Nucleic Acids Res, 1991 Nov 25, 19(22), 6295 - 300 Effect of RecF protein on reactions catalyzed by RecA protein; Madiraju MV et al.; RecF protein is one of at least three single strand DNA (ssDNA) binding proteins which act in recombination and repair in Escherichia coli . In this paper we show that our RecF protein preparation complexes with ssDNA so as to retard its electrophoretic movement in an agarose gel . The apparent stoichiometry of RecF-ssDNA-binding measured in this way is one RecF molecule for every 15 nucleotides and the binding appears to be cooperative . Interaction of the other two ssDNA-binding proteins, RecA and Ssb proteins, has been studied extensively; so in this paper we begin the study of the interaction of RecF and RecA proteins . We found that the RecF protein preparation inhibits the activity of RecA protein in the formation of joint molecules whether added before or after addition of RecA protein to ssDNA . It, therefore, differs from Ssb protein which stimulates joint molecule formation when added to ssDNA after RecA protein . We found that our RecF protein preparation inhibits two steps prior to joint molecule formation: RecA protein binding to ssDNA and coaggregate formation between ssDNA-RecA complexes and dsDNA . We found that it required a much higher ratio of RecF to RecA protein than normally occurs in vivo to inhibit joint molecule formation . The insight that these data give to the normal functioning of RecF protein is discussed. J Biol Chem, 1991 Nov 25, 266(33), 22386 - 91 Nitrate-inducible formate dehydrogenase in Escherichia coli K-12 . II . Evidence that a mRNA stem-loop structure is essential for decoding opal (UGA) as selenocysteine; Berg BL et al.; fdnG, encoding the selenopeptide of Escherichia coli formate dehydrogenase-N, contains an in-frame opal (UGA) codon at amino acid position 196 that directs selenocysteine incorporation . We have identified sequences that contribute to the mRNA context required for decoding this UGA as selenocysteine . We identified a potential stem-loop structure immediately downstream of UGA196 that is comparable in size and structure to a stem-loop predicted to form in fdhF, which encodes the selenopeptide of E . coli formate dehydrogenase-H . Mutational analysis of the fdnG stem-loop structure suggests that it is critical for decoding UGA196 as selenocysteine . Our data indicate that both stability and specific nucleotide sequences of the stem-loop likely contribute to the appropriate mRNA context for selenocysteine incorporation into the fdnG gene product. J Biol Chem, 1991 Nov 25, 266(33), 22380 - 5 Nitrate-inducible formate dehydrogenase in Escherichia coli K-12 . I . Nucleotide sequence of the fdnGHI operon and evidence that opal (UGA) encodes selenocysteine; Berg BL et al.; The fdnGHI operon of Escherichia coli encodes nitrate-inducible formate dehydrogenase . We report here the entire nucleotide sequence of fdnGHI . The sequence contains three open reading frames of sizes appropriate to encode the three subunits of formate dehydrogenase-N . fdnG contains an in-frame UGA codon that specifies selenocysteine incorporation, and the predicted amino acid sequence of FdnG shows similarity to two other bacterial formate dehydrogenase enzymes . FdnH contains 4 cysteine clusters typical of those found in iron-sulfur proteins . FdnG also contains a cysteine cluster . Evidence from sequence and spectral analyses suggest that FdnI encodes cytochrome bFdn556 . Implications for the membrane topology of formate dehydrogenase-N and its mechanism of proton translocation are discussed. J Biol Chem, 1991 Nov 25, 266(33), 22141 - 6 One-step purification of Escherichia coli H(+)-ATPase (F0F1) and its reconstitution into liposomes with neurotransmitter transporters; Moriyama Y et al.; About 30% of the protein in the inner membrane of Escherichia coli strain DK8/pBWU13 is H(+)-ATPase (F0F1), and practically homogeneous F0F1 could be obtained by gradient centrifugation after solubilization of these membranes . The recombinant plasmid pBWU13 carries the unc operon for F0F1 . When reconstituted into liposomes, F0F1 formed an ATP-dependent proton gradient and membrane potential . Proteoliposomes reconstituted with F0F1 and solubilized transporters from chromaffin granules or synaptic vesicle membranes could transport serotonin, dopamine, and norepinephrine dependent on ATP hydrolysis . F0F1 can be obtained rapidly from DK8/pBWU13, and its reconstitution into liposomes with transporters may be useful for monitoring these transporters during their purification. J Theor Biol, 1991 Nov 21, 153(2), 255 - 85 Control theory of regulatory cascades; Kahn D et al.; We have extended Metabolic Control Theory to include cascades consisting of several modules controlling each other solely via regulatory effects . We derive several theorems that determine how the control properties of a cascade derive from (1) the control properties of each module, taken in isolation and (2) the regulatory interactions between the modules . Two cases are treated explicitly . The first concerns cascades in the absence of feed-back: in this case the internal control behaviour of each module is unaffected by external regulatory interactions . The second includes one feed-back loop and gives a quantitative expression of how feed-back modifies control properties: the internal control matrix within one module can be calculated as if the elasticity matrix of this module was the sum of its intrinsic elasticity matrix and a cyclic regulation matrix . More complex cascades can be analysed recursively by subdividing them into simpler modules, which can be treated individually . The theoretical framework developed here should facilitate quantitative experimental analysis of the control of cell physiology where the latter involves regulatory cascades. J Mol Biol, 1991 Nov 20, 222(2), 373 - 90 Two-dimensional 1H nuclear magnetic resonance study of the (5-55) single-disulphide folding intermediate of bovine pancreatic trypsin inhibitor; van Mierlo CP et al.; An analogue of the bovine pancreatic trypsin inhibitor (BPTI) folding intermediate that contains only the disulphide bond between Cys5 and Cys55 has been prepared in Escherichia coli by protein engineering methods, with the other four Cys residues replaced by Ser . Two-dimensional 1H nuclear magnetic resonance studies of the analogue have resulted in essentially complete resonance assignments of the folded form of the protein . The folded protein has a compact conformation that is structurally very similar to that of native BPTI, although there are subtle differences and the folded conformation is not very stable . Approximately half of the protein molecules are unfolded at 3 degrees C, and this proportion increases at higher temperatures . The folded and unfolded conformations are in slow exchange . The conformational properties of the analogue can explain many aspects of the kinetic role that the normal (5-55) intermediate plays in the folding of BPTI. J Mol Biol, 1991 Nov 20, 222(2), 353 - 71 (14-38, 30-51) double-disulphide intermediate in folding of bovine pancreatic trypsin inhibitor: a two-dimensional 1H nuclear magnetic resonance study; van Mierlo CP et al.; An analogue of the BPTI folding intermediate that contains only the disulphide bonds between Cys14 and Cys38 and between Cys30 and Cys51 has been prepared in Escherichia coli by protein engineering methods . The other two Cys residues of native BPTI (at positions 5 and 55) have been replaced by Ser . Essentially complete proton resonance assignments of the analogue were obtained by employing two-dimensional 1H nuclear magnetic resonance techniques . The intermediate has a more extended conformation in the N-terminal (residues 1 to 7) region and there are other differences in the C-terminal (residues 55 to 58) region . The remainder of the protein is substantially identical to native BPTI . The conformational properties of the analogue can explain several aspects of the kinetic role that the normal (14-38, 30-51) intermediate plays in the folding of BPTI. J Mol Biol, 1991 Nov 20, 222(2), 265 - 80 Absolute in vivo translation rates of individual codons in Escherichia coli . The two glutamic acid codons GAA and GAG are translated with a threefold difference in rate; Sorensen MA et al.; We have determined the absolute translation rates for four individual codons in Escherichia coli . We used our previously described system for direct measurements of in vivo translation rates using small, in-frame inserts in the lacZ gene . The inserts consisted of multiple synthetic 30 base-pair DNA oligomers with high densities of the four individual codons, GAA (Glu), GAG (Glu), CCG (Pro) and CGA (Arg) . Our method is independent of expression level, of mRNA half-life and of transcription rate . Codon GAA was found to be translated with a rate of 21.6 codons/second whereas codon GAG was translated 3.4-fold slower (6.4 codons/s) . These two codons are read by the same tRNA species . Codon CCG and CGA are both read by abundant tRNA species but nevertheless we found them to be translated slowly with rates of 5.8 and 4.2 codons/second, respectively . The context of these codons were varied, but we found no significant influence of context on their translation rates and we suggest a mechanism for why context may not affect translation rates . One insert with a low translation rate gave results that most readily can be explained by assuming queue formation of ribosomes on the insert . Such a queue was found to reduce the expression level by approximately 35% . Our experiments allowed us to estimate the average distance between ribosomes and thereby the translation initiation frequency on the wild-type lacZ mRNA . This was found to be one per three seconds. J Mol Biol, 1991 Nov 20, 222(2), 251 - 64 Attachment sites of primary binding proteins L1, L2 and L23 on 23 S ribosomal RNA of Escherichia coli; Egebjerg J et al.; The attachment sites of the primary binding proteins L1, L2 and L23 on 23 S ribosomal RNA of Escherichia coli were examined by a chemical and ribonuclease footprinting method using several probes with different specificities . The results show that the sites are confined to localized RNA regions within the large ribonuclease-protected ribonucleoprotein fragments that were characterized earlier . They are as follows: (1) L1 recognizes a tertiary structural motif in domain V centred on two interacting internal loops; the main protein interaction sites occur at the internal loop/helix junctions . (2) The L2 site constitutes a single irregular stem/loop structure in the centre of domain IV where non-Watson-Crick pairing is likely to occur . (3) L23 recognizes a tertiary structural motif involving a single terminal loop structure and part of an adjacent internal loop at the centre of domain III . Each of the three primary binding proteins, whose presence is essential for ribosomal assembly, has been associated with important ribosomal functions: L1 lies in the E-site for deacylated tRNA binding while L2 and L23 have been implicated in the P and A substrate sites, respectively, of the peptidyl transferase centre . Moreover, each of the protein sites, but particularly those of L2 and L23, lies at the centre of RNA domains where they can maximally influence both the assembly of secondary binding proteins and the function of the RNA region. J Mol Biol, 1991 Nov 20, 222(2), 139 - 42 Expression of the protease inhibitor ecotin and its co-crystallization with trypsin; McGrath ME et al.; We have expressed the serine protease inhibitor ecotin to high levels (greater than 400 mg/l of cell culture) in its natural mileau, the Escherichia coli periplasm, using the endogenous signal peptide and the heterologous tac promoter . After induction, functional, soluble ecotin comprises 15% of total cellular protein . This expression system has facilitated initiation of a crystallographic study to determine the structural basis for inhibition of the pancreatic serine proteases by ecotin . Ecotin was co-crystallized with rat trypsin mutant D102N . Preliminary crystallographic analysis of co-crystals showed that they diffract to at least 2.7 A, and indicate that they belong to the monoclinic space group, P21 . The cell constants are a = 52.0 A, b = 93.3 A, c = 160.7 A, and beta = 96 degrees . Four molecules each of trypsin and ecotin are found in the asymmetric unit. J Mol Biol, 1991 Nov 20, 222(2), 189 - 96 An Escherichia coli rpoB mutation that inhibits transcription of catabolite-sensitive operons; Rockwell P et al.; The Escherichia coli rpoB636 mutant is defective in the transcription of lac and other catabolite-sensitive operons . The lac promoter variant, UV5, which is independent of cyclic AMP and the cyclic AMP receptor protein, CRP, was also defective in rpoB636 mutants . The activity of the lac UV5 promoter was restored to wild-type levels by deletion of cya (adenylate cyclase) or crp . Cyclic AMP and CRP apparently act as inhibitors of the rpoB636 RNA polymerase. Biochemistry, 1991 Nov 19, 30(46), 11092 - 103 Investigation of the functional role of tryptophan-22 in Escherichia coli dihydrofolate reductase by site-directed mutagenesis; Warren MS et al.; We have applied site-directed mutagenesis methods to change the conserved tryptophan-22 in the substrate binding site of Escherichia coli dihydrofolate reductase to phenylalanine (W22F) and histidine (W22H) . The crystal structure of the W22F mutant in a binary complex with the inhibitor methotrexate has been refined at 1.9-A resolution . The W22F difference Fourier map and least-squares refinement show that structural effects of the mutation are confined to the immediate vicinity of position 22 and include an unanticipated 0.4-A movement of the methionine-20 side chain . A conserved bound water-403, suspected to play a role in the protonation of substrate DHF, has not been displaced by the mutation despite the loss of a hydrogen bond with tryptophan-22 . Steady-state kinetics, stopped-flow kinetics, and primary isotope effects indicate that both mutations increase the rate of product tetrahydrofolate release, the rate-limiting step in the case of the wild-type enzyme, while slowing the rate of hydride transfer to the point where it now becomes at least partially rate determining . Steady-state kinetics show that below pH 6.8, kcat is elevated by up to 5-fold in the W22F mutant as compared with the wild-type enzyme, although kcat/Km(dihydrofolate) is lower throughout the observed pH range . For the W22H mutant, both kcat and kcat/Km(dihydrofolate) are substantially lower than the corresponding wild-type values . While both mutations weaken dihydrofolate binding, cofactor NADPH binding is not significantly altered . Fitting of the kinetic pH profiles to a general protonation scheme suggests that the proton affinity of dihydrofolate may be enhanced upon binding to the enzyme . We suggest that the function of tryptophan-22 may be to properly position the side chain of methionine-20 with respect to N5 of the substrate dihydrofolate. Biochemistry, 1991 Nov 19, 30(46), 11073 - 80 Purification and characterization of selenomethionyl thymidylate synthase from Escherichia coli: comparison with the wild-type enzyme; Boles JO et al.; Replacement of methionine (Met) residues by selenomethionine (SeMet) was recently shown to facilitate the crystallographic analysis of protein structure through the application of multi-wavelength anomalous diffraction techniques {Yang et al . (1990) Science (Washington, D.C.) 249, 1398-1405} . The availability of SeMet-containing proteins provides an excellent opportunity to evaluate the effects of the complete replacement of Met by SeMet . We chose to compare the properties of selenomethionyl thymidylate synthase isolated from Escherichia coli DL41 (a methionine auxotroph) and wild-type (wt) enzyme obtained from E . coli Rue10 . An improved purification procedure for thymidylate synthase was developed which permitted the isolation of 25 mg of pure protein from 2 g of E . coli in 90% yield in no more than 8 h . The pure wt and SeMet enzymes exhibited specific activities 40% higher than published values . Thermal stability studies at 30 degrees C in degassed buffer showed that the SeMet enzyme (t1/2 67 h) was 8-fold less stable than wt enzyme (t1/2 557 h) . The half-lives for the latter enzymes in nondegassed buffers at 30 degrees C were decreased by 2-fold, thus indicating the sensitivity of the enzyme to dissolved oxygen . Both enzymes exhibited essentially the same kinetic and binding properties, including Km(dUMP) (1.2 x 10(-6) M), specificity constant (1.6 x 10(6) s-1 M-1), and Kd for 5-fluorodeoxyuridylate binding (1.2 nM) in covalent inhibitory ternary complexes . In addition, X-ray crystallographic analysis by difference Fourier synthesis showed there was no significant difference in conformation between the SeMet enzyme and the wt enzyme. Biochemistry, 1991 Nov 19, 30(46), 11046 - 54 The function of amino acid residues contacting the nicotinamide ring of NADPH in dihydrofolate reductase from Escherichia coli; Adams JA et al.; The importance of three amino acid residues contacting the nicotinamide ring of NADPH in Escherichia coli dihydrofolate reductase has been defined using site-directed mutagenesis and detailed steady-state and pre-steady-state kinetic experiments . Replacement of Tyr-100 with either glycine or isoleucine (Y100G or Y100I) disrupts an aromatic-aromatic interaction between the phenolic side chain and the nicotinamide ring . Both mutations remove the differential binding of the oxidized and reduced coenzymes implicating Tyr-100 as a major determinant for coenzyme specificity . Replacement of Ser-49 for alanine (S49A), designed to either displace or reduce the polarizability of a bound water molecule contacting the N1 of the nicotinamide ring, affects only the rate of release of NADP+ . Replacement of Ile-14 with alanine (I14A), designed to alter both a weakly polar and a hydrogen bonding interaction with the periphery of the nicotinamide ring, affects only the binding of NADPH . Y100I, Y100G, and I14A all increase the activation barrier for the chemical step by approximately 2 kcal/mol . The lack of an effect for S49A suggests that water structure is not important for stabilizing the hydride transfer transition state . In addition, the nominal effects observed for these mutations disfavor the hypothesis that neighboring amino acid residues participate in the stabilization of the reaction transition state through polar or weakly polar contacts. Biochemistry, 1991 Nov 19, 30(46), 11054 - 63 Functional consequences of engineering the hydrophobic pocket of carbonic anhydrase II; Fierke CA et al.; Twelve amino acid substitutions of varying size and hydrophobicity were constructed at Val 143 in human carbonic anhydrase II (including Gly, Ser, Cys, Asn, Asp, Leu, Ile, His, Phe and Tyr) to examine the catalytic roles of the hydrophobic pocket in the active site of this enzyme . The CO2 hydrase and p-nitrophenyl acetate (PNPA) esterase activities, the pKa of the zinc-water ligand, the inhibition constant for cyanate (KOCN), and the binding constants for sulfonamide inhibitors were measured for various mutants and correlated with the size and hydrophobicity of the substituted amino acid . The kcat/KM for PNPA hydrolysis and KOCN are linearly dependent on the hydrophobicity of the amino acid at position 143 . All of the activities of CAII are decreased by more than a factor of 10(3) when large amino acids (Phe and Tyr) are substituted for Val 143, but the CO2 hydrase activity is the most sensitive to the size and structure of the substituted amino acid . Addition of a single methyl group (V143I) decreases the activity 8-fold, while substitution of valine by tyrosine essentially destroys the enzyme function (kcat/KM for CO2 hydration is decreased by more than 10(5)-fold) . KOCN does not increase until Phe is substituted for Val 143, suggesting that the cyanate and CO2 binding sites are not identical . The functional data in conjunction with X-ray crystallographic studies of four of the mutants {Alexander et al., 1991 (following paper in this issue)} allow interpretation of the mutants at a molecular level and mapping of the region of the active site important for CO2 association . The hydrophobic pocket, including residues Val 121 and Val 143, is important for CO2 and PNPA association; if the pocket is blocked, substrates cannot approach the zinc-hydroxide with the correct orientation to react . The interaction between Val 143 and CO2 is relatively weak (less than or equal to 0.5 kcal/mol) and nonspecific; the association site does not tightly hold CO2 in one fixed orientation for reaction with the zinc-hydroxide . This mechanism of catalysis may reflect a decreased requirement for specific orientation by CO2 since it is a symmetrical molecule. FEBS Lett, 1991 Nov 18, 293(1-2), 21 - 4 P26-calcium binding protein from bovine retinal photoreceptor cells; Kutuzov MA et al.; The primary structure of bovine retinal calcium binding protein P26 has been determined by parallel analysis of protein and corresponding cDNA . This protein is identical to recovering and shares 59% homology with visinin, a cone specific calcium binding protein from chicken retina . Preliminary data are presented on expression of P26 as a fusion protein in E . coli. FEBS Lett, 1991 Nov 18, 293(1-2), 164 - 8 Bacterioferritins and ferritins are distantly related in evolution . Conservation of ferroxidase-centre residues; Andrews SC et al.; Iron-storage proteins can be divided into two classes; the bacterioferritins and ferritins . In spite of many apparent structural and functional analogies, no significant amino acid sequence similarity has been detected previously . This report now reveals a distant evolutionary relationship between bacterioferritins and ferritins derived by 'Profile Analysis' . Optimum alignment of bacterioferritin and ferritin sequences suggests that key residues of the ferroxidase centres of ferritins are conserved in bacterioferritins. FEBS Lett, 1991 Nov 18, 293(1-2), 160 - 3 Impaired affinity for phenylalanine in Escherichia coli phenylalanyl-tRNA synthetase mutant caused by Gly-to-Asp exchange in motif 2 of class II tRNA synthetases; Kast P et al.; Phenylalanyl-tRNA synthetase (PheRS; alpha 2 beta 2 subunit structure) is a member of class II of tRNA synthetases . We report here the genetic analysis of an Escherichia coli mutant strain which is auxotrophic for phenylalanine because it has a PheRS with a decreased affinity for phenylalanine . The mutant pheS gene encoding the PheRS alpha subunit was cloned and sequenced, and the deviation from the wild-type gene was found to result in a Gly191-to-Asp191 exchange . This alteration is located within motif 2, one of 3 conserved sequence motifs characteristic for class II aminoacyl-tRNA synthetases . Motif 2 may thus participate in the formation of the phenylalanine binding site in PheRS. FEBS Lett, 1991 Nov 18, 293(1-2), 106 - 10 Purification and characterization of human T-cell leukemia virus type I protease produced in Escherichia coli; Kobayashi M et al.; Human T-cell leukemia virus type I (HTLV-I) protease has been purified to homogeneity from a strain of recombinant Escherichia coli . The protease was expressed as a larger precursor, which was autoprocessed to form a mature protease . Protein chemical analyses revealed the coding sequence of mature protease, which agreed with the putative sequence predicted from the sequence of bovine leukemia virus protease . The purified protease processed the natural substrate gag precursor (p53) to form gag p19 and gag p24 . The protease activity was inhibited by pepstatin A . These results provide direct evidence that this protease belongs to the aspartic protease family and has an activity consistent with the protease in HTLV-I virion. FEBS Lett, 1991 Nov 18, 293(1-2), 34 - 6 Mutational analysis of the putative PLA2-inhibiting sequence of annexin 1; Trave G et al.; Annexin 1 has been proposed to inhibit phospholipase A2 by direct interaction through a specific amino acid sequence spanning residues 246-254 . The possible role of this region was investigated by protein engineering . Three point mutations and a deletion have been performed . The four mutant proteins have been expressed in E . coli, purified and tested for calcium and lipid binding, and for phospholipase inhibition . All mutant proteins conserved the properties of the wild-type recombinant protein . This result clearly demonstrates that this part of the molecule is not involve in the inhibition of phospholipase A2. Tijdschr Diergeneeskd, 1991 Nov 15, 116(22), 1122 - 9 {Colibacillosis in poultry: etiology, pathology and therapy}; Goren E; Colibacillosis is a secondary infection caused by inhalation, particularly in young broilers and poults . The severity of this septicaemic disease is due to a combination of factors as the virulence, exposure to aerogenic infection, the E . coli strains involved and the intensity of the predisposing alteration of the respiratory epithelium by viruses . Mycoplasmas and/or CO2, NH3 and dust in the atmosphere . Despite preventive vaccination against several predisposing viral infections and the successful eradication of M . gallisepticum in the Netherlands, colibacillosis continues to be an important disease in the broiler industry . Treatment is confronted with a number of serious problems: high costs when treating flocks with the correct (relatively high) dosage for a sufficiently long period, resistance to bacterial drugs and drug residues . Despite negligible resorption from the gastrointestinal tract, very satisfactory therapeutic results were obtained by oral treatment, even against in vitro resistant E . colli strains . To explain the mode of action it is suggested that well-absorbed metabolites or degradation products of spectinomycin block the adhesion of bacteria to tissue receptors. Proc Natl Acad Sci U S A, 1991 Nov 15, 88(22), 9909 - 13 Mutant profiles of selectable genetic elements; Wurst H et al.; A method is presented that allows simultaneous analysis of the effects of all possible point mutations within a specific mutation window of at least 50 base pairs on a DNA fragment that codes for a selectable function . It relies on the detection of mismatched base pairs with hydroxylamine and osmium tetroxide . A mutant plasmid library of randomly distributed point mutations within the lacZ' gene of Escherichia coli was selected for functional alpha-complementation by growth on lactose . The DNA fragments of the selected and unselected library were each heat denatured and again renatured, thereby generating a randomly distributed set of all possible mismatches within the mutagenesis window . Cytidine-containing mismatches were then detected with hydroxylamine, and thymidine-containing mismatches were detected with osmium tetroxide . When this procedure was performed for both DNA strands, all mismatches could be detected . A comparison of the results of the unselected and selected library leads to an estimation of the effects of each detectable mutation on alpha-complementation in vivo . This method, called "mutant profiling," should be applicable to all selectable genetic elements. Proc Natl Acad Sci U S A, 1991 Nov 15, 88(22), 10193 - 7 Replication of acetylaminofluorene-adducted plasmids in human cells: spectrum of base substitutions and evidence of excision repair; Mah MC et al.; In rats fed the liver carcinogen 2-acetylaminofluorene (AAF), the two most abundant types of DNA adduct are N-(deoxyguanosin-8-yl)-2-acetylaminofluorene and its deacetylated derivative . When plasmids carrying AAF adducts replicate in bacteria, the predominant mutations are frameshifts, whereas with deacetylated (AF) adducts, they are mainly base substitutions, just as we found when plasmids carrying AF adducts replicated in human cells . We have investigated the frequency and spectrum of mutations induced when a shuttle vector carrying AAF adducts (85% bound to the C8 position of guanine, 15% to the N2 position) replicated in human cells . The frequency induced per initial AAF adduct was higher than with AF adducts, but the kinds of mutations were similar--i.e., 85% base substitutions, principally G.C----T.A transversions . There was good correlation between the "hot spots" for mutations and hot spots for AAF adduct formation, suggesting that mutational hot spots reflect preferential binding of the carcinogen to DNA . 32P-postlabeling analysis of the adducts before and after the DNA was transfected into the human cells showed that there was no deacetylation of AAF adducts and that 85% of both types of adducts were removed within 3.5 hr, most probably by excision repair. Proc Natl Acad Sci U S A, 1991 Nov 15, 88(22), 10049 - 53 Characterization of G-protein alpha subunits in the Gq class: expression in murine tissues and in stromal and hematopoietic cell lines; Wilkie TM et al.; Murine G alpha 14 and G alpha 15 cDNAs encode distinct alpha subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) . These alpha subunits are related to members of the Gq class and share certain sequence characteristics with G alpha q, G alpha 11, and G alpha 16, such as the absence of a pertussis toxin ADP-ribosylation site . G alpha 11 and G alpha q are ubiquitously expressed among murine tissues but G alpha 14 is predominantly expressed in spleen, lung, kidney, and testis whereas G alpha 15 is primarily restricted to hematopoietic lineages . Among hematopoietic cell lines, G alpha 11 mRNA is found in all cell lines tested, G alpha q is expressed widely but is not found in most T-cell lines, G alpha 15 is predominantly expressed in myeloid and B-cell lineages, and G alpha 14 is expressed in bone marrow adherent (stromal) cells, certain early myeloid cells, and progenitor B cells . Polyclonal antisera produced from synthetic peptides that correspond to two regions of G alpha 15 react with a protein of 42 kDa expressed in B-cell membranes and in Escherichia coli transformed with G alpha 15 cDNA . The expression patterns that were observed in mouse tissues and cell lines indicate that each of the alpha subunits in the Gq class may be involved in pertussis toxin-insensitive signal-transduction pathways that are fundamental to hematopoietic cell differentiation and function. J Biol Chem, 1991 Nov 15, 266(32), 22057 - 62 Evaluation of the role of conserved His and Met residues among lipoxygenases by site-directed mutagenesis of recombinant human 5-lipoxygenase; Nguyen T et al.; The 5-, 12-, and 15-lipoxygenases contain a highly conserved sequence of the form His-(X)4-His-(X)4-His-(X)17-His-(X)8-His which represents a potential binding site for non heme iron to the protein . The importance of selected amino acids within this His cluster for the activity of human 5-lipoxygenase was investigated by site-directed mutagenesis using bacteria and insect cells expression systems . After single mutation of each of the 5 His residues at positions 363, 368, 373, 391, and 400 by Ser, Cys, or Lys, measurable levels of 5-lipoxygenase activity could be recovered in Escherichia coli only for the Ser363 and Cys363 mutants, with most amino acid substitutions causing a decrease in the levels of expression of the soluble protein . In contrast, 25-80% of soluble 5-lipoxygenase activity was recovered after the replacement of several of the hydrophobic amino acids in this region: Tyr384 by Ser or Phe; Phe394 by Trp and Val375 by Ala . Met436 could be replaced by Leu with little effect on 5-lipoxygenase activity or turnover inactivation half-time . High levels of mutant 5-lipoxygenases containing a Ser residue instead of His at each of the five positions were also expressed in Spodoptera frugiperda (Sf9) cells infected with recombinant baculovirus . The specific activity (58-75% of control) and the reaction time course of the Ser363, Ser391, and Ser400 mutants were comparable with that of native 5-lipoxygenase whereas inactive proteins were obtained for the Ser368 and Ser373 mutants . These results show that His368 and His373 residues are important for 5-lipoxygenase activity and that the other conserved His363, His391, His400, and Met436 residues are not crucial for the catalytic cycle or for the mechanism of self-inactivation of 5-lipoxygenase. J Biol Chem, 1991 Nov 15, 266(32), 21657 - 65 Mechanism-based inactivation of alanine racemase by 3-halovinylglycines; Thornberry NA et al.; Alanine racemase, an enzyme important to bacterial cell wall synthesis, is irreversibly inactivated by 3-chloro- and 3-fluorovinylglycine . Using alanine racemase purified to homogeneity from Escherichia coli B, the efficient inactivation produced a lethal event for every 2.2 +/- 0.2 nonlethal turnovers, compared to 1 in 800 for fluoroalanine . The mechanism of inhibition involves enzyme-catalyzed halide elimination to form an allenic intermediate that partitions between reversible and irreversible covalent adducts, in the ratio 3:7 . The reversible adduct (lambda max = 516 nm) decays to regenerate free enzyme with a half-life of 23 min . The lethal event involves irreversible alkylation of a tyrosine residue in the sequence -Val-Gly-Tyr-Gly-Gly-Arg . The second-order rate constant for this process with D-chlorovinylglycine (122 +/- 14 M-1 s-1), the most reactive analog examined, is faster than the equivalent rate constant for D-fluoroalanine (93 M-1 s-1) . The high killing efficiency and fast turnover of these mechanism-based inhibitors suggest that their design, employing the haloethylene moiety to generate a reactive allene during catalysis, could be extended to provide useful inhibitors of a variety of enzymes that conduct carbanion chemistry. J Biol Chem, 1991 Nov 15, 266(32), 21608 - 15 Functional characteristics of the rrnD promoters of Escherichia coli; Langert W et al.; The function of the tandem rrnD promoters (P1, P2) of Escherichia coli, which are highly efficient in directing rRNA synthesis, was studied in vitro using the strong hybrid promoter PtacI as a reference . One of the characteristics of the rrnD promoters is a pronounced instability of binary and initiating complexes formed with RNA polymerase . The rate of productive complex formation and of chain initiation at these promoters was found to be limited by a step in binary complex transitions with an apparent first-order rate constant equal to 3.9 x 10(-2) s-1 . A comparison of this rate with that determined previously by filter binding assays (Gourse, R . (1988) Nucleic Acids Res . 16, 9789-9809) suggests that the rate-limiting step is a conversion of an intermediate species of open complex to one that is efficient in productive initiation . The slow rate of this reaction and the instability of open complexes account for the relatively low competitive strengths of the rrnD promoters . However, this limitation of rrn promoter function changes with promoter occupancy because the rate of chain initiation increased after completion of the first round of initiation . Despite their poor competitive strength, the rrnD promoters are more productive than PtacI at nonlimiting RNA polymerase concentrations . This can be ascribed to the different rates with which RNA polymerases leave PtacI and the rrnD promoters . These functional differences of the promoters are consistent with a "stressed intermediate" model of chain initiation (Straney, D.C., and Crothers, D.M . (1987) J . Mol . Biol . 193, 267-278) which predicts that rapid clearance of the rrn promoters is mechanistically related to the instability of the binary complexes. J Biol Chem, 1991 Nov 15, 266(32), 21458 - 65 Expression of calreticulin in Escherichia coli and identification of its Ca2+ binding domains; Baksh S et al.; Recombinant calreticulin and discrete domains of calreticulin were expressed in Escherichia coli, using the glutathione S-transferase fusion protein system, and their Ca2+ binding properties were determined . Native calreticulin bound 1 mol of Ca2+/mol of protein with high affinity, and also bound approximately 20 mol of Ca2+/mol of protein with low affinity . Both Ca2+ binding sites were present in the recombinant calreticulin indicating that proper folding of the protein was achieved using this system . Calreticulin is structurally divided into three distinct domains: the N-domain encompassing the first 200 residues; the P-domain which is enriched in proline residues (residue 187-317); and the C-domain which covers the carboxyl-terminal quarter of the protein (residues 310-401), and contains a high concentration of acidic residues . These domains were expressed in E . coli, isolated, and purified, and their Ca2+ binding properties were analyzed . The C-domain bound approximately 18 mol of Ca2+/mol of protein with a dissociation constant of approximately 2 mM . The P-domain bound approximately 0.6-1 mol of Ca2+/mol of protein with a dissociation constant of approximately 10 microM . The P-domain and the C-domain, when expressed together as the P+C-domain, bound Ca2+ with both high affinity and low affinity, reminiscent of both full length recombinant calreticulin and native calreticulin . In contrast the N-domain, did not bind any detectable amount of 45Ca2+ . We conclude that calreticulin has two quite distinct types of Ca2+ binding sites, and that these sites are in different structural regions of the molecule . The P-domain binds Ca2+ with high affinity and low capacity, whereas the C-domain binds Ca2+ with low affinity and high capacity. Eur J Biochem, 1991 Nov 15, 202(1), 53 - 66 The solution structures of Escherichia coli trp repressor and trp aporepressor at an intermediate resolution; Arrowsmith C et al.; We have determined the solution structures and examined the dynamics of the Escherichia coli trp repressor (a 25-kDa dimer), with and without the co-repressor L-tryptophan, from NMR data . This is the largest protein structure thus far determined by NMR . To obtain a set of data sufficient for a structure determination it was essential to resort to isotopic spectral editing . Line broadening observed in this molecular mass range precludes for the most part the measurement of coupling constants and stereospecific assignments, with the inevitable result that the attainable resolution of the final structure will be somewhat lower than the resolution reported for smaller proteins and peptides . Nevertheless the general topology of the protein can be deduced from the subsets of NOEs defining the secondary and tertiary structure, providing a basis for further refinement using the full set of NOEs and energy minimization . We report here (a) an intermediate resolution structure that can be deduced from NMR data, covalent, angular and van-der-Waals constraints only, without resort to detailed energy calculations, and (b) the limits of uncertainty within which this structure is valid . An examination of these structures combined with backbone amide exchange data shows that even at this resolution three important conclusions can be drawn: (a) the protein structure changes upon binding tryptophan; (b) the putative DNA binding region is much more flexible than the core of the molecule, with backbone amide proton exchange rates 1000 times faster than in the core; (c) the binding of tryptophan stabilizes the repressor molecule, which is reflected in both the appearance of additional NOEs, and in the slowing of backbone proton exchange rates by factors of 3-10 . Sequence-specific 1H-NMR assignments and the secondary structure of the holopressor (L-tryptophan-bound form) have been reported previously {C . H . Arrowsmith, R . Pachter, R . B . Altman, S . B . Iyer & O . Jardetzky (1990) Biochemistry 29, 6332-6341} . Those for the trp aporepressor (L-tryptophan-free form), made using the same methods and conditions as described in the cited paper, are reported here . The secondary structure of the aporepressor was calculated from sequential and medium-range NOEs and is the same as reported for the holorepressor except that helix E is shorter . The tertiary solution structures for both forms of the repressor were calculated from long-range NOE data.(ABSTRACT TRUNCATED AT 400 WORDS) Arch Biochem Biophys, 1991 Nov 15, 291(1), 107 - 12 Studies of ligand binding to Escherichia coli adenylosuccinate synthetase; Soans C et al.; Dissociation constants of Escherichia coli adenylosuccinate synthetase with IMP, GTP, adenylosuccinate, and AMP (a competitive inhibitor for IMP) were determined by measuring the extent of quenching of the intrinsic tryptophan fluorescence of the enzyme . The enzyme has one binding site for each of these ligands . Aspartate and GDP did not quench the fluorescence to any great extent, and their dissociation constants could not be determined . These ligand binding studies were generally supportive of the kinetic mechanism proposed earlier for the enzyme . Cys291 was modified with the fluorescent chromophores N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonate and tetramethylrhodamine maleimide in order to measure enzyme conformational changes attending ligand binding . The excitation and emission spectra of these fluorophores are not altered by the addition of active site binding ligands . TbGTP and TbGDP were used as native reporter groups, and changes in their fluorescence on complexing with the enzyme and various ligands made it possible to detect conformational changes occurring at the active site . Evidence is presented for abortive complexes of the type: enzyme-TbGTP-adenylosuccinate and enzyme-TbGTP-adenylosuccinate-aspartate . These results suggest that the IMP and aspartate binding sites are spatially separated. Cell, 1991 Nov 15, 67(4), 807 - 14 Cloning and expression of genes for the Oxytricha telomere-binding protein: specific subunit interactions in the telomeric complex; Gray JT et al.; Telomeres of Oxytricha nova macronuclear chromosomes consist of a repeated T4G4 sequence, single-stranded at the 3' terminus, bound by a heterodimeric protein . The cloning of genes for the two polypeptides and their separate expression in E . coli have enabled evaluation of their individual contributions to DNA binding . The 56 kd alpha subunit binds single-stranded DNA by itself, one polypeptide per T4G4 block; multiple subunits can coat a (T4G4)n multimer . The derived amino acid sequence of alpha does not reveal any known DNA-binding motif, so it appears to represent a novel type of DNA-binding protein . The previously cloned 41 kd beta subunit does not by itself protect DNA from methylation, but is required along with alpha to recreate the pattern of methylation protection indicative of telomeres in vivo . The unusual ability of the protein to engage in two different interactions with the same telomeric DNA sequence might provide the versatility necessary for diverse telomere functions. Proc Natl Acad Sci U S A, 1991 Nov 15, 88(22), 9989 - 93 Conversion of the E1A Cys4 zinc finger to a nonfunctional His2,Cys2 zinc finger by a single point mutation; Webster LC et al.; Trans-activation by the adenovirus E1A 289R protein requires a zinc finger defined by Cys-154, Cys-157, Cys-171, and Cys-174 . Whereas individually replacing the four cysteine residues with serines resulted in a loss of transactivation, only three of the Cys----Ser mutants (C157S, C171S, and C174S) lost the ability to bind Zn(II) . X-ray absorption fine structure analysis revealed that, in the wild-type protein, Zn(II) is coordinated by four cysteine residues whereas in the C154S mutant, Zn(II) is coordinated by two histidines and two cysteines . The mutant protein probably retains, as ligands, two cysteines on the right side of the zinc finger (Cys-171 and Cys-174) and recruits two of the four histidines on the left side (His-149, His-152, His-158, and His-160), despite the presence of Cys-157 . This finding may shed light on the general structural requirements of zinc fingers. Proc Natl Acad Sci U S A, 1991 Nov 15, 88(22), 10153 - 7 Relationship between alternatively spliced exons and functional domains in tropomyosin; Cho YJ et al.; Smooth and striated muscle alpha-tropomyosins differ as a consequence of alternative splicing of exons 2 and 9 encoding amino acid residues 39-80 and 258-284, respectively {Ruiz-Opazo, N., Weinberger, J . & Nadal-Ginard, B . (1985) Nature (London) 315, 67-70} . To understand the relationship between alternatively spliced exons and functional domains in tropomyosin, recombinant unacetylated striated muscle, smooth muscle, and chimeric rat alpha-tropomyosins (+H3N-tropomyosins) expressed in and purified from Escherichia coli were analyzed . The functional differences between the isoforms can be primarily ascribed to exon 9 . +H3N-Tropomyosins with the smooth muscle exon 9 bound to skeletal muscle filamentous actin with at least a 5-fold higher affinity than +H3N-tropomyosins with the striated muscle exon 9 . On the other hand, in the presence of Ca2+, troponin increased the affinity of +H3N-tropomyosins with the striated muscle exon 9 at least 50-fold, whereas it had little effect on +H3N-tropomyosins with the smooth muscle exon 9 . The unique striated muscle alpha-tropomyosin exon 9 seems to be specialized for Ca(2+)-insensitive interaction with troponin on the thin filament . The unique smooth muscle alpha-tropomyosin exon 2 was associated with a slightly lower actin affinity than the striated muscle exon 2 . Although the regions encoded by exons 2 and 9 correspond to functional domains, they are not recognizable as independent units or structural domains in the extended coiled-coil structure of this fibrous actin binding protein. J Biol Chem, 1991 Nov 15, 266(32), 21700 - 8 Early events in the transport of proteins into mitochondria . Import competition by a mitochondrial presequence; Cyr DM et al.; Studies with a synthetic presequence peptide, F1 beta 1-20, corresponding to the NH2-terminal 20 amino acids of the F1-ATPase beta-subunit precursor (pF1 beta) show that although this peptide binds avidly to phospholipid bi-layers it does not efficiently compete for import of full-length precursor into mitochondria, Ki approximately 100 microM (Hoyt, D.W., Cyr, D.M., Gierasch, L.M., and Douglas, M.G . (1991) J . Biol . Chem . 266, 21693-21699) . Herein we report that longer F1 beta presequence peptides F1 beta 1-32 + 2, F1 beta 1-32SQ + 2, and F1 beta 21-51 + 3 compete for mitochondrial import at 1000-, 250-, and 25-fold lower concentrations, respectively, than F1 beta 1-20 . A longer peptide, F1 beta 1-51 + 3, was no more effective as an import competitor than F1 beta 1-32 + 2 . Both minimal length and amphiphilic character appear required in order for F1 beta peptides to block mitochondrial import . Import competition by longer F1 beta peptides seems to occur at a step common to all precursors since they blocked import of precursors to F1-ATPase alpha- and beta-subunits and the ADP/ATP carrier protein . Dissipation of membrane potential (delta psi) across the inner mitochondrial membrane is observed in the presence of F1 beta-peptides, but this mechanism alone does not account for the observed import inhibition . F1 beta 1-32 + 2 and 21-51 + 3 block import of pF1 beta 100% at peptide concentrations which dissipate delta psi less than 25% . In contrast, experiments with valinomycin demonstrate that when mitochondrial delta psi is reduced 25% import of pF1 beta is inhibited only 25% . Therefore, at least 75% of maximal import inhibition observed in the presence of F1 beta 1-32 + 2 and F1 beta 21-51 + 3 does not result from dissipation of delta psi . Import inhibition by F1 beta-peptides is reversible and can be overcome by increasing the amount of full-length precursor in import reactions . F1 beta presequence peptides and full-length precursor are therefore likely to compete for a common import step . Presequence dependent binding of pF1 beta to trypsin-sensitive elements on the outer mitochondrial membrane is insensitive to inhibitory concentrations of F1 beta presequence peptide . We conclude that import inhibition by F1 beta presequence peptides is competitive and occurs at a site beyond initial interaction of precursor proteins with mitochondria. J Biol Chem, 1991 Nov 15, 266(32), 21693 - 9 Interaction of peptides corresponding to mitochondrial presequences with membranes; Hoyt DW et al.; The transport of the F1-ATPase beta-subunit precursor into mitochondria is dependent upon a presequence at its amino terminus . Within the mitochondrial membrane translocation site the potential amphiphilic character of the presequence region may be necessary to stabilize binding to the mitochondrial inner membrane . To better understand its role in protein import, the interaction of the F1 beta-presequence with lipid membranes was measured using circular dichroism and surface tensiometry . These studies reveal that a 20-residue peptide containing the F1 beta-presequence binds to phospholipid vesicles (Kd = 4.5-6.0 x 10(-8)M and adopts a predominantly alpha-helical structure . Although the presequence peptide binds avidly to lipids, it does not appear to penetrate deeply into the bilayer to perturb a reporter probe in the membrane interior . Compared with the effect of the peptides with demonstrated membrane insertion and lytic properties, the F1-beta-presequence appears to displace phospholipid head groups but not insert deeply into the bilayer . High concentrations (greater than 50 microM) of presequence peptides are required to noticibly perturb import of the full length F1 alpha- or F1 beta-subunit precursors . Thus, the F1 beta-presequence alone is not sufficient to efficiently compete for import but may require a protein context or a minimal length to assist insertion into the transport site . These observations are discussed in light of the different requirements for import of various presequence containing precursors into mitochondria. Anal Biochem, 1991 Nov 15, 199(1), 119 - 24 A chemiluminescent assay for quantitation of beta-galactosidase in the femtogram range: application to quantitation of beta-galactosidase in lacZ-transfected cells; Jain VK et al.; An optimized chemiluminescent assay for beta-galactosidase using a chemiluminescent substrate AMPGD (3-(4-methoxyspiro{1,2-dioxetane-3,2'-tricyclo-{3.3.1 . 1(3,7)}decan}-4- yl)phenyl-beta-D-galactopyranoside) is described . This assay is rapid and sensitive and can detect as little as 2 fg of beta-galactosidase . Its use for the quantitation of beta-galactosidase in cells transfected with lacZ-expressing vectors is described . It is possible to detect a single cell stably expressing lacZ by this technique. Gene, 1991 Nov 15, 107(2), 241 - 6 Three individual regulatory elements of the promoter positively activate the transcription of the murine gene encoding granulocyte colony-stimulating factor; Asano M et al.; At least three regulatory elements GPE1, GPE2 and GPE3 (G-CSF promoter elements) controlling the gene (G-CSF) encoding granulocyte colony-stimulating factor (G-CSF) are indispensable for the constitutive expression of the G-CSF gene in human CHU-2 cells and for its lipopolysaccharide(LPS)-inducible expression in macrophages . The enhancer activities of each regulatory element were examined with or without the SV40 enhancer element placed downstream from the reporter gene . A GPE1 tetramer mediated the constitutive expression in CHU-2 cells, and the LPS-inducible expression in macrophage cell lines, while the GPE2 element was active in CHU-2 and LPS-treated macrophage cell lines only in combination with the SV40 enhancer . A GPE3 tetramer had efficient enhancer activity in CHU-2 cells but not in macrophage cell lines without the SV40 enhancer . In combination with the SV40 enhancer, GPE3 worked as an LPS-inducible enhancer element in macrophage BAM3 cells . Gel retardation assay indicated that the CHU-2 and the macrophage cells contained nuclear factors which specifically bound to each GPE sequence. Biochem J, 1991 Nov 15, 280 ( Pt 1), 187 - 91 Isolation and overexpression in Escherichia coli of the flavodoxin gene from Anabaena PCC 7119; Fillat MF et al.; The gene coding for flavodoxin from Anabaena PCC 7119 was cloned by using the polymerase chain reaction (PCR) . The gene is transcribed into a 1250-base transcript . The expression of the flavodoxin gene was analysed and found to be regulated at the transcriptional level by the availability of iron . The PCR-amplified gene was cloned into the expression vector pTrc 99b and expressed in Escherichia coli . High concentrations of flavodoxin were found (20% of total protein) . The recombinant protein was purified from the cytosolic fraction of the cells and it exhibited properties identical with those of the wild-type Anabaena flavodoxin. J Biol Chem, 1991 Nov 15, 266(32), 21777 - 83 Cloning of a human alpha(1,3)-fucosyltransferase gene that encodes ELFT but does not confer ELAM-1 recognition on Chinese hamster ovary cell transfectants; Kumar R et al.; In previous studies, Chinese hamster ovary (CHO) cell genomic DNA transfectants that expressed a human alpha(1,3)-fucosyltransferase (alpha(1,3)Fuc-T) gene were isolated and shown to possess a common approximately 7.5-kilobase (kb) EcoRI fragment that hybridized to an Alu probe (Potvin, B., Kumar, R., Howard, D . R., and Stanley, P . (1990) J . Biol . Chem . 265, 1615-1622) . One of these transfectants was used to make a genomic DNA library in lambda ZAP-II from EcoRI-digested, size-selected (6-8 kb) DNA, and plaques that hybridized to an Alu probe were purified . After in vivo excision, two plasmids with DNA inserts greater than or equal to 6 kb were obtained and one of these (D2.1) conferred human alpha(1,3)-Fuc-T activity on CHO transfectants . A partial restriction map of this clone revealed an approximately 3.6-kb PstI fragment that contained an Alu sequence . This fragment was subcloned into pGEM-3Zf(+) and compared by restriction analyses with a previously described approximately 3.6-kb PstI DNA fragment isolated from a human peripheral blood lymphocyte library and shown to encode an alpha(1,3)-Fuc-T gene (Lowe, J . B., Stoolman, L . M., Nair, R . P., Larsen, R . D., Berhend, T . L., and Marks, R . M . (1990) Cell 63, 475-484) . Both approximately 3.6-kb fragments gave identical restriction patterns . In addition, they both caused CHO transfectants to synthesize the Lex determinant Gal beta(1,4){Fuc alpha(1,3)}GlcNAc beta 1 but not the alpha(2,3)-sialyl-Lex determinant . As expected, these transfectants did not bind to ELAM-1 on activated endothelial cells, since sialyl-Lex is a carbohydrate ligand recognized by ELAM-1 . Surprisingly, however, an open reading frame encoded within the approximately 3.6-kb PstI fragment had a sequence identical to that of ELFT, an alpha(1,3)-Fuc-T previously reported to confer ELAM-1 binding on a previously reported to confer ELAM-1 binding on a CHO transfectant (Goelz, S . E., Hession, C., Goff, D., Griffiths, B., Tizard, R., Newman, B., Chi-Rosso, G., and Lobb, R., (1990) Cell 63, 1349-1356) . Possible explanations for these apparently disparate results are discussed. Eur J Biochem, 1991 Nov 15, 202(1), 95 - 9 Recombinant aprotinin homologue with new inhibitory specificity for cathepsin G; Brinkmann T et al.; The substitution of amino acids in the reactive site of aprotinin, a bovine serine proteinase inhibitor with potent activity against trypsin, plasmin and tissue kallikrein, led to a change in specificity of the inhibitor . Twelve new aprotinin variants prepared by recombinant DNA technology and expressed in Escherichia coli clearly demonstrated that the neighbouring groups of the P1 residue, in particular P'2, contribute to the specificity of the inhibitor, while earlier investigations on semisynthetically prepared variants revealed the importance of the P1 residue in dominating the inhibitory specificity . Recombinant aprotinin variants which act specifically against chymotrypsin-like proteinases, were obtained by substitution of the amino acids in position P1 and P'2 by hydrophobic amino acids like phenylalanine, tyrosine and leucine . Some of these variants, particularly those with phenylalanine or leucine substitutions, were also found to exhibit inhibitory activity against cathepsin G with an equilibrium constant of dissociation Ki of 10(-8) M . Inhibitory specificity against cathepsin G was not found in any semisynthetic variant prepared earlier. FEMS Microbiol Lett, 1991 Nov 15, 68(2), 227 - 30 Amplification by the polymerase chain reaction of a specific target sequence in the gene coding for Escherichia coli verotoxin (VTe variant); Johnson WM et al.; Synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) protocol to target a specific sequence in the gene coding for the A subunit of Escherichia coli verotoxin (VTe-variant, VTev) . This PCR protocol permits the VTe-variant target sequence to be distinguished from closely related sequences in the same coding regions for type 1, type 2, and type 2 variant E . coli verotoxins . This procedure will be a valuable adjunct to other DNA amplification techniques currently being used for molecular epidemiological studies of verotoxigenic E . coli. Biochim Biophys Acta, 1991 Nov 15, 1080(3), 259 - 63 Low- and high-activity forms of glutamine synthetase from Rhodospirillum rubrum: sensitivity to feed-back effectors and activation of the low-activity form; Hammarstrom A et al.; Glutamine synthetase from Rhodospirillum rubrum can be isolated in two forms, with low and high activity, respectively, depending on the concentration of combined nitrogen in the medium before harvest . The two forms have been studied with respect to their dependence on Mn2+ and Mg2+ in both the transferase and the biosynthetic assay . There is no difference in pH optimum between the forms in the biosynthetic assay . In addition the pH-optima for the two cations studied are very close, 7.4 (Mg2+) and 7.2 (Mn2+) . It also shows that the activity of the low-activity form is higher than that of the high-activity form in the Mn(2+)-dependent biosynthetic assay . The two forms of Rsp . rubrum glutamine synthetase have also been studied with respect to their sensitivity towards feed-back effectors . In the transferase assay both forms are inhibited to essentially the same degree by alanine, glycine, histidine, AMP, CTP and UTP, CTP being the most effective of the nucleotides and of the amino acids alanine causes the highest inhibition . In the biosynthetic assay these effectors show different degrees of inhibition on the two different forms; the high-activity form being the most sensitive . The results are discussed in relation to properties of glutamine synthetase from Escherichia coli and other phototropic bacteria in which regulation of glutamine synthetase is known to be due to adenylylation . It is also shown that the low-activity form of Rsp . rubrum glutamine synthetase can be activated in crude extracts in a reaction that is inhibited by glutamine. FEMS Microbiol Lett, 1991 Nov 15, 68(2), 135 - 8 Isolation and preliminary characterization of the Porphyromonas gingivalis prtC gene expressing collagenase activity; Takahashi N et al.; A gene, prtC, has been isolated from Porphyromonas gingivalis ATCC 53977 in Escherichia coli utilizing the plasmid vector pPL-lambda . The resultant protease positive clone NHS1, harboring plasmid pS1 with a 5.9-kilobase P . gingivalis insert, expressed an enzyme capable of hydrolyzing the synthetic collagenase substrate PZ-PLGPA as well as solubilized type I collagen . Subcloning and deletion analysis located the prtC gene at one end of the P . gingivalis DNA insert on plasmid pS1. Gene, 1991 Nov 15, 107(2), 307 - 12 The hygromycin-resistance-encoding gene as a selection marker for vaccinia virus recombinants; Zhou J et al.; Hygromycin B (Hy), an inhibitor of RNA translation, was shown to block the replication of vaccinia virus (VV) in cultured cell lines . Insertion of the Escherichia coli Hy resistance-encoding gene (hph) into the VV genome under control of early or late synthetic VV promoters could overcome inhibition of viral replication . When hph was inserted into VV in tandem with the human papillomavirus type 16 (HPV16) L1 open reading frame, hph recombinant viruses could be selected which expressed HPV16 L1. Biochem J, 1991 Nov 15, 280 ( Pt 1), 233 - 41 The cysteine-rich domain of human proteins, neuronal chimaerin, protein kinase C and diacylglycerol kinase binds zinc . Evidence for the involvement of a zinc-dependent structure in phorbol ester binding; Ahmed S et al.; Diacylglycerol (DG) and its analogue phorbol 12-myristate 13-acetate (PMA) activate the ubiquitous phospholipid/Ca2(+)-dependent protein kinase, protein kinase C (PKC), and cause it to become tightly associated with membranes . DG is produced transiently as it is rapidly metabolized by DG kinase (DGK) to phosphatidic acid . Phorbol esters such as PMA are not metabolized and induced a prolonged membrane association of PKC . Until recently, PKC was the only known phorbol ester receptor . We have shown that a novel brain-specific cDNA, neuronal chimaerin (NC), expressed in Escherichia coli, binds phorbol ester with high affinity, stereospecificity and a phospholipid requirement {Ahmed, Kozma, Monfries, Hall, Lim, Smith & Lim (1990) Biochem . J . 272, 767-773} . The proteins NC, PKC and DGK possess a cysteine-rich domain with the motif HX11/12CX2CXnCX2CX4HX2CX6/7C (where n varies between 12 and 14) . The partial motif, CX2CX13CX2C, is present in a number of transcription factors including the steroid hormone receptors and the yeast protein, GAL4, in which zinc plays a structural role of co-ordinating cysteine residues and is essential for DNA binding (protein-nucleic acid interactions) . The cysteine-rich domain of NC and PKC is required for phospholipid-dependent phorbol is required for phospholipid-dependent phorbol ester binding, suggesting an involvement of this domain in protein-lipid interactions . We have expressed recombinant NC, PKC and DGK glutathione S-transferase and TrpE fusion proteins in E . coli to investigate the relationship between the cysteine-rich motif, HX11/12CX2CX10-14CX2CX4HX2CX6/7C, zinc and phorbol ester binding . The cysteine-rich domain of NC, PKC and DGK bound 65Zn2+ but only NC and PKC bound {3H}phorbol 12,13-dibutyrate . When NC and PKC were subjected to treatments known to remove metal ions from GAL4 and the human glucocorticoid receptor, phorbol ester binding was inhibited . These data provide evidence for the role of a zinc-dependent structure in phorbol ester binding. Biochem J, 1991 Nov 15, 280 ( Pt 1), 125 - 9 Identification of residues essential for catalysis and binding of calmodulin in rat brain inositol 1,4,5-trisphosphate 3-kinase; Takazawa K et al.; In order to identify the amino acid residues involved in calmodulin (CaM) binding and catalytic activity, rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase was expressed in Escherichia coli as a beta-galactosidase fusion protein {clone C5; Takazawa, Vandekerckhove, Dumont & Erneux (1990) Biochem . J . 272, 107-112} . Three deletion mutants in the plasmid of clone C5 were generated using convenient restriction enzymes . The results show that the removal of 34 amino acids from the C-terminal end of InsP3 3-kinase resulted in an inactive protein which still interacted with CaM-Sepharose in a Ca2(+)-dependent way . The catalytic domain is thus located at the C-terminal end of the protein . A series of 5' deletion mutants was prepared and used to produce proteins with the same C-terminal end but shortened N-termini, varying in length by over 80 amino acids . Assay of InsP3 3-kinase activity in bacterial extracts indicated that a maximum of 275 amino acids in the C-terminal region may be sufficient for the construction of a catalytically active domain . Affinity chromatography on CaM-Sepharose of 5' and 3' deletion mutants revealed that the sequence stretching from Ser-156 to Leu-189 is involved in CaM binding and enzyme stimulation. Biochim Biophys Acta, 1991 Nov 15, 1080(3), 271 - 4 Prediction of the general structure of OmpF and PhoE from the sequence and structure of porin from Rhodobacter capsulatus . Orientation of porin in the membrane; Welte W et al.; By comparing the hydrophilicity profiles and sequences of porin from Rhodobacter capsulatus with those of OmpF and PhoE from Escherichia coli, a set of insertions and deletions for alignment of the sequences has been deduced . With this alignment a similar folding of OmpF and PhoE has been predicted as found by X-ray structure analysis of porin from Rhodobacter capsulates . Furthermore, the orientation of the porin trimer in the outer membrane was inferred from topological data on PhoE . According to this result a single channel of approx . 30 A diameter starts at the outer surface . Near the middle of the outer membrane bilayer this channel branches out into three separate channels, each running within a single porin monomer to the periplasmic surface. Proc Natl Acad Sci U S A, 1991 Nov 15, 88(22), 10297 - 301 Cloning and characterization of a 20-kDa ubiquitin carrier protein from wheat that catalyzes multiubiquitin chain formation in vitro; Van Nocker S et al.; Recent evidence indicates that the commitment to degrade cellular proteins by the ubiquitin proteolytic pathway is dependent on the covalent attachment of multiubiquitin chains to the target protein {Chau, V., Tobias, J . W., Bachmair, A., Marriott, D., Ecker, D . J., Gonda, D . K . & Varshavsky, A . (1989) Science 243, 1576-1583} . We have isolated a 20-kDa ubiquitin carrier protein {E2(20 kDa)} from wheat by using ubiquitin covalent affinity chromatography and anion-exchange HPLC that catalyzes multiubiquitin chain formation in vitro . This reaction is blocked by the addition of a mutant ubiquitin in which arginine has been substituted for lysine at residue 48, demonstrating that the coupling of ubiquitin to ubiquitin is likely to be through an isopeptide linkage between the C-terminal glycine and Lys48 of ubiquitin . By immunoscreening a wheat cDNA expression library with anti-E2(20 kDa) antibodies, a cDNA encoding the complete protein was isolated . The clone (designated UBC7) was confirmed as encoding E2(20 kDa) by comparison of the derived amino acid sequence with peptide sequences of E2(20 kDa) tryptic fragments . The encoded protein contains a single cysteine at position 91, which is presumably the active site, and has regions of amino acid sequence similarity to other known E2s from plants and yeast . Expression of this cDNA in Escherichia coli produced an active E2 capable of catalyzing multiubiquitin chain formation in vitro . By virtue of its activity, E2(20 kDa) may have a pivotal role in protein degradation by the ubiquitin-dependent proteolytic pathway. Proc Natl Acad Sci U S A, 1991 Nov 15, 88(22), 10168 - 72 Directed mutagenesis of an iron-sulfur protein of the photosystem I complex in the filamentous cyanobacterium Anabaena variabilis ATCC 29413; Mannan RM et al.; In oxygenic photosynthetic organisms the PSI-C polypeptide, encoded by the psaC gene, provides the ligands for two {4Fe-4S} centers, FA and FB, the terminal electron acceptors in the photosystem I (PSI) complex . An insertion mutation introduced in the psaC locus of the filamentous cyanobacterium Anabaena variabilis ATCC 29413 resulted in the creation of a mutant strain, T398-1, that lacks the PSI-C polypeptide . In medium supplemented with 5 mM fructose, the mutant cells grew well in the dark . However, when grown in the same medium under light, the doubling rate of T398-1 cells was significantly decreased . In intact cells of T398-1, bicarbonate-dependent whole-chain electron transport (PSII and PSI) could not be detected, although partial electron transport reactions involving either one of the two photosystems could be measured at significant rates . The low-temperature EPR signals attributed to the {4Fe-4S} centers FA and FB were absent in the mutant cells . Chemical titration measurements indicated that the ratios of chlorophyll to the primary donor P700 were virtually identical in membranes from the wild-type and mutant cells . Moreover, room-temperature optical spectroscopic analysis of the thylakoid membranes isolated from T398-1 showed flash-induced P700 oxidation followed by dark rereduction, indicating primary photochemistry in PSI . Thus stable assembly of the reaction center of PSI can occur in the absence of the Fe-S cluster cofactors FA and FB . These studies demonstrate that Anabaena 29413 offers a useful genetic system for targeted mutagenesis of the PSI complex. J Biol Chem, 1991 Nov 15, 266(32), 21753 - 9 Expression of a sodium proton antiporter (NhaA) in Escherichia coli is induced by Na+ and Li+ ions; Karpel R et al.; Regulation of expression of nhaA, the gene which encodes a Na+/H+ antiporter in Escherichia coli has been studied . Two promoters have been identified in the upstream sequence of the gene and the corresponding start point of transcription mapped by primer extension . Monitoring the beta-galactosidase activity of a chromosomal translation fusion of nhaA'-'lacZ show that at pH 7.5 the gene is induced, within 1 h, by 100 mM of either Li+ or Na+ . Change of pH between 6.5 and 8.5 by itself does not increase expression of the gene but it markedly increases the sensitivity of the expression system to the ions . At pH 7.5 maximal induction is obtained by 100 mM NaCl, whereas at pH 8.6, 10 mM NaCl elicit similar response . The pattern of regulation of nhaA reflects its importance in adaptation to high salinity and alkaline pH in E . coli. J Biol Chem, 1991 Nov 15, 266(32), 21736 - 44 Purification of TnsB, a transposition protein that binds to the ends of Tn7; Arciszewska LK et al.; We have purified TnsB, a transposition protein encoded by the bacterial transposon Tn7 . The purification procedure involves three chromatographic steps (DNA-cellulose, norleucine-Sepharose, and phosphocellulose) and yields milligram quantities of highly purified protein . The apparent molecular mass of denatured TnsB protein is approximately 85 kDa . Gel filtration chromatography and sucrose gradient sedimentation studies indicate that in solution, native TnsB is a monomer of nonspherical shape . Using DNase I protection analysis, we established that TnsB is a sequence-specific DNA-binding protein that recognizes multiple sites in both ends of the transposon . The TnsB binding sites, three in the left end of Tn7 and four in the right end, are highly related in nucleotide sequence and are located in DNA segments that we have previously shown contain cis-acting sequences important for Tn7 transposition . Our results also show that one of the TnsB binding sites overlaps a proposed promoter for the transposition genes of Tn7 . These studies suggest that the specific binding of TnsB to the ends of Tn7 mediates recombination and may also regulate the expression of Tn7-encoded transposition genes. J Biol Chem, 1991 Nov 15, 266(32), 21404 - 8 Interferon-inducible mouse Mx1 protein that confers resistance to influenza virus is GTPase; Nakayama M et al.; The murine Mx1 protein is an interferon-inducible nuclear protein and confers resistance to influenza virus infection even though the resistance mechanism is yet unclear . The Mx1 protein contains a tripartite GTP-binding domain consisting of GXXXXGKS, DXXG, and T/NKXD motifs . In the GTPase gene superfamily such as p21ras protein, signal-transducing G protein, and translation elongation factor, the GTPase activity plays a key role in each protein function . Here we show that GTPase activity is indeed associated with the intact Mx1 protein purified from Escherichia coli expressing Mx1 cDNA . Amino acid substitution within the GTP-binding motif led to significant reduction in the GTPase activity . Yeast vacuolar protein sorting (VPS1) protein and the rat microtubule-associated mechanochemical enzyme dynamin were found to be homologous to Mx1 not only in the tripartite GTP-binding motif, but also in the amino-terminal region of approximately 300 amino acids in length . The function of Mx1 is discussed in comparison with these proteins. Biochim Biophys Acta, 1991 Nov 14, 1115(1), 49 - 53 Radiofrequency dielectric spectroscopy of ribosome suspensions; Bonincontro A et al.; Dielectric measurements on different ribosome suspensions were carried out in the frequency range from 10 kHz to 1 GHz . In intact ribosomes two dispersions were detected: one around 100 kHz and the other one in the MHz region . In separated ribosomal subunits and in ribosomes resuspended in a buffer with no magnesium ions (relaxed ribosomes) only the MHz dispersion was observed . Electrical conductivities of the samples at 1 kHz were also measured . The temperature dependence of the two dispersions was investigated and a tentative attribution was proposed. Biochim Biophys Acta, 1991 Nov 14, 1115(1), 36 - 41 Synthesis and accumulation of thiamin triphosphate in Escherichia coli cells expressing chicken cytosolic adenylate kinase; Shioda T et al.; To examine whether cytosolic adenylate kinase (AK1, EC 2.7.4.3) catalyzes synthesis of thiamin triphosphate (TTP) in vivo, chicken AK1 was expressed in Escherichia coli, and cellular AK1 activity and TTP content were determined . E . coli harboring the vector plasmid was used as a control . Chicken AK1 was expressed in the producer strain at a high level (83 U/mg protein) even without inducers, and this expression was doubled (153 U/mg protein) by beta-D-isopropylthiogalactopyranoside (IPTG) . TTP was accumulated in the producer cells at a high level (5.7 nmol/g dry weight) without IPTG and this was also doubled (10.2 nmol/g dry weight) by IPTG . TTP content in the control strain was very low (0.2-0.9 nmol/g dry weight) and was unaffected by IPTG . Neither bacterial growth curve nor cellular content of AMP, ADP, ATP, thiamin diphosphate or total thiamin (sum of thiamin and its phosphate esters) was different between the producer and the control strains . These results indicate that chicken AK1 expressed in E . coli catalyzed the synthesis and accumulation of TTP within the bacterial cells. Biochem Biophys Res Commun, 1991 Nov 14, 180(3), 1408 - 15 Engineering by PCR-based exon amplification of the genomic porcine interferon-gamma DNA for expression in Escherichia coli; Vandenbroeck K et al.; Interferon-gamma (IFN-gamma) is coded for by a single gene containing three introns, localized within the coding region . We have previously cloned the IFN-gamma gene from a pig genomic DNA lambda library and have determined its nucleotide sequence . In order to construct the porcine IFN-gamma DNA without intervening sequence, the four exons were separately amplified by the polymerase chain reaction (PCR) using primers matching the exon-termini . From the amplified exon-fragments the complete intron-free DNA was obtained by a strategy consisting of alternate rounds of PCR and ligation . The sequence so-obtained was used for expression in E . coli . The recombinant protein appeared as inclusion bodies which were solubilized and refolded in order to obtain biologically active IFN-gamma. Biochem Biophys Res Commun, 1991 Nov 14, 180(3), 1222 - 6 ClpB proteins copurify with the anaerobic Escherichia coli reductase; Pontis E et al.; Two proteins, called alpha and beta 3, copurify with the anaerobic ribonucleotide reductase from Escherichia coli (Eliasson et al . (1990) Proc . Natl . Acad . Sci . U.S.A . 87, 3314-3318) . Both are now identified as products of the clpB gene that is presumed to code for a subunit of an ATP dependent protease . The tight associations suggest the possibility that the ClpB proteins are involved in the regulation of the anaerobic reductase. Nature, 1991 Nov 14, 354(6349), 161 - 4 FtsZ ring structure associated with division in Escherichia coli; Bi EF et al.; Genes for cell division have been identified in Escherichia coli by the isolation of conditional lethal mutations that block cell division, but do not affect DNA replication or segregation . Of these genes, ftsZ is of great interest as it acts earliest in the division pathway, is essential, its level dictates the frequency of division, and it is thought to be the target of two cell-division inhibitors, SulA, produced in response to DNA damage, and MinCD, which prevents division at old sites . Here we have used immunoelectronmicroscopy to localize the FtsZ protein to the division site . The results suggest that FtsZ self-assembles into a ring structure at the future division site and may function as a cytoskeletal element . The formation of this ring may be the point at which division is regulated. Biochem Biophys Res Commun, 1991 Nov 14, 180(3), 1476 - 82 Isolation and sequence of rat testis cDNA for a calcium binding polypeptide similar to the regulatory subunit of calcineurin; Sugimoto M et al.; We have cloned and sequenced rat testis cDNAs coding for a calcium binding polypeptide similar to calcineurin beta subunit, the Ca(2+)-binding subunit of the Ca2+/calmodulin stimulated protein phosphatase . Rat testis cDNA library was screened with a monoclonal antibody Va1 raised against bovine brain calcineurin beta subunit . The deduced amino acid sequence is similar to that of human brain calcineurin beta subunit with respect to containing four putative calcium binding sites . However, distinct differences were found: 1) The cloned cDNA had six amino acids polypeptide tail at carboxy-terminal which is absent in human brain calcineurin beta subunit . This amino acids tail makes the carboxy-terminal highly hydrophilic in contrast to the human brain beta subunit which is hydrophobic at carboxy-terminal; 2) eleven amino acids at the N terminal of the cloned cDNA were completely different from the corresponding region of the brain calcineurin beta subunit. Biochemistry, 1991 Nov 12, 30(45), 10920 - 4 RNA folding during transcription by T7 RNA polymerase analyzed using the self-cleaving transcript assay; Tyagarajan K et al.; We have used a self-cleaving RNA molecule (a "hammerhead") to study the length-dependent folding of RNA produced during transcription by T7 RNA polymerase . Transcript elongation is arrested at defined positions using chain-terminating ribonucleoside triphosphate analogues, 3'-deoxynucleoside triphosphates . When the nascent transcript attains the minimum length required for the "hammerhead" domain of the transcript to fully emerge from the ternary complex, the "hammerhead" structure forms and self-cleaves, producing a truncated product . The experiment yields an RNA sequencing ladder which terminates at the length at which cleavage becomes possible; the sequencing ladder is compared to that generated by using a noncleaving control template . We have shown that 13 nucleotides past the cleavage point must be synthesized before the transcript can self-cleave in the ternary complex whereas RNA freed from the complex by heating can cleave with only 3 or more nucleotides present beyond the cleavage site . The results indicate that the RNA in T7 RNA polymerase is not free of steric interactions in the ternary complex and not available for structure formation until it is at least 10 bases away from the site of polymerization . The results suggest that the maximum possible length of the RNA-DNA hybrid in the ternary complexes is 10 . The relevance of the results in comparisons with other RNA polymerases, especially Escherichia coli RNA polymerase, is discussed. Biochemistry, 1991 Nov 12, 30(45), 10914 - 20 An isotope edited classical Raman difference spectroscopic study of the interactions of guanine nucleotides with elongation factor Tu and H-ras p21; Manor D et al.; We have measured the Raman spectrum of GDP bound to the elongation factor protein, EF-Tu, and the c-Harvey-ras protein, p21, two proteins of the guanine nucleotide binding family . In order to separate the Raman spectrum of the nucleotide from the much more intense protein spectrum, we investigate the feasibility of "tagging" the normal modes of the nucleotide by isotopic substitution, here by incoporating deuterium-labeled guanine at the C8 position into the active site . A difference spectrum between the labeled and unlabeled protein-nucleotide complex shows the changes in the Raman spectrum of the bound nucleotide that arise from the isotopic exchange . We find that surprisingly good Raman spectra of bound ligands can be obtained with this method and that the method can be easily generalized to other systems . The data show that the guanine amino group of the nucleotide interacts differently with both EF-Tu and p21 than it does with water, showing a change in hydrogen-bonding properties upon binding . On the other hand, no change in hydrogen bonding is observed at guanine's N7 . The data strongly suggest that the conformation of the nucleotide when bound to EF-Tu and that p21 is the C2' endo pucker of the ribose ring and anti about the glycosidic bond . These results are compared to previous structural and chemical studies. Biochemistry, 1991 Nov 12, 30(45), 10872 - 7 NMR study of the phosphate-binding elements of Escherichia coli elongation factor Tu catalytic domain; Lowry DF et al.; The phosphoryl-binding elements in the GDP-binding domain of elongation factor Tu were studied by heteronuclear proton observe methods . Five proton resonances were found below 10.5 ppm . Two of these were assigned to the amide groups of Lys 24 and Gly 83 . These are conserved residues in each of the consensus sequences . Their uncharacteristic downfield proton shifts are attributed to strong hydrogen bonds to phosphate oxygens as for resonances in N-ras-p21 {Redfield, A . G., & Papastavros, M . Z . (1990) Biochemistry 29, 3509-3514} . The Lys 24 of the EF-Tu G-domain has nearly the same proton and nitrogen shifts as the corresponding Lys 16 in p21 . These results suggest that this conserved lysine has a similar structural role in proteins in this class . The tentative Gly 83 resonance has no spectral analogue in p21 . A mutant protein with His 84 changed to glycine was fully 15N-labeled and the proton resonance assigned to Gly 83 shifted downfield by 0.3 ppm, thereby supporting the assignment. Biochemistry, 1991 Nov 12, 30(45), 10832 - 8 Selecting high-affinity binding proteins by monovalent phage display; Lowman HB et al.; Variants of human growth hormone (hGH) with increased affinity and specificity for the hGH receptor were isolated using an improved phage display system . Nearly one million random mutants of hGH were generated at 12 sites previously shown to modulate binding to the hGH receptor or human prolactin (hPRL) receptor . The mutant hormones were displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle . After three to six cycles of enrichment for hGH-phage particles that bound to hGH receptor beads, we isolated hGH mutants that exhibited consensus binding sequences for the hGH receptor . Residues previously identified as important for hGH receptor binding by alanine-scanning mutagenesis were more highly conserved by this selection method . However, other residues nearby were not optimal, and by mutating them, hormone variants having greater affinity and selectivity for the hGH receptor were isolated . This approach should be useful for those who wish to modify and understand the energetics of protein-ligand interfaces. Biochemistry, 1991 Nov 12, 30(45), 10987 - 91 Nucleotide-dependent and dicyclohexylcarbodiimide-sensitive conformational changes in the epsilon subunit of Escherichia coli ATP synthase; Mendel-Hartvig J et al.; The rate of trypsin cleavage of the epsilon subunit of Escherichia coli F1F0 (ECF1F0) is shown to be ligand-dependent as measured by Western analysis using monoclonal antibodies . The cleavage of the epsilon subunit was rapid in the presence of ADP alone, ATP + EDTA, or AMP-PNP + Mg2+, but slow when Pi was added along with ADP + Mg2+ or when ATP + Mg2+ was added to generate ADP + Pi (+Mg2+) in the catalytic site . Trypsin treatment of ECF1Fo was also shown to increase enzymic activity on a time scale corresponding to that of the cleavage of the epsilon subunit, indicating that the epsilon subunit inhibits ATPase activity in ECF1Fo . The ligand-dependent conformational changes in the epsilon subunit were also examined in cross-linking experiments using the water-soluble carbodiimide 1-ethyl-3-{3-(dimethylamino)propyl}-carbodiimide (EDC) . In the presence of ATP + Mg2+ or ADP + Pi + Mg2+, the epsilon subunit cross-linked product was much reduced . Prior reaction of ECF1Fo with dicyclohexylcarbodiimide (DCCD), under conditions in which only the Fo part was modified, blocked the conformational changes induced by ligand binding . When the enzyme complex was reacted with DCCD in ATP + EDTA, the cleavage of the epsilon subunit was rapid and yield of cross-linking of beta to epsilon subunit low, whether trypsin cleavage was conducted in ATP + EDTA or ATP + Mg2+ . When enzyme was reacted with DCCD in ATP + Mg2+, cleavage of the epsilon subunit was slow and yield of cross-linking of beta to epsilon high, under all nucleotide conditions for proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1991 Nov 12, 30(45), 10844 - 9 Gene structure and expression of the rat cytochrome P450IIC13, a polymorphic, male-specific cytochrome in the P450IIC subfamily; Eguchi H et al.; The male-specific CYP2C13 gene has been isolated from two independent rat genomic libraries . This gene spans more than 50 kb and contains eight introns which are subject to the GT-AG rule . Two allelic forms of the CYP2C13 gene were identified . Determination of the exonic sequences revealed that one of them encodes cytochrome P450(+g) and the other encodes cytochrome P450(-g) . Using allele-specific restriction enzyme sites, a good correlation between the genotype and the phenotype of CYP2C13 was shown . Nucleotide substitutions between the (+g) and the (-g) genes exist not only in the exons but also in the introns and the 5'-flanking region . Although five nucleotide differences were identified within 287 base pairs of the (+g) and (-g) 5'-flanking regions, the transcription initiation sites were identical . In addition to a canonical TATA box located 31 base pairs upstream of the start site of transcription, putative binding sites for the liver-enriched and liver-specific transcription factors HNF1/LF-B1/APF, HNF3, HNF4/AF-1, C/EBP, LAP, and eH-TF/TGT3 and the ubiquitous factors NF-1 and OTF-1 were identified. Am J Obstet Gynecol, 1991 Nov, 165(5 Pt 1), 1568 - 74 A rabbit model for bacterially induced preterm pregnancy loss: intervention studies with ampicillin-sulbactam; McDuffie RS Jr et al.; We conducted experiments with a previously described rabbit model of Escherichia coli-induced preterm pregnancy loss . Does at 70% gestation were inoculated hysteroscopically with 0.2 ml of Escherichia coli (10(5) colony-forming units per milliliter) or saline solution . Animals were randomly assigned to either receive treatment with ampicillin-sulbactam (begun 1 to 2 hours before inoculation and continued for up to 7 days) or to receive no therapy . Animals were killed after delivery or after 7 days . Saline solution-inoculated animals had no pregnancy loss . Of the Escherichia coli-inoculated animals, those treated with ampicillin-sulbactam had significantly fewer deliveries, fewer positive cultures, and more live fetuses than the untreated animals (p less than or equal to 0.001) . Cultures from multiple sites, amniotic fluid prostaglandin levels, and maternal progesterone levels were obtained, and the placenta, uterus, and fetal lung were histologically evaluated . In the second phase of the study, the Escherichia coli-inoculated animals were treated with ampicillin-sulbactam at one of three times: at inoculation or 2 or 4 hours after inoculation . The Escherichia coli-inoculated does treated with ampicillin-sulbactam at or before inoculation had significantly fewer deliveries, fewer positive cultures, and more live fetuses than the Escherichia coli-inoculated does in which treatment was delayed 4 hours (p less than or equal to 0.01). J Bacteriol, 1991 Nov, 173(21), 6991 - 7 Isolation and expression of a gene cluster responsible for biosynthesis of the glycopeptidolipid antigens of Mycobacterium avium; Belisle JT et al.; Bacteria within the Mycobacterium avium complex are prominent in the environment and are a source of serious disseminated infections in patients with AIDS . Serovars of the M . avium complex are distinguished from all other mycobacteria and from one another by the presence of highly antigenic glycolipids, the glycopeptidolipids, on their surfaces . A genomic library of DNA from serovar 2 of the M . avium complex was constructed in the Escherichia coli-Mycobacterium shuttle cosmid, pYUB18, and used to clone and express in Mycobacterium smegmatis the genes responsible for the biosynthesis of the oligosaccharide segment of the M . avium serovar 2-specific glycopeptidolipid . The responsible gene cluster was mapped to a 22- to 27-kb functional region of the M . avium genome . The recombinant glycolipid was also isolated by high-pressure liquid chromatography and chemically characterized, by gas chromatography-mass spectrometry and fast atom bombardment-mass spectrometry, to demonstrate that the lipopeptide core originated in M . smegmatis, whereas the oligosaccharide segment arose from the cloned M . avium genes . This first-time demonstration of the cloning and expression, in a nonpathogenic mycobacterium, of the genes encoding complex cell wall glycoconjugates from a pathogenic mycobacterium presents a new approach for studying the role of such products in disease processes. Res Microbiol, 1991 Nov-Dec, 142(9), 943 - 9 Differences in codon usage among genes encoding proteins of different function in Rhodobacter capsulatus; Wu LF et al.; Codon usage in Rhodobacter was evaluated and found to be strikingly different from that in Escherichia coli . While codon usage for genes concerned with nitrogen utilization and carotenoid biosynthesis corresponded to expectation, based on codon usage for Rhodobacter in general, that for the fructose utilization (fru) operon and for the photosynthetic genes encoding the reaction centre and light harvesting proteins exhibited significant deviation from expectation and from each other for specific amino acids . The differences in codon usage for the fru operon versus the photosynthetic genes may reflect different proportions of the various tRNA specific for certain amino acids when cells are grown under heterotrophic versus phototropic conditions . In addition, preferential use of the initiation codon, GTG, was found for the first cistrons of Rhodobacter operons. Nucleic Acids Res, 1991 Nov 11, 19(21), 6033 - 40 SQUIRREL: Sequence QUery, Information Retrieval and REporting Library . A program package for analyzing signals in nucleic acid sequences for the VAX; Gartmann CJ et al.; A computer tool is described for comparison, analysis and search of genetic signals . The method is based on sequence consensus matrices . It assumes that a genetic signal (such as a promoter, enhancer or whatever) is composed of several signal blocks separated from each other by variable distances . A set of programs is presented to perform the analysis . The result of such an analysis is a description of the investigated signal including matrices for each signal block, distances between each block and distribution of the values . Programs are provided to search for a signal using results from previous analysis . The method is able to align large sets of sequences within a few minutes and to check the quality of the alignment . An analysis of E.coli promoters is provided as an example. Nucleic Acids Res, 1991 Nov 11, 19(21), 6007 - 13 Construction of synthetic genes using PCR after automated DNA synthesis of their entire top and bottom strands; Ciccarelli RB et al.; A new method is described for the direct construction of synthetic genes by applying a modified version of the polymerase chain reaction (PCR) to crude oligonucleotide mixtures made by automated solid phase DNA synthesis . Construction of the HIV-1 393 bp rev gene and the 655 bp nef gene by this method is illustrated . The sequences for the entire top and bottom strands of rev were each programmed into an automated DNA synthesizer . Following DNA synthesis, the two crude oligonucleotide solutions were mixed together, specific primers were added, and the target gene was amplified by a modified PCR technique . Although the longer (greater than 200 bases) strands comprise a very small percentage of the total DNA after solid phase synthesis, this method uses PCR to 'find' and amplify such strands to create the target gene . The rev gene constructed by this method was found to contain 4 sequence errors, which were subsequently corrected by site-directed mutagenesis . In order to evaluate the source of sequence errors, several nef genes were made from the top and bottom strand DNA synthesis solutions using independent PCR's . Results suggest that sequence errors arose from both DNA synthesis and PCR . The utility of this method in producing a functional gene is demonstrated by expression of rev in E.coli. Nucleic Acids Res, 1991 Nov 11, 19(21), 5839 - 42 W (A or T) sequences as probes and primers suitable for genomic mapping and fingerprinting; Drmanac R et al.; A limitation to the use of oligonucleotide probes as tools for genetic and physical mapping has been the low hybridization positive frequency obtained by oligonucleotides of sufficient length to hybridize preferentially to cloned insert DNA (and not host E . coli genomic DNA) . Both computer and experimental results now indicate that oligonucleotide probes composed of W (A or T) sequence are preferentially found in eukaryotic DNA, and can be used to provide high frequency, discriminative hybridization . Such W sequences may be useful as either probes or PCR primers in molecular diagnostic applications as well as in genetic and physical mapping. Nucleic Acids Res, 1991 Nov 11, 19(21), 5863 - 70 A novel RNA product of the tyrT operon of Escherichia coli; Bosl M et al.; The tyrT operon of E . coli and several other tRNA operons of E . coli show striking structural features: they contain repeated sequence units including a 19bp motif resembling the 3' end of the corresponding mature tRNA . A novel RNA, encoded by the repeated sequence of the tyrT operon, was identified . The RNA, characterized by primer extension and Nuclease-S1 analysis, contained 171 nucleotides and terminated with the 19bp motif of the CCA-end of tRNA(1Tyr) . The RNA, designated as rtT RNA, is probably released from the primary transcript of tyrT during tRNA processing, it includes the coding capacity for the arginine rich peptide Tpr . Predictions of secondary folding resulted a rather stable RNA structure with a free energy of -44.3kcal/mol . A weak ribosome binding site was found, preceding the second possible AUG initiator codon for Tpr . The comparison of rtT RNA with putative transcripts from the repeated sequences associated with related tRNA genes showed common features with respect to primary structure, arrangement and secondary folding . In E . coli cultures the lag-phase during growth, caused by transient glycine or by isoleucine limitation, was found to be overcome or markedly shortened in the presence of rtT RNA . These and previously reported results suggest a modulatory effect of rtT RNA on stringent response. Biochim Biophys Acta, 1991 Nov 11, 1090(3), 293 - 8 Differential expression in Escherichia coli of the alpha and beta forms of heparin-binding acidic fibroblast growth factor-1: potential role of RNA secondary structure; Forough R et al.; Synthetic DNA fragments encoding the entire open-reading frame of human heparin-binding growth factor-1 (HBGF-1 beta) and its NH2-terminal truncated form (HBGF-1 alpha) were constructed . When both constructs were expressed in Escherichia coli under control of the trp-lac promoter, biologically active HBGF-1 alpha, but not HBGF-1 beta was produced in high yield . However, high level expression of HBGF-1 beta was obtained using the T7 polymerase expression vector . Computer analysis of HBGF-1 beta predicts the potential for the formation of exaggerated RNA secondary structure near the translation initiation codon and this could be implicated in contributing to the poor translation of HBGF-1 beta under the trp-lac promoter. Nucleic Acids Res, 1991 Nov 11, 19(21), 5907 - 14 Two distinct human DNA diesterases that hydrolyze 3'-blocking deoxyribose fragments from oxidized DNA; Chen DS et al.; Mammalian cells were investigated for enzymes that help correct oxidative damages in DNA . We focused on 3'-repair diesterases, which process DNA ends at oxidative strand breaks by removing 3'-blocking fragments of deoxyribose that prevent DNA repair synthesis . Two enzymes were found in a variety of mouse, bovine and human tissues and cultured cells . The two activities were purified to differing degrees from HeLa cells . One enzyme had the properties of the known HeLa AP endonuclease (Mr approximately 38,000, with identical substrate specificity and reaction requirements, and cross-reactivity with anti-HeLa AP endonuclease antiserum) and is presumed identical to that protein . The second activity did not interact with anti-HeLa AP endonuclease antibodies and had relatively less AP endonuclease activity . This second enzyme may have been detected in other studies but never characterized . In addition to the 3'-repair diesterase and AP endonuclease, this partially purified preparation also harbored DNA 3'-phosphatase and 3'-deoxyribose diesterase activities . It is unknown whether all activities detected in the second preparation are due to a single protein, although activity against undamaged DNA was not detected . The in vivo roles of these two widely distributed 3'-repair diesterase/AP endonucleases have not been determined, but with the characterizations presented here such questions may now be focused. Nucleic Acids Res, 1991 Nov 11, 19(21), 5889 - 94 Rapid mapping by transposon mutagenesis of epitopes on the muscular dystrophy protein, dystrophin; Sedgwick SG et al.; Antibody-binding epitopes in the central helical region of the muscular dystrophy protein, dystrophin, have been mapped using a new strategy of transposon mutagenesis . Tn1000 transposons carrying translation termination codons were introduced randomly by bacterial mating into a large fragment of dystrophin cDNA in a pEX2 plasmid to produce a library of transformants expressing truncated dystrophin fusion proteins . Epitopes were progressively lost as the expressed sequences were shortened, enabling the epitopes recognised by 22 monoclonal antibodies to be placed in order along the dystrophin molecule without in vitro manipulation of DNA . The C-terminus of each truncated fusion protein was precisely located within the dystrophin sequence by direct sequencing of pEX2 transformants using transposon-specific primers . Sequences as short as 7 and 17 amino-acids have been identified as essential for antibody binding in this way . Nineteen of the 22 monoclonal antibodies had been selected for their ability to bind both native and SDS-denatured dystrophin and 15 of these bind to one sequence of 74 amino-acids (residues 1431-1505 of the 3684 residue sequence) . This may be an area of high immunogenicity or of close structural similarity between native dystrophin and the SDS-treated recombinant fragment used for immunization. Biochim Biophys Acta, 1991 Nov 11, 1090(3), 317 - 25 Binding characteristics of Escherichia coli biotin repressor-operator complex; Lin KC et al.; The genes of the operon bioA.BFCD are transcribed divergently from a single regulatory region between the bioA and, bioB genes . Transcription in both directions is co-repressed by biotinyl-5'-adenylate and the biotin repressor . The multimeric state of the biotin repressor bound to DNA and how it affects transcription have not been fully characterized . Therefore, we isolated the BirA protein from a recombinant strain which overproduced the biotin repressor and studied the repressor-operator binding characteristics through restriction enzyme site protection experiments and the mobility shift assay . We also measured the stoichiometry of the biotin repressor-operator complex directly . The results of restriction enzyme site protection studies are consistent with the postulation that the biotin operator is approx . 40 bp long . Only one retarded DNA band appeared in the mobility shift experiments, suggesting that the repressor-operator binding could be a single step reaction . The repressor-operator binding stoichiometry determination revealed that two repressor monomers may occupy the wild type or half palindromic biotin operator sequences. Biochem Pharmacol, 1991 Nov 6, 42(11), 2131 - 9 Formation of different reaction products with single- and double-stranded DNA by the ortho-quinone and the semi-quinone free radical of etoposide (VP-16-213); Mans DR et al.; In this report, the types of DNA damage introduced by the ortho-quinone and the semiquinone free radical of 4'-demethylepipodophyllotoxin-9-(4-6-O-ethylidene-beta-D- glucopyranoside) (etoposide) and their relevance for the inactivation of single-stranded (ss) and double-stranded (ds) replicative form (RF) phi X174 DNA have been examined in vitro . The ortho-quinone yielded in both ss and ds DNA only chemical adducts, of which on the average about 1 out of 3 and 1 out of 12 per DNA molecule led to inactivation of ss or RF phi X174 DNA, respectively . The semi-quinone free radical, on the other hand, generated both frank and alkali-labile strand-breaks in ss and in ds DNA which, however, did not contribute significantly to DNA inactivation . The radical introduced, in addition, chemical DNA adducts . Unlike the ortho-quinone adducts, however, each of the semi-quinone adducts was lethal in ss phi X174 DNA, while more than 40 were required for the inactivation of RF DNA . The excision repair system of Escherichia coli did not operate on semi-quinone-modified RF DNA but removed about half of the ortho-quinone adducts {van Maanen JMS, Lafleur MVM, Mans DRA, van den Akker E, de Ruiter C, Koostra PR, Pappie D, de Vries J, Retel J and Pinedo HM, Biochem Pharmacol 37: 3579-3589, 1988} . When ortho-quinone-modified ss or ds DNA was subjected to a post-alkaline treatment, the adducts remained stably bound to the DNA and the degree of biological inactivation was not influenced . In contrast, post-alkaline treatment removed about 70 and 60% of the semi-quinone adducts from ss and ds DNA, respectively, which, in the case of ss phi X174 DNA, resulted in a partial restoration of the biological activity . It is concluded that the ortho-quinone and the semi-quinone free radical of etoposide produce different types of damage in DNA which have different effects on the biological activity. J Mol Biol, 1991 Nov 5, 222(1), 99 - 124 Amino acid substrate specificity of Escherichia coli phenylalanyl-tRNA synthetase altered by distinct mutations; Kast P et al.; Neither the tertiary structure nor the location of active sites are known for phenylalanyl-tRNA synthetase (PheRS; alpha 2 beta 2 structure), a member of class II aminoacyl-tRNA synthetases . In an attempt to detect the phenylalanine (Phe) binding site, two Escherichia coli PheRS mutant strains (pheS), which were resistant to p-fluorophenylalanine (p-F-Phe) were analysed genetically . The pheS mutations were found to cause Ala294 to Ser294 exchanges in the alpha subunits from both independent strains . This alteration (S294) resided in the well-conserved C-terminal part of the alpha subunit, precisely within motif 3, a typical class II tRNA synthetase sequence . We thus propose that motif 3 participates in the formation of the Phe binding site of PheRS . Mutation S294 was also the key for proposing a mechanism by which the substrate analogue p-F-Phe is excluded from the enzymatic reaction; this may be achieved by steric interactions between the para-position of the aromatic ring and the amino acid residue at position 294 . The Phe binding site model was then tested by replacing the alanine at position 294 as well as the two flanking phenylalanines (positions 293 and 295) by a number of selected other amino acids . In vivo and in vitro results demonstrated that Phe293 and Phe295 are not directly involved in substrate binding, but replacements of those residues affected PheRS stability . However, exchanges at position 294 altered the binding of Phe, and certain mutants showed pronounced changes in specificity towards Phe analogues . Of particular interest was the Gly294 PheRS in which presumably an enlarged cavity for the para position of the aromatic ring allowed an increased aminoacylation of tRNA with p-F-Phe . Moreover, the larger para-chloro and para-bromo derivatives of Phe could interact with this enzyme in vitro and became highly toxic in vivo . The possible exploitation of the Gly294 mutant PheRS for the incorporation of non-proteinogenic amino acids into proteins is discussed. J Biol Chem, 1991 Nov 5, 266(31), 21174 - 8 Mutation of a single amino acid converts germ cell alkaline phosphatase to placental alkaline phosphatase; Watanabe T et al.; Human placental and germ cell alkaline phosphatases (PLAP and GCAP, respectively), are characterized by their differential sensitivities to inhibition by L-leucine, EDTA, and heat . Yet, they differ by only 7 amino acids at positions 15, 67, 68, 84, 241, 254, and 429 within their respective 484 residues . To determine the structural basis and the amino acid(s) involved in these physicochemical differences, we constructed three GCAP mutants by site-directed mutagenesis and six GCAP/PLAP chimeras and then expressed these alkaline phosphatase mutants in COS-1 cells . We report that the differential reactivity of PLAP and GCAP depends critically on a single amino acid at position 429 . GCAP with Gly-429 is strongly inhibited by L-leucine, EDTA, and heat, whereas PLAP with Glu-429 is resistant . By substituting Gly-429 of GCAP with a series of amino acids, we demonstrate that the relative sensitivities of these mutants to L-leucine, EDTA, and heat inhibition are, in general, parallel . Mutants in the order of resistance to these treatments are: Glu (most resistant), Asp/Ile/Leu, Gln/Val/Lys, Ser/His, and Arg/Thr/Met/Cys/Phe/Trp/Tyr/Pro/Asn/Ala/Gly (least resistant) . However, the Ser-429 and His-429 mutants were more resistant to EDTA and heat inhibition than the wild-type GCAP, but were equally sensitive to L-leucine inhibition . Structural analysis of mammalian alkaline phosphatase modeled on the refined crystal structure of Escherichia coli alkaline phosphatase indicates that the negative charge of Glu-429 of PLAP, which simultaneously stabilizes the protein as a whole and the metal binding specifically, probably acts through interactions with the metal ligand His-320 (His-331 in E . coli alkaline phosphatase) . Replacement of codon 429 with Gly in GCAP leads to destabilization and loosening of the metal binding . The data suggest that the natural binding site for L-leucine may be near position 429, with the amino and carboxyl groups of L-leucine interacting with bound phosphate and His-432 (His-412 in E . coli alkaline phosphatase), respectively. J Biol Chem, 1991 Nov 5, 266(31), 21118 - 24 Cytotoxic properties of DAB486EGF and DAB389EGF, epidermal growth factor (EGF) receptor-targeted fusion toxins; Shaw JP et al.; Elevated expression of the receptor for epidermal growth factor (EGF) is a characteristic of several malignancies including those of the breast, bladder, prostate, lung, and neuroglia . To therapeutically target the cytotoxic action of diphtheria toxin to EGF receptor-expressing tumor cells, we have constructed a hybrid gene in which the sequences for the binding domain of diphtheria toxin have been replaced by those for human EGF . The resulting fusion toxins, DAB486EGF and DAB389EGF, bind specifically to the EGF receptor and inhibit protein synthesis in a variety of EGF receptor expressing human tumor cell lines with an IC50 as low as 0.1 pM . Comparisons of DAB486EGF and DAB389EGF showed that DAB389EGF was consistently 10- to 100-fold more cytotoxic than DAB486EGF . Like diphtheria toxin, the cytotoxic action of DAB389EGF results from ADP-ribosylation of elongation factor-2 and is sensitive to the action of chloroquine . Studies of the kinetics of cellular intoxication showed that a 15-min exposure of EGF receptor-expressing A431 cells to DAB389EGF results in complete protein synthesis inhibition within 4 h . Furthermore, inhibition of protein synthesis results in elimination of human tumor cell colonies . These findings show that DAB389EGF is a potential therapeutic agent for a wide variety of EGF receptor-expressing solid tumors. J Biol Chem, 1991 Nov 5, 266(31), 20946 - 52 Site-directed mutagenesis studies on the recombinant thioesterase domain of chicken fatty acid synthase expressed in Escherichia coli; Pazirandeh M et al.; The animal fatty acid synthase is a multifunctional protein with a subunit molecular weight of 260,000 . We recently reported the expression and characterization of the acyl carrier protein and thioesterase domains of the chicken liver fatty acid synthase in Escherichia coli . In order to gain insight into the mechanism of action of the thioesterase domain, we have replaced the putative active site serine 101 with alanine and cysteine and the conserved histidine 274 with alanine by site-directed mutagenesis . While both the Ser101----Ala and His274----Ala mutant proteins were inactive, the Ser101----Cys mutant enzyme (thiol-thioesterase) retained considerable activity, but the properties of the enzyme were changed from an active serine esterase to an active cysteine esterase, providing strong evidence for the role of Ser101 as the active site nucleophile . In order to further probe into the role of His274, a double mutant was constructed containing both the Ser101----Cys and the His274----Ala mutations . The double-mutant protein was inactive and exhibited diminished reactivity of the Cys-SH to iodoacetamide as compared to that of the Ser101----Cys-thioesterase, suggesting a role of His274 as a general base in withdrawing the proton from the Cys-SH in the thiol-thioesterase or Ser101 in the wild-type enzyme . Incubation of the recombinant thioesterases with {1-14C} palmitoyl-CoA resulted in the incorporation of {1-14C} palmitoyl into the enzyme only in the double mutant, suggesting that Cys-SH of the double mutant is reactive enough to form the palmitoyl-S-enzyme intermediate . This intermediate is not hydrolyzed because of the lack of His274, which is required for the attack of H2O on the acyl enzyme . These results suggest that the catalytic mechanism of the thioesterases may be similar to that of the serine proteases and lipases, which employ a serine-histidine-aspartic acid catalytic triad as part of their catalytic mechanism. J Biol Chem, 1991 Nov 5, 266(31), 20928 - 33 Characteristics of the gene for a spermidine and putrescine transport system that maps at 15 min on the Escherichia coli chromosome; Furuchi T et al.; The nucleotide sequence of the gene for the spermidine and putrescine transport system that maps at 15 min on the Escherichia coli chromosome was determined . It contained four open reading frames encoding A, B, C, and D proteins . By making several subclones, we showed that expression of all the four proteins was necessary for maximal spermidine and putrescine transport activity . A single transport system was involved in the transport of both spermidine and putrescine . The A protein (Mr 43K) was found to be associated with membranes, as shown by Western blot analysis of the cell fractions . In addition, it had consensus amino acid sequences for the nucleotide binding site . B (Mr 31K) and C (Mr 29K) proteins consisted of six putative transmembrane spanning segments linked by hydrophilic segments of variable length as shown by cell localization of the proteins synthesized in maxicells and by hydropathy profiles . D protein (Mr 39K) was inferred to be a polyamine binding protein existing in a periplasmic fraction from the results of Western blot analysis of the cell fractions and from measurements of polyamine binding to the protein . These results indicate that the spermidine and putrescine transport system can be defined as a bacterial periplasmic transport system. J Biol Chem, 1991 Nov 5, 266(31), 20922 - 7 Coexistence of the genes for putrescine transport protein and ornithine decarboxylase at 16 min on Escherichia coli chromosome; Kashiwagi K et al.; The nucleotide sequence of one of the putrescine transport operon