorescein di-beta-D galactopyranoside as a fluorescent probe . A tremendous induction of lacZ expression was triggered by these drugs . Analysis of cell cycles showed little disruptions with 5-azacytidine and sodium butyrate, but an important blockage in the S-phase following 5-aza 2'-deoxycytidine treatment was observed . This approach allows a rapid identification and study of environmental demethylating agents.

Appl Microbiol Biotechnol, 1992 Oct, 38(1), 77 - 83
Gene-dose-dependent expression of soluble mammalian cytochrome b5 in Escherichia coli; Gallagher J et al.; A synthetic structural gene encoding a mammalian cytochrome b5, carrying an optimised ribosomal binding sequence, was tandemly polymerised ranging from one (n = 1) to six (n = 6) gene copies . The gene, placed in p lambda-ncyt under the control of the lambda PL promoter, transcribed mono- to hexahomocistronic mRNA, expressing one to six copies of cytochrome b5 . The expressed levels of cytochrome b5 in Escherichia coli p lambda-ncyt corresponded linearly with the gene dose when up to five copies were present; saturating build-up of the recombinant protein was reached at six gene copies . Cells bearing p lambda-6cyt produced 75 micrograms cytochrome b5/ml of unit optical density at 600 nm culture, constituting 55% of the soluble bacterial protein . The recombinant protein accumulated predominantly in a haem-deficient, apoform, together with lesser amounts of the holocytochrome b5 . Whereas the overall expressed protein (apo and holo forms) was gene dose dependent, there was an inverse relationship between holocytochrome b5 production and gene dose . Incubation of the thermally induced bacterial lysates with exogenous haem a converted all of the soluble apocytochrome b5 into holocytochrome b5 that was spectrally indistinguishable with its native counterpart . Culture supplementation with the likely metabolic precursors of haem synthesis, 5-aminolevulinic acid, glycine/succinate or glutamate, significantly alleviated the protoporphyrin deficiency during hyperproduction of cytochrome b5 in E . coli.

Curr Opin Biotechnol, 1992 Oct, 3(5), 481 - 5
Protein export in Escherichia coli; Johnson K et al.; The export of protein from Escherichia coli has been studied by genetic, biochemical and biophysical techniques . These studies have defined a number of steps in the export pathway and have identified the cellular components required for the translocation process . New information is presented on the function of some of these components.

Curr Opin Biotechnol, 1992 Oct, 3(5), 468 - 73
Folding and assembly of oligomeric proteins in Escherichia coli; Teschke CM et al.; High levels of expression of oligomeric proteins in heterologous systems are frequently associated with misfolding and accumulation of the polypeptides in inclusion bodies . This reflects aspects of the folding and assembly pathways of oligomeric proteins, which generally proceed from either folding intermediates or native-like metastable species that are not in their final conformation . Methods for optimizing the yield of correctly assembled oligomers are discussed.

Bioessays, 1992 Oct, 14(10), 661 - 70
On the nature of origins of DNA replication in eukaryotes; Benbow RM et al.; Chromosomal origins of DNA replication in higher eukaryotes differ significantly from those of E . coli (oriC) and the tumor virus, SV40 (ori sequence) . Initiation events appear to occur throughout broad zones rather than at specific origin sequences . Analysis of four chromosomal origin regions reveals that they share common modular sequence elements . These include DNA unwinding elements, pyrimidine tracts that may serve as strong DNA polymerase-primase start sites, scaffold associated regions, transcriptional regulatory sequences, and, possibly, initiator protein binding sites and inherently destabilized regions . Based on the novel organization of chromosomal origin regions, we propose a model for initiation of DNA replication in higher eukaryotes . Unwinding of duplex DNA during initiation may be uncoupled, both temporally and spatially, from DNA synthesis, resulting in transient single-stranded intermediates that function in lieu of conventional replication forks during chromosomal DNA replication . DNA synthesis begins subsequently at multiple sites within the unwound regions rather than at specific origin sequences.

Acta Virol, 1992 Oct, 36(5), 488 - 90
The use of consensus sequence for amplification and oriented cloning of human alpha interferon genes; Kudela O et al.; A clone from human cDNA library was amplified in polymerase chain reaction (PCR) by the synthetic oligonucleotides . The final construct after linker ligation and digestion with restriction endonucleases was suitable for oriented cloning into E . coli expression vector . The interferon (IFN) expression could be detected by SDS-PAGE electrophoresis . Western blotting and biological antiviral assay . This set of oligonucleotides can be used also for amplification of genomic DNA or cDNA libraries.

Biull Eksp Biol Med, 1992 Oct, 114(10), 429 - 32
{The ultrastructural characteristics of the endocrine cells of the normal murine cecum and in experimental escherichiosis}; Barkhina TG et al.; Ultrastructural investigations and a quantitative analysis of caecum endocrine cells were performed in the period from 15 minutes to 2 weeks after inoculation, using the model of experimental escherichiosis . The authors identified 5 types of endocrinocytes in the caecum of mice and showed the reaction of these cells: degranulation, extrusion of granules and their accumulation dependent on the time of the exposure.

Biotechniques, 1992 Oct, 13(4), 550 - 4
Preparation of plasmid DNA by sequential enzymatic digestion; Hyman ED; A new method for the preparation of plasmid DNA from Escherichia coli, sequential enzymatic digestion, is described . The method is based on sequential and selective enzymatic digestion of all components of E . coli except for the supercoiled plasmid DNA . The key enzymes are exonuclease I and exonuclease III that specifically hydrolyze linear chromosomal DNA and are unable to attack supercoiled plasmid DNA under controlled conditions . Isolated plasmid DNA can be sequenced and digested with restriction enzymes.

Comp Immunol Microbiol Infect Dis, 1992 Oct, 15(4), 285 - 92
Occurrence of fimbriae and enterotoxins in Escherichia coli strains isolated from piglets in Poland; Osek J et al.; 1125 and 1146 E . coli strains isolated from suckling and weaned piglets with diarrhea, respectively, and 724 strains from healthy piglets were tested for the presence of fimbriae and production of enterotoxins . The fimbriae were determined by hemagglutination and slide agglutination tests, enterotoxins--by the use of ileal loop test in piglets (LT and STb enterotoxins) and suckling mouse assay (STa enterotoxin) . It was found that 72.8 and 53.0% strains, isolated from diseased suckling and weaned piglets, respectively, possessed specific fimbrial hemagglutinins, in most cases with K88 antigen . Additionally, 987P fimbriae were detected in 14.0 and 0.7% strains isolated from piglets with diarrhea . Only 5 strains (0.7%) recovered from healthy piglets had specific fimbriae, usually with undetermined antigenic structure . F1 fimbriae (called common or unspecific) were found in strains isolated both from diseased (15.2 and 16.3% strains, respectively) and healthy piglets (27.1% strains) . It was noted that the strains isolated from suckling and weaned piglets with diarrhea in most cases were enterotoxigenic (90.5 and 69.1% strains, respectively) and most frequently produced heat-labile toxin LT alone or with STb . 18.5% of enterotoxigenic strains isolated from healthy piglets produced STa toxin.

Proc Natl Acad Sci U S A, 1992 Oct 1, 89(19), 9141 - 5
Leukotriene A4 hydrolase: abrogation of the peptidase activity by mutation of glutamic acid-296; Wetterholm A et al.; The metal-binding motif in the sequence of leukotriene A4 (LTA4) (EC 3.3.2.6), a bifunctional zinc metalloenzyme, contains a glutamic acid that is conserved in several zinc hydrolases . To study its role for the two catalytic activities, Glu-296 in mouse leukotriene A4 hydrolase was replaced by a glutamine or alanine residue by site-directed mutagenesis . Wild-type and mutated cDNAs were expressed four or five times in Escherichia coli, and the resulting proteins were purified to apparent homogeneity . With respect to their epoxide hydrolase activities--i.e., the conversion of LTA4 into leukotriene B4--the mutated enzymes {Gln296}LTA4 hydrolase and {Ala296}LTA4 hydrolase exhibited specific activities of 1070 +/- 160 and 90 +/- 30 nmol of LTB4 per mg of protein per min (mean +/- SD; n = 4 or 5), respectively, corresponding to 150% and 15% of unmutated enzyme . In contrast, when the mutated proteins were assayed for peptidase activity toward alanine-4-nitroanilide, they were found to be virtually inactive (less than or equal to 0.2% of unmutated enzyme) . To serve as a positive control, we also replaced Ser-298 with an alanine residue, which resulted in a protein ({Ala298}LTA4 hydrolase) with catalytic properties almost indistinguishable from the wild-type enzyme . Substitution of Glu-296 by glutamine or alanine was also carried out with human LTA4 hydrolase, and the mutated human enzymes displayed specific activities similar to the corresponding mouse proteins . Zinc analyses of the purified mouse and human proteins confirmed that the mutations did not significantly influence their zinc content . In conclusion, the results of the present study indicate a direct catalytic role for Glu-296 in the peptidase reaction of LTA4 hydrolase, where it presumably acts as a base to polarize water, whereas its function, if any, is apparently not essential in the epoxide hydrolase reaction.

J Bacteriol, 1992 Oct, 174(20), 6394 - 403
Murein-metabolizing enzymes from Escherichia coli: existence of a second lytic transglycosylase; Engel H et al.; In addition to the soluble lytic transglycosylase, a murein-metabolizing enzyme with a molecular mass of 70 kDa (Slt70), Escherichia coli possesses a second lytic transglycosylase, which has been described as a membrane-bound lytic transglycosylase (Mlt; 35 kDa; EC 3.2.1.-) . The mlt gene, which supposedly encodes Mlt, was cloned, and the complete nucleotide sequence was determined . The open reading frame, identified on a 1.7-kb SalI-PstI fragment, codes for a protein of 323 amino acids (M(r) = 37,410) . Two transmembrane helices and one membrane-associated helix were predicted in the N-terminal half of the protein . Lysine and arginine residues represent up to 15% of the amino acids, resulting in a calculated isoelectric point of 10.0 . The deduced primary structure did not show significant sequence similarity to Slt70 from E . coli . High-level expression of the presumed mlt gene was not paralleled by an increase in murein hydrolase activity . To clarify the identity of the second transglycosylase, we purified an enzyme with the specificity of a transglycosylase from an E . coli slt deletion strain . The completely soluble transglycosylase, with a molecular mass of approximately 35 kDa, was designated Slt35 . Its determined 26 N-terminal amino acids showed similarity to a segment in the middle of the Slt70 primary structure . Polyclonal anti-Mlt antibodies, which had been used for the isolation of the mlt gene, were found to cross-react with Mlt as well as with Slt35, suggesting that the previously described Mlt preparation was contaminated with Slt35 . We conclude that the second transglycosylase of E . coli is not a membrane-bound protein but rather is a soluble protein.

J Bacteriol, 1992 Oct, 174(19), 6061 - 70
Role of phosphorylated metabolic intermediates in the regulation of glutamine synthetase synthesis in Escherichia coli; Feng J et al.; Transcription of the Ntr regulon is controlled by the two-component system consisting of the response regulator NRI (NtrC) and the kinase/phosphatase NRII (NtrB), which both phosphorylates and dephosphorylates NRI . Even though in vitro transcription from nitrogen-regulated promoters requires phosphorylated NRI, NRII-independent activation of NRI also occurs in vivo . We show here that this activation likely involves acetyl phosphate; it is eliminated by mutations that reduce synthesis of acetyl phosphate and is elevated by a mutation expected to cause accumulation of acetyl phosphate . With purified components, we investigated the mechanism by which acetyl phosphate stimulates glutamine synthetase synthesis . Acetyl phosphate, carbamyl phosphate, and phosphoramidate but not ATP or phosphoenolpyruvate acted as substrates for the autophosphorylation of NRI in vitro . Phosphorylated NRI produced by this mechanism exhibited the properties associated with NRI phosphorylated by NRII, including the activated ATPase activity of the central domain of NRI and the ability to activate transcription from the nitrogen-regulated glutamine synthetase glnAp2 promoter.

J Bacteriol, 1992 Oct, 174(19), 6033 - 45
Characterization of the complex pdxH-tyrS operon of Escherichia coli K-12 and pleiotropic phenotypes caused by pdxH insertion mutations; Lam HM et al.; We report the first molecular genetic analysis of a pyridoxine 5'-phosphate oxidase, the PdxH gene product of Escherichia coli K-12 . Chromosomal insertions in and around pdxH were generated with various transposons, and the resulting phenotypes were characterized . The DNA sequence of pdxH was determined, and the promoters of pdxH and the downstream gene tyrS, which encodes tyrosyl-tRNA synthetase, were mapped by RNase T2 protection assays of chromosomal transcripts . These combined approaches led to the following conclusions: (i) pdxH is transcribed from a sigma 70-type promoter and shares its transcript with tyrS; (ii) tyrS is additionally transcribed from a relatively strong, nonconventional internal promoter that may contain an upstream activating sequence but whose expression is unaffected by a fis mutation; (iii) PdxH oxidase is basic, has a molecular mass of 25,545 Da, and shares striking homology (greater than 40% identity) with the developmentally regulated FprA protein of Myxococcus xanthus; (iv) mild pyridoxal 5'-phosphate limitation of pdxH mutants inhibits cell division and leads to formation of unsegregated nucleoids; (v) E . coli PdxH oxidase is required aerobically and anaerobically, but second-site suppressors that replace pdxH function entirely can be isolated; and (vi) pdxH mutants excrete significant amounts of L-glutamate and a compound, probably alpha-ketoisovalerate, that triggers L-valine inhibition of E . coli K-12 strains . These findings extend earlier observations that pyridoxal 5'-phosphate biosynthetic and aminoacyl-tRNA synthetase genes are often members of complex, multifunctional operons . Our results also show that loss of pdxH function seriously disrupts cellular metabolism in unanticipated ways.

Eur J Biochem, 1992 Oct 1, 209(1), 453 - 8
Purification, cDNA cloning and Northern-blot analysis of mitochondrial chaperonin 60 from pumpkin cotyledons; Tsugeki R et al.; Two different cDNA clones, pMCPN60-1 and pMCPN60-2, encoding the mitochondrial homologues of chaperonin 60 (Cpn60) were isolated from a cDNA library of germinating pumpkin cotyledons by use of mixtures of synthetic oligonucleotides based on the N-terminal amino acid sequence of the protein . Determination of the complete nucleotide sequences of the two cDNA revealed that pMCPN60-1 and pMCPN60-2 each contain one open reading frame that encodes a protein of 575 amino acids with molecular masses of 61052 Da and 61127 Da, respectively . The deduced amino acid sequences of the two polypeptides include a 32-residue N-terminal putative mitochondrial presequence attached to the mature polypeptides, and they are 95.3% identical . From a comparison of deduced amino acid sequences with other Cpn60, it appears that the mature polypeptides of pumpkin mitochondrial Cpn60 are 44-59% identical to the other Cpn60, namely, GroEL of Escherichia coli, the 60-kDa heat-shock protein (Hsp60) of mitochondria in the yeast Saccharomyces cerevisiae, P1 protein of mammalian mitochondria and the Ribulose-1,5-bisphosphate carboxylase/oxygenase subunit-binding proteins alpha and beta of plastids in higher plants . Genomic Southern-blot analysis identified at least two copies of the gene for mitochondrial Cpn60 in the pumpkin genome . The levels of mRNA for mitochondrial Cpn60 in cotyledons, hooks and hypocotyls of pumpkin seedlings increased in response to heat stress, as deduced from Northern-blot analysis, indicating that pumpkin mitochondrial Cpn60 is a heat-induced stress protein.

Eur J Biochem, 1992 Oct 1, 209(1), 249 - 55
Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation . Structure/activity studies and mechanistic implications; Almas B et al.; Three isozymes of human tyrosine hydroxylase (hTH1, hTH2 and hTH4) were expressed in Escherichia coli and purified to homogeneity . Natural catecholamines and related synthetic compounds were found to be potent inhibitors, competitive to the tetrahydrobiopterin cofactor, of all the isozymes . Combining visible spectroscopy and equilibrium-binding studies, it was found that catecholamines bind to hTH1 and hTH2 with a stoichiometry of about 1.0 mol/mol enzyme subunit, interacting with the catalytic iron at the active site . All the isozymes tested were excellent substrates for cAMP-dependent protein kinase (Km = 5 microM, Vmax = 9.5 mumol.min-1.mg kinase-1) . The incorporation of about 1.0 mol phosphate/subunit at Ser40 decreased the affinity of dopamine binding by a factor of 10 . Conversely, the addition of stoichiometric amounts of Fe(II) and dopamine to the apoenzymes reduced both the affinity and stoichiometry of phosphorylation by cAMP-dependent protein kinase by 2-3-fold . These data provide evidence for a mutual interaction between the presumed regulatory and catalytic domains of hTH, and show that activation of the enzyme by phosphorylation and inactivation by binding of catecholamines are related events, which probably represent important mechanisms for the regulation of the enzyme activity in vivo.

Plant Mol Biol, 1992 Oct, 20(1), 61 - 70
Characterization of the Ac/Ds behaviour in transgenic tomato plants using plasmid rescue; Rommens CM et al.; We describe the use of plasmid rescue to facilitate studies on the behaviour of Ds and Ac elements in transgenic tomato plants . The rescue of Ds elements relies on the presence of a plasmid origin of replication and a marker gene selective in Escherichia coli within the element . The position within the genome of modified Ds elements, rescued both before and after transposition, is assigned to the RFLP map of tomato . Alternatively to the rescue of Ds elements equipped with plasmid sequences, Ac elements are rescued by virtue of plasmid sequences flanking the element . In this way, the consequences of the presence of an (active) Ac element on the DNA structure at the original site can be studied in detail . Analysis of a library of Ac elements, rescued from the genome of a primary transformant, shows that Ac elements are, infrequently, involved in the formation of deletions . In one case the deletion refers to a 174 bp genomic DNA sequence immediately flanking Ac . In another case, a 1878 bp internal Ac sequence is deleted.

Mem Inst Oswaldo Cruz, 1992 Oct-Dec, 87(4), 487 - 92
Disseminated American muco-cutaneous leishmaniasis caused by Leishmania braziliensis braziliensis in a patient with AIDS: a case report; Machado ES et al.; The authors report a case of culture-proven disseminated American muco-cutaneous leishmaniasis caused by Leishmania braziliensis braziliensis in an HIV positive patient . Lesions began in the oropharynx and nasal mucosa eventually spreading to much of the skin surface . The response to a short course of glucantime therapy was good.

Clin Pharm, 1992 Oct, 11(10), 834 - 50; quiz 892-4
Recombinant interferon gamma for treatment of chronic granulomatous disease and other disorders; Bolinger AM et al.; The chemistry, biological activity, and pharmacokinetics of gamma-interferon and recombinant interferon gamma are reviewed, and the agent's clinical efficacy, adverse effects, and dosage and administration for the treatment of chronic granulomatous disease (CGD) and other disorders are described . Endogenous gamma-interferon is a 166-amino-acid protein encoded by a single gene on chromosome 12 . Recombinant human interferon gamma is purified from Escherichia coli as a monomer containing 139 amino acids . Gamma-interferon has antiviral, immunomodulatory, and antiproliferative activity . Serum concentrations of recombinant interferon gamma increase in proportion to the dose . Clearance after i.m . or s.c . administration fits a two-compartment model . The half-life is 3.5-7.5 hours, and bioavailability is 89% . Evidence that recombinant interferon gamma can enhance phagocytic oxidative metabolism led to its evaluation for use in the treatment of CGD . Clinical studies showed that the agent decreases the frequency of serious infections in patients with CGD . Recombinant interferon gamma has shown only limited success in the treatment of metastatic renal cell carcinoma (RCC), both as a single agent and in combination with recombinant interferon alfa . Similarly, although interferons appear to be able to change cytogenetic abnormalities in some patients with Philadelphia chromosome-positive chronic myelogenous leukemia, therapy with recombinant interferon gamma has led to minimal success . However, the agent has produced some encouraging results in atopic dermatitis . The adverse effects of recombinant interferon gamma in patients with CGD usually consist only of fever, chills, headache, and erythema . The recommended dosage in CGD-afflicted children whose body surface area is greater than 0.5 sq m is 50 micrograms/sq m given by s.c . injection three times a week for life . Recombinant interferon gamma has given new hope to patients with CGD . Although the drug is very expensive, the cost may be offset by fewer hospitalizations to treat infection.

J Biochem (Tokyo), 1992 Oct, 112(4), 508 - 13
Expression of Ca(2+)-induced Ca2+ release channel activity from cardiac ryanodine receptor cDNA in Chinese hamster ovary cells; Imagawa T et al.; We constructed an expression plasmid (pMAMCRR51) that carried the entire protein-coding sequence of the rabbit cardiac ryanodine receptor cDNA, linked to the dexamethasone-inducible mouse mammary tumor virus promoter and Escherichia coli xanthine-guanine phosphoribosyltransferase (gpt) . Chinese hamster ovary (CHO) cells were transfected with pMAMCRR51 and mycophenolic acid-resistant cells showing caffeine-induced intracellular Ca2+ transients were selected . Immunoprecipitation with a monoclonal antibody against the canine cardiac ryanodine receptor revealed that the cell clones thus selected exhibited Ca(2+)-dependent {3H}ryanodine binding activity, which was stimulated by 5 mM ATP or 1 M KCl . The apparent dissociation constant (Kd) for {3H}ryanodine was 6.6 nM in 1 M KCl, which was similar to the Kd obtained with cardiac microsomes . Immunoprecipitation also demonstrated that these cell clones expressed a protein indistinguishable in M(r) from the ryanodine receptor in canine cardiac microsomes . The ryanodine binding activity expressed in CHO cells increased significantly after dexamethasone induction . In saponin-skinned CHO cells transfected with pMAMCRR51, micromolar Ca2+ or millimolar caffeine evoked rapid Ca2+ release from the intracellular Ca2+ stores . In skinned control CHO cells, we did not observe such Ca2+ release activity . These results clearly demonstrate that the cardiac ryanodine receptor is stably expressed in internal membranes of CHO cells and functions as Ca(2+)-induced Ca2+ release channels.

Pathol Biol (Paris), 1992 Oct, 40(8), 767 - 72
{Biotechnology of beta-adrenergic receptors}; Strosberg D; This article discusses the structural and functional features of a new family of membrane receptors including alpha-adrenergic and beta-adrenergic receptors for catecholamines, muscarinic receptors for acetylcholine, and receptors for histamine, dopamine, serotonin, and neuropeptides such as angiotensin . All these receptors are coupled to GTP-binding proteins, called G proteins . All G proteins have similar membrane topologies with a single peptide chain composed of seven membrane-spanning domains separating a series of successive extracellular and intracellular portions . Results of studies of beta-adrenergic receptors suggest that some amino acids in the seven membrane-spanning domains are part of the ligand-binding site, whereas the protein G-coupling site seems to involve amino acids located in the third cytoplasmic loop and in the C-terminal extremity, which also contains the phosphorylation sites . Genes encoding this family of receptors exhibit sequence homologies and also contain sequences which may be involved in the regulation of the level of expression of these receptors . Recent development of a receptor-expressing bacterial system can be expected to provide significant advances in the accurate investigation of receptor structure-function correlations.

J Gen Microbiol, 1992 Oct, 138 ( Pt 10), 2083 - 91
Filamentation promotes F'lac loss in Escherichia coli K12; Debbia EA; The stability of plasmid F'lac in Escherichia coli strain SP45 (a temperature conditional mutant which grows as spherical cells at 42 degrees C and as a rod at 30 degrees C) was studied . F'lac elimination was demonstrated when bacteria exposed to subinhibitory concentrations of various chemicals were induced to form filaments . No plasmid loss was found when spherical cells were subjected to the same treatments . Plasmid loss was also observed in dnaA46 and lexA41 mutants when cell filamentation was induced at 42 degrees C, but not when they were cultured at 30 degrees C . Nalidixic acid promoted F'lac elimination at 0.25 micrograms ml-1 in a recA13 mutant and at 1.5 micrograms ml-1 in the recA+ counterpart . A marked difference was found in the rate of F'lac elimination from thermosensitive DNA gyrase mutants {gyrA43(Ts) and gyrB41(Ts)} between rods and their spherical (rodA51) derivatives growing at semipermissive temperature (36.5 degrees C) . Plasmids carrying the ccd segment of F in DNA gyrase mutants were lost after 2.5 generations from rods and after 6 generation from spherical cells . Plasmid segregation into non-viable minicell-like elements was found after induction of filaments . These data suggest that plasmid stability is correlated with cell shape and that curing is more easily achieved when bacteria can elongate normally.

J Biomol Struct Dyn, 1992 Oct, 10(2), 295 - 309
Specific binding of cyclic-AMP receptor protein to DNA . Effect of the sequence and of the introduction of a nick in the binding site; Giraud-Panis MJ et al.; The binding of Escherichia coli Cyclic AMP Receptor Protein (CRP) to several DNA fragments of about 45 base pairs, bearing the natural lactose or galactose sites, as well as several synthetic related sites, was investigated using fluorescence spectroscopy and gel retardation experiments . The salt dependence of the equilibrium binding constant indicates that CRP makes an identical number of ion pairs with the lac, lacL8 and gal sites although the binding constants are drastically different . However increasing the symmetry of the gal site leads to an increase of the number of ion pairs between the protein and the DNA . A single strand nick was introduced at the centre of a symmetrized gal site and this reduces the binding energy of CRP by about 0.6 Kcal . These results are discussed with respect to the bending constraints imposed on the DNA by the binding of CRP . The results are in agreement with the recently published crystal structure of the CRP complexed with DNA {Schutz, S.C., Shields, G.C . and Steitz, T.A., Science 253, 1001-1007 (1991)} showing that the 90 degrees bending of the DNA in the complex results from two kinks.

Protein Expr Purif, 1992 Oct, 3(5), 417 - 20
Preparation and properties of an affinity support for purification of cyclic AMP receptor protein from Escherichia coli; Corrie JE et al.; Reaction of cyclic AMP with 1,1'-carbonyldiimidazole produces an intermediate which reacts with primary amines to provide a stable 2'-O-carbamyl derivative . This chemistry has been used to tether cyclic AMP to a Sepharose gel . The resulting affinity support has been used to effect a simple, nondenaturing purification of cyclic AMP receptor protein from crude cell extracts.

Biotechnol Appl Biochem, 1992 Oct, 16(2), 211 - 5
A procedure for large-scale plasmid isolation without using ultracentrifugation; Chakrabarti A et al.; An expedient procedure for large-scale plasmid isolation from Escherichia coli strains without using ultracentrifugation or special setups or reagents is described . The protocol, which utilizes a modified alkaline extraction procedure as well as differential precipitations by isopropanol and lithium chloride, is simple and rapid and yet produces plasmid DNA with a yield of about 2 mg/liter culture . The isolated plasmids consisted of mostly monomeric and dimeric covalently closed circular DNA . The plasmids could be digested by various restriction endonucleases and were compatible with gene cloning, transfection-gene expression, and viral production.

Vet Microbiol, 1992 Oct, 32(3-4), 281 - 92
Recombinant polypeptide from the gp48 region of the bovine viral diarrhea virus (BVDV) detects serum antibodies in vaccinated and infected cattle; Kwang J et al.; To characterize the immune response of cattle to bovine viral diarrhea virus (BVDV) glycoprotein gp48, we have produced a large amount of recombinant glutathione-s-transferase-gp48 (GST-gp48) fusion protein in Escherichia coli . Antibodies to gp48 were present in cattle vaccinated with killed or modified-live virus vaccination, or following natural infection . These results were in agreement with results of serum neutralization (SN) test which detected gp53 of BVDV.

Plant Cell, 1992 Oct, 4(10), 1273 - 81
The S-locus receptor kinase gene in a self-incompatible Brassica napus line encodes a functional serine/threonine kinase; Goring DR et al.; An S-receptor kinase (SRK) cDNA, SRK-910, from the active S-locus in a self-incompatible Brassica napus W1 line has been isolated and characterized . The SRK-910 gene is predominantly expressed in pistils and segregates with the W1 self-incompatibility phenotype in an F2 population derived from a cross between the self-incompatible W1 line and a self-compatible Westar line . Analysis of the predicted amino acid sequence demonstrated that the extracellular receptor domain is highly homologous to S-locus glycoproteins, whereas the cytoplasmic kinase domain contains conserved amino acids present in serine/threonine kinases . An SRK-910 kinase protein fusion was produced in Escherichia coli and found to contain kinase activity . Phosphoamino acid analysis confirmed that only serine and threonine residues were phosphorylated . Thus, the SRK-910 gene encodes a functional serine/threonine receptor kinase.

Plant Cell, 1992 Oct, 4(10), 1263 - 71
The TMK1 gene from Arabidopsis codes for a protein with structural and biochemical characteristics of a receptor protein kinase; Chang C et al.; Genomic and cDNA clones that code for a protein with structural and biochemical properties similar to the receptor protein kinases from animals were obtained from Arabidopsis . Structural features of the predicted polypeptide include an amino-terminal membrane targeting signal sequence, a region containing blocks of leucine-rich repeat elements, a single putative membrane spanning domain, and a characteristic serine/threonine-specific protein kinase domain . The gene coding for this receptor-like transmembrane kinase was designated TMK1 . Portions of the TMK1 gene were expressed in Escherichia coli, and antibodies were raised against the recombinant polypeptides . These antibodies immunodecorated a 120-kD polypeptide present in crude extracts and membrane preparations . The immunodetectable band was present in extracts from leaf, stem, root, and floral tissues . The kinase domain of TMK1 was expressed as a fusion protein in E . coli, and the purified fusion protein was found capable of autophosphorylation on serine and threonine residues . The possible role of the TMK1 gene product in transmembrane signaling is discussed.

Arch Oral Biol, 1992 Oct, 37(10), 807 - 12
Cloning of the gene encoding a glycylprolyl aminopeptidase from Porphyromonas gingivalis; Nakamura S et al.; A genomic library of Porphyromonas gingivalis 381 was constructed in the cosmid vector pHC79 . A clone, pSN1, was identified by the expression of glycylprolyl-naphthylamide hydrolysing activity . The DNA insert contained within the cosmid pSN1 was subcloned into the plasmid vector pBR328 to create the recombinant plasmid pSN11 containing a 2.9 kb EcoRV insert . An Escherichia coli transformant containing pSN11 produced a protein having a molecular weight of 75 kDa . Southern-blot hybridization revealed that the 2.9 kb EcoRV DNA hybridized with an identical sized Eco RV DNA fragment in the chromosomal DNA of P . gingivalis 381.

J Bioenerg Biomembr, 1992 Oct, 24(5), 485 - 91
H+ transport and coupling by the F0 sector of the ATP synthase: insights into the molecular mechanism of function; Fillingame RH; The F0 sector of the ATP synthase complex facilitates proton translocation through the membrane, and via interaction with the F1 sector, couples proton transport to ATP synthesis . The molecular mechanism of function is being probed by a combination of mutant analysis and structural biochemistry, and recent progress on the Escherichia coli F0 sector is reviewed here . The E . coli F0 is composed of three types of subunits (a, b, and c) and current information on their folding and organization in F0 is reviewed . The structure of purified subunit c in chloroform-methanol-H2O resembles that in native F0, and progress in determining the structure by NMR methods is reviewed . Genetic experiments suggest that the two helices of subunit c must interact as a functional unit around an essential carboxyl group as protons are transported . In addition, a unique class of suppressor mutations identify a transmembrane helix of subunit a that is proposed to interact with the bihelical unit of subunit c during proton transport . The role of multiple units of subunit c in coupling proton translocation to ATP synthesis is considered . The special roles of Asp61 of subunit c and Arg210 of subunit a in proton translocation are also discussed.

Mol Reprod Dev, 1992 Oct, 33(2), 124 - 30
Genetic manipulation of mammalian dictyate oocytes: factors affecting transient expression of microinjected DNA templates; Bevilacqua A et al.; Transcription of exogenous DNA templates in mouse ovarian oocytes was investigated by microinjecting constructs encoding for the Escherichia coli lacZ gene under control of promoters from: 1) the mouse hsp68 gene; 2) the human beta-actin gene; and 3) simian virus 40 (SV40) early genes . Various amounts of circular or linear DNA constructs were injected into dictyate oocyte nuclei at different stages of follicle growth, and the beta-galactosidase activity was then cytochemically evaluated in single cells . In middle-sized growing oocytes, expression of circular constructs was observed with amounts of DNA ranging from 50 to 10(3) plasmid copies/nucleus and was first observed 10-12 hr after injection . Maximal expression levels were reached by 17 hr after injection and were specific for the constructs used . Circular constructs containing the hsp68 and early SV40 promoters were expressed at similar levels in small- and middle-sized growing oocytes, while the construct carrying the beta-actin promoter was expressed only in small-sized cells . In contrast to growing oocytes, these constructs were never expressed in fully grown oocytes . DNA linearization depressed construct activity regardless of the site of cleavage . These results show that: 1) lacZ is a valuable reporter gene in the analysis of eukaryotic promoter activity in dictyate mouse oocytes; 2) transient construct expression requires the injection of DNA in circular form; and 3) the expression efficiency of different DNA templates is dependent on the presence of a specific promoter and on the differentiation stage of oocytes analyzed.

J Exp Med, 1992 Oct 1, 176(4), 1015 - 24
Functional characterization of the human tumor necrosis factor receptor p75 in a transfected rat/mouse T cell hybridoma; Vandenabeele P et al.; We investigated the biological role of the human tumor necrosis factor p75 (hTNF-R75), making use of the species specificity of TNF responses in murine (m) T cell lines . Several TNF-mediated activities on mouse T cells, such as cytokine induction or proliferation, showed a 100-500-fold difference in specific biological activity between mTNF and hTNF . After transfection of hTNF-R75 cDNA in a rat/mouse T cell hybridoma (PC60), however, the 100-fold lower specific biological activity of hTNF was converted to the same specific biological activity as mTNF . The TNF-mediated induction of granulocyte/macrophage colony-stimulating factor was strongly synergized by the addition of interleukin 1 . In the presence of the latter cytokine, ligand-competing monoclonal antibodies against hTNF-R75 (utr-1, utr-2, utr-3) were agonistic on transfected PC60 cells . This agonistic activity was further enhanced by crosslinking with sheep anti-murine immunoglobulin antibodies . These data provide direct evidence for a functional role of TNF-R75, without ligand-dependent TNF-R55 involvement, in the induction of cytokine secretion in T cells.

J Bacteriol, 1992 Oct, 174(20), 6685 - 7
Two transcriptionally active OmpR mutants that do not require phosphorylation by EnvZ in an Escherichia coli cell-free system; Bowrin V et al.; D55Q-T83A and D55Q-G94S, two pseudorevertants of the D55Q mutant OmpR, an Escherichia coli transcriptional activator, were isolated previously by R . Brissette, K . Tsung, and M . Inouye (J . Bacteriol . 173:3749-3755, 1991) . These pseudorevertant OmpR proteins were purified and examined for their function as transcriptional activators in a cell-free system with an ompF DNA fragment . These proteins were transcriptionally active, even after acid treatment, whereas the wild-type OmpR was completely inactive after the same treatment . Phosphorylation of acid-treated wild-type OmpR with an EnvZ11 membrane fraction and ATP restored transcriptional activity, whereas the activities of the mutant OmpR proteins did not change after phosphorylation.

J Bacteriol, 1992 Oct, 174(20), 6554 - 62
arc-dependent thermal regulation and extragenic suppression of the Escherichia coli cytochrome d operon; Wall D et al.; In a screen for Escherichia coli genes whose products are required for high-temperature growth, we identified and characterized a mini-Tn10 insertion that allows the formation of wild-type-size colonies at 30 degrees C but results in microcolony formation at 36 degrees C and above (Ts- phenotype) . Mapping, molecular cloning, and DNA sequencing analyses showed that the mini-Tn10 insertion resides in the cydB gene, the distal gene of the cydAB operon (cytochrome d) . The Ts- growth phenotype was also shown to be associated with previously described cyd alleles . In addition, all cyd mutants were found to be extremely sensitive to hydrogen peroxide . Northern (RNA) blot analysis showed that cyd-specific mRNA levels accumulate following a shift to high temperature . Interestingly, this heat shock induction of the cyd operon was not affected in an rpoH delta background but was totally absent in an arcA or arcB mutant background . Extragenic suppressors of the Cyd Ts- phenotype are found at approximately 10(-3) . Two extragenic suppressors were shown to be null alleles in either arcA or arcB . One interpretation of our results is that in the absence of ArcA or ArcB, which are required for the repression of the cyo operon (cytochrome o), elevated levels of Cyo are produced, thus compensating for the missing cytochrome d function . Consistent with this interpretation, the presence of the cyo gene on a multicopy plasmid suppressed the Ts- and hydrogen peroxide-sensitive phenotypes of cyd mutants.

J Bacteriol, 1992 Oct, 174(20), 6411 - 7
Cloning, sequencing, and expression of the pantothenate kinase (coaA) gene of Escherichia coli; Song WJ et al.; Pantothenate kinase catalyzes the rate-controlling step in coenzyme A (CoA) biosynthesis . The structural gene (coaA) located at 90 min of the Escherichia coli chromosome was cloned and sequenced . The coaA gene was transcribed in the opposite direction to the flanking genes birA and thrU and produced a single 1.1-kb transcript . Translation of the coaA gene produced two protein products (36.4 and 35.4 kDa) that differed by eight amino acids at the amino terminus . The poor homology of the coaA promoter region to consensus E . coli promoter sequences and the low frequency of optimal codon usage (0.565) were consistent with the low abundance of pantothenate kinase . Strains containing multiple copies of the coaA gene possessed 76-fold-higher specific activity of pantothenate kinase; however, there was only a 2.7-fold increase in the steady-state level of CoA . These data corroborate the conclusion that regulation of pantothenate kinase activity by feedback inhibition is the critical factor controlling the intracellular CoA concentration.

Infect Immun, 1992 Oct, 60(10), 4213 - 20
Glycoprotein receptors for a heat-stable enterotoxin (STh) produced by enterotoxigenic Escherichia coli; Hirayama T et al.; Glycoprotein receptors for heat-stable enterotoxin STh of enterotoxigenic Escherichia coli in the rat intestinal cell membrane were identified and characterized . Incubation of rat intestinal cell membranes with radioiodinated N-5-azidonitrobenzoyl-STh{5-19} (125I-ANB-STh{5-19}) followed by photolysis resulted in specific radiolabeling of two distinct proteins with M(r)s of 200,000 (designated STR-200A and STR-200B) . STR-200A was found to be composed of two molecules of a protein with an M(r) of 70,000 (70-kDa protein), whereas STR-200B was composed of two different protein molecules with M(r)s of 53,000 (53-kDa protein) and 77,000 (77-kDa protein) . These proteins showed no guanylate cyclase activity . The 70-kDa protein was labeled most with 125I-ANB-STh{5-19}, suggesting that STR-200A is the main receptor protein in the rat intestinal cell membrane . The carbohydrate moieties of STR-200A and STR-200B were examined by enzymatic deglycosylation . The 70-kDa protein of STR-200A was found to contain N-linked high-mannose-type and/or hybrid-type oligosaccharides, and results suggested that it possesses at least three N glycosylation sites . The 53-kDa protein of STR-200B was found to have an N-linked complex-type oligosaccharide side chain . The deglycosylated 70-kDa protein retained activity for binding to STh, suggesting that the carbohydrate moieties of these receptor proteins are not important for binding with STh.

Gene, 1992 Oct 1, 119(2), 155 - 61
Mammalian expression vectors with modulatable promoters and two multiple cloning sites; Wang Q et al.; To facilitate the use of a wide range of selectable markers in transfection studies with human cells, in conjunction with the use of modulatable promoters for regulated expression of the genes of interest, we constructed two pUC19-based mammalian expression vectors, each containing two lacZ alpha-based multiple cloning sites (MCS) . Selectable markers can be inserted into the MCS derived from pUC19, and the recombinants can be screened by lacZ complementation . The genes of interest can be inserted into the second MCS . The new MCS contains an amber stop codon in-frame with translation of the LacZ alpha-peptide . The presence of insert in the second MCS can also be screened on XGal plates, but in an Escherichia coli host containing an amber suppressor gene . Expression of the genes of interest can be modulated through transcription from the promoter of the mouse metallothionein-I-encoding gene or the long terminal repeat of the mouse mammary tumor virus . These vectors, as well as several of the intermediate plasmids described in this report, can be used to clone any two genetic elements into a single plasmid.

EMBO J, 1992 Oct, 11(10), 3747 - 57
Role of the open reading frames of Rous sarcoma virus leader RNA in translation and genome packaging; Donze O et al.; The Rous sarcoma virus (RSV) RNA leader sequence carries three open reading frames (uORFs) upstream of the AUG initiator of the gag gene . We studied, in vivo, the role of these uORFs by changing two or three nucleotides of the three AUGs or by deleting the first uORF . Our results show that (i) unlike most previously characterized uORFs, which decrease translation, the first uORF (AUG1) of RSV acts as an enhancer of translation, since absence of the first AUG decreased translation; AUG3 also modulates translation, probably by interfering with scanning ribosomes as described for other upstream ORFs, and mutation of AUG2 had no effect on translation . (ii) Mutation of each of the upstream AUGs lowered the infectivity of progeny virions . (iii) Unexpectedly, mutation of AUG1 and/or AUG3 dramatically reduced RNA packaging by 50-to 100-fold, unlike mutation of AUG2 which did not alter RNA packaging efficiency . Additional mutants in the vicinity of uORF1 and uORF3 were constructed in order to elucidate the mechanism by which uORFs affect RNA packaging: a translation model requiring uORFs 1 and 3, and involving ribosome pausing at AUG 3 is discussed.

EMBO J, 1992 Oct, 11(10), 3635 - 43
The cAMP-CRP/CytR nucleoprotein complex in Escherichia coli: two pairs of closely linked binding sites for the cAMP-CRP activator complex are involved in combinatorial regulation of the cdd promoter; Holst B et al.; Transcription initiation at CytR regulated promoters in Escherichia coli is controlled by a combinatorial regulatory system in which the cAMP receptor protein (CRP) functions as both an activator and a co-repressor . By combining genetic studies and footprinting analyses, we demonstrate that regulated expression of the CytR controlled cdd promoter requires three CRP-binding sites: a high affinity site (CRP-1) and two overlapping low affinity sites (CRP-2 and CRP-3) centred at positions -41, -91 and -93, respectively . In the absence of CytR, cAMP-CRP interacts at one set of sites (CRP-1 and CRP-2) and both of these binding sites are required for full promoter activation . In the presence of CytR, however, the two regulators bind cooperatively to cddP forming a nucleoprotein complex in which cAMP-CRP binds to CRP-1 and CRP-3 and CytR occupies the sequence between these sites . Thus, association of the two regulators involves a repositioning of the cAMP-CRP complex . Moreover, mutant cdd promoters in which CRP-2 and CRP-3 have been deleted are partially regulated by CytR, and cAMP-CRP and CytR still bind cooperatively to these promoters . These findings provide clues to an understanding of how cAMP-CRP and CytR interact at a structurally diverse set of promoters.

Virology, 1992 Oct, 190(2), 724 - 32
Independent association of antibodies against human papillomavirus type 16 E1/E4 and E7 proteins with cervical cancer; Kanda T et al.; The E4 open reading frame (ORF) of human papillomaviruses (HPVs) is transcribed in abundant mRNAs encoding an E1/E4 fusion gene during the productive infection, and the HPV 16 E7 ORF encodes an oncoprotein detectable in the cell lines derived from cervical carcinoma . We examined 421 human sera, which included 108 samples from the patients with cervical carcinoma, for the presence of IgG antibodies against the HPV 16 E4 and E7 proteins by enzyme-linked immunosorbent assay . Bacterially expressed fusion protein lac-E1/E4 and nonfusion protein E7 were purified and used as antigens . All of the 22 serum samples positive for anti-E7 antibody and the 11 out of 15 samples positive for anti-E1/E4 antibody were from the patients with cervical carcinoma, but only one sample was found to contain both anti-E1/E4 and anti-E7 antibodies . These findings show specific and independent association of these antibodies with cervical carcinoma.

Virology, 1992 Oct, 190(2), 702 - 15
Genetic analysis of the herpes simplex virus type 1 UL9 gene: isolation of a LacZ insertion mutant and expression in eukaryotic cells; Malik AK et al.; HSV-1 host range mutants in complementation group 1-36 (hr27 and hr156) whose mutations map in the UL9 gene, encoding the origin binding protein, are unable to form plaques or synthesize viral DNA or late viral proteins when grown in nonpermissive Vero cells (Carmichael, E . P., Kosovsky, M . J., Weller, S . K., 1988, J . Virol . 62, 91-99) . These defects are complemented efficiently by growth in the permissive cell line, S22, which contains the wild type version of several HSV genes including UL9 . In this report the precise nature and location of the lesions in host range mutants hr27 and hr156 were determined by DNA sequencing; both mutants were found to contain identical single-base-pair substitutions at codons 309 and 311 in the UL9 open reading frame . This region lies within the putative helicase domain of the UL9 protein . The UL9 gene was disrupted by the insertion of an insertional mutagen ICP6::lacZ in which the Escherichia coli lacZ gene is expressed under control of the viral ICP6 promoter . Hr94, a viral mutant containing this insertion, does not form plaques or synthesize viral DNA when grown in Vero cells, although both defects are complemented efficiently on permissive cell lines . These results confirm that the UL9 gene product is essential for viral growth and DNA replication . Furthermore, since no detectable UL9 protein is synthesized in hr94-infected cells, this virus provides a useful genetic background for further structure-function analysis since no potentially interfering nonfunctional UL9 protein will be expressed . We have expressed the UL9 open reading frame under the control of the strong and inducible HSV-1 ICP6 promoter and have derived Vero cell lines containing variable copy numbers of the ICP6::UL9 construct . Cells whose copy number of this construct exceeded approximately 120 are unable to support efficient plaque formation by wild-type virus . Cell lines with low copy numbers of this construct are able to complement hr27, hr156, and hr94.

Plant Mol Biol, 1992 Oct, 20(1), 37 - 47
Molecular cloning and expression of the gene encoding ADP-glucose pyrophosphorylase from the cyanobacterium Anabaena sp . strain PCC 7120; Charng YY et al.; Previous studies have indicated that ADP-glucose pyrophosphorylase (ADPGlc PPase) from the cyanobacterium Anabaena sp . strain PCC 7120 is more similar to higher-plant than to enteric bacterial enzymes in antigenicity and allosteric properties . In this paper, we report the isolation of the Anabaena ADPGlc PPase gene and its expression in Escherichia coli . The gene we isolated from a genomic library utilizes GTG as the start codon and codes for a protein of 48,347 Da which is in agreement with the molecular mass determined by SDS-PAGE for the Anabaena enzyme . The deduced amino acid sequence is 63, 54, and 33% identical to the rice endosperm small subunit, maize endosperm large subunit, and the E . coli sequences, respectively . Southern analysis indicated that there is only one copy of this gene in the Anabaena genome . The cloned gene encodes an active ADPGlc PPase when expressed in an E . coli mutant strain AC70R1-504 which lacks endogenous activity of the enzyme . The recombinant enzyme is activated and inhibited primarily by 3-phosphoglycerate and Pi, respectively, as is the native Anabaena ADPGlc PPase . Immunological and other biochemical studies further confirmed the recombinant enzyme to be the Anabaena enzyme.

Tohoku J Exp Med, 1992 Oct, 168(2), 175 - 82
Complex regulation of a tumor marker expression . Enhancer and silencer of the GST-P gene; Muramatsu M et al.; Glutathione transferase P (GST-P) is expressed at high levels in precancerous lesions and hepatocellular carcinomas from a very early stage of chemically-induced hepatocarcinogenesis in the rat . To explore the molecular mechanisms of its specific activation, we are investigating the regulation mechanisms of the GST-P gene expression . By using gene technology, we have identified a strong enhancer, GPEI, at 2.5 Kb and a silencer region at about 400 bp upstream from the transcription start site . GPEI has a palindromic structure composed of two TPA-responsive element (TRE)-like sequences and binds at least three proteins including AP-1 (c-jun/c-fos) . The silencer is composed of several sequences resembling each other and binds at least three proteins including SF-B/LAP/LIP . To determine whether the GST-P gene is activated together with a putative hepato-oncogene because they are located close to each other (cis-mechanism), or because they share a trans-acting factor that can activate both genes simultaneously (trans-mechanism), transgenic rats were produced with GST-P control region connected to the CAT reporter . The results unequivocally demonstrate that GST-P gene is activated position-independently by a trans-mechanism.

Protein Sci, 1992 Oct, 1(10), 1298 - 307
Inactivation and covalent modification of CTP synthetase by thiourea dioxide; Robertson JG et al.; Thiourea dioxide was used in chemical modification studies to identify functionally important amino acids in Escherichia coli CTP synthetase . Incubation at pH 8.0 in the absence of substrates led to rapid, time dependent, and irreversible inactivation of the enzyme . The second-order rate constant for inactivation was 0.18 M-1 s-1 . Inactivation also occurred in the absence of oxygen and in the presence of catalase, thereby ruling out mixed-function oxidation/reduction as the mode of amino acid modification . Saturating concentrations of the substrates ATP and UTP, and the allosteric activator GTP prevented inactivation by thiourea dioxide, whereas saturating concentrations of glutamine (a substrate) did not . The concentration dependence of nucleotide protection revealed cooperative behavior with respect to individual nucleotides and with respect to various combinations of nucleotides . Mixtures of nucleotides afforded greater protection against inactivation than single nucleotides alone, and a combination of the substrates ATP and UTP provided the most protection . The Hill coefficient for nucleotide protection was approximately 2 for ATP, UTP, and GTP . In the presence of 1:1 ratios of ATP:UTP, ATP:GTP, and UTP:GTP, the Hill coefficient was approximately 4 in each case . Fluorescence and circular dichroism measurements indicated that modification by thiourea dioxide causes detectable changes in the structure of the protein . Modification with {14C}thiourea dioxide demonstrated that complete inactivation correlates with incorporation of 3 mol of {14C}thiourea dioxide per mole of CTP synthetase monomer . The specificity of thiourea dioxide for lysine residues indicates that one or more lysines are most likely involved in CTP synthetase activity . The data further indicate that nucleotide binding prevents access to these functionally important residues.

Res Microbiol, 1992 Oct, 143(8), 755 - 65
DNA fingerprinting of Chlamydia trachomatis by use of ribosomal RNA, oligonucleotide and randomly cloned DNA probes; Scieux C et al.; DNA fingerprinting of 15 reference strains and 24 clinical isolates of Chlamydia trachomatis, 2 strains of C . psittaci and one strain of C . pneumoniae was studied by use of universal 16 + 23S RNA from Escherichia coli, 16S rDNA-directed oligonucleotide and randomly cloned chlamydial DNA probes . The rRNA-gene restriction patterns (ribotypes) enabled the differentiation of chlamydial species . Following DNA cleavage by restriction endonuclease PvuII, lymphogranuloma venereum and trachoma biovars of C . trachomatis could be differentiated . An oligonucleotide, designed to hybridize the C . trachomatis 16S rDNA, also allowed for both species-specific identification and biovar typing of C . trachomatis human strains . Molecular typing system using 3 lambda clones containing C . trachomatis serotype E random DNA inserts, combined to ribotyping, revealed 12 groups of variable banding patterns within C . trachomatis, and could provide an alternative epidemiological tool.

Res Microbiol, 1992 Oct, 143(8), 743 - 53
Characterization of high molecular weights of complexes and polymers of cytoplasmic proteins in Escherichia coli; Imamura R et al.; To search for filamentous polymers of cytoplasmic proteins of Escherichia coli, high molecular weights (> 670 kDa) of protein complexes of cell extracts were fractionated by gel filtration and ion-exchange column chromatography . Proteins of 100, 77 and 52 kDa were co-purified . The 100- and 52-kDa proteins were identified to be pyruvate dehydrogenase and lipoamide dehydrogenase, respectively, by determining the N-terminal amino acid sequences . Experimental results indicate that the 77-kDa protein is identical to dihydrolipoamide acyltransferase . The 100-kDa protein was found to be identical to the 100-kDa protein described by Tomioka (1991), and was related to the formation of filaments and sheets in the presence of 100 mM KCl . However, neither long filaments nor sheets were observed in our sample containing these enzymes, which was not consistent with Tomioka's conclusion . Another 100-kDa protein which forms spirosome-like particles was purified and identified to be alcohol dehydrogenase based on the N-terminal sequence.

Genet Anal Tech Appl, 1992 Oct-Dec, 9(5-6), 151 - 8
Histochemical techniques for locating Escherichia coli beta-galactosidase activity in transgenic organisms; Fire A; Escherichia coli beta-galactosidase is a commonly used reporter molecule for analyzing gene expression . Recently, beta-galactosidase fusions have been applied to a variety of eukaryotic systems . The techniques for constructing and introducing beta-galactosidase fusion constructs as well as soluble assays for total enzyme function have been described in detail elsewhere . This article describes histochemical techniques for analyzing organisms that contain a functional beta-galactosidase fusion construct . The object is to determine semiquantitatively which cells are expressing the beta-galactosidase fusion protein, as well as the subcellular localization of the protein . Due to its prevalence in the author's laboratory, Caenorhabditis elegans is used as a canonical organism for the detailed methods described.

Genet Anal Tech Appl, 1992 Oct-Dec, 9(5-6), 134 - 9
High-throughput DNA preparation system; Garner HR et al.; A system demonstrating the feasibility of high-throughput, centrifugation-based DNA separations and purifications has been constructed and tested . Samples are currently processed at a rate of 96 in approximately 2-3 h . The device implements an automation-optimized alkaline lysis protocol for the rapid extraction of plasmid or cosmid DNA from 1-ml bacteria cultures . The conditions for optimal culturing in deep-well (96 x 1 ml) microwell plates have been developed, and all sample manipulations are done within these plates . The use of microwell plates was essential to obtain high throughput and make manipulations following the DNA preparation (prep) easier because they can then be manipulated using a variety of commercially available robots . The entire prep system is constructed above a Beckman GPR centrifuge and operated under Macintosh IIcx control . This device has systems for fluid handling, microwell-plate manipulations, and centrifuge rotor alignment.

Genet Anal Tech Appl, 1992 Oct-Dec, 9(5-6), 127 - 33
Analysis of protocol variations on DNA yield; Armstrong B et al.; A modified alkaline lysis method for preparing plasmid DNA from bacterial cells has been developed for automation implementation . The objective of this study was to develop a simplified centrifuge-based protocol that can process samples in a microwell plate . These manual experiments and parametric studies show that the alkaline lysis method can be modified significantly to enable DNA preparations to be done rapidly and reliably by an automated system . The conclusions of this study were (a) centrifugation at < 1500 g is sufficient, (b) centrifugation times need not be extended to compensate for the reduced force, (c) reactions can be done at room temperature, (d) reagent volumes can be reduced over those typically used, and (e) certain reagents can be combined to simplify the handling of fluids.

Chin Med J (Engl), 1992 Oct, 105(10), 833 - 8
Protective effect of Re-LPS antiserum on experimental multiple system organ failure; Yao YM et al.; This study examines the possible beneficial effect of Re-LPS (F515) antiserum on experimental multiple system organ failure (MSOF) in rabbits . The results showed that the plasma LPS level was significantly decreased, and it took a shorter period to clear up LPS in experimental than in control rabbits after receiving Re-LPS antiserum . Pretreatment with antiserum can markedly improve the function of the liver, lungs, kidneys, blood and gastrointestinal tract . The MSOF incidence in the group of rabbits receiving immune sera was only 11.2% and the survival rate was raised by about 40.0% . The results suggest that early passive immunotherapy may neutralize gut-derived endotoxin, inhibit endotoxin-induced mediators release and prevent development of severe complications due to sepsis . It is therefore postulated that LPS core antiserum may provide a prophylactic effect on the development of experimental MSOF.

Biull Eksp Biol Med, 1992 Oct, 114(10), 442 - 4
{The morphofunctional characteristics of the cecum in experimental escherichiosis}; Ali-Riza AE et al.; Using the model of experimental escherichiosis in mice by means of morphological, immunomorphological, morphometrical and electron microscopy methods, the authors give morphofunctional characteristics of caecum 15 minutes to 2 weeks after inoculation . The authors show the dynamics of infectious process, characterized by changes of microcirculation, increasing lymphoplasmocellular infiltration, dystrophic changes in cells of neuroplexes and degranulation of mast and endocrine cells . The data obtained show that pathological process in caecum during experimental escherichiosis has an immune character, that the above portion of the intestine is a part of endocrine system.

Zhongguo Zhong Xi Yi Jie He Za Zhi, 1992 Oct, 12(10), 620 - 1, 582
{Experimental study on tea in inhibiting mutational specificity of 6 antineoplastic drugs}; Zhao ZZ et al.; According to the principles of SOS response, the authors tested the mutational specificity of tea and its inhibitory effects to the mutational specificity of 6 antineoplastic drugs by using the method of mutational and anti-mutational synchronous test . The results revealed that the tea had no mutational toxicity but anti-mutation effect . It also had the inhibitory effect on mutational toxicity of 6 antineoplastic drugs, including Mitomycin C, Bleomycin, Fluorouracil, Cisdiaminodichoroplatinum, Arabinosylcytosin and Mustargen . These results have provided referential basis for further study on anti-cancer effect and clinical use of tea.

Exp Hematol, 1992 Oct, 20(9), 1118 - 24
Canine stem cell factor (c-kit ligand) supports the survival of hematopoietic progenitors in long-term canine marrow culture; Shull RM et al.; The cDNA for canine stem cell factor (cSCF, c-kit ligand) was cloned and expressed in Escherichia coli . The recombinant protein (rcSCF), 165 amino acids in length, is very similar structurally to the soluble form of previously cloned and sequenced rodent and human SCFs . The biological effects of rcSCF were studied in a day-10 granulocyte-macrophage colony-forming unit (CFU-GM) clonogenic assay and in long-term liquid bone marrow culture of non-adherent hematopoietic cells in the absence of a stromal underlayer . Synergism in the stimulation of growth of CFU-GM was demonstrated between rcSCF and both recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) and naturally occurring colony-stimulating activity present in the serum of a neutropenic dog . Alone, rcSCF was nonstimulatory for committed marrow precursors in methylcellulose cultures and had minimal effect on hematopoietic progenitor cell survival in stromaless, liquid cultures . When rcSCF was combined with phytohemagglutinin-stimulated canine lymphocyte-conditioned medium (PHA-LCM) or rh interleukin 6 (IL-6), with or without rhGM-CSF, CFU-GM survived for up to 5 weeks . The combination of rcSCF and rhGM-CSF, without rhIL-6, led to an early increase in CFU-GM in liquid cultures that declined more rapidly than in flasks that included rhIL-6 . Survival of progenitor cells was negligible beyond 1 week in flasks with growth factor combinations lacking rcSCF . Sustained production of nonadherent cells in long-term cultures also was dependent on rcSCF in combination with canine PHA-LCM or recombinant human growth factors . It appears that rcSCF, like that from rodent and primate species, has the ability to influence the survival and proliferation of CFU-GM, and perhaps earlier progenitor cells, in hematopoietic tissues . In a long-term liquid culture system in which growth factor production by stromal cells is limited, rcSCF possesses a unique ability to maintain the viability of progenitor cells for up to 5 weeks.

Fukuoka Igaku Zasshi, 1992 Oct, 83(10), 367 - 76
Anti-tolA antibody in non-A, non-B chronic liver disease; Nakamuta M et al.; We constructed a cDNA library against the plasma obtained from the patient with acute exacerbation of non-A, non-B liver cirrhosis, and immunoscreened this library with the sera obtained from the patients with non-A, non-B chronic liver disease . One positive clone lambda 22C containing about 1.2 kb cDNA insert was isolated from 10(6) clones . Nucleotide sequence determination and subsequent homology search revealed its identity to the tolA gene of Escherichia coli . Anti-tolA antibody was detected in 54.5% of the patients with NANB chronic liver disease whose sera were negative for antibody to hepatitis C virus (anti-C100) . In contrast, anti-tolA was detected only of 14.6% patients with anti-C100 positive NANB chronic liver disease, 10.5% with hepatitis B surface antigen-positive chronic liver disease, 7.7% with alcoholic liver disease and 4.2% in normal control, and no positive case in acute hepatitis of etiology and in primary biliary cirrhosis . However, antibody to the core protein of hepatitis C virus (anti-JCC) was detected 50% of the patients whose sera were negative for anti-C100 but positive for anti-tolA . Recently, it has been reported that hepatitis C virus is rich in mutations and has some variants . These results indicated the presence of a common epitope between the tolA protein and some agent related to non-A, non-B hepatitis, especially to anti-C100 negative non-A, non-B hepatitis such as variants of hepatitis C virus which have mutations in C100 coded region.

Mol Microbiol, 1992 Oct, 6(19), 2815 - 24
An SOS-inducible defective retronphage (phi R86) in Escherichia coli strain B; Kirchner J et al.; In Escherichia coli, RecA protein regulates the DNA damage-inducible survival-enhancing SOS response . Mutant allele recA730, which causes constitutive SOS expression, is lethal at high temperatures in B/r, a derivative of wild-type B, but not in K-12 or in certain B/r--K-12 hybrids . We present evidence that killing is due to SOS induction of a defective retronphage, phi R86, which is integrated into the B/r chromosome at 19 min, but is absent in K-12 . phi R86 contains retron EC-86 which encodes reverse transcriptase and a small multicopy DNA-RNA complex, msDNA-RNA . Induction of phi R86 in recA730 B/r strains results in inhibition of host DNA replication before cell death . A retronphage 'killer' gene, ORF336, when overexpressed from a plasmid, causes similar effects without SOS induction . phi R86 is not detectably u.v.-inducible in recA+ strains.

Biochemistry, 1992 Sep 29, 31(38), 9304 - 9
Equilibrium denaturation of recombinant porcine growth hormone; Bastiras S et al.; Equilibrium denaturation of recombinant porcine growth hormone (pGH) derived from Escherichia coli using the denaturant guanidine hydrochloride (GuHCl) was followed by ultraviolet absorption spectroscopy, intrinsic fluorescence, far-ultraviolet circular dichroism, and size-exclusion chromatography . The normalized denaturation transition curves for each of the above methods were not coincident; denaturation resulted in an initial disruption of the tertiary structure, whereas secondary structure and degree of compactness were disrupted at higher concentrations of denaturant . Size-exclusion chromatography also detected an associated form of pGH at intermediate GuHCl concentrations . These findings conclusively show that pGH does not follow a simple two-state folding mechanism but are consistent with the framework model of folding . Stable intermediates observed were similar to those seen in other nonhuman growth hormones and are characterized as compact and largely alpha-helical yet lacking nativelike tertiary structure.

Biochemistry, 1992 Sep 29, 31(38), 9288 - 93
Structural and functional characterization of the mutant Escherichia coli glutaredoxin (C14----S) and its mixed disulfide with glutathione; Bushweller JH et al.; Glutaredoxin is essential for the glutathione (GSH)-dependent synthesis of deoxyribonucleotides by ribonucleotide reductase, and in addition, it displays a general GSH disulfide oxidoreductase activity . In Escherichia coli glutaredoxin, the active site contains a redox-active disulfide/dithiol of the sequence Cys11-Pro12-Tyr13-Cys14 . In this paper, we have prepared and characterized the Cys14----Ser mutant of E . coli glutaredoxin and its mixed disulfide with glutathione . The Cys14----Ser mutant of glutaredoxin is shown to retain 38% of the GSH disulfide oxidoreductase activity of the wild-type protein with hydroxyethyl disulfide as substrate but to be completely inactive with ribonucleotide reductase, demonstrating that dithiol glutaredoxin is the hydrogen donor for ribonucleotide reductase . The covalent structure of the mixed disulfide of glutaredoxin(C14S) with GSH prepared with 15N-labeling of the protein was confirmed with nuclear magnetic resonance (NMR) spectroscopy, establishing a basis for NMR structural studies of the glutathione binding site on glutaredoxin.

Biochemistry, 1992 Sep 29, 31(38), 9237 - 42
Site-directed mutagenesis identifies catalytic residues in the active site of Escherichia coli phosphofructokinase; Berger SA et al.; Six active site mutants of Escherichia coli phosphofructokinase have been constructed and characterized using steady-state kinetics . All but one of the mutants (ES222) have significantly lower maximal activity, implicating these residues in the catalytic process . Replacement of Asp127, the key catalytic residue in the forward reaction with Glu, results in an enzyme with wild-type cooperative and allosteric behavior but severely decreased Fru6P binding . Replacement of the same residue with Tyr abolishes cooperativity while retaining sensitivity to allosteric inhibition and activation . Thus, this mutant has uncoupled homotropic from heterotropic allostery . Mutation of Asp103 to Ala results in an enzyme which retains wild-type Fru6P-binding characteristics with reduced activity . GDP, which allosterically activates the wild-type enzyme, acts as a mixed inhibitor for this mutant . Mutation of Thr125 to Ala and Asp129 to Ser produces mutants with impaired Fru6P binding and decreased cooperativity . In the presence of the activator GDP, both these mutants display apparent negative cooperativity . In addition, ATP binding is now allosterically altered by GDP . These results extend the number of active site residues known to participate in the catalytic process and help to define the mechanisms behind catalysis and homotropic and heterotropic allostery.

Biochemistry, 1992 Sep 29, 31(38), 9183 - 9
Specific valylation of turnip yellow mosaic virus RNA by wheat germ valyl-tRNA synthetase determined by three anticodon loop nucleotides; Dreher TW et al.; The valylation by wheat germ valyl-tRNA synthetase of anticodon loop mutants of turnip yellow mosaic virus RNA has been studied . RNA substrates 264 nucleotides long were made by T7 RNA polymerase from cDNA encompassing the 3' tRNA-like region of genomic RNA . Substitution singly, or in combination, of three nucleotides in the anticodon loop resulted in very poor valylation (Vmax/KM less than 10(-3) relative to wild type) . These nucleotides thus represent the major valine identity determinants recognized by wheat germ valyl-tRNA synthetase; their relative contribution to valine identity, in descending order, was as follows: the middle nucleotide of the anticodon (A56 in TYMV RNA), the 3' anticodon nucleotide (C55), and the 3'-most anticodon loop nucleotide (C53) . Substitutions in the wobble position (C57) had no significant effect on valylation kinetics, while substitutions of the discriminator base (A4) resulted in small decreases in Vmax/Km . Mutations in the major identity nucleotides resulted in large increases in KM, suggesting that wheat germ valyl-tRNA synthetase has a lowered affinity for variant substrates with low valine identity . Comparison with other studies using valyl-tRNA synthetases from Escherichia coli and yeast indicates that the anticodon has been phylogenetically conserved as the dominant valine identity region, while the identity contribution of the discriminator base has been less conserved . The mechanism by which anticodon mutations are discriminated also appears to vary, being affinity-based for the wheat germ enzyme, and kinetically-based for the yeast enzyme {Florentz et al . (1991) Eur . J . Biochem . 195, 229-234}.

J Biol Chem, 1992 Sep 25, 267(27), 19650 - 4
Escherichia coli mutant SELD enzymes . The cysteine 17 residue is essential for selenophosphate formation from ATP and selenide; Kim IY et al.; Synthesis of a labile selenium donor compound, selenophosphate, from selenide and ATP by the Escherichia coli SELD enzyme was reported previously from this laboratory . From the gene sequence, SELD is a 37-kDa protein that contains 7 cysteine residues, 2 of which are located at positions 17 and 19 in the sequence -Gly-Ala-Cys-Gly-Cys-Lys-Ile- (Leinfelder, W., Forchhammer, K., Veprek, B., Zehelein, E., and Bock, A . (1990) Proc . Natl . Acad . Sci . U.S.A . 73, 543-547) . Inactivation of the enzyme by alkylation with iodoacetamide indicated that at least 1 cysteine residue in the protein is essential for enzyme activity . To test the possibility that the Cys17 and/or Cys19 residue might be essential, these were changed to serine residues by site-specific mutagenesis . The biological activities of the wild type and mutant proteins were studied using E . coli MB08 (selD-) transformed with plasmids containing the selD genes . The plasmid containing the Cys17-mutated gene failed to complement MB08, whereas the Cys19-mutated gene was indistinguishable from wild type . The mutant proteins, like the wild type enzyme, bound to an ATP-agarose matrix, showing that their affinities for ATP were unimpaired . Selenide-dependent formation of AMP from ATP was abolished by mutation of Cys17, but the Cys19 mutation had no effect on the ability of the enzyme to catalyze the reaction . These results indicate that Cys17 has an essential role in the catalytic process that leads to the formation of selenophosphate from ATP and selenide.

J Biol Chem, 1992 Sep 25, 267(27), 19631 - 5
Neither lipid modification nor processing of prolipoprotein is essential for the formation of murein-bound lipoprotein in Escherichia coli; Zhang WY et al.; The relationship between the modification and processing of prolipoprotein and the formation of murein-bound lipoprotein has been investigated using Escherichia coli mutants altered in the signal sequence of prolipoprotein and an E . coli strain producing OmpF-Lpp hybrid protein . The glyceride-modified prolipoprotein in mutant lppT20 and in globomycin-treated wild-type strain were covalently attached to the peptidoglycan . Likewise, the unmodified prolipoproteins in mutants lppL20, lppV20, and lppG21 were attached to the peptidoglycan . The OmpF-Lpp hybrid protein that is processed but not modified with lipid due to the absence of the cysteine-containing modification site in the hybrid protein was also covalently linked to the peptidoglycan . These results indicate that neither lipid modification nor the processing of prolipoprotein is essential for the formation of murein-bound lipoprotein in E . coli . In contrast, introduction of a charged amino acid residue such as Asp or Arg at the 14th position of prolipoprotein affected not only the lipid modification and processing of the mutant prolipoprotein but also the formation of murein-bound lipoprotein . Replacement of the Gly14 with Glu or Lys partially affected the lipid modification and processing of prolipoprotein; the peptidoglycan of the lppE14 and lppK14 mutants contained a reduced amount of mature lipoprotein but no mutant prolipoprotein . In addition, lpp mutants A20I23I24 and A20I23K24 were found to be defective in both lipid modification/processing of prolipoprotein and the formation of murein-bound lipoprotein . The defective formation of murein-bound lipoprotein in the latter mutants may be related to an alteration in the secondary structure at the modification/processing site of the mutant prolipoproteins.

J Biol Chem, 1992 Sep 25, 267(27), 19600 - 6
Characterization and site-directed mutagenesis of a low M(r) GTP-binding protein, ram p25, expressed in Escherichia coli; Nagata K et al.; The ram gene encodes a GTP-binding protein with a M(r) of 25,068 (Nagata, K., Satoh, T., Itoh, H., Kozasa, T., Okano, Y., Doi, T., Kaziro, Y., and Nozawa, Y . (1990) FEBS Lett . 275, 29-32) . It has a putative effector domain very similar to that of yeast SEC4 protein, and shares 40% identity and 60% homology with it, respectively . In order to analyze the biochemical properties, ram cDNA was engineered and inserted into a bacterial expression vector; this allowed the production at a high level of soluble recombinant ram p25 in Escherichia coli . The purified ram p25 contained an equimolar amount of GDP . The purified protein bound approximately 1 mol of {35S}guanosine 5'-O-(thiotriphosphate) GTP gamma S)/mol of protein, with a Kd value of 120 nM . {35S}GTP gamma S binding to this protein was inhibited by GTP and GDP, but not by ATP and ADP . In the presence of 10 mM Mg2+, the dissociation of {8,5'-3H}GDP and {35S}GTP gamma S from ram p25 occurred with rates of 0.015 min-1 and 0.004 min-1, respectively, showing that the ram p25 has a higher affinity for GTP than GDP . The rate of release of Pi from {gamma-32P}GTP-bound ram p25 was calculated to be 0.011 min-1 . The contribution of guanine nucleotide-binding and GTP-hydrolysis domains of the protein to its biochemical activities was investigated by site-directed mutagenesis . Substitution of Val for Gly at position 19 resulted in disappearance of {35S}GTP gamma S- and {3H}GDP-binding activity in spite of good expression of the protein . Mutations of Thr41 to Ser, Ala76 to Thr, and Asn133 to His slightly increased the rates of {35S} GTP gamma S binding and {3H}GDP dissociation, but had almost no effects on the manner of {gamma-32P}GTP hydrolysis . Replacement of Gln78 with Leu significantly increased the {3H}GDP dissociation rate (7-fold) and decreased GTP hydrolytic activity considerably.

J Biol Chem, 1992 Sep 25, 267(27), 19560 - 4
Alterations of the carboxyl-terminal amino acid residues of Escherichia coli lipoprotein affect the formation of murein-bound lipoprotein; Zhang WY et al.; Mutations in the Escherichia coli lpp gene resulting in the alterations of the COOH-terminal region of the lipoprotein have been isolated by oligonucleotide-directed mutagenesis . As might be expected, substitution of Lys78 with Arg78 completely abolished the formation of murein-bound lipoprotein . Each of the following single amino acid substitutions did not significantly affect the formation of bound-form lipoprotein: Asp70 to Glu70 or Gly70; Lys75 to Thr75; and Tyr76 to His76, Ile76, or Leu76 . In contrast, mutational alterations of Tyr76 to Cys76, Gly76, Asn76, Pro76, or Ser76 resulted in a reduction of the bound-form lipoprotein to levels of 14-32% of that in the wild-type strain . A common feature of these lpp COOH-terminal mutations affecting the formation of bound-form lipoprotein is the presence of a beta-turn secondary structure at the COOH-terminal region of all these mutant lipoproteins . In addition, substitution of Tyr76 to Asp76 or Glu76, and Arg77 to Asp77 or Leu77 also resulted in a reduced formation of the bound-form lipoprotein . These results suggest that the formation of murein-bound lipoprotein requires a COOH-terminal Lys residue and a positively charged COOH-terminal region . Furthermore, a beta-turn secondary structure in the COOH-terminal random coil region interferes with the attachment of the lipoprotein to the peptidoglycan.

J Biol Chem, 1992 Sep 25, 267(27), 19529 - 35
Site-specific mutagenesis of residues in the Escherichia coli mannitol permease that have been suggested to be important for its phosphorylation and chemoreception functions; Weng QP et al.; The Escherichia coli mannitol permease is an integral membrane protein that catalyzes the concomitant transport and phosphorylation of D-mannitol and also acts as the chemoreceptor for chemotaxis of E . coli to this hexitol . At least 4 aminoacyl residues in this protein have been suggested to be important in these activities: His-195, His-256, Cys-384, and His-554 . Previous evidence has implicated His-554 and Cys-384 as residues that are covalently phosphorylated, in sequence, as intermediates in phosphotransfer to mannitol . We have constructed a number of site-specific mutants of the mannitol permease at these positions . The properties of proteins in which His-554 or Cys-384 has been changed are consistent with their essential roles in phosphorylation . We also used these mutants to show that intermolecular phosphotransfer between His-554 and Cys-384 can occur in vivo in membrane-bound heterodimers consisting of different mutant subunits . The properties of proteins with mutations at position 195 suggest an important role for this residue involving hydrogen bonding, while His-256 performs no significant function in the mannitol permease . Finally, the phosphorylation and chemoreception activities for each mutant protein were each roughly in the same proportion to these activities in the wild-type protein, showing that these functions of the mannitol permease are tightly coupled under normal physiological conditions.

J Biol Chem, 1992 Sep 25, 267(27), 19334 - 40
Defective replication activity of a dominant-lethal dnaB gene product from Escherichia coli; Marszalek J et al.; dnaB protein of Escherichia coli is an essential replication protein . A missense mutant has been obtained which results in replacement of an arginine residue with cysteine at position 231 of the protein (P . Shrimankar, L . Shortle, and R . Maurer, unpublished data) . This mutant displays a dominant-lethal phenotype in strains that are heterodiploid for dnaB . Biochemical analysis of the altered form of dnaB protein revealed that it was inactive in replication in several purified enzyme systems which involve specific and nonspecific primer formation on single-stranded DNAs, and in replication of plasmids containing the E . coli chromosomal origin . Inactivity in replication appeared to be due to its inability to bind to single-stranded DNA . The altered dnaB protein was inhibitory to the activity of wild type dnaB protein in replication by sequestering dnaC protein which is also required for replication . By contrast, it was not inhibitory to dnaB protein in priming of single-stranded DNA by primase in the absence of single-stranded DNA binding protein . Sequestering of dnaC protein into inactive complexes may relate to the dominant-lethal phenotype of this dnaB mutant.

J Biol Chem, 1992 Sep 25, 267(27), 19095 - 100
Functional role of arginine 302 within the lactose permease of Escherichia coli; Matzke EA et al.; Within the lactose permease, an arginine residue is found on a transmembrane segment at position 302 . Based upon the effects of mutations at or in the vicinity of Arg-302, this residue has been implicated to be involved with H+ and/or sugar recognition . To further elucidate the role of this residue, we have substituted Arg-302 with serine, histidine, and leucine via site-directed mutagenesis . All three of these substitutions result in an impaired ability to transport galactosides as evidenced by their poor growth on minimal plates supplemented with lactose or melibiose . Furthermore, in vitro transport assays revealed substantial alterations in the kinetic constants for downhill lactose transport . The wild-type strain exhibited a Km for lactose transport of 0.30 mM and a Vmax of 267 nmol of lactose/min.mg of protein . The Ser-302, His-302, and Leu-302 were observed to have Km values of 0.18, 2.3, and 2.8 mM, and Vmax values of 11.6, 56.4, and 22.0 nmol of lactose/min.mg of protein, respectively . In uphill transport assays, all three mutants were unable to accumulate beta-methyl-D-thiogalactoside . However, both the Ser-302 and His-302 mutants were able to accumulate lactose against a concentration gradient . During H+ transport assays, all three mutants were shown to transport H+ in conjunction with thiodigalactoside . In addition, the Ser-302 and His-302 strains exhibited small alkalinizations upon the addition of lactose . However, for the Leu-302 mutant, the addition of lactose did not result in a significant level of H+ transport . Finally, experiments were conducted which were aimed at measuring the ability of the mutant permeases to catalyze an H+ leak . In this regard, a comparison was made between the wild-type and mutant strains concerning their steady state pH gradient and their rates of H+ influx following oxygen pulses . The results of these experiments suggest that mutations at position 302 cause a sugar-dependent H+ leak.

J Biol Chem, 1992 Sep 25, 267(27), 19075 - 81
Characterization and RNA-binding properties of a chloroplast S1-like ribosomal protein; Franzetti B et al.; Control of translation is an important step in chloroplast gene expression . A first control can be exerted during the initiation complex formation which, in Escherichia coli, involves the ribosomal protein (r-protein) S1 . A cDNA clone have been characterized which codes for the precursor of the chloroplast r-protein CS1 . The mature protein consists of a central core which shows 31.5% amino acid homology to the E . coli protein S1 . The CS1 is considerably shorter (40 kDa) than the protein S1 (61 kDa) . The core fragment contains three degenerated repeats which show homology to both the ribosome- and the RNA-binding domain of S1 . RNA-protein CS1 interactions were studied by UV cross-linking and toe-printing . CS1 has been over-expressed in E . coli, and after purification its RNA-binding properties were studied in vitro . We conclude that the CS1 exhibits an RNA-binding activity which is actively involved in the chloroplast initiation complex formation . It is shown that the CS1 binds to poly(A) in contrast with S1 which binds strongly to poly(U) . These results are interpreted in relation to the presence of poly(A)-rich regions in chloroplast transcripts of higher plants.

Biochem Pharmacol, 1992 Sep 25, 44(6), 1165 - 70
Inhibition of enzymatic activity of phospholipases A2 by minocycline and doxycycline; Pruzanski W et al.; Extracellular phospholipases A2 play an important role in articular and extra-articular inflammatory processes . Secretory non-pancreatic phospholipase A2 (PLA2) has been implicated in the pathogenesis of articular inflammation in rheumatoid arthritis, whereas pancreatic PLA2 contributes to the tissue damage associated with acute pancreatitis . Since in experimental models lipophilic tetracyclines such as minocycline and doxycycline are antiinflammatory, we examined their effects on PLA2 activity using two assay systems in vitro . We found that minocycline and to a lesser degree doxycycline were markedly inhibitory to both pancreatic and non-pancreatic PLA2 . Using {14C}oleic acid labeled Escherichia coli membrane phospholipids as substrate, the IC50 values for minocycline and doxycycline were 3.6 x 10(-5) M (18 micrograms/mL) and 0.98 x 10(-4) M (47 micrograms/mL), respectively . In a scooting mode assay using the synthetic phospholipid 1-palmitoyl-2-(10-pyrenedecanoyl)-3-L-phosphatidylmethanol as substrate, IC50 values for minocycline were 5 microM (2.47 micrograms/mL) for non-pancreatic PLA2 and 8 microM (3.95 micrograms/mL) for pancreatic PLA2 . Addition of excess calcium up to 50 mM did not reverse the inhibitory activity of tetracyclines . We conclude that lipophilic tetracyclines inhibit PLA2, probably by interaction with the substrate, and may be a useful adjunct in the therapy of inflammatory conditions in which PLA2 is implicated pathogenetically.

Nucleic Acids Res, 1992 Sep 25, 20(18), 4859 - 64
Two regions in human DNA polymerase beta mRNA suppress translation in Escherichia coli; Date T et al.; Although human DNA polymerase beta (DNA pol beta) shows 96% identity with rat DNA pol beta at the amino acid level, it is weakly expressed in Escherichia (E.) coli relative to the rat enzyme . The mechanism of this suppression was investigated . Pulse-chase protein labeling and steady state mRNA analysis showed that mature human DNA pol beta protein is relatively stable in E . coli and the levels of human and rat DNA pol beta mRNA were comparable indicating that the human DNA pol beta expression is suppressed at the translational level . By systematic expression analysis of a number of chimeric genes composed of human and rat cDNAs, two strong translational suppression regions were mapped in the human DNA pol beta mRNA; one was named TSR-1, corresponding to CGG encoding arginine (arg) at position 4 and the other, termed TSR-2, is located between codons 153 and 199 . Since substitution of the rat Arg-4 codon with synonymous codons showed strong effects upon the expression level, we propose that the arg codon at the N-terminal coding region plays a role in modulating expression.

Nucleic Acids Res, 1992 Sep 25, 20(18), 4811 - 6
How M.MspI and M.HpaII decide which base to methylate; Mi S et al.; The HpaII methylase (M.HpaII) recognizes the sequence CCGG and methylates the inner cytosine residue . The MspI methylase (MspI) recognizes the same sequence but methylates the outer cytosine residue . Both methylases have the usual architecture of 10 well-conserved motifs surrounding a variable region, responsible for sequence specific recognition, that is quite different in the two methylases . We have constructed hybrids between these two methylases and studied their methylation properties . A hybrid containing the variable region and C-terminal sequences from M.MspI methylates the outer cytosine residue . A second hybrid identical to the first except that the variable region derives from the M.HpaII methylates the inner cytosine residue . Thus the choice of base to be methylated within the recognition sequence is determined by the variable region.

Nucleic Acids Res, 1992 Sep 25, 20(18), 4789 - 93
Repair of UV-induced (6-4)photoproducts measured in individual genes in the Drosophila embryonic Kc cell line; de Cock JG et al.; The nucleotide excision repair (NER; dark-repair) of (6-4)photoproducts ((6-4)PPs) was assayed in cells from a permanent Drosophila melanogaster embryonic cell line, Kc, after exposure to 20 or 40 J/m2 ultraviolet (UV) light . Induction rates in the transcriptionally active genes Gart and Notch as well as in the inactive white locus is similar . They are formed with a frequency of about one-third of that of cyclobutane pyrimidine dimers (CPDs) . In all three genes, (6-4)PPs are repaired with the same rate and to the same extent: 31% of the (6-4)PPs are removed in 4 hours post-irradiation and after 16 hours repair is nearly complete . In none of the three genes strand-specific repair was found . Exposure of cells that were irradiated with 40 J/m2 UV to photoreactivating light for 1 hour prior to dark-repair incubation, resulted in enhanced repair of (6-4)PPs.

Nucleic Acids Res, 1992 Sep 25, 20(18), 4773 - 9
Transcription frequency modulates the efficiency of an attenuator preceding the rpoBC RNA polymerase genes of Escherichia coli: possible autogenous control; Steward KL et al.; Expression of the rpoBC genes encoding the beta and beta' RNA polymerase subunits of Escherichia coli is autogenously regulated . Although previous studies have demonstrated a post-transcriptional feedback mechanism, complex transcriptional controls of rpoBC expression may also contribute . We show that an attenuator (rpoBa) separating the ribosomal protein (rpl) genes from the rpoBC genes in the rplKAJLrpoBC gene cluster is modulated in its efficiency in response to changes in the frequency of transcription initiated by promoters located upstream . A series of rplJLrpoBalacZ transcriptional fusions was constructed on lambda vectors in which transcription into the rpoBa attenuator was varied by using a variety of promoters with different strengths . beta-galactosidase assays performed on monolysogens of the recombinant phage show that with transcription increasing over a 40-fold range, readthrough of rpoBa decreases from 61% to 19% . In contrast, two other well-characterized terminators show nearly constant efficiencies over a similar range of transcription frequencies . Using a set of phage P22 ant promoter variants with single-nucleotide changes in the promoter consensus sequences also demonstrates that the modulation of rpoBa function appears to be unrelated to the phenomenon of 'factor-independent antitermination' reported by others . The implications for autogenous control of RNA polymerase synthesis are discussed.

J Biol Chem, 1992 Sep 25, 267(27), 19272 - 7
Mutagenesis of the C-terminal nucleotide-binding site of an anion-translocating ATPase; Kaur P et al.; An oxyanion-translocating ATPase encoded by a bacterial plasmid confers resistance to antiomonials and arsenicals in Escherichia coli by extrusion of the toxic oxyanions from the cytosol . The anion pump is composed of two polypeptides, the ArsA and ArsB proteins . Purified ArsA protein is an oxyanion-stimulated ATPase with two nucleotide-binding consensus sequences, one in the N-terminal half and one in the C-terminal half of the protein . The ArsA protein can be labeled with {alpha-32P}ATP by a UV-catalyzed reaction . Previously reported mutations in the N-terminal site abolish photoadduct formation . Using site-directed mutagenesis the glycine-rich region of the C-terminal putative nucleotide-binding sequence was altered . Three C-terminal site mutant proteins (GR337, KE340, KN340) were analyzed, as well as one additional N-terminal mutant protein (KE21) . Strains bearing the mutated plasmids were arsenite sensitive to varying degrees . The purified ArsA protein from mutant KE340 retained approximately 20% of the wild type oxyanion-stimulated ATPase activity, while the purified proteins from the other mutants were catalytically inactive . The KE21 mutation in the N-terminal nucleotide-binding site eliminated photoadduct formation with {alpha-32P} ATP, while the purified proteins with mutations in the C-terminal site retained the ability to form a photoadduct . Each mutant protein was capable of forming a membrane-bound complex in arsB expressing strains . These results suggest first that both sites are required for resistance and ATPase activity, and second that the conserved lysyl residue in the glycine-rich loop of the C-terminal nucleotide-binding site is not essential for catalytic activity.

J Biol Chem, 1992 Sep 25, 267(27), 19082 - 8
Effects of decreased cytosine content on rho interaction with the rho-dependent terminator trp t' in Escherichia coli; Zalatan F et al.; We have introduced multiple cytosine-to-uracil mutations in the rho-dependent transcription terminator trp t' and have characterized a subset of the resulting mutant derivatives in vitro for termination efficiency, affinity for rho, and stimulation of rho-ATPase activity . No specific cytosine residue appears to be required for termination, and at least 13 of the 28 cytosine residues in the 104 nucleotide trp t' region can be mutated with little effect on termination efficiency . One derivative with 11 mutations is significantly less efficient than other derivatives with a similar number of mutations, implying that the pattern of alterations, as well as the number, can affect termination efficiency . Derivatives with 20 and 28 cytosines mutated are non-functional in termination, consistent with the idea that cytosines are required for rho-dependent termination . Our results are inconsistent, however, with the hypothesis that the termination efficiency is directly related to the cytosine/guanine ratio in the nascent transcript . The results support the idea that neither RNA binding affinity nor ATPase activation per se are accurate predictors of rho-dependent termination efficiency.

J Biol Chem, 1992 Sep 25, 267(27), 19060 - 5
Location of the cytoplasmic epitope for a K(+)-competitive antibody of the (H+,K+)-ATPase; Bayle D et al.; The monoclonal antibody (mAb) 95-111 binds the alpha subunit of (H+,K+)-ATPase and inhibits the K(+)-ATPase activity . To map the epitope, all of the partial sequences of the alpha subunit were expressed in Escherichia coli HB101 using rabbit alpha subunit cDNA restriction fragments ligated into PuEx vector . Bacterial recombinant lysates were separated by sodium dodecyl sulfate-gel electrophoresis, and the epitope was detected by Western blotting . The antibody site was mapped between Cys529 and Glu561 . This is close to the Lys517 that binds fluorescein isothiocyanate (FITC) and is considered to be between M4 and M5 close to the ATP binding domain . However, the mAb inhibition of ATPase is not ATP-competitive but is K(+)-competitive with a KI of 2 x 10(-9) M . The mAb also inhibits K+ quench of FITC fluorescence competitively with a KI of 8 x 10(-9) M . The K+ activation of ATPase activity and quench of FITC fluorescence are dependent on K+ binding to an E2 form of the enzyme from the extracytoplasmic surface . The mAb epitope is cytoplasmic since the K(+)-ATPase activity of ion-tight gastric vesicles is inhibited . The 125I-mAb 95-111 binds to a single class of sites with an apparent KD of 2.3 +/- 0.8 x 10(-9) M and K+ does not displace bound mAb . Hence, antibody binding to a cytoplasmic Cys529-Glu561 epitope allosterically competes with K(+)-dependent reactions at extracytoplasmic sites.

Nucleic Acids Res, 1992 Sep 25, 20(18), 4897 - 901
Mutagenesis by O6 meG residues within codon 12 of the human Ha-ras proto-oncogene in monkey cells; Pletsa V et al.; The first or/and the second guanines of the human Ha-ras codon 12 (normally GGC) were substituted by O6 meG residues and the modified sequence was subsequently introduced into an SV40-based shuttle vector able to replicate in both simian cells and bacteria . After replication in simian COS7 cells (proficient in O6-alkyl-guanine transferase), plasmid DNA was extracted and mutations were screened in E . coli DH5 alpha cells . The vast majority of the mutations induced by O6 meG were G----A transitions . The mutation frequency observed at the second guanine of codon 12 (12G2 position: 3.75% +/- 0.4) was higher than the one observed at the first guanine (12G1 position: 1.09% +/- 0.6) . This difference was confirmed by the results obtained when two adjacent O6 meG residues were positioned within codon 12 . The higher mutation frequency observed for the 12G2 position could be attributed to differential repair or/and variation in polymerase fidelity . These results are in agreement with animal experiments where alkylating agents gave rise to mutation on G2 position of codon 12.

Nucleic Acids Res, 1992 Sep 25, 20(18), 4699 - 703
DNA deoxyribophosphodiesterase of Escherichia coli is associated with exonuclease I; Sandigursky M et al.; DNA deoxyribophosphodiesterase (dRpase) of E . coli catalyzes the release of deoxyribose-phosphate moieties following the cleavage of DNA at an apurinic/apyrimidinic (AP) site by either an AP endonuclease or AP lyase . Exonuclease I is a single-strand specific DNA nuclease which affects the expression of recombination and repair pathways in E . coli . We show here that a major dRpase activity in E . coli is associated with the exonuclease I protein . Highly purified exonuclease I isolated from an over-producing stain contains high levels of dRpase activity; it catalyzes the release of deoxyribose-5-phosphate from an AP site incised with endonuclease IV of E . coli and the release of 4-hydroxy-2-pentenal-5-phosphate from an AP site incised by the AP lyase activity of endonuclease III of E . coli . A strain containing a deletion of the sbcB gene showed little dRpase activity; the activity could be restored by transformation of the strain with a plasmid containing the sbcB gene . The dRpase activity isolated from an overproducing stain was increased 70-fold as compared to a normal sbcB+ strain (AB3027) . These results suggest that the dRpase activity may be important in pathways for both DNA repair and recombination.

J Biol Chem, 1992 Sep 25, 267(27), 19163 - 71
Arg-257 and Arg-307 of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase bind the C-2 phospho group of fructose-2,6-bisphosphate in the fructose-2,6-bisphosphatase domain; Lin K et al.; Rat liver fructose-2,6-bisphosphatase, which catalyzes its reaction via a phosphoenzyme intermediate, is evolutionarily related to the phosphoglycerate mutase enzyme family (Bazan, F., Fletterick, R., and Pilkis, S.J . (1989) Proc . Natl . Acad . Sci . U.S.A . 86, 9642-9646) . Arg-7 and Arg-59 of the yeast phosphoglycerate mutase have been postulated to be substrate-binding residues based on the x-ray crystal structure . The corresponding residues in rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, Arg-257 and Arg-307, were mutated to alanine . The Arg257Ala and Arg307Ala mutants and the wild-type enzyme were expressed in Escherichia coli and then purified to homogeneity . Both mutant enzymes had identical far and near UV circular dichroism spectra and 6-phosphofructo-2-kinase activities when compared with the wild-type enzyme . However, the Arg257Ala and Arg307Ala mutants had altered steady state fructose-2,6-bisphosphatase kinetic properties; the Km values for fructose-2,6-bisphosphate of the Arg257Ala and Arg307Ala mutants were increased by 12,500- and 760-fold, whereas the Ki values for inorganic phosphate were increased 7.4- and 147-fold, respectively, as compared with the wild-type values . However, the Ki values for the other product, fructose-6-phosphate, were unchanged for the mutant enzymes . Although both mutants exhibited parallel changes in kinetic parameters that reflect substrate/product binding, they had opposing effects on their respective maximal velocities; the maximal velocity of Arg257Ala was 11-fold higher, whereas that for Arg307Ala was 700-fold lower, than that of the wild-type enzyme . Pre-steady state kinetic studies demonstrated that the rate of phosphoenzyme formation for Arg307Ala was at least 4000-fold lower than that of the wild-type enzyme, whereas the rate for Arg257Ala was similar to the wild-type enzyme . Furthermore, consistent with the Vmax changes, the rate constant for phosphoenzyme breakdown for Arg257Ala was increased 9-fold, whereas that for Arg307Ala was decreased by a factor of 500-fold, as compared with the wild-type value . The results indicate that both Arg-257 and Arg-307 interact with the reactive C-2 phospho group of fructose 2,6-bisphosphate and that Arg-307 stabilizes this phospho group in the transition state during phosphoenzyme breakdown, whereas Arg-257 stabilizes the phospho group of the ground state phosphoenzyme intermediate.

J Biol Chem, 1992 Sep 25, 267(27), 19155 - 62
Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10 . The role of a conserved sequence motif, GXXXXRXGRR, in a putative cytoplasmic loop between helices 2 and 3; Yamaguchi A et al.; The region including the conserved Ser65-Asp66 dipeptide in the tetracycline/H+ antiporter (TET) encoded by transposon Tn10 is thought to play a gating role (Yamaguchi, A., Ono, N., Akasaka, T., Noumi, T., and Sawai, T . (1990) J . Biol . Chem . 265, 15525-15530) . The dipeptide is in putative interhelix loop2-3, which also includes the conserved sequence motif, GXXXXRXGRR, found in all TET proteins and sugar/H+ symporters . Through the combination of localized random and site-directed mutagenesis, each residue in loop2-3 was replaced . Among 10 residues in putative loop2-3, the important residues, of which substitution resulted in significant reduction or complete loss of the transport activity, were Gly62, Asp66, Gly69, and Arg70 . The defect in the transport activity of the Gly62 and Gly69 substitution mutants corresponded to the steric hindrance by the substituents as to the putative beta-turn structure of the peptide backbone containing these glycines . Of 3 conserved Arg residues, the replacement of only Arg70 caused complete loss of the activity except for replacement with Lys, indicating the importance of a positive charge at this position, which is similar to the essentiality of a negative charge at Asp66 . A "charge-neutralizing" intra-loop salt bridge between Asp66 and Arg70 was not likely because the double mutant in which Asp66 and Arg70 were replaced with asparagine and leucine, respectively, showed no transport activity . A triple mutant with only one positive charge at Arg70 in this loop showed about half the wild-type activity, indicating that the polycationic nature of the loop was not critical for the activity . Cys mutants as to the unessential residues in the loop were modifiable with N-ethylmaleimide, except for the Met64----Cys and Arg71----Cys mutants; however, the modification of only the Ser65----Cys mutant caused significant inhibition of the transport activity, indicating that position 65 is a unique position in the structure of loop2-3.

Biochim Biophys Acta, 1992 Sep 23, 1159(2), 223 - 6
Allosteric properties of haemoglobin beta 41 (C7) Phe-->Tyr: a stable, low-oxygen-affinity variant synthesized in Escherichia coli; Baudin V et al.; In human deoxy haemoglobin, the alpha 42(C7)Tyr-residue is hydrogen-bonded to beta 99(G1)Asp which stabilizes the low-oxygen-affinity deoxy conformation . We engineered a haemoglobin with Tyr for Phe at the homologous C7 position in beta-chains . The oxygen affinity of the variant is decreased about two-fold relative to Hb A while keeping similar KR and KT values . This mutant may be a candidate for the development of an artificial oxygen carrier, as it would not require an external effector for significant oxygen unloading in vivo.

Mol Cell Biochem, 1992 Sep 22, 115(1), 19 - 26
Effect of spermine on peptide-bond formation, catalyzed by ribosomal peptidyltransferase; Kalpaxis DL et al.; The effect of spermine on the binding of AcPhe-tRNA to poly(U)-programmed ribosomes (step 1) and on the puromycin reaction (step 2) has been studied in a cell-free system, derived from E . coli . In the absence of ribosomal wash (FWR fraction) and at suboptimal concentration of Mg++ (6 mM), spermine stimulated the binding of AcPhe-tRNA at least five fold, while at 10 mM Mg++ there was a three fold stimulation . The above stimulatory effect was decreased at 6 mM Mg++, or was abolished at 10 mM Mg++ by the presence of FWR during the binding . Beside the stimulatory effect, spermine enhanced the stability of initiation complex AcPhe-tRNA-poly(U)-ribosome . In step 2, spermine affected the final degree of puromycin reaction and the activity status of peptidyltransferase . Both stimulatory and inhibitory effects have been observed, depending on the experimental conditions followed during the binding of the donor and during the peptide bond formation.

Biochemistry, 1992 Sep 22, 31(37), 9063 - 72
Characterization of the protonation and hydrogen bonding state of the histidine residues in IIAmtl, a domain of the phosphoenolpyruvate-dependent mannitol-specific transport protein; Van Dijk AA et al.; The A domain of the mannitol-specific EII, IIAmtl, was subcloned and proven to be functional in the isolated form (Van Weeghel et al., 1991) . It contains a histidine phosphorylation site, the first of two phosphorylation sites in the parent protein . In this paper, we describe the characterization of the three histidine residues in IIAmtl with respect to their protonation and hydrogen bonding state, using 1H{15N} heteronuclear NMR techniques and protein selectively enriched with {delta 1,epsilon 2-15N}histidine . The active site residue has a low pKa (less than 5.8) and shows no hydrogen bond interactions . The proton in the neutral ring is located at the N epsilon 2 position, which also proved to be the site of phosphorylation . The phosphorylation raises the pKa of the active site histidine considerably but does not change the hydrogen bond situation . The other two histidine residues, one of which is probably located on the surface of the protein, were also characterized . Both show hydrogen bond interactions in the unphosphorylated protein, but these are disturbed by the phosphorylation process . These observations, combined with small changes in pKa and titration behavior, indicate that the IIAmtl changes its conformation upon phosphorylation.

Biochemistry, 1992 Sep 22, 31(37), 8892 - 7
Site-directed mutagenesis of lysine 319 in the lactose permease of Escherichia coli; Persson B et al.; Lys319, which is on the same face of putative helix X as His322 and Glu325 in the lactose permease of Escherichia coli, has been replaced with Leu by oligonucleotide-directed, site-specific mutagenesis . Although previous experiments suggested that the mutation does not alter permease activity, we report here that K319L permease is unable to catalyze active lactose accumulation or lactose efflux down a concentration gradient . The mutant does catalyze facilitated influx down a concentration gradient at a significant rate; however, the reaction occurs without concomitant H+ translocation . The mutant also catalyzes equilibrium exchange at about 50% of the wild-type rate, but it exhibits poor counterflow activity . Finally, flow dialysis and photoaffinity labeling experiments with p-nitrophenyl alpha-D-galactopyranoside indicate that K319L permease probably has a markedly decreased affinity for substrate . The alterations described are not due to diminished levels of the mutated protein in the membrane, since immunological studies reveal comparable amounts of permease in wild-type and K319L membranes . It is proposed that Lys319, like Arg302, His322, and Glu325, plays an important role in active lactose transport, as well as substrate recognition.

FEBS Lett, 1992 Sep 21, 310(1), 9 - 12
Differential role of four cysteines on the activity of a low M(r) phosphotyrosine protein phosphatase; Chiarugi P et al.; In this paper we describe the construction of five mutants of a bovine liver low M(r) phosphotyrosine protein phosphatase (PTPase) expressed as a fusion protein with the maltose binding protein in E . coli . Almost no changes in the kinetic parameters were observed in the fusion protein with respect to the native PTPase . Using oligonucleotide-directed mutagenesis Cys-17, Cys-62 and Cys-145 were converted to Ser while Cys-12 was converted to both Ser and Ala . The kinetic properties of the mutants, using p-nitrophenyl phosphate as substrate, were compared with those of the normal protein fused with the maltose binding protein of E . coli; both of the Cys-12 mutants showed a complete loss of enzymatic activity while the specific activity of the Cys-17 mutant was greatly decreased (200-fold) . The Cys-62 mutant showed a 2.5-fold decrease in specific activity, while the Cys-145 mutant remained almost unchanged . These data confirm the involvement of Cys-12 and Cys-17 in the catalytic site and suggest that Cys-62 and Cys-145 mutations may destabilise the structure of the enzyme.

FEBS Lett, 1992 Sep 21, 310(1), 1 - 4
Interaction of thioredoxin with oxidized aminobutyrate aminotransferase . Evidence for the formation of a covalent intermediate; Park J et al.; Pig brain 4-aminobutyrate aminotransferase is inactivated by pre-incubation with pyrroloquinoline quinone (2,7,9-tricarboxy-1H-pyrrolo{2,3,f}quinoline- 4,5-dione; PQQ) at pH 7 . The reaction of approximately 2 SH residues/dimer is sufficient to inactivate the enzyme . Reoxidized aminotransferase is reactivated by E . coli thioredoxin . Similar results were obtained with E . coli 4-aminobutyrate aminotransferase . The spectroscopic properties of thioredoxin, tagged with the fluorescence probe, anthraniloyl, were used to monitor its interaction with re-oxidized 4-aminobutyrate aminotransferase . During the regeneration of native aminotransferase by thioredoxin, the substrate forms a covalent intermediate with the oxidoreductase, as revealed by gel filtration chromatography . It is postulated that the substrate (oxidized aminotransferase) forms a covalent intermediate with thioredoxin through disulfide linkages.

Gene, 1992 Sep 21, 119(1), 151 - 2
Sequence of the 5-aminolevulinic acid dehydratase-encoding gene from the hyperthermophilic methanogen, Methanothermus sociabilis; Brockl G et al.; We report on the sequence of the Methanothermus sociabilis aladh gene, which encodes the 5'-aminolevulinic acid dehydratase . The identity of the enzyme was determined by sequence comparison and by expression in Escherichia coli.

Gene, 1992 Sep 21, 119(1), 149 - 50
Vectors for generating nested deletions and facilitating subcloning G+C-rich DNA between Escherichia coli and Streptomyces sp; Gewain KM et al.; New multiple cloning sites (MCS), which facilitate the subcloning of G+C-rich DNA, were added to pUC18, M13mp18, pVE616 (a pBR322-derived insertion vector), and the low-copy-number Streptomyces vector, pIJ922 . The MCS in these vectors contain sites found infrequently in Streptomyces DNA, facilitating the exchange of subclones between the vectors . The MCS added to M13mp18 and pUC18 was also designed to generate nested deletions within subcloned fragments.

Gene, 1992 Sep 21, 119(1), 101 - 6
The Escherichia coli rna gene encoding RNase I: sequence and unusual promoter structure; Zhu L et al.; A clone containing the Escherichia coli rna gene encoding the nonspecific endoribonuclease, RNase I, was isolated and sequenced . The sequence of the 1070-nucleotide (nt) fragment agreed completely with that of a rna clone recently reported by Meador and Kennell {Gene 95 (1990) 1-7} . The transcription start point (tsp) of rna was identified using primer extension analysis, and its promoter sequence was established by comparison of RNase I expression levels in various deletion mutants . Our results indicate that the rna promoter is highly unusual . Its -35 region shows a poor match to the consensus sequence, and moreover, it is located within a stem-loop structure that apparently is a Rho-independent transcription termination site for an upstream gene.

Gene, 1992 Sep 21, 119(1), 95 - 100
Sequences of the genes encoding the minor tip components of Pap-3 pili of Escherichia coli; Klann AG et al.; We report the sequence of the papE, papF and papG genes from the O75:K5 uropathogenic P pili variant, Pap-3 . Comparison of the deduced amino acid sequences with those of other P pili variants reveals regions of complete homology, as well as regions of variation . Analysis of the variations in the hydrophilic domains of these proteins will help elucidate the residues which determine binding specificity.

Gene, 1992 Sep 21, 119(1), 127 - 9
The construction of Streptomyces cyaneus genomic libraries in Escherichia coli is dependent upon the use of Mcr-deficient strains; Wang P et al.; Streptomyces cyaneus genomic DNA ligated into either lambda phage or plasmid vectors was very inefficiently cloned into standard Escherichia coli host strains . However, the same material could be efficiently cloned using Mcr-deficient E . coli strains . These results suggest that the S . cyaneus genome contains 5-methylcytosine residues, some of which occur within the recognition sequences of the E . coli Mcr restriction system.

J Mol Biol, 1992 Sep 20, 227(2), 396 - 406
cAMP-CRP activator complex and the CytR repressor protein bind co-operatively to the cytRP promoter in Escherichia coli and CytR antagonizes the cAMP-CRP-induced DNA bend; Pedersen H et al.; Initiation of transcription from the cytRP promoter in Escherichia coli is activated by the cAMP-CRP complex and negatively regulated by the CytR repressor protein . By combining gel retardation and footprinting assays, we show that cAMP-CRP binds to a single site centered at position -64 and induces a considerable bend in the DNA . CytR binds to a region immediately downstream from, and partially overlapping, the CRP site, and induces a modest bend into the DNA . In combination, cAMP-CRP and CytR bind co-operatively to cytRP forming a nucleoprotein complex in which the proteins directly interact with each other and bind to the same face of the DNA helix . CytR binding concomitantly antagonizes the cAMP-CRP-induced bend . This study indicates that the minimal DNA region required to obtain CytR regulation consists of a single binding site for each of cAMP-CRP and CytR . The case described here, in which a protein-induced DNA bend is modulated by a second protein, may illustrate a mechanism that applies to other regulatory systems.

J Immunol Methods, 1992 Sep 18, 154(1), 11 - 20
The detection of intracytoplasmic interleukin-1 alpha, interleukin-1 beta and tumour necrosis factor alpha expression in human monocytes using two colour immunofluorescence flow cytometry; de Caestecker MP et al.; Two colour flow cytometry was used to analyse in situ cytokine expression by human monocytes . Whole blood was cultured in siliconised glass bottles, with or without E . coli lipopolysaccharide (LPS), for various times, and the mononuclear cells (MNCs) then exposed to a variety of permeabilisation procedures prior to flow cytometric analysis . Paraformaldehyde (PF)/saponin fixation preserved cellular morphology, and caused a reproducible degree of permeabilisation (estimated by propidium iodide inclusion: mean 94%, range 86-99% (n = 33)) . After fixation with 4% PF and permeabilisation with 1% saponin at 0 degrees C in PBS containing 20% human serum, MNCs were incubated with phycoerythrin(PE)-conjugated mouse anti-CD14 (monocyte phenotype) and polyclonal rabbit anti-human interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumour necrosis factor alpha (TNF-alpha), or control rabbit IgG . Binding of rabbit antibodies was detected using goat anti-rabbit IgG fluorescein isothiocyanate (FITC) . FITC fluorescence was increased in CD14 PE positive cells with the three anti-cytokine antibodies following LPS stimulation, compared with controls . There was a reproducible dose related response in monocyte IL-1 beta and TNF-alpha expression following LPS stimulation, with early peaks in TNF-alpha (2 h), compared with IL-1 beta (4 h), and IL-1 alpha (12 h) . Specificity of this cytokine detection system was confirmed by inhibition studies using the corresponding recombinant human cytokines, by an absence of staining in CD14 negative or unpermeabilised MNCs, and by the characteristic cytoplasmic localisation of the different cytokines visualised with UV immunochemistry . Hence, the methods described here provide a reproducible, semiquantitative and specific assay for the detection of cell associated monokines . The technique may be applicable to the analysis of a variety of different cytokines in other phenotypically defined cell populations.

Nature, 1992 Sep 17, 359(6392), 251 - 4
Escherichia coli cell-division gene ftsZ encodes a novel GTP-binding protein; RayChaudhuri D et al.; Escherichia coli divides by forming a septum across the middle of the cell . The biochemical mechanism underlying this process is unknown . Genetic evidence suggests that of all the fts (filamentation temperature sensitive) genes involved in E . coli cell division, ftsZ plays a central role at the earliest known step of septation . Here we show that FtsZ protein binds GTP in vitro using unusual sequence elements . In contrast, such binding to the product of the conditional-lethal ftsZ84 allele is impaired . Purified FtsZ displays a Mg(2+)-dependent GTPase activity which is markedly reduced in the FtsZ84 protein . FtsZ copurifies with near stoichiometric amounts of noncovalently-bound GDP, implying the presence of a GTPase cycle in vivo, similar to that known for signal-transducing GTP-binding proteins . We also show that a small fraction of FtsZ exists as a distinct membrane-associated species that binds GTP . The membrane association of FtsZ and the known ability of GTPases to act as molecular switches implicate FtsZ in a GTP-activated signal transduction pathway that may regulate the start of septation in E . coli.

Biochem Biophys Res Commun, 1992 Sep 16, 187(2), 970 - 5
Association of oriC region of Escherichia coli chromosome with outer membrane: effects of culture condition; Kambe-Honjoh H et al.; We isolated complexes containing oriC region DNA and outer membrane, named origin complex heavy and origin complex light, from the cells of Escherichia coli cultured in media with poor or rich of nutrients, and found the different nature of association between origin DNA and outer membrane . The ratio of origin complex light to origin complex heavy prepared from the cells cultured in rich media was lower than that of those from minimal medium culture . Outer membrane preparations from the cells grown in nutritious media had high abilities of association with origin complex light in the presence of magnesium . These results indicated that the number of binding sites on outer membrane with origin region DNA increase, or the binding between outer membrane and origin region DNA become more rigid, when cells grow faster and DNA replication initiate more frequently in a nutritious medium.

Biochem Biophys Res Commun, 1992 Sep 16, 187(2), 1106 - 12
Maintenance of repression control of the ilvGMEDA operon in a temperature-sensitive leucyl-transfer RNA synthetase mutant of Escherichia coli K-12 at a restrictive temperature; Whittaker JJ et al.; The mechanism of L-leucine regulation of ilvGMEDA is thought to be by ribosome-mediated attenuation that is dependent upon the concentration of Leu-tRNA(Leu) which results from leucyl-tRNA synthetase (LeuRS) activity . The requirement for LeuRS activity in attenuation control was tested in an Escherichia coli K-12 strain containing a temperature-sensitive LeuRS and the ilvGMEDA operon with an active ilvGM . Growth of this strain at 30 degrees C followed by a shift to 37 degrees C to inactivate the LeuRS revealed that ilvGM expression decreased at the restrictive temperature whereas the downstream gene expression was slightly elevated . We suggest that ilvGM does not respond to a deattenuation signal, and that, possibly, a secondary repression/derepression mechanism exists.

Biochem Biophys Res Commun, 1992 Sep 16, 187(2), 737 - 43
Mammalian and viral DNA sequences which interfere with the maintenance of a centromeric vector in yeast; Blangy A et al.; We constructed a recombinant plasmid by inserting into the pRS314 yeast centromeric plasmid vector the mouse DNA sequence responsible for the maintenance in transgenic mice of plasmid p12B1 (1) . Such constructs could constitute convenient shuttle vectors between yeast and mouse cells . However, the recombinant molecule could not be established as a stable plasmid in Saccharomyces cerevisiae . A region with a limited similarity to the yeast centromere (CEN element) is present in this mouse sequence as well as in two other sequences subsequently identified in a data bank search using the CEN consensus . One of them is localized in Bovine Papillomavirus Type 1 DNA, and the other one in the human beta-globin locus . Once inserted in pRS314, these two sequences showed the same inhibitory effect on plasmid maintenance as the p12B1 mouse DNA fragment . This effect appears to depend on the simultaneous presence in the construct of one of the "CEN-like regions" and of an authentic CEN element . Non-centromeric yeast plasmids carrying one of the three sequences could replicate autonomously, and were even stabilized to a significant extent . These results identify in the genomes of higher eukaryotes and their viruses a family of sequences which cannot be simply cloned in centromeric yeast vectors.

Proc Natl Acad Sci U S A, 1992 Sep 15, 89(18), 8794 - 7
Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector; Shizuya H et al.; A bacterial cloning system for mapping and analysis of complex genomes has been developed . The BAC system (for bacterial artificial chromosome) is based on Escherichia coli and its single-copy plasmid F factor . It is capable of maintaining human genomic DNA fragments of greater than 300 kilobase pairs . Individual clones of human DNA appear to be maintained with a high degree of structural stability in the host, even after 100 generations of serial growth . Because of high cloning efficiency, easy manipulation of the cloned DNA, and stable maintenance of inserted DNA, the BAC system may facilitate construction of DNA libraries of complex genomes with fuller representation and subsequent rapid analysis of complex genomic structure.

Proc Natl Acad Sci U S A, 1992 Sep 15, 89(18), 8701 - 5
In vivo requirement of integration host factor for nar (nitrate reductase) operon expression in Escherichia coli K-12; Rabin RS et al.; The nitrate reductase operon (narGHJI) of Escherichia coli encodes an anaerobic respiratory enzyme . Previous work has identified two cis-acting sites in the nar operon control region: a proximal site required for anaerobic induction mediated by the activator Fnr and a remote upstream site required for nitrate induction mediated by the activator NarL {Li, S . & DeMoss, J . A . (1988) J . Biol . Chem . 263, 13700-13705} . Our search for nar regulatory mutants yielded one strain with a mutation in himD, the structural gene for one of the subunits of integration host factor (IHF) . Strains carrying null alleles of the IHF structural genes, himD and himA, had severe defects in nitrate induction of the nar operon but were normal for nitrate induction of the coordinately regulated fdn operon . Anaerobic expression of both operons was normal in him mutants . Gel-mobility-shift and DNase I protection experiments revealed a single IHF binding site in the nar operon control region, located midway between the upstream activation site and the promoter . We conclude that an IHF-mediated DNA bend is essential for efficient nitrate induction of the sigma 70-dependent nar operon promoter . This requirement of IHF for transcriptional activation had been noted for several sigma 54-dependent promoters.

Proc Natl Acad Sci U S A, 1992 Sep 15, 89(18), 8651 - 5
Engineered iron oxide-adhesion mutants of the Escherichia coli phage lambda receptor; Brown S; Escherichia coli able to specifically adhere to iron oxide and not adhere to other metal oxides were constructed by genetic engineering . Concatamers of random oligonucleotides were introduced into a portion of a plasmid-borne lamB gene encoding an external domain of the phage lambda receptor . Bacteria able to adhere to iron oxide were selected by serial enrichment from the population of plasmid transformants . The concatameric nature of the inserted DNA allows a genetic analysis analogous to exons shuffling . Results of this genetic analysis indicate that in some isolates, part of the binding site is encoded by flanking vector sequences . This strategy may prove generally useful for identifying protein sequences able to recognize specific surfaces.

Proc Natl Acad Sci U S A, 1992 Sep 15, 89(18), 8419 - 23
Either of two functionally redundant sensor proteins, NarX and NarQ, is sufficient for nitrate regulation in Escherichia coli K-12; Rabin RS et al.; Nitrate acts through the response regulator NarL to activate and repress anaerobic respiratory gene expression in Escherichia coli . The narX gene product encodes a cognate sensor (histidine protein kinase) . However, previous work discovered that NarL-mediated nitrate regulation is essentially normal in delta narX deletion mutants . In other two-component regulatory systems studied, the cognate sensor gene is essential for normal regulation . We suggested that NarX-mediated signal transduction reactions are also provided by a functionally redundant nitrate sensor, NarQ . We report here the identification and analysis of narQ insertion mutants . In narX+ strains, a narQ::Tn10 insertion had no perceptible effect on nitrate regulation . However, the same narQ::Tn10 insertion eliminated nitrate regulation when present in delta narX deletion strains . Thus, either narX+ or narQ+ was sufficient for essentially normal NarL-mediated nitrate regulation . The narQ gene mapped to 53 minutes on the E . coli genetic map, a location distinct from all known nitrate regulatory or target genes . The predicted NarQ sequence shares substantial similarity with NarX, particularly in the histidine protein kinase region and in a region of shared similarity with the methyl-accepting chemotaxis proteins . Both NarQ and NarX apparently have N-terminal periplasmic domains, but the primary structures of these regions are largely dissimilar in the two sequences . Analysis of narX* and narL missense alleles in narQ+ versus narQ::Tn10 backgrounds suggests that NarQ and NarX may have subtle functional differences.

J Biol Chem, 1992 Sep 15, 267(26), 18940 - 5
Structural studies on human glutathione S-transferase pi . Substitution mutations to determine amino acids necessary for binding glutathione; Manoharan TH et al.; In order to identify amino acids involved in binding the co-substrate glutathione to the human glutathione S-transferase (GST) pi enzyme, we assembled three criteria to implicate amino acids whose role in binding and catalysis could be tested . Presence of a residue in the highly conserved exon 4 of the GST gene, positional conservation of a residue in 12 glutathione S-transferase amino acid sequences, and results from published chemical modification studies were used to implicate 14 residues . A bacterial expression vector (pUC120 pi), which enabled abundant production (2-26% of soluble Escherichia coli protein) of wild-type or mutant GST pi, was constructed, and, following nonconservative substitution mutation of the 14 implicated residues, five mutants (R13S, D57K, Q64R, I68Y, L72F) showed a greater than 95% decrease in specific activity . A quantitative assay was developed which rapidly measured the ability of wild-type or mutant glutathione S-transferase to bind to glutathione-agarose . Using this assay, each of the five loss of function mutants showed a greater than 20-fold decrease in binding glutathione, an observation consistent with a recent crystal structure analysis showing that several of these residues help to form the glutathione-binding cleft.

J Biol Chem, 1992 Sep 15, 267(26), 18356 - 60
Immunological characterization of the complex forms of chloroplast translational initiation factor 2 from Euglena gracilis; Ma L et al.; Euglena gracilis chloroplast translational initiation factor 2 (IF-2chl) occurs in several complex forms ranging in molecular mass from 200 to 800 kDa . Subunits of 97 to greater than 200 kDa have been observed in these preparations . Two monoclonal antibodies were prepared against the 97-kDa subunits of IF-2chl . Both of these antibodies recognize all of the higher molecular mass forms of this factor, suggesting that these subunits are closely related . Gel filtration chromatography indicates that the higher molecular mass subunits of IF-2chl are present in the higher molecular mass complexes, whereas the smaller subunits are present in the 200-400 kDa forms of IF-2chl . Probing extracts of light-induced and dark-grown cells with the antibodies indicates that the light induction of this chloroplast factor results from the synthesis of new polypeptide rather than from the activation of an inactive precursor form of the protein . Both the higher and lower molecular mass subunits of IF-2chl are present in 30 S initiation complexes as indicated by Western analysis . The binding of IF-2chl to chloroplast 30 S ribosomal subunits requires the presence of GTP, but does not require fMet-tRNA, messenger RNA, or other initiation factors . Neither polyclonal nor monoclonal antibodies against E . gracilis IF-2chl cross-react with Escherichia coli IF-2 or with animal mitochondrial IF-2.

J Biol Chem, 1992 Sep 15, 267(26), 18284 - 90
Molecular cloning of a cDNA encoding chicken T-protein of the glycine cleavage system and expression of the functional protein in Escherichia coli . Effect of mRNA secondary structure in the translational initiation region on expression; Okamura-Ikeda K et al.; DNA clones encoding chicken T-protein of the glycine cleavage system were isolated from chicken liver lambda gt10 cDNA libraries . Three overlapping clones provided an open reading frame of 1176 nucleotides that predicts a polypeptide of 392 amino acids (M(r) 42,056) comprised of a 16-residue mitochondrial targeting sequence and a 376-residue mature protein (M(r) 40,292) . The amino acid sequence predicted for the mature protein showed 67% identity with that of bovine T-protein . A cDNA encoding mature T-protein was constructed, and the nucleotide sequence just downstream of the initiation codon was modified without amino acid substitution to reduce the free energy of formation for the folded mRNA . Expression plasmids containing these cDNA variants produced large amounts of T-protein in Escherichia coli, while very low expression was observed with a plasmid containing wild type cDNA . Enzymatically active T-protein was obtained when the expression was conducted at 30 degrees C with 25 microM isopropyl-1-thio-beta-D-galactopyranoside . Under the full inducing condition (at 37 degrees C and 1 mM inducer), the expressed T-protein was recovered as insoluble and inactive protein . The recombinant T-protein was purified to near homogeneity with a yield of about 30% . Apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is approximately 40,000, similar to the size of T-protein purified from chicken liver . NH2-terminal amino acid sequence analysis (9 residues) revealed 100% identity with chicken T-protein determined chemically . The kinetic properties of the recombinant T-protein resembled those of the native chicken T-protein.

Biochim Biophys Acta, 1992 Sep 15, 1117(2), 232 - 4
Carbohydrate specificity of the receptor sites of mistletoe toxic lectin-I; Wu AM et al.; The carbohydrate specificity of mistletoe toxic lectin-I (ML-I) was studied by haemagglutination-inhibition assay . The results indicated that ML-I has a broad range of affinity for Gal alpha,beta linked sequences . The galabiose (E, Gal alpha 1----4Gal) sequence, a receptor of the uropathogenic E . coli ligand, was one of the best disaccharide inhibitors tested . The lectin also exhibits affinity for Lac(Gal beta 1----4Glc), T(Gal beta 1----3GalNAc), I/II(Gal beta 1----3/4GlcNAc) and B(Gal alpha 1----3Gal) sequences . Gal alpha 1----4Gal and Gal beta 1----4Glc are frequently occurring sequences of many glycosphingolipids located at the mammalian cell membranes, such as intestinal and red blood cell membranes, for ligand binding and toxin attachment . This finding provides important information concerning the possible mechanism of intoxication of cells by the mistletoe preparation.

J Immunol, 1992 Sep 15, 149(6), 1896 - 904
The beta 2-microglobulin dissociation rate is an accurate measure of the stability of MHC class I heterotrimers and depends on which peptide is bound; Parker KC et al.; Stable, recombinant, water-soluble complexes of HLA-A2 and HLA-B27 were reconstituted from 125I-labeled beta 2-microglobulin (beta 2m), a synthetic peptide, and HLA H chain fragments expressed as inclusion bodies in the Escherichia coli cytoplasm . Using this system, we were able to show: 1) the t1/2 of beta 2m dissociation from HLA complexes at 37 degrees C varied from approximately 40 h to less than 1 h, depending on the peptide employed for reconstitution . Peptide length and composition were found to be critical factors in determining the beta 2m dissociation rate . Endogenous peptides form complexes that are about as stable as those formed with typical antigenic peptides . 2) Peptide exchange reactions, in which an exogenous peptide replaces the peptide that is already bound by the class I molecule, proceed readily for complexes that have rapid beta 2m dissociation rates . Thus, difficulties in demonstrating peptide binding to complexes that contain endogenous peptides can be attributed to the stability of the endogenous peptide/class I molecule complex . 3) The peptide exchange reaction does not require concomitant beta 2m dissociation . 4) Distal parts of the class I molecule, which are not directly involved in peptide binding or beta 2m binding, have a major impact on the stability of class I molecules . Thus, these studies show that the dissociation rate of beta 2m is an excellent measure of how tightly a given peptide binds to class I MHC molecules, that the ability to bind peptide is tightly coupled to the binding of beta 2m and vice versa, and that regions of the molecule distal from the binding site influence the stability of peptide binding.

Thromb Res, 1992 Sep 15, 67(6), 677 - 85
Reduction of the anticoagulant activity of glycosaminoglycans on the surface of the vascular endothelium by endotoxin and neutrophils: evaluation by an amidolytic assay; Heyderman RS et al.; The processes that underlie the coagulopathy observed in severe infection are not fully understood, but seem to be due to an imbalance in the antithrombotic, and prothrombotic properties of the vascular endothelium . Sulphated glycosaminoglycans (GAGs) present on the vessel wall represent an important component of the non-thrombogenic nature of the endothelium . We have modified an amidolytic assay to study the functional ability of GAGs on human umbilical vein endothelial cells (HUVECS), and investigate the effect of E . coli endotoxin and neutrophils on HUVEC surface anticoagulant activity (SAA) . Neither endotoxin alone, nor separated neutrophils at lower concentrations (less than 10(6) neutrophils per ml), had major effects on endothelial SAA . When activated neutrophils were incubated with HUVECS pre-stimulated with endotoxin, a significant decrease in SAA was seen using either plasma (mean percentage of control 67.8% +/- sem 7.8; p < 0.02) or purified ATIII (mean percentage of control 69% +/- sem 4.6; p < 0.001) . We suggest that alterations in endothelial surface GAGs may occur during sepsis and inflammation, and that this may have important consequences for vascular function . This system will allow the further study of the role of GAGs in the intravascular thrombosis of severe sepsis, and other inflammatory diseases.

Biochem J, 1992 Sep 15, 286 ( Pt 3), 825 - 8
Human interleukin-5 expressed in Escherichia coli has N-terminal modifications; Rose K et al.; Recombinant human interleukin-5 exists as four major isoforms all possessing N-terminal methionine . Peptide mapping and subsequent analysis by fast-atom-bombardment mass spectrometry (f.a.b.-m.s.) have shown that N-terminal modifications are the cause of the charge heterogeneity . In order of decreasing abundance, these are unmodified methionine, retention of N-terminal formyl group, oxidation of N-terminal methionine to sulphoxide and carbamoylation of the N-terminus . These results were confirmed by analysis of the reduced and alkylated intact protein by electrospray-ionization mass spectrometry . The implications of these findings for the production and characterization of recombinant proteins are briefly discussed.

Biochem J, 1992 Sep 15, 286 ( Pt 3), 721 - 7
Binding energy and catalysis . Fluorinated and deoxygenated glycosides as mechanistic probes of Escherichia coli (lacZ) beta-galactosidase; McCarter JD et al.; Kinetic parameters for the hydrolysis of a series of deoxy and deoxyfluoro analogues of 2',4'-dinitrophenyl beta-D-galactopyranoside by Escherichia coli (lacZ) beta-galactosidase have been determined and rates found to be two to nine orders of magnitude lower than that for the parent compound . These large rate reductions result primarily from the loss of transition-state binding interactions due to the replacement of sugar hydroxy groups, and such interactions are estimated to contribute at least 16.7 kJ (4 kcal).mol-1 to binding at the 3, 4 and 6 positions and more than 33.5 kJ (8 kcal).mol-1 at the 2 position . The existence of a linear free-energy relationship between log(kcat./Km) for these compounds and the logarithm of the first-order rate constant for their spontaneous hydrolysis demonstrates that electronic effects are also important and provides direct evidence for oxocarbonium ion character in the enzymic transition state . A covalent intermediate which turns over only extremely slowly (t1/2 = 45 h) accumulates during hydrolysis of the 2-deoxyfluorogalactoside, and kinetic parameters for its formation have been determined . This intermediate is nonetheless catalytically competent, since it re-activates much more rapidly in the presence of the transglycosylation acceptors methanol or glucose, thereby providing support for the notion of a covalent intermediate during hydrolysis of the parent substrates.

FEMS Microbiol Lett, 1992 Sep 15, 75(2-3), 203 - 6
Cosmid pJAR4, a novel Streptomyces-Escherichia coli shuttle vector for the cloning of Streptomyces operons; Tercero JA et al.; A novel shuttle cosmid vector (pJAR4), based on pK505, was constructed for the cloning of Streptomyces DNA . It is a low-copy-number vector which determines hygromycin B-resistance as a selective marker and was used to clone the puromycin biosynthesis pathway from Streptomyces alboniger . Cosmids pJAR4 and pKC505 (which determines apramycin-resistance) stably co-transform both Streptomyces lividans and Streptomyces griseofuscus.

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