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 act