J Theor Biol, 1994 Dec 7, 171(3), 239 - 49 Fractal geometry study of DNA binding proteins; Xu J et al.; The main aim of this paper is to search for three-dimensional structure homology between DNA-binding proteins . The run of a protein's main chain is depicted by a sequence, which is constituted by the fractal indices, where each index describes how twisted or extended a partial protein segment is . Two proteins are compared by constructing a dot matrix between two such sequences, and the matrix is searched for dot-concentrated rectangles which is an indication of tertiary homology . This new method is applied to DNA-binding proteins which are selected from Brookhaven Protein Data Bank, among which most contain a helix-turn-helix motif . The dot-matrix shows that there is tertiary homology between two motifs . Other interesting results are also presented in this paper. Proc Natl Acad Sci U S A, 1994 Dec 6, 91(25), 12218 - 22 A molecular chaperone, ClpA, functions like DnaK and DnaJ; Wickner S et al.; The two major molecular chaperone families that mediate ATP-dependent protein folding and refolding are the heat shock proteins Hsp60s (GroEL) and Hsp70s (DnaK) . Clp proteins, like chaperones, are highly conserved, present in all organisms, and contain ATP and polypeptide binding sites . We discovered that ClpA, the ATPase component of the ATP-dependent ClpAP protease, is a molecular chaperone . ClpA performs the ATP-dependent chaperone function of DnaK and DnaJ in the in vitro activation of the plasmid P1 RepA replication initiator protein . RepA is activated by the conversion of dimers to monomers . We show that ClpA targets RepA for degradation by ClpP, demonstrating a direct link between the protein unfolding function of chaperones and proteolysis . In another chaperone assay, ClpA protects luciferase from irreversible heat inactivation but is unable to reactivate luciferase. Proc Natl Acad Sci U S A, 1994 Dec 6, 91(25), 12213 - 7 Substrate spectrum of human excinuclease: repair of abasic sites, methylated bases, mismatches, and bulky adducts; Huang JC et al.; Nucleotide-excision repair is the repair system for removing bulky lesions from DNA . Humans deficient in this repair pathway suffer from xeroderma pigmentosum (XP), a disease characterized by photodermatoses, including skin cancers . At the cellular level, XP patients fail to remove cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts induced by UV light, as well as other bulky DNA lesions caused by various genotoxic agents . XP cells are not particularly sensitive to ionizing radiation or to alkylating agents that cause mostly nonbulky DNA lesions . Therefore, it has generally been assumed that the human nucleotide-excision repair enzyme (excinuclease) is specific for bulky adducts . To determine the substrate range of human excinuclease we used the highly sensitive excision assay and tested bulky adducts, synthetic apurinic/apyrimidinic sites, N6-methyladenine, O6-methylguanine, and mismatches as potential substrates . We found that all of these "lesions" were removed by human excinuclease, although with vastly different efficiencies. Proc Natl Acad Sci U S A, 1994 Dec 6, 91(25), 12130 - 4 Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)-anchored proteins: GPI-anchored proteins in liposomes and cells show similar behavior; Schroeder R et al.; Proteins anchored by GPI are poorly solubilized from cell membranes by cold nonionic detergents because they associate with detergent-resistant membranes rich in cholesterol and sphingolipids . In this study, we demonstrated that cholesterol and sphingolipid-rich liposomes were incompletely solubilized by Triton X-100 . GPI-anchored placental alkaline phosphatase incorporated in these liposomes was also not solubilized by cold Triton X-100 . As sphingolipids have much higher melting temperatures (Tm) than cellular phospholipids, a property correlated with Tm might cause detergent inextractability . In support of this idea, we found that the low-Tm lipid dioleoyl phosphatidylcholine (DOPC) was efficiently extracted from detergent-resistant liposomes by Triton X-100, whereas the high-Tm lipid dipalmitoyl phosphatidylcholine (DPPC) was not . The fluorescence polarization of liposome-incorporated diphenylhexatriene was measured to determine the "fluidity" of the detergent-resistant liposomes . We found that these liposomes were about as fluid as DPPC/cholesterol liposomes, which were present in the liquid-ordered phase, and much less fluid than DOPC or DOPC/cholesterol liposomes . These findings may explain the behavior of GPI-anchored proteins, which often have saturated fatty acyl chains and should prefer a less-fluid membrane . Therefore, we propose that acyl chain interactions can influence the association of GPI-anchored proteins with detergent-resistant membrane lipids . The affinity of GPI-anchored proteins for a sphingolipid-rich membrane phase that is not in the liquid crystalline state may be important in determining their cellular localization. Proc Natl Acad Sci U S A, 1994 Dec 6, 91(25), 12036 - 40 Topology of the RNA polymerase active center probed by chimeric rifampicin-nucleotide compounds; Mustaev A et al.; Spatial organization of the binding sites for the priming substrate, the template DNA, and the transcription inhibitor rifampicin (Rif) in Escherichia coli RNA polymerase (EC 2.7.7.6) was probed with chimeric compounds in which Rif is covalently attached to a ribonucleotide . The compounds bind to RNA polymerase in bifunctional manner and serve as substrates for RNA chain extension, yielding chains up to 8 nucleotides in length, with Rif linked to their 5' termini . These products act as potent inhibitors of normal transcription . Using the linker between the two ligands as ruler, we determined the distance between the sites for Rif and the priming nucleotide to be approximately 15 A . A reactive side group placed in the linker next to Rif crosslinks to the template strand of DNA at the -2 or -3 position of the promoter . Thus, bound Rif is juxtaposed to DNA immediately upstream of the start site, suggesting that Rif plugs the channel leading RNA out of the active center. Proc Natl Acad Sci U S A, 1994 Dec 6, 91(25), 11899 - 903 Mutagenic potency of exocyclic DNA adducts: marked differences between Escherichia coli and simian kidney cells; Moriya M et al.; A single-stranded shuttle vector containing a single 3,N4-etheno-2'-deoxycytidine (epsilon dC) or 1,N2-(1,3-propano)-2'- deoxyguanosine (PdG) DNA adduct was used to investigate translesional DNA synthesis in Escherichia coli and simian kidney (COS) cells . The presence of either exocyclic adduct was associated with reduced numbers of transformants . In E . coli, this inhibitory effect could be overcome partially by irradiating cells with UV light before transformation . Translesional synthesis past both exocyclic lesions was accompanied by targeted mutations . For PdG, the primary mutagenic events observed in both hosts were PdG-->T transversions; in preirradiated E . coli, PdG-->A transitions were also observed . The targeted mutation frequency for single-stranded DNA that contained PdG was 100% in nonirradiated E . coli, 68% in preirradiated cells, and 8% in COS cells . In contrast, the targeted mutation frequency for single-stranded DNA that contained epsilon dC was 2% in nonirradiated E . coli, 32% in preirradiated cells, and 81% in COS cells . The primary mutations generated by epsilon dC in both E . coli and COS cells were epsilon dC-->A and epsilon dC-->T base substitutions . These observations appear to reflect the variable specificity of DNA replication complexes in incorporating bases opposite certain adducts . We conclude that DNA synthesis past the same DNA adduct can have strikingly different consequences in bacteria and mammalian cells, underscoring the importance of establishing the intrinsic mutagenic potential of DNA adducts in mammalian cells. Proc Natl Acad Sci U S A, 1994 Dec 6, 91(25), 11826 - 30 Solution scattering from 50S ribosomal subunit resolves inconsistency between electron microscopic models; Svergun DI et al.; Models of the 50S ribosomal subunit from electron microscopy on isolated particles and on ordered sheets display significantly different features . A model of the shape of the native Escherichia coli 50S subunit in solution and of its RNA-rich core at 4-nm resolution has been produced by using methods for joint interpretation of x-ray and neutron small-angle scattering data obtained by contrast variation . The good agreement between the shape of the entire 50S subunit and the electron microscopic models of isolated particles and between the RNA-rich core and the model obtained from ordered sheets leads to the conclusion that the latter, which is based on the subjective contouring of density maps, is heavily biased toward the RNA. Biochemistry, 1994 Dec 6, 33(48), 14565 - 78 Kinetic mechanism of adenine nucleotide binding to and hydrolysis by the Escherichia coli Rep monomer . 2 . Application of a kinetic competition approach; Moore KJ et al.; The Escherichia coli Rep protein is a DNA helicase that functions as a homodimer to catalyze the unwinding of duplex DNA during DNA replication in a reaction that is coupled to the binding and hydrolysis of ATP . As a first step toward a molecular understanding of the interactions of Rep with adenine nucleotides, we have investigated the kinetic mechanism of adenine nucleotide binding to the Rep monomer, which is the state of the protein in the absence of DNA . Although ATP binding to Rep does not significantly change the intrinsic tryptophan fluorescence, the binding of the fluorescent nucleotide analogue, 2'(3')-O-(N-methylanthraniloyl)-ATP (mantATP) is associated with a large increase in mant nucleotide fluorescence intensity {lambda ex = 290 nm, lambda em > 420 nm; Moore, K . J . M., & Lohman, T . M . (1994) Biochemistry (preceding article in this issue)} . We have used the fluorescence signal from mantATP binding to monitor the kinetics of nonfluorescent nucleotide binding to Rep by a kinetic competition approach . The simultaneous and parallel binding of a mixture of mantATP and ATP to the Rep monomer is associated with a complex triphasic fluorescence transient during the approach to equilibrium . Global analysis of the fluorescence transients over a range of {ATP} by numerical integration techniques was used to define the kinetic mechanism of ATP binding and to determine the elementary rate constants . Using this approach, the kinetic rate constants for ADP, ATP gamma S, AMPPNP, AMP, adenosine, and inorganic phosphate were also determined at 4 degrees C in 20 mM Tris.HCl (pH 7.5), 6 mM NaCl, 10% (v/v) glycerol, and 5 mM MgCl2 . The kinetics of adenine nucleotide binding to the Rep monomer are similar to those observed with the mant nucleotides under identical experimental conditions (Moore & Lohman, 1994) . The kinetic competition data are consistent with the following two-step mechanism for the binding of ATP, ADP, and ATP gamma S, where P is the Rep monomer and A is the adenine nucleotide: P+A {formula: see text} P-A {formula: see text} (P-A) . In the presence of 5 mM MgCl2, the values of K+1 (approximately 10(7) M-1 s-1) and k+2 (approximately 10 s-1) are comparable for each nucleotide, whereas k+2 > k-1 for ATP and ATP gamma S while for ADP k+2 << k-1; hence, differences in the overall equilibrium binding affinities of these nucleotides are primarily due to changes in k-1.(ABSTRACT TRUNCATED AT 400 WORDS) Biochemistry, 1994 Dec 6, 33(48), 14550 - 64 Kinetic mechanism of adenine nucleotide binding to and hydrolysis by the Escherichia coli Rep monomer . 1 . Use of fluorescent nucleotide analogues; Moore KJ et al.; The Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA in a reaction that is coupled to ATP binding and hydrolysis . The Rep protein is a stable monomer in the absence of DNA but dimerizes upon binding either single-stranded or duplex DNA, and the dimer appears to be the functionally active form of the Rep helicase . As a first step toward understanding how ATP binding and hydrolysis are coupled energetically to DNA unwinding, we have investigated the kinetic mechanism of nucleotide binding to the Rep monomer (P) using stopped-flow techniques and the fluorescent ATP analogue, 2'(3')-O-(N-methylanthraniloyl-ATP (mantATP) . The fluorescence of mantATP is enhanced upon Rep binding due to energy transfer from tryptophan . The results are consistent with the following two-step mechanism, in which the bimolecular association step is followed by a conformational change in the P-mantATP complex: P + mantATP {formula: see text} P-mantATP {formula: see text} (P-mantATP) . The following rate and equilibrium constants were determined at 4 degrees C in 20 mM Tris.HCl (pH 7.5), 6 mM NaCl, 5 mM MgCl2, and 10% (v/v) glycerol: k+1 = (1.1 +/- 0.2) x 10(7) M-1 s-1; k-1 = 3.2 (+/- 0.5) s-1; k+2 = 2.9 (+/- 0.5) s-1; k-2 = 0.04 (+/- 0.005) s-1; K1 = k+1/k-1 = (3.4 +/- 0.8) x 10(6) M-1; K2 = k+2/k-2 = 73 (+/- 10); Koverall = K1K2 = (2.30 +/- 0.6) x 10(8) M-1 . Similar rate and equilibrium constants are obtained with mantATP gamma S, whereas the apparent rate constant for mantAMPPNP binding is 15-fold lower than for mantATP and equilibrium binding is weaker (Koverall approximately 10(6) M-1) . Rep monomer does bind mantATP in the absence of Mg2+ (Koverall approximately 5 x 10(5) M-1), although the four rate constants in the above reaction increase by at least 8-fold (k-1 and k-2 increase by approximately 100- and approximately 1000-fold, respectively) . The affinities of Mg2+ for P-mantATP and (P-mantATP)* are 10- and 1000-fold higher than those for nucleotide-free Rep monomer, indicating that the second step in the reaction is associated with a marked increase in Mg2+ affinity . The bound Mg2+ in a (P-mantATP)*-Mg2+ complex dissociates at a rate that is comparable to the rate of mantATP release.(ABSTRACT TRUNCATED AT 400 WORDS) FEBS Lett, 1994 Dec 5, 355(3), 229 - 32 APS-sulfotransferase activity is identical to higher plant APS-kinase (EC 2.7.1.25); Schiffmann S et al.; A cDNA from Arabidopsis thaliana L . Heynh encoding the APS-kinase (EC 2.7.1.25) was modified by deletion of a plastidic transit peptide to enable its expression in Escherichia coli . The resultant protein (MW 25,761) is enzymatically active as APS-kinase and restores prototrophic growth in an APS-kinase mutant . All transformants harbouring the modified plant DNA also acquired APS-sulfotransferase activity . In the absence of ATP but provided with DTT, a tetrameric form of recombinant APS-kinase exhibits APS-sulfotransferase activity . Monospecific polyclonal antibodies raised against the APS-kinase as immunogen also reacted against APS-sulfotransferase . We propose that APS-sulfotransferase activity is a nonphysiological side reaction of APS-kinase. FEBS Lett, 1994 Dec 5, 355(3), 220 - 2 In vivo and in vitro effects of mutagenesis of active site tyrosine residues of mercuric reductase; Rennex D et al.; X-ray crystal structure analysis of mercuric reductase suggested that the binding site for Hg2+ consisted of two tyrosine residues, Tyr264 and Tyr605, as well as two cysteine residues, Cys207 and Cys628 . We have previously shown that mutagenesis of Tyr605 to Phe lowered the kcat of the enzyme 6-fold, whereas the same mutation of Tyr264 resulted in a reduction of 160-fold {(1993) Biochemistry 32, 7475-7478} . Tyr605 occupies the same position in mercuric reductase as the active site His residue in the related enzyme glutathione reductase . The mutation of Tyr605 of mercuric reductase to a His residue produced a 24-fold decrease in kcat and a 15-fold decrease in Km . The in vivo resistance to Hg2+ of E . coli strains carrying wild type or mutant merA genes correlated with the in vitro measurements of kcat/Km for mercuric reductase activity. Cell, 1994 Dec 2, 79(5), 767 - 78 Isolation of a protein that is essential for the first step of nuclear protein import; Gorlich D et al.; We have purified a cytosolic protein from Xenopus eggs that is essential for selective protein import into the cell nucleus . The purified protein, named importin, promotes signal-dependent binding of karyophilic proteins to the nuclear envelope . We have cloned, sequenced, and expressed a corresponding cDNA . Importin shows 44% sequence identity with SRP1p, a protein associated with the yeast nuclear pore complex . Complete, signal-dependent import into HeLa nuclei can be reconstituted by combining importin purified from Xenopus eggs or expressed in E . coli with Ran/TC4 . Evidence for additional stimulatory factors is provided. J Biol Chem, 1994 Dec 2, 269(48), 30726 - 33 Triple hydroxylation of tetracenomycin A2 to tetracenomycin C in Streptomyces glaucescens . Overexpression of the tcmG gene in Streptomyces lividans and characterization of the tetracenomycin A2 oxygenase; Shen B et al.; Nucleotide sequence analysis of the tcmG gene has suggested that the TcmG protein is responsible for the triple-hydroxylation of tetracenomycin (Tcm) A2 to Tcm C in Streptomyces glaucescens (Decker, H., Motamedi, H., and Hutchinson, C.R . (1993) J . Bacteriol . 175, 3876-3886) . The heterologous expression of the tcmG gene in Streptomyces lividans and the purification and characterization of TcmG protein, which we have named Tcm A2 oxygenase, are described here . NH2-terminal amino acid analysis of the purified enzyme led to the revision of the translational start site of tcmG to a TTG, 33 base pairs downstream of the GTG site assigned initially on the basis of nucleotide sequence analysis . Tcm A2 oxygenase is a monomeric protein in solution and contains 1 mol of non-covalently bound FAD; the apoenzyme can be partially reconstituted in vitro by addition of FAD . Tcm A2 oxygenase exhibits an optimal pH of 9.0-9.5 and prefers NADPH over NADH as an electron donor . The apparent K'm of the enzyme for Tcm A2, NADH, and NADPH are 1.81 +/- 0.38, 260 +/- 19, and 82.1 +/- 17 microM, respectively, and the apparent V'max for the reaction is 14.7 +/- 1.1 nmol Tcm C/min.mg . Purification and characterization of Tcm A2 oxygenase provide direct evidence to support the notion that the angular hydroxy groups of naphthacenequinones like Tcm C are introduced from 18O2 via a mono- or dioxygenase process. J Biol Chem, 1994 Dec 2, 269(48), 30713 - 7 How the ribosome moves along the mRNA during protein synthesis; Beyer D et al.; The movement of a ribosome along the mRNA was assessed by the following experimental strategy . mRNAs were synthesized which contained a short coding sequence with at least four codons and a 32P label at one end and an oligo(C) sequence at the other end . When these mRNAs were fixed on the ribosome with tRNAs specific for the defined codons, the oligo(C) stretches were partially outside of the ribosome, whereas the labeled ends were inside the ribosome and thus protected . The overhanging oligo(C) regions were trimmed with the cytidyl-specific RNase CL3 identifying the nucleotides of the mRNAs, which emerged from the ribosome . An mRNA enters the ribosome at nucleotide 18 +/- 1, when counting starts at the first nucleotide of the P-site codon, and leaves the ribosome at nucleotide -21 +/- 2 . The ribosome does not move at either side upon A-site occupation but does so at both sides simultaneously upon translocation . The results further indicate that most of the mRNA stretches downstream and upstream of the coding region are not required for the translocation reaction . It is therefore likely that the tRNAs are pulling the mRNA through the ribosome via codon-anticodon interactions in the course of translocation. J Biol Chem, 1994 Dec 2, 269(48), 30489 - 95 DNA glycosylase activities for thymine residues oxidized in the methyl group are functions of the AlkA enzyme in Escherichia coli; Bjelland S et al.; The alkA gene of Escherichia coli encodes a DNA glycosylase involved in base excision repair of DNA alkylation damage . In an attempt to define the reactions of the AlkA enzyme with methylated DNA, we discovered that the enzyme released substantial amounts of radioactivity from {methyl-3H}thymidine-labeled DNA even without any exposure of the DNA to methylating agents . The excised material was identified by chromatography as two different oxidized derivatives of thymine, 5-hydroxymethyluracil and 5-formyluracil . These products are formed in such DNA by one and two consecutive decays, respectively, of the tritiums of the labeled methyl group . Kinetic analysis showed that both the apparent Km and Vmax values for 5-formyluracil removal are within the same range as found for 3-methyladenine removal, suggesting that this catalytic property of AlkA is also significant under in vivo conditions . Removal of 5-hydroxymethyluracil proceeds at a rate that is 1-3 orders of magnitude slower . Since both 5-formyluracil and 5-hydroxymethyluracil are major products formed in DNA by exposure to ionizing radiation, these results implicate the alkA gene function also in the repair of oxidative DNA damage . Neither of the two other enzymes involved in the repair of oxidative DNA damage in E . coli, i.e . endonuclease III and formamidopyrimidine DNA glycosylase, has any affinity for oxidized unsaturated pyrimidines in DNA. J Biol Chem, 1994 Dec 2, 269(48), 30470 - 8 Common and divergent peptide binding specificities of hsp70 molecular chaperones; Fourie AM et al.; We have studied the binding of synthetic peptides to three hsp70 molecular chaperones, DnaK, BiP, and hsc70, as a model for the interaction of hsp70 proteins with unfolded regions of target polypeptides . We measured the ability of 53 peptides to inhibit the formation of complexes between the hsp70 proteins and denatured lactalbumin . Peptides that bound with highest affinity to all three hsp70 proteins contained stretches of at least 7 residues that included large hydrophobic and basic amino acids, but few or no acidic residues . Amino acid substitutions within one heptameric peptide showed that an important feature for its binding to all three chaperones was a large hydrophobic residue in position 4, while specificity differences between the chaperones were revealed by substitutions at positions 2 and 6 . Such specificity differences were frequently observed with other peptides, the most extreme example being a peptide rich in basic residues that bound with high affinity to DnaK, intermediate affinity to hsc70, and negligible affinity to BiP . Substitution of a lysine residue at position 2 in this peptide by tyrosine abolished the specificity difference by increasing the affinities of the DnaK and hsc70 proteins 5- and 20-fold, respectively, and that of BiP by greater than 2 orders of magnitude . Thus, hsp70 proteins can exhibit common or exclusive binding specificities, depending on the peptide sequence. J Biol Chem, 1994 Dec 2, 269(48), 30457 - 60 Active recombinant rat calpain II . Bacterially produced large and small subunits associate both in vivo and in vitro; Graham-Siegenthaler K et al.; cDNA for the C-terminal Ca(2+)-binding domain of rat calpain small subunit was cloned by means of the polymerase chain reaction . The encoded protein (21 kDa), which corresponds closely to the natural autolysis product of the small subunit, was produced in soluble form in Escherichia coli at a level of 20 mg/liter of cell culture . Rat calpain II large subunit (80 kDa) was produced from a cDNA clone in E . coli in soluble form at a level of approximately 1 mg/liter . The 80-kDa subunit alone had no proteinase activity, with or without Ca2+, but Ca(2+)-dependent proteinase activity was obtained following association of the two subunits, which was achieved either by co-expression of the two subunit cDNAs in E . coli, or by mixing the two partially purified subunits in the presence of 1 M NaSCN followed by dialysis . The heterodimeric (80 + 21 kDa) proteinase had a Ca2+ requirement for 50% activity of 0.35 mM and a specific activity at 2 mM Ca2+ of approximately 1 unit/microgram, values essentially identical to those of natural (80 + 30 kDa) calpain II . The results establish association and biological activity of the bacterially produced subunits and provide a system for studying structure-function relationships in calpain by means of mutagenesis. J Biol Chem, 1994 Dec 2, 269(48), 30364 - 9 A mechanism of proton translocation by F1F0 ATP synthases suggested by double mutants of the a subunit; Vik SB et al.; Three amino acid residues in the a subunit of the Escherichia coli F1F0 ATP synthase are essential for proton translocation: Arg210, Glu219, and His245 . In this study, the essential glutamic acid has been relocated to position 252 with retention of function . It had been known that Gln252 can be replaced by Glu without significant effect . To test whether Q252E would function in the absence of Glu219, a "site-directed second-site suppressor" experiment was designed . Saturation mutagenesis was applied to residue Glu219, and 14 different amino acid substitutions were isolated, five of which permitted growth on succinate minimal medium at 37 degrees C: Asp, Lys, Gly, Ala, and Ser . These results indicate that Q252E can provide the essential carboxyl group normally provided by Glu219, but that strict requirements are placed on the residue at position 219 . We interpret these results to mean that the Q252E must occupy, at least partially, the normal position of Glu219 . We present a novel mechanism of proton translocation by F1F0 ATP synthases that includes a rotating oligomer of c subunits, in which the Asp61 of two c subunits simultaneously interact with Glu219 and Arg210 of the a subunit . This mechanism can be adapted for both mitochondrial and sodium-driven bacterial ATP synthases. J Biol Chem, 1994 Dec 2, 269(48), 30313 - 9 Affinity labeling of aryl sulfotransferase IV . Identification of a peptide sequence at the binding site for 3'-phosphoadenosine-5'-phosphosulfate; Zheng Y et al.; 2'-O-{(R)-Formyl(adenin-9-yl)-methyl}-(S)-glyceraldehyde 3'-triphosphate (also designated as ATP dialdehyde or ATPDA) was utilized as an affinity label for the 3'-phosphoadenosine 5'-phosphosulfate (PAPS) binding site of an aryl sulfotransferase . The sulfotransferase employed in these studies was rat hepatic aryl sulfotransferase (AST) IV (also known as tyrosine-ester sulfotransferase, EC 2.8.2.9), for which a cDNA had been previously cloned and expressed in Escherichia coli and the resulting enzyme purified to homogeneity . ATPDA was a time-dependent irreversible inhibitor of the recombinant AST IV, and this inhibition was prevented by including either PAPS or adenosine 3',5'-diphosphate (PAP) in the incubation of AST IV with ATPDA . Experiments relating covalent binding of {2,8-3H}ATPDA with catalytic activity indicated that 1 nmol of the affinity label was bound per nmol of AST IV subunit . Incubation of {2,8-3H}ATPDA with the enzyme followed by reduction with sodium cyanoborohydride, proteolysis with trypsin, and separation of the resulting peptides by high pressure liquid chromatography yielded two labeled peptide fractions . Automated sequence analysis showed that both modified peptide fractions were derived from the same sequence in AST IV: 63-Leu-Glu-Lys-Cys-Gly-Arg-68 . Both the sequencing results and examination of the two peptide fractions by matrix-assisted laser desorption ionization mass spectrometry indicated that the ATPDA affinity label was bound to the hexapeptide at both lysine 65 and cysteine 66 . These affinity labeled amino acids are located within a region of sequence in AST IV that shows considerable homology with various sulfotransferases that possess diverse specificities for acceptor substrates, and this may provide insight into PAPS binding in other sulfotransferases. J Biol Chem, 1994 Dec 2, 269(48), 30187 - 94 Structural and kinetic studies of the 10 S<==>6 S transition in smooth muscle myosin; Rosenfeld SS et al.; The conformational transitions that smooth muscle myosin undergoes after nucleotide binding have been examined using fluorescently labeled nucleotides and regulatory light chain . The 10 S conformation of smooth muscle myosin could be induced by addition of 1-N6-ethenoadenosine or mant ADP plus beryllium fluoride, as well as by mant adenosine 5'-(beta,gamma-iminotriphosphate) (AMPPNP) . Fluorescence lifetime studies using 1-N6-ethenoadenosine plus beryllium fluoride reveal two components for both (10 S)- and (6 S)-myosins, with little difference in the values of these lifetimes, their fractional amplitudes, or solute accessibilities . Anisotropy decay studies of myosin-mant nucleotide complexes demonstrate that the rotational correlation time for (10 S)-myosin is nearly 4-fold longer than that for (6 S)-myosin . Qualitatively similar results were obtained with a 5-{{{2(iodoacetyl)amino}ethyl}amino}naphthalene-1-sulfonic acid fluorescent probe attached to the regulatory light chain . Mant AMPPNP can be trapped in the active site by (10S)-myosin . Actin accelerates this release rate by 40-50-fold . These studies reveal: 1) reduction in nucleotide release rate by converting (6S) to (10S)-myosin is not due to a reduction in solute accessibility of the nucleotide 2) the heads in (10 S)-myosin are rigidly attached to the rest of the molecule, while in (6 S)-myosin, they have segmental flexibility, 3) regulatory light chain phosphorylation mimics the effect of high salt in enhancing segmental flexibility of the myosin heads, and 4) actin can induce the unfolding of (10 S)-myosin in the absence of regulatory light chain phosphorylation. J Mol Biol, 1994 Dec 2, 244(3), 319 - 31 Chaperone-dependent folding and activation of ribosome-bound nascent rhodanese . Analysis by fluorescence; Kudlicki W et al.; Fluorescently labeled rhodanese was synthesized by coupled transcription/translation in a cell-free Escherichia coli system . A derivative of coumarin was co-translationally incorporated at the N terminus of the polypeptide . Molecules released from the ribosomes during the incubation are enzymatically active; however, continued incubation results in accumulation of enzymatically inactive full-length rhodanese polypeptides on the ribosomes . These can be activated and released in the presence of the added chaperones, DnaJ, DnaK, GrpE, GroEL, GroES and ATP . Fluorescence parameters (quantum yield, anisotropy and the emission maximum) of ribosome-bound coumarin-labeled rhodanese are affected differentially by addition of the chaperones individually or sequentially . Rhodanese released from the ribosomes in the presence of all chaperones (enzymatically active) differs in fluorescence properties from rhodanese released by GroES or DnaK only or by puromycin (enzymatically inactive) indicating a difference in conformation . Using sparsomycin, an inhibitor of the peptidyl transferase reaction, full-length rhodanese can be trapped on the ribosomes . A ribosome-bound intermediate formed by DnaJ or DnaJ plus DnaK was demonstrated by the effect of these chaperones on fluorescence spectra resulting from binding of anticoumarin antibodies to the N terminus of newly synthesized rhodanese . The results support the hypothesis that folding of nascent proteins can take place on the ribosome. Gene, 1994 Dec 2, 150(1), 57 - 61 Instability of plasmid DNA maintenance caused by transcription of poly(dT)-containing sequences in Escherichia coli; Kiyama R et al.; A series of pUC19-derived plasmids which contain a (dA)34 x (dT)34 tract was constructed to examine the effect of this sequence on plasmid maintenance in Escherichia coli . When this sequence was placed downstream from the lacZ promoter (lacZp), plasmids transcribing the (dT)34 strand were rapidly lost from the cells irrespective of the orientation relative to the replication origin, while plasmids transcribing the (dA)34 strand were stably maintained . A plasmid with a deletion within the lacZp region, preventing transcription from the (dT)34 strand, exhibited no such instability . The apparent transcription-dependent plasmid instability was not observed for the TcR (tetracycline-resistance gene) promoter which has weaker activity than lacZp . Similar strand-specific instability was observed for another microsatellite sequence, (dG)34 x (dC)34 . These findings suggest that accumulation of transcription products of the poly(dT) sequences could block DNA replication. Gene, 1994 Dec 2, 150(1), 197 - 8 Positive selection of recombinant plasmids based on the EcoK restriction activity of Escherichia coli K-12; De Backer O et al.; We have constructed a pTZ19R-derived vector which allows efficient positive selection of recombinant plasmids . The system uses the EcoK restriction activity of Escherichia coli K-12 to select against non-recombinant plasmids . The vector contains an EcoK site which, if deleted or disrupted by ligating a DNA fragment, yields recombinant plasmids that are no longer susceptible to EcoK restriction when transformed into a restriction-proficient E . coli host. Gene, 1994 Dec 2, 150(1), 1 - 8 Direct recognition of the trp operator by the trp holorepressor--a review; Youderian P et al.; Two different X-ray co-crystal structures of the Escherichia coli trp holorepressor complexed with DNA suggest that the TrpR protein recognizes specific DNA sequences primarily with a network of water-mediated H-bonds . However, the more recent nuclear magnetic resonance (NMR) solution structures of the holorepressor-operator complex show no long-lived, ordered water molecules at the protein-DNA interface and place amino acids in intimate contact with nucleotide bases . Both genetic and biochemical studies support a model in which the trp repressor recognizes specific DNA sequences by a direct mechanism, as seen in the NMR solution structures, not by the 'indirect readout' mechanism initially proposed on the basis of X-ray studies. Carbohydr Res, 1994 Dec 2, 265(1), 41 - 7 Structural determination of the capsular antigen of Escherichia coli O8:K25:H9; Anderson AN et al.; The primary structure of the acidic capsular antigen of Escherichia coli O8:K25:H9 was shown by monosaccharide analysis, methylation analysis, and by 1D and 2D 1H and 13C NMR spectroscopy to be composed of linear tetrasaccharide repeating units having the structure: Carbohydr Res, 1994 Dec 2, 265(1), 113 - 20 Structural studies of the enterotoxigenic Escherichia coli (ETEC) O153 O-antigenic polysaccharide; Ratnayake S et al.; The O-specific side-chain of the lipopolysaccharide from Escherichia coli O153 has been investigated using methylation analysis, Smith degradation, partial hydrolysis, FABMS, and NMR spectroscopy as the principal methods . It is concluded that the polysaccharide is composed of pentasaccharide repeating-units having the following structure. J Biol Chem, 1994 Dec 2, 269(48), 30378 - 85 Signaling-induced association of a tyrosine-phosphorylated 36-kDa protein with p50csk; Ford CE et al.; The protein-tyrosine kinase, p50csk, is thought to participate in the regulation of signal transduction pathways by catalyzing the phosphorylation of the Src-related protein-tyrosine kinases on a negative regulatory tyrosine residue located near the COOH terminus . To study possible mechanisms by which the activity of p50csk might be regulated, we searched for p50csk-interacting proteins in human erythroleukemia cells . We found that in response to the treatment of cells with pervanadate, a potent inhibitor of protein tyrosine phosphatases, or to the cross-linking of Fc gamma RIIA receptors, p50csk becomes tightly associated with a 36-kDa protein (p36) . This association is dependent on the tyrosine phosphorylation of p36 and involves its interaction with the SH2 domain of p50csk.p36 can be phosphorylated in vitro by p50csk or by a full-length GST-Csk fusion protein expressed in Escherichia coli . Tyrosine-phosphorylated p36 is found exclusively in the particulate membrane fraction of the cell . Conditions that induce the formation of the p50csk.p36 complex promote the appearance of p50csk in the particulate fraction . These data suggest that the association between p50csk and p36 serves to translocate the normally cytosolic p50csk to the membrane, where it presumably interacts with its physiologically relevant substrates. J Bacteriol, 1994 Dec, 176(24), 7730 - 4 Molecular cloning and expression of an isomalto-dextranase gene from Arthrobacter globiformis T6; Iwai A et al.; The gene encoding an extracellular isomalto-dextranase, designated imd, was isolated from the chromosomal DNA of Arthrobacter globiformis T6 and cloned and expressed in Escherichia coli . A single open reading frame consisting of 1,926 base pairs that encoded a polypeptide composed of a signal peptide of 39 amino acids and a mature protein of 602 amino acids (M(r), 65,900) was found . The primary structure had no significant homology with the structures of any other reported carbohydrases, including two other dextranases . Transformed E . coli cells carrying the 2.3-kb fragment overproduced isomalto-dextranase into the periplasmic space under control of the promoter of the imd gene itself. J Bacteriol, 1994 Dec, 176(24), 7653 - 8 Importance of anaerobic superoxide dismutase synthesis in facilitating outgrowth of Escherichia coli upon entry into an aerobic habitat; Kargalioglu Y et al.; The manganese-containing isozyme of superoxide dismutase (MnSOD) is synthesized by Escherichia coli only during aerobiosis, in accordance with the fact that superoxide can be formed only in aerobic environments . In contrast, E . coli continues to synthesize the iron-containing isozyme (FeSOD) even in the absence of oxygen . A strain devoid of FeSOD exhibited no deficits during either anaerobic or continuously aerobic growth, but its growth lagged for 2 h during the transition from anaerobiosis to aerobiosis . Complementation of this defect with heterologous SODs established that anaerobic SOD synthesis per se is necessary to permit a smooth transition to aerobiosis . The growth deficit was eliminated by supplementation of the medium with branched-chain amino acids, indicating that the growth interruption was due to the established sensitivity of dihydroxyacid dehydratase to endogenous superoxide . Components of the anaerobic respiratory chain rapidly generated superoxide when exposed to oxygen in vitro, suggesting that this transition may be a period of acute oxidative stress . These results show that facultative bacteria must preemptively synthesize SOD during anaerobiosis in preparation for reaeration . The data suggest that evolution has chosen FeSOD for this function because of the relative availability of iron, in comparison to manganese, during anaerobiosis. J Bacteriol, 1994 Dec, 176(24), 7646 - 52 Wild-type gas vesicle formation requires at least ten genes in the gvp gene cluster of Halobacterium halobium plasmid pNRC100; DasSarma S et al.; To study the functions of the 13 gvp genes, gvpMLKJIHGFEDACN, on plasmid pNRC100 of Halobacterium halobium in gas vesicle formation, we carried out linker scanning mutagenesis of the gene cluster . We constructed a 24.5-kb Escherichia coli-H . halobium shuttle plasmid, pFL2, containing the gvp gene cluster and introduced a kanamycin resistance (kappa) cassette into each gene (except for gvpA) . Transformation of H . halobium SD109, which had the entire gvp gene cluster deleted, with pFL2 and mutated pFL2 derivatives showed that while the unmutated gene cluster successfully programmed gas vesicle formation, derivatives with insertion of the kappa cassette in any of the gvp genes, except gvpM, did not lead to production of normal gas vesicles . Insertions in gvpL, -K, -J, -I, and -F resulted in a complete block in gas vesicle synthesis, while insertions in gvpH, -G, -E, -D, -C, and -N resulted in greatly reduced gas vesicle synthesis . In most cases, the block in gas vesicle synthesis did not result from polar effects, since similar results were obtained for derivatives of the insertion mutants in which most of the internal portion of the kappa cassette was deleted and only small (15 to 54-bp) insertions remained . The only exceptions were for gvpH and gvpD, where deletion of the internal portion of the kappa insertions resulted in phenotypic reversion . Electron microscopic analysis of the kappa mutants revealed that interruptions of gvpC and gvpN result in the formation of smaller gas vesicle than in the wild type, while interruptions of gvpF, -G, -H, -J, -K, and -L produce no discernible vesicle intermediates . These results indicate the gvpA, -C, and -N, which have the rightward transcriptional orientation, encode structural proteins, with gvpC and gvpN necessary for late stages of vesicle formation, and gvpL, -K, -J, -I, -H, -G, and -F, which have the leftward transcriptional orientation encode proteins involved in early steps in the assembly of gas vesicles. J Bacteriol, 1994 Dec, 176(24), 7638 - 45 Induction of the Escherichia coli aidB gene under oxygen-limiting conditions requires a functional rpoS (katF) gene; Volkert MR et al.; The Escherichia coli aidB gene is regulated by two different mechanisms, an ada-dependent pathway triggered by methyl damage to DNA and an ada-independent pathway triggered when cells are grown without aeration . In this report we describe our search for mutations affecting the ada-independent aidB induction pathway . The mutant strain identified carries two mutations affecting aidB expression . These mutations are named abrB (aidB regulator) and abrD . The abrB mutation is presently poorly characterized because of instability of the phenotype it imparts . The second mutation, abrD1, reduces the expression of aidB observed when aeration is ceased and oxygen becomes limiting . Genetic and phenotypic analysis of the abrD1 mutation demonstrates that it is an allele of rpoS . Thus, aidB is a member of the family of genes that are transcribed by a sigma S-directed RNA polymerase holoenzyme . Examination of aidB expression in an rpoS insertion mutant strain indicates that both rpoS13::Tn10 and abrD1 mutations reduce aidB expression under oxygen-limiting conditions that prevail in unaerated cultures, reduce aidB induction by acetate at a low pH, but have little or no effect on the ada-dependent alkylation induction of aidB. J Bacteriol, 1994 Dec, 176(24), 7601 - 13 Nucleotide sequence of the afimbrial-adhesin-encoding afa-3 gene cluster and its translocation via flanking IS1 insertion sequences; Garcia MI et al.; The afa gene clusters encode afimbrial adhesins (AFAs) that are expressed by uropathogenic and diarrhea-associated Escherichia coli strains . The plasmid-borne afa-3 gene cluster is responsible for the biosynthesis of the AFA-III adhesin that belongs to the Dr family of hemagglutinins . Reported in this work is the nucleotide sequence of the 9.2-kb insert of the recombinant plasmid pILL61, which contains the afa-3 gene cluster cloned from a cystitis-associated E . coli strain (A30) . The afa-3 gene cluster was shown to contain six open reading frames, designated afaA to afaF . It was organized in two divergent transcriptional units . Five of the six Afa products showed marked homologies with proteins encoded by previously described adhesion systems that allowed us to attribute to each of them a putative function in the biogenesis of the AFA-III adhesin . AfaE was identified as the structural adhesin product, whereas AfaB and AfaC were recognized as periplasmic chaperone and outer membrane anchor proteins, respectively . The AfaA and AfaF products were shown to be homologous to the PapI-PapB transcriptional regulatory proteins . No function could be attributed to the AfaD product, the gene of which was previously shown to be dispensable for the synthesis of a functional adhesin . Upstream of the afa-3 gene cluster, a 1.2-kb region was found to be 96% identical to the RepFIB sequence of one of the enterotoxigenic E . coli plasmids (P307), suggesting a common ancestor plasmid . This region contains an integrase-like gene (int) . Sequence analysis revealed the presence of an IS1 element between the int gene and the afa-3 gene cluster . Two other IS1 elements were detected and located in the vicinity of the afa-3 gene cluster by hybridization experiments . The afa-3 gene cluster was therefore found to be flanked by two IS1 elements in direct orientation and two in opposite orientations . The afa-3 gene cluster, flanked by two directly oriented IS1 elements, was shown to translocate from a recombinant plasmid to the E . coli chromosome . This translocation event occurred via IS1-specific recombination mediated by a recA-independent mechanism. J Bacteriol, 1994 Dec, 176(24), 7524 - 31 Hyperrecombination in the terminus region of the Escherichia coli chromosome: possible relation to nucleoid organization; Louarn J et al.; The terminus region of the Escherichia coli chromosome is the scene of frequent homologous recombination . This can be demonstrated by formation of deletions between directly repeated sequences which flank a genetic marker whose loss can be easily detected . We report here that terminal recombination events are restricted to a relatively large terminal recombination zone (TRZ) . On one side of the TRZ, the transition from the region with a high excision rate to the normal (low) excision rates of the rest of the chromosome occurs along a DNA stretch of less than 1 min . No specific border of this domain has been defined . To identify factors inducing terminal recombination, we examined its relation to two other phenomena affecting the same region, site-specific recombination at the dif locus and site-specific replication pausing . Both the location and the efficiency of terminal recombination remained unchanged after inactivation of the dif-specific recombination system . Similarly, inactivation of site-specific replication pausing or displacement of the replication fork trap so that termination occurs about 200 kb away from the normal region had no clear effect on this phenomenon . Therefore, terminal recombination is not a direct consequence of either dif-specific recombination or replication termination . Furthermore, deletions encompassing the wild-type TRZ do not eliminate hyperrecombination . Terminal recombination therefore cannot be attributed to the activity of some unique sequence of the region . A possible explanation of terminal hyperrecombination involves nucleoid organization and its remodeling after replication: we propose that post replicative reconstruction of the nucleoid organization results in a displacement of the catenation links between sister chromosomes to the last chromosomal domain to be rebuilt . Unrelated to replication termination, this process would facilitate interactions between the catenated molecules and would make the domain highly susceptible to recombination between sister chromosomes. J Bacteriol, 1994 Dec, 176(24), 7439 - 46 Novel ionizing radiation-sensitive mutants of Deinococcus radiodurans; Udupa KS et al.; Two new loci, irrB and irrI, have been identified in Deinococcus radiodurans . Inactivation of either locus results in a partial loss of resistance to ionizing radiation . The magnitude of this loss is locus specific and differentially affected by inactivation of the uvrA gene product . An irrB uvrA double mutant is more sensitive to ionizing radiation than is an irrB mutant . In contrast, the irrI uvrA double mutant and the irrI mutant are equally sensitive to ionizing radiation . The irrB and irrI mutations also reduce D . radiodurans resistance to UV radiation, this effect being most pronounced in uvrA+ backgrounds . Subclones of each gene have been isolated, and the loci have been mapped relative to each other . The irrB and irrI genes are separated by approximately 20 kb of intervening sequence that encodes the uvrA and pol genes. Exp Parasitol, 1994 Dec, 79(4), 489 - 505 Brugia malayi: the diagnostic potential of recombinant excretory/secretory antigens; Kumari S et al.; The diagnostic potential of recombinant E/S antigens of the lymphatic filaria Brugia malayi was investigated by Western blot . A cDNA expression library was constructed using B . malayi male adult worm mRNA, and E/S recombinants were identified with a rabbit antiserum raised against E/S products collected in vitro from B . malayi male and female adult worms . Two of these recombinants, Bm12 and Bm14L, were studied after subcloning the cDNA inserts in an Escherichia coli plasmid expression and purification vector, obtaining the inserts' nucleotide sequence, and purifying the expressed proteins . By homology of their deduced amino acid sequence with that of previously identified proteins, Bm12 was identified as the B . malayi gp 15/400 antigen, and Bm14 as a member of the hsp90 family of heat shock proteins . The antigenic cross-reactivity of the purified recombinant proteins was assessed with 28 serum samples from patients infected with Ascaris, Trichuris, or hookworm, and also with a few samples from patients with onchocerciasis and loiasis . For Bm12, the specificity for all of the intestinal helminthiasis together was 75% . Bm14L, on the other hand, cross-reacted with all of the ascariasis serum samples with which it was tested . Presence of antibodies cross-reactive with B . malayi was confirmed in all of these serum samples by examining their antibody reactivity with Western blots of extracts of whole B . malayi adult worms . A semiquantitative (+ or -) assessment of the sensitivity of Bm12 for antibody detection was performed using 6 serum samples from patients with chronic filariasis and 24 samples from patients with microfilaremia . All of these serum samples contained anti-Bm12 antibody (sensitivity of 100%) . Finally, the ability of Bm12 to detect antibody before the onset of patency was established with a longitudinal collection of serum samples obtained from 2 African green vervets (Cercopithecus aethiops) and 3 rhesus macaques (Macaca mulatta), all of which were infected with B . malayi . Anti-Bm12 antibodies were detectable in all animals between 4 and 11 weeks before patency. Eur J Biochem, 1994 Dec 1, 226(2), 731 - 7 The reaction of hydrogen peroxide with pulsed cytochrome bo from Escherichia coli; Moody AJ et al.; The reaction of hydrogen peroxide (H2O2) with pulsed cytochrome bo leads to characteristic spectral changes in the enzyme . The difference spectrum shows minima at 401, 494 and 628 nm, and maxima at 420, approximately 468, 526 and 556 nm . delta epsilon 420-epsilon 401 is in the range 73-86 mM-1.cm-1 and delta epsilon 556-epsilon 628 is 7.7-9.6 mM-1.cm-1 (taking delta epsilon 560-epsilon 580 for the reduced minus oxidised spectrum to be 20.5 mM-1.cm-1) . The stoichiometry of the reaction, determined by titration of the spectral changes, is 1:1 . The second order rate constant for the reaction, which is 1.0-1.5 x 10(3) M-1.s-1 at 20 degrees C, is independent of pH over the range 6.5-8.0 . The product of the reaction decays with a first-order rate constant in the range 1-4 x 10(-4) s-1, so the Kd value is apparently in the range 0.05-0.40 microM . The spectral changes observed immediately after quinol-induced turnover, or during steady-state turnover induced by hydrazine or by carbon monoxide, are qualitatively the same as those induced by H2O2 though of lower amplitude . H2O2 addition perturbs the hydrazine-induced or CO-induced steady states by increasing the amplitude of the spectral changes, but there is no qualitative change . From this observation, and the 1:1 stoichiometry of the reaction, we conclude that the intermediate induced by H2O2, which we term F., requires donation of only two electrons to the enzyme from an external source. Eur J Biochem, 1994 Dec 1, 226(2), 681 - 9 Interaction of plant profilin with mammalian actin; Giehl K et al.; The mode of interaction of birch and bovine profilins with actin was compared using a number of techniques . Birch profilin was purified from pollen or as a recombinant protein from Escherichia coli, using poly(L-proline) affinity chromatography and a monoclonal antibody for the identification of the isolated product . On two-dimensional gels, the genuine and recombinant proteins were identical in molecular mass and isoelectric point and revealed that birch profilin, in contrast to the basic profilins found in mammals, is an acidic protein, analogous to maize profilins . Bovine profilin was obtained from calf thymus . In viscometric assays, the birch protein was seen to modulate actin filament formation analogous to animal profilin . Birch profilin increased the critical concentration required for muscle and brain actin polymerization in a concentration-dependent manner, supporting the notion of the formation of a heterologous complex between the plant protein and animal actin . The effect was Mg(2+)-sensitive, as had been described for homologous complexes . The dissociation constants obtained for the plant/vertebrate and the vertebrate/vertebrate system were both in the micromolar range . The affinity of birch profilin for muscle actin was slightly lower than that for nonmuscle (brain) actin . A binary complex of birch profilin and skeletal muscle actin could be isolated by gel chromatography . Cross-linking experiments with actin, birch profilin, the G-actin binding peptide thymosin beta 4 and gelsolin segment 1, the N-terminal fragment of an actin capping protein, showed that profilin competed with thymosin beta 4, but had no effect on segment 1 binding to actin . These data indicate that the actin-binding domains in plant and animal profilins are functionally highly conserved, although the overall sequence similarity is less than 25%. Eur J Biochem, 1994 Dec 1, 226(2), 673 - 80 Isolation of a novel Plasmodium falciparum gene encoding a protein homologous to the Tat-binding protein family; Hirtzlin J et al.; We have cloned a Plasmodium falciparum gene that belongs to the nuclear Tat-binding protein (TBP) gene family . This gene, PfTBP, is (A + T)-rich and encodes a 49.5-kDa protein . The predicted protein encoded by this gene has highest similarity to the slime mold protein DdTBP10 (86%) and to the yeast protein SUG1 (81.8%), both of which belong to the Tat-binding protein family . In agreement with the characteristics of this family, PfTBP contains a highly conserved domain of approximately 200 amino acids, in which are found the motifs A and B of ATPases, and amino acid sequences characteristic of a large family of RNA or DNA helicases, suggesting a role in RNA or DNA unwinding . Like DdTBP10, the PfTBP protein has a heptad repeat of four leucine residues, reminiscent of a leucine zipper motif known to mediate dimerization . We have further characterized PfTBP gene expression by Northern-blot analysis . This gene is expressed in a stage-specific manner, with higher expression in the late trophozoite stage . The recombinant PfTBP gene has been expressed in Escherichia coli and a polyclonal antiserum has been raised in rabbits against the recombinant protein . This antibody has been used to study the protein in the parasite . The gene product is expressed in a stage-specific manner with higher expression in the late trophozoite and schizont stages, and is localized in the nucleus of the erythrocytic stage parasite . Thus the protein might have a function in DNA synthesis and/or in transcription, as is the case for other Tat-binding proteins. Eur J Biochem, 1994 Dec 1, 226(2), 657 - 64 The folding of the bifunctional TRP3 protein in yeast is influenced by a translational pause which lies in a region of structural divergence with Escherichia coli indoleglycerol-phosphate synthase; Crombie T et al.; The yeast TRP3 gene encodes a bifunctional protein with anthranilate synthase II and indoleglycerol-phosphate synthase activities . Replacing ten consecutive non-preferred codons in the indoleglycerol-phosphate synthase region of the TRP3 gene with synonymous preferred codons (to create the TRP3pr gene; translational pause replaced) causes a 1.5-fold reduction in relative indoleglycerol-phosphate synthase activity {Crombie, T., Swaffield, J.C . & Brown, A.J.P . (1992) J . Mol . Biol . 228, 7-12} . Here, we report that both the anthranilate synthase II and indoleglycerol-phosphate synthase domains are affected to similar extents when the translational pause is removed . Also, structural modelling of the yeast indoleglycerol-phosphate synthase domain against the X-ray crystal structure of indoleglycerol-phosphate synthase from Escherichia coli indicates that the translational pause lies in a region of structural divergence between similar structures . To probe the role of cytoplasmic heat-shock protein 70 (Hsp 70) chaperones in Trp3 protein folding, anthranilate synthase and indoleglycerol-phosphate synthase activities were measured in ssa and ssb mutants . Neither indoleglycerol-phosphate synthase nor anthranilate synthase were affected significantly in the ssb mutant . However, depletion of Hsp70 proteins encoded by the SSA genes led to decreased anthranilate synthase and indoleglycerol-phosphate synthase activities from the TRP3 gene, suggesting that both domains depend to some extent upon the SSA chaperone family . The data are consistent with roles for both the translational pause and Ssa chaperones in Trp3 protein folding in vivo. Eur J Biochem, 1994 Dec 1, 226(2), 619 - 26 Pathogen-defence gene prp1-1 from potato encodes an auxin-responsive glutathione S-transferase; Hahn K et al.; Genetic studies have previously implicated the prp1 gene family in the defence of potato against infection with the late blight fungus Phytophthora infestans . Here, we show that the concentrations of PRP1 mRNA as well as protein rapidly increase in potato leaves after fungal infection and stay at high levels during an extended period of the infection cycle . After separation of subcellular components by differential centrifugation, PRP1 protein was identified in the cytosolic fraction . Expression studies with chimeric promoter/beta-glucuronidase gene constructs in transgenic potato plants provided evidence that transcription of the prp1-1 gene, representing one member of the prp1 gene family, is at least partly responsible for the accumulation of PRP1 mRNA and protein upon fungal infection . After expression of the prp1-1-coding sequence in Escherichia coli, the corresponding 26-kDa protein exhibited glutathione S-transferase activity with Km values of 9.8 mM and 0.11 mM for the artificial standard substrate 1-chloro-2,4-dinitrobenzene and glutathione, respectively . Photoaffinity labeling of the protein with tritiated 5-azido-indole-3-acetic acid suggested that the phytohormone indole-3-acetic acid or a structurally related compound serve as a regulator or substrate of the prp1-1 encoded glutathione S-transferase . This assumption was further supported by the inhibitory effect of the phytohormone on the enzyme activity in vitro . The implications of these findings for a potential involvement of indole-3-acetic acid in the control of defence reactions are discussed. Eur J Biochem, 1994 Dec 1, 226(2), 459 - 63 The NADPH: sulfite reductase of Escherichia coli is a paraquat reductase; Gaudu P et al.; The NADPH:sulfite reductase of Escherichia coli is a soluble enzyme that has a subunit structure alpha 8 beta 4, where the alpha subunit is a flavoprotein and the beta subunit is a metalloprotein . Overexpression of the holoenzyme in E . coli has greatly simplified the purification of this enzyme . Under aerobic conditions, recombinant sulfite reductase catalyzes the reduction of 1,1'-dimethyl-4,4'-bipyridinium dichloride (paraquat) by NADPH, with Km values for paraquat and NADPH of approximately 70 microM and 80 microM, respectively . Since pure flavoprotein alpha subunit, encoded by the cysJ gene, has similar catalytic activities, it is suggested that paraquat receives electrons directly from the alpha subunit . A mutant strain lacking an active cysJ gene is resistant to paraquat . The NADPH:ferredoxin reductase of E . coli is also a paraquat reductase but with much higher Km values for paraquat and lower enzyme activities . These results suggest that the sulfite reductase is a major paraquat reductase in E . coli and is responsible for the toxic activation of the drug. Eur J Biochem, 1994 Dec 1, 226(2), 403 - 12 Purification and biochemical characterization of TrwC, the helicase involved in plasmid R388 conjugal DNA transfer; Grandoso G et al.; TrwC is an essential protein in conjugative DNA transfer of the broad-host-range plasmid R388 . TrwC was purified in two chromatographic steps from TrwC-overproducing bacteria . The purification procedure resulted in > 90% pure TrwC protein, which was free of contaminating nuclease activities . TrwC behaved as a dimer in gel-filtration chromatography in the presence of 550 mM NaCl, and had a pI of 10.1 . The purified protein showed in-vitro ssDNA-dependent nucleoside-5'-triphosphatase and DNA helicase activities . ATP was the preferred substrate for the NTP hydrolysis reaction, which required Mg2+ . The helicase activity was dependent on ATP and Mg2+ . The efficiency of the unwinding reaction catalyzed by TrwC ranged from > 90% of fragment displaced for a 93-nucleotide sequence to < 5% for a 365-nucleotide sequence . Unwinding was unidirectional in the 5' to 3' direction . The enzyme turned over very slowly from one DNA substrate molecule to another . TrwC is only the second DNA helicase to be described which is involved in conjugative DNA transfer . The biochemical properties of TrwC described here confirm its functional relatedness to helicase I (TraI) encoded by plasmid F of E . coli. Eur J Biochem, 1994 Dec 1, 226(2), 361 - 7 In vitro proteolytic activity and active-site identification of the human cytomegalovirus protease; Stevens JT et al.; Human cytomegalovirus (HCMV) encodes a protease that cleaves itself and the HCMV assembly protein . Two proteolytic processing sites within the protease were identified at Ala 256-Ser 257 (release site) and Ala 643-Ser 644 (maturation site) . Identification of rP5-P4' and mP4-P6' as the minimal peptide substrates spanning the release and maturation cleavage sites, respectively, demonstrated a requirement for residues flanking the conserved core in substrate recognition and hydrolysis, which are unique to HCMV . Kinetic parameters determined for release-site-derived and maturation-site-derived peptides revealed a 10-fold increase in kcat/Km for a maturational peptide (mP4-P8') over release-site peptide (rP5-P5') . Experimental results with a panel of class-specific protease inhibitors were consistent with the protease being a member of the serine or cysteine family of proteases . Further investigation revealed that the HCMV protease activity decreased with incorporation of {14C}iodoacetic acid, but when approximately 4.5 mol 14C were incorporated/mol enzyme, the enzyme retained approximately 20% of its original activity, indicating that hydrolysis does not require a cysteine nucleophile . Analysis of diisopropyl-fluorophosphate-inactivated protease by mass spectrometry indicated a stoichiometry of 1 diisopropyl phosphate/protease molecule, suggesting that hydrolysis requires a single serine nucleophile . The residue modified by diisopropyl fluorophosphate was identified as Ser132 by modification with 3H-labeled diisopropyl fluorophosphate, peptide mapping and Edman degradation . This residue and the region in which it is found is highly conserved among the herpes virus proteases . These data demonstrates that HCMV protease is a serine protease and that Ser132 is the active-site nucleophile. Carcinogenesis, 1994 Dec, 15(12), 2805 - 9 Tissue-specific mutational spectra of 2-amino-3,4-dimethylimidazo{4,5-f}quinoline in the liver and bone marrow of lacI transgenic mice; Ushijima T et al.; 2-Amino-3,4-dimethylimidazo{4,5-f}quinoline (MeIQ) is a food-borne heterocyclic amine, so clarification of its mutational spectrum is important for evaluation of its carcinogenic risk to humans . The mutational spectrum of MeIQ was investigated in the liver and bone marrow of transgenic mice carrying the lacI gene . By PCR--single-strand conformation polymorphism analysis and sequencing of the lacI gene, 81 and 61 mutations were identified in 80 and 59 mutants obtained from the liver and bone marrow respectively of three transgenic mice given food containing 300 p.p.m . MeIQ . In the liver, G-->T transversions were the most frequent, accounting for 46% of the total mutations, followed by G-->A transitions (25%) . In the bone marrow, four types of mutations, G-->T transversions, G-->A transitions, complex mutations and one base deletions, each accounted for 21-23% of the total mutations . Of the total mutations, 10% were found at nucleotide 92 in the liver and at nucleotide 222 in the bone marrow . Analysis of 27 and 13 mutants from the liver and bone marrow respectively of control mice showed frequent G-->A transitions at CpG sites . These findings suggest a tissue-specific mechanism of mutagenesis. Biochem J, 1994 Dec 1, 304 ( Pt 2), 477 - 83 Mechanisms of endotoxin-induced haem oxygenase mRNA accumulation in mouse liver: synergism by glutathione depletion and protection by N-acetylcysteine; Rizzardini M et al.; In in vitro systems haem oxygenase-1 (HO-1) mRNA increases after exposure to agents causing oxidative stress . We lowered cellular antioxidant defence systems in vivo by giving mice increasing doses (0.15 g/kg-1.6 g/kg) of DL-buthionine-(S,R)-sulphoximine (BSO), a specific inhibitor of glutathione synthesis . Maximum glutathione depletion (80%) coincided with maximum hepatic HO-1 mRNA accumulation (about 20 times), whereas with 50% depletion, accumulation was only doubled . It has been suggested that reactive oxygen and nitrogen intermediates are involved in hepatic toxicity of endotoxin (lipopolysaccharide, LPS); LPS even at low doses {0.1 mg/kg, intraperitoneally (i.p.)} induces HO-1 mRNA about 25-fold after 1 h . Hepatic glutathione depletion (respectively 40% and 80%) after a low (0.3 g/kg) or a high (1.6 g/kg) BSO dose, resulted in potentiation of the HO-1 mRNA accumulation induced by LPS (0.1 mg/kg, i.p.) . In the absence of BSO, N-acetylcysteine (NAC) (1 g/kg orally) reduced LPS-induced HO-1 mRNA accumulation to one fourth . Under the same experimental conditions S-adenosylmethionine (SAM) was not effective . NAC also reduced HO-1 mRNA accumulation when administered to mice in which glutathione was depleted and its synthesis blocked by BSO (1.6 g/kg) . Thus reactive oxygen intermediates are likely mediators of LPS-induced HO-1 mRNA accumulation, and glutathione content appears to be one of the factors regulating this accumulation in the liver . Our findings are compatible with the theory that HO-1 induction might have a protective function in vivo when defence mechanisms against oxidants are challenged. J Gen Virol, 1994 Dec, 75 ( Pt 12), 3503 - 10 Identification of a second protein encoded by influenza C virus RNA segment 6; Hongo S et al.; Influenza C virus matrix protein (M1) is encoded by a spliced mRNA derived from RNA segment 6 . Unspliced mRNA from this RNA segment, which has not been previously identified, can potentially encode a polypeptide that contains an additional 132 amino acids on the carboxy terminus of the M1 protein . Here the nucleotide sequences of RNA segment 6 of four influenza C strains, isolated in Japan between 1964 and 1988, were compared with the previously determined sequence of C/Ann Arbor/1/50 . The results indicated that the deduced amino acid sequence of the carboxy-terminal 132 amino acid domain is conserved fairly well although it is more divergent than the M1 protein sequence . Examination of RNA segment 6-specific mRNAs also showed that unspliced mRNA is present, although in small quantities (approximately 13% of spliced mRNA), in influenza C virus-infected cells . To search for a polypeptide encoded by the unspliced mRNA, the extra carboxy-terminal domain was expressed in Escherichia coli as the glutathione S-transferase fusion protein, and rabbit immune serum was raised against the purified fusion protein . Immunoprecipitation experiments with this antiserum revealed that a previously unrecognized protein of apparent M(r) approximately 18,000, designated CM2, is synthesized in influenza C virus-infected cells. J Gen Virol, 1994 Dec, 75 ( Pt 12), 3375 - 83 Analysis of type-restricted and cross-reactive epitopes on virus-like particles of human papillomavirus type 33 and in infected tissues using monoclonal antibodies to the major capsid protein; Sapp M et al.; A panel of six monoclonal antibodies recognizing at least three different antigenic regions has been raised against the L1 major capsid protein of human papillomavirus type 33 (HPV-33), which is associated with cervical carcinoma . The antigenic sites defined by these antibodies have been mapped and classified as type-restricted or broadly cross-reactive using bacterially expressed L1 fusion proteins of a variety of HPV types . Conformational and linear epitopes have been distinguished using native and denatured virus-like particles . HPV infection of genital lesions has been analysed using both monoclonal antibodies and DNA amplification by PCR . The antibodies obtained should be useful to probe the structure of HPV capsids and to develop a general assay for the detection and classification of productive HPV infections. J Gen Virol, 1994 Dec, 75 ( Pt 12), 3365 - 74 Adenovirus protein-protein interactions: hexon and protein VI; Matthews DA et al.; A variety of mastadenoviruses were denatured, their polypeptides separated by electrophoresis on SDS-polyacrylamide gels and transferred to nitrocellulose . The immobilized polypeptides were washed, incubated with buffers containing hexons from human adenoviruses (Ad) types 2, 5 and 12 and the location of bound hexons was detected with anti-hexon antibodies . It was found that hexons from any of the three human adenovirus types bound to protein VI from all the mastadenoviruses examined . Furthermore we found that hexon-VI binding was significantly greater than the interaction between hexon and the precursor to VI, pVI . This binding was susceptible to detergents and to changes in pH or salt concentration . A rabbit polyclonal antibody was raised against a recombinant protein derived from the middle third of pVI from Ad2 and was used to quantify the difference in binding and to demonstrate the presence of a single intermediate (designated iVI) in the processing of pVI to VI . The affinity between iVI and hexon was considerably greater in our assay than that of pVI but was less than that between hexon and VI . A complementary binding of recombinant iVI to immobilized hexons was also demonstrated . This latter interaction, however, was only observed when hexon preparations were not boiled prior to electrophoresis, substantiating the proposition that the recognition motif on the hexon was conformation-dependent . These results are discussed in the context of understanding further the molecular basis of protein-protein interactions between the structural proteins of adenoviruses and the factors involved in virion maturation. J Gen Virol, 1994 Dec, 75 ( Pt 12), 3353 - 63 Polydnavirus of the parasitic wasp Chelonus inanitus (Braconidae): characterization, genome organization and time point of replication; Albrecht U et al.; Ultrastructural analysis of the polydnavirus of the braconid wasp Chelonus inanitus revealed that virions consist of one cylindrical nucleocapsid enveloped by a single unit membrane . Nucleocapsids have a constant diameter of 33.7 +/- 1.4 nm and a variable length of between 8 and 46 nm . Spreading of viral DNA showed that the genome consists of circular dsDNA molecules of variable sizes and measurement of the contour lengths indicated sizes of between 7 and 31 kbp . When virions were exposed to osmotic shock conditions to release the DNA, only one circular molecule was released per particle suggesting that the various DNA molecules are singly encapsidated in this bracovirus . The viral genome was seen to consist of at least 10 different segments and the aggregate genome size is in the order of 200 kbp . By partial digestion of viral DNA with HindIII or EcoRI in the presence of ethidium bromide and subsequent ligation with HindIII-cut pSP65 or EcoRI-cut pSP64 and transfection into Escherichia coli, libraries of 103 HindIII and 23 EcoRI clones were obtained . Southern blots revealed that complete and unrearranged segments were cloned with this approach, and restriction maps for five segments were obtained . Part of a 16.8 kbp segment was sequenced, found to be AT-rich (73%) and to contain six copies of a 17 bp repeated sequence . The development of the female reproductive tract in the course of pupal-adult development of the wasp was investigated and seen to be strictly correlated with the pigmentation pattern . By the use of a semiquantitative PCR, replication of viral DNA was observed to initiate at a specific stage of pupal-adult development. J Gen Virol, 1994 Dec, 75 ( Pt 12), 3327 - 35 Identification of structural domains within the large subunit of herpes simplex virus ribonucleotide reductase; Conner J et al.; The large subunit (R1) of herpes simplex virus (HSV) ribonucleotide reductase is a bifunctional protein consisting of a unique N-terminal protein kinase domain and a ribonucleotide reductase domain . Previous studies showed that the two functional domains are linked by a protease sensitive site . Here we provide evidence for two subdomains, of 30K and 53K, within the reductase domain . The two fragments, which were produced by limited proteolysis and were resistant to further degradation, remained tightly associated in a complex containing two molecules of each . They were capable of binding the R2 subunit of HSV ribonucleotide reductase with approximately the same affinity as the intact protein but the complex did not complement the small subunit (R2) to give an active enzyme . At low concentrations (0.4 micrograms/ml) of trypsin or V8 protease, cleavage between the subdomains was prevented by the presence of the N-terminal protein kinase domain . At higher protease concentrations (1 micrograms/ml) the N-terminal domain is extensively proteolysed and the 30K and 53K domains were generated . Identical results were obtained using purified R1 isolated from infected cell extracts or following expression in Escherichia coli . The origin of the two domains was investigated by N-terminal sequencing of the 53K fragment and by examining their reactivity with a panel of R1-specific monoclonal antibodies which we isolated and epitope mapped for that purpose . The trypsin cleavage site was found to lie between arginine 575 and asparagine 576, and proteolysis in this region was not prevented by the presence of R2 or the nonapeptide YAGAVVNDL . We propose that the ribonucleotide reductase region of HSV R1 exists in a two domain structure, and that the interdomain linking region is protected by the unique N terminus. J Infect Dis, 1994 Dec, 170(6), 1610 - 3 Isolates of Escherichia coli O44:H18 of diverse origin are enteroaggregative; Smith HR et al.; One hundred thirteen strains of Escherichia coli O44:H18 isolated in several countries over 25 years were examined for adhesion to tissue culture cells and for hybridization with DNA probes . Fifty-nine strains were from sporadic cases of infection; 54 were from 12 outbreaks . Of the 113 strains, 85 showed aggregative adhesion to HEp-2 cells; 36 were from sporadic cases and 49 were from 9 outbreaks . All adhesive strains hybridized with the probe for enteroaggregative E . coli (EAggEC) and 1 nonadhesive strain was also positive . However, 80 of the 86 EAggEC probe-positive strains also hybridized with the probe for diffusely adherent E . coli (DAEC) derived from the daaC gene of strain F1845 . The EAggEC and DAEC probes hybridized to different fragments of a large plasmid in the O44:H18 strains. J Infect Dis, 1994 Dec, 170(6), 1498 - 507 Intestinal kinetics and dynamics of Escherichia coli heat-stabile enterotoxin in suckling mice; Waldman SA et al.; The heat-stabile enterotoxin produced by Escherichia coli (ST) induces diarrhea by binding to receptors on intestinal cells, activating guanylyl cyclase, and increasing cyclic GMP . High- and low-affinity receptors for this toxin have been identified previously . ST induces intestinal secretion in suckling mice in picomole doses, suggesting a role for high-affinity receptors in this process . The present studies examine the relative roles of high- and low-affinity receptors in this process . The time course of changes in free ST concentrations in suckling mouse intestine was determined after intragastric inoculation . Also, binding characteristics of high- and low-affinity receptors and their coupling to guanylyl cyclase were defined in intestinal membranes from suckling mice . Intestinal concentrations of toxin and receptor binding characteristics empirically determined were used in a dynamic model correlating fractional occupancy of high- and low-affinity receptors with intestinal secretion to estimate their relative contributions to ST-induced diarrhea. Genes Dev, 1994 Dec 1, 8(23), 2928 - 38 Regulation of rDNA transcription in chloroplasts: promoter exclusion by constitutive repression; Iratni R et al.; Spinach chloroplasts contain two types of RNA polymerases . One is multimeric and Escherichia coli-like . The other one is not E . coli-like and might represent a monomeric enzyme of 110 kD . The quantitative relation of the two polymerases changes during plant development . This raises the question, how are plastid genes transcribed that contain E . coli-like and non-E . coli-like promoter elements during developmental phases when both enzymes are present? Transcription of the spinach plastid rrn operon promoter is initiated at three sites: P1, PC, and P2 . P1 and P2 are preceded by E . coli-like promoter elements that are recognized by E . coli RNA polymerase in vitro . However, in vivo, transcription starts exclusively at PC . We analyzed different promoter constructions using in vitro transcription and gel mobility-shift studies to understand why P1 and P2 are not used in vivo . Our results suggest that the sequence-specific DNA-binding factor CDF2 functions as a repressor for transcription initiation of the E . coli-like enzyme at P1 and P2 . We propose a mechanism of constitutive repression to keep the rrn operon in all developmental phases under the transcriptional control of the non-E . coli-like RNA polymerase. Genes Dev, 1994 Dec 1, 8(23), 2857 - 67 The WD repeats of Tup1 interact with the homeo domain protein alpha 2; Komachi K et al.; Tup1 and Ssn6 transcriptionally repress a wide variety of genes in yeast but do not appear to bind DNA . We provide genetic and biochemical evidence that the DNA-binding protein alpha 2, a regulator of cell-type-specific genes, recruits the Tup1/Ssn6 repressor by directly interacting with Tup1 . This interaction is mediated by a region of Tup1 containing seven copies of the WD repeat, a 40 amino acid motif of unknown function found in many other proteins . We have found that a single WD repeat will interact with alpha 2, indicating that the WD repeat is a protein-protein interaction domain . Furthermore, a fragment of Tup1 containing primarily WD repeats provides at least partial repression in the absence of Ssn6, suggesting that the repeats also mediate interaction between Tup1 and other components of the repression machinery. EMBO J, 1994 Dec 1, 13(23), 5779 - 85 The mechanism of the stringent control of lambda plasmid DNA replication; Szalewska-Palasz A et al.; Lambda plasmid DNA replication is inhibited in amino acid-starved wild type Escherichia coli strains (stringent response) but not in amino acid-starved relA mutants (relaxed response) . This replication is perpetuated by the replication complex containing the lambda O protein (which is protected from proteases by other elements of the complex) and inherited by one of two daughter copies after a replication round . Since a fraction of stable lambda O protein was observed in relA- and relA+ strains, and negative regulation by the lambda Cro repressor does not seem to be important in the stringent or relaxed response of lambda plasmid replication to amino acid starvation, the inhibition of lambda plasmid replication in amino acid-starved wild type strains was investigated . lambda plasmids were unable to replicate in amino acid-starved relA- bacteria treated with rifampicin . Moreover, transcription from pR, which produces mRNA for replication protein synthesis and serves as transcriptional activation of ori lambda, was significantly decreased during the stringent response as well as in non-starved cells containing increased levels of ppGpp . However, it was little or totally not affected by the relaxed response . The replacement of pR with plac (which is known to be uninhibited by ppGpp) in a lambda plasmid resulted in its DNA replication during relaxed and stringent responses as well as during overproduction of ppGpp in unstarved bacteria . We conclude that ppGpp-mediated inhibition of transcriptional activation of ori lambda is responsible for inhibition of lambda plasmid DNA replication in amino acid-starved wild type strains.(ABSTRACT TRUNCATED AT 250 WORDS) EMBO J, 1994 Dec 1, 13(23), 5679 - 88 Regulation of mammalian spliceosome assembly by a protein phosphorylation mechanism; Mermoud JE et al.; Splicing of mRNA precursors (pre-mRNA) is preceded by assembly of the pre-mRNA with small nuclear ribonucleoprotein particles (snRNPs) and protein factors to form a splicesome . Here we show that stimulating Ser/Thr-specific protein dephosphorylation selectively inhibits an early step during mammalian spliceosome assembly . Treatment of HeLa nuclear splicing extracts with human protein phosphatase 1 (PP1) expressed in Escherichia coli, or PP1 purified from rabbit skeletal muscle, prevents pre-spliceosome E complex (early complex) formation and stable binding of U2 and U4/U6.U5 snRNPs to the pre-mRNA . PP1 does not inhibit splicing catalysis if added after spliceosome assembly has taken place . Addition of purified SR protein splicing factors restores spliceosome formation and splicing to PP1-inhibited extracts, consistent with SR proteins being targets regulated by phosphorylation . These data extend earlier observations showing that splicing catalysis, but not spliceosome assembly, is blocked by inhibiting protein phosphatases . It therefore appears that pre-mRNA splicing, in common with other biological processes, can be regulated both positively and negatively by reversible protein phosphorylation. EMBO J, 1994 Dec 1, 13(23), 5647 - 55 Modulation of tyrT promoter activity by template supercoiling in vivo; Bowater RP et al.; We have found that initiation of RNA synthesis at the tyrT promoter of Escherichia coli can be stimulated on a plasmid by a factor of 4-6 by elevation of DNA supercoiling in vivo . Increased unconstrained plasmid supercoiling was achieved by inserting the tyrT promoter upstream of the tetracycline resistance gene tetA and transformation into a topA host . Under these conditions there is marked oversupercoiling of the plasmid DNA and we have shown previously that this can lead to increased promoter activity in the topological domain created . A critical element in the formation of this domain is the coupled transcription, translation and membrane insertion of tetA and we show that all of these events are important in the stimulation of tyrT promoter activity . The magnitude of the stimulation is in reasonable agreement with that measured in vitro as a function of plasmid supercoiling, if the unconstrained level of negative supercoiling in vivo is increased from a basal level of -sigma approximately 0.022 to -sigma approximately -0.052 by transcription-induced supercoiling . The induced supercoiling is very efficient, indicating that the tyrT promoter is itself contributing to the steady-state level despite a total lack of membrane anchorage for the tyrT transcription unit . This study provides a new example of the topological coupling of promoters. Arch Surg, 1994 Dec, 129(12), 1296 - 300 Endotoxin-induced nitric oxide production in pulmonary artery endothelial cells is regulated by cytokines; Cendan JC et al.; BACKGROUND: L-Arginine is the sole precursor of nitric oxide (NO) . Bacterial lipopolysaccharide (endotoxin) (LPS) stimulates carrier-mediated L-arginine transport in porcine pulmonary artery endothelial cells (PAECs) through an autocrine pathway that involves interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor alpha (TNF-alpha) . OBJECTIVES: To determine if Escherichia coli LPS stimulates NO synthesis in PAECs and, if so, if LPS stimulation of NO production is also mediated by autocrine secretion of IL-1 alpha and TNF-alpha . DESIGN: Monolayers of PAECs were incubated with various concentrations of LPS, recombinant human TNF-alpha, or IL-1 alpha, and total nitrate-nitrite accumulation was measured at different time points with the Greiss reagent following cadmium reduction . Release of TNF-alpha and IL-1 alpha release by LPS-stimulated PAECs were measured using the WEHI (for TNF-alpha) and A375.S2 (for IL-1 alpha) bioassays . The PAECs were then incubated with saline solution or LPS in the presence or absence of either a polyclonal antibody to human TNF or IL-1 receptor antagonist, and nitrate-nitrite accumulation was measured at 48 hours . RESULTS: Production of NO by PAECs was increased 230% by LPS (1 microgram/mL), 350% by TNF-alpha (1000 U/mL), and 240% by IL-1 alpha (1000 U/mL) (P < .05 vs control) . The LPS-stimulated NO production was inhibited by IL-1 receptor antagonist (100 micrograms/mL) or antibody to TNF (10 micrograms/mL) to control levels (P < .05 vs LPS; difference vs saline solution was not significant) . The LPS-stimulated TNF-alpha secretion by PAECs and TNF-alpha activity were maximal at 6 hours (400 +/- 42 pg/mL) . The IL-1 alpha activity was not detectable in LPS-stimulated PAECs by the A375.S2 bioassay . CONCLUSIONS: Endotoxin, TNF-alpha, and IL-1 alpha stimulated NO synthesis in PAECs . Endotoxin-stimulated NO synthesis through an autocrine pathway involving the cytokines TNF-alpha and IL-1 alpha. Arch Surg, 1994 Dec, 129(12), 1290 - 4; discussion 1294-5 Endotoxin stimulates interleukin-6 production in intestinal epithelial cells . A synergistic effect with prostaglandin E2; Meyer TA et al.; OBJECTIVE: To test the hypothesis that endotoxin stimulates the release of interleukin-6 (IL-6) from intestinal epithelial cells and that this effect of endotoxin is regulated by prostaglandin E2 (PGE2) . DESIGN: A rat intestinal crypt cell line, IEC-6, was cultured in the presence of lipopolysaccharide (LPS), 0.1 to 1.0 microgram/mL, and/or PGE2, 1 mumol/L . In other experiments, indomethacin, 20 mumol/L, was added to LPS-treated cells to block the effects of prostaglandins . Control wells contained medium alone . Levels of IL-6 were determined by the B9 murine hybridoma bioassay . Polymerase chain reaction was performed on RNA from control and LPS-treated cells to examine IL-6 message . RESULTS: Lipopolysaccharide and PGE2 induced IL-6 release from IEC-6 cells in a dose- and time-dependent fashion, and the substances interacted synergistically . Addition of indomethacin blunted the effect of endotoxin on IL-6 production, consistent with a stimulatory role of PGE2 . Polymerase chain reaction demonstrated increased IL-6 messenger RNA in endotoxin-treated cells . CONCLUSIONS: Endotoxin and PGE2 stimulate IL-6 production in IEC-6 cells and interact synergistically . The endotoxin-stimulated IL-6 release may be regulated at the transcriptional level. Arch Surg, 1994 Dec, 129(12), 1263 - 9; discussion 1270 Macrophage endotoxin tolerance . Tumor necrosis factor and interleukin-1 regulation by lipopolysaccharide pretreatment; Seatter SC et al.; OBJECTIVE: To correlate cytokine gene expression with the release of protein product by murine peritoneal macrophages rendered tolerant by sequential endotoxin stimulation in vitro . DESIGN: In vitro investigation of the regulation of endotoxin-stimulated cytokine production following endotoxin pretreatment using cytokine bioassays, polymerase chain reaction, and Northern blot analyses . SETTING: In vitro cell culture model of sequential endotoxin stimulation of murine macrophages . INTERVENTIONS: Macrophages were pretreated with 0 or 100 ng/mL of lipopolysaccharide (LPS1) for 24 hours and then stimulated with 0 or 100 ng/mL of LPS (LPS2) for 4 or 24 hours . After stimulation, supernatant tumor necrosis factor (TNF) and interleukin-1 (IL-1) levels were measured by bioassay . Total RNA was extracted and messenger RNA (mRNA) corresponding to TNF and IL-1 was amplified by reverse transcription-polymerase chain reaction or analyzed by Northern blot . RESULTS: Endotoxin pretreatment resulted in the augmentation of IL-1 (mean +/- SD, 78 +/- 9 vs 596 +/- 42 pg/mL, P < .01) and the inhibition of TNF (274 +/- 63 vs 61 +/- 3 pg/mL, P < .01) release 4 hours after stimulation with 100 ng/mL of LPS2 . A similar pattern of cytokine release was observed 24 hours after LPS2 stimulation . Pretreatment produced an increased IL-1 message in response to 100 ng/mL of LPS2 . The TNF message was detectable in all groups receiving LPS2 alone, but the highest levels of TNF mRNA were seen in LPS1-pretreated cells stimulated with LPS2 . CONCLUSIONS: Endotoxin pretreatment produced increased IL-1 message that paralleled the augmentation of IL-1 protein, whereas abundant TNF message was present even though TNF protein release was significantly inhibited . In this model of in vitro endotoxin tolerance, pretreatment initiates divergent pathways of cytokine regulation. Arch Surg, 1994 Dec, 129(12), 1233 - 9 The pulmonary effect of nitric oxide synthase inhibition following endotoxemia in a swine model; Ogura H et al.; OBJECTIVE: To evaluate the pulmonary effect of treatment with N-nitro-L-arginine methyl ester (NAME) with and without inhaled nitric oxide (NO) in a swine model of endotoxemia . DESIGN: Randomized controlled trial . SETTING: Laboratory . INTERVENTIONS: Following a 20-minute intravenous infusion of Escherichia coli lipopolysaccharide (LPS) (200 micrograms/kg), animals were resuscitated with saline solution (1 mL/kg per minute) and observed for 3 hours while mechanically ventilated (fraction of inspired oxygen {FIO2}, 0.6; tidal volume, 12 mL/kg; positive end-expiratory pressure, 5 cm H2O) . Group 1 (LPS, n = 6) received no additional treatment; group 2 (NAME, n = 5) received NAME (3 mg/kg per hour) for the last 2 hours; group 3 (NO, n = 6) received NAME (3 mg/kg per hour) and inhaled NO (40 ppm) for the last 2 hours; and group 4 (control, n = 5) received only saline solution without LPS . MAIN OUTCOME MEASURES: Cardiopulmonary variables and blood gases were measured serially . The multiple inert gas elimination technique was performed at 3 hours . The wet-to-dry lung weight ratio was measured following necropsy . RESULTS: Administration of LPS resulted in pulmonary arterial hypertension, pulmonary edema, and hypoxemia with increased ventilation perfusion ratio mismatching . None of these changes were attenuated by NAME treatment alone but all were significantly improved by the simultaneous administration of inhaled NO . CONCLUSIONS: Systemic NO synthase inhibition failed to restore hypoxic pulmonary vasoconstriction following LPS administration . The deleterious effects of endotoxemia on pulmonary function can be improved by inhaled NO but not by systemic inhibition of NO synthase. Arch Biochem Biophys, 1994 Dec, 315(2), 527 - 32 Expression of a plant sesquiterpene cyclase gene in Escherichia coli; Back K et al.; 5-Epi-aristolochene synthase is a sesquiterpene cyclase activity found in pathogen-challenged tobacco cells, but not in nonchallenged tissues, and appears to be encoded by a complex gene family . As a prerequisite to assessing the functional significance of these multiple genes, bacterial expression systems were examined for their capacity to express a tobacco sesquiterpene cyclase cDNA . Insertion of full-length 5-epi-aristolochene synthase cDNA into two commonly used expression vectors, pET-11d and pGBT-T19, resulted in high level expression of the cyclase activity . The highest level of expression occurred 3 h after induction with low concentrations (0.1-0.5 mM) of IPTG, incubation at 27 degrees C instead of 37 degrees C, and in the bacterial host strain BL21(DE3) . Under these conditions, the cyclase protein constituted 5 to 8% of the soluble and 35% of the total Escherichia coli proteins . Enzyme reaction products of the native tobacco and recombinant enzyme were identical, based on argentation-thin layer chromatography . Deletion mutants of the cyclase gene corresponding to the amino and carboxy termini of the enzyme were prepared . The cyclase proteins resulting from bacterial expression of these mutant constructs were found largely in the insoluble protein fraction and no soluble enzyme activity was detected. Arch Biochem Biophys, 1994 Dec, 315(2), 445 - 53 Cloning and expression of rabbit and human brain tryptophan hydroxylase cDNA in Escherichia coli; Tipper JP et al.; Rabbit and human brain tryptophan hydroxylase were cloned and expressed in Escherichia coli . Each of the respective cDNAs, including the complete coding sequence of tryptophan hydroxylase, was obtained by reverse transcription of rabbit or human brain mRNA and subcloned into the expression vector pET-3C . The expressed rabbit brain tryptophan hydroxylase activity, measured in the presence of tetrahydrobiopterin, represents approximately a 50-fold enhancement in yield (units/g tissue (wet wt) over that of a rabbit brain extract . Likewise, the level of expressed human brain tryptophan hydroxylase is approximately 57 times the average yield previously reported for a human brain homogenate and approximately 10-times the activity of homogenates of human raphe nucleus . The rabbit brain and pineal-derived tryptophan hydroxylase sequences varied by disparities in six amino acid residues (99% identity) . The human carcinoid and brain peptide sequences varied by disparities in 18 amino acid residues (96% identity) . Several properties of both expressed enzymes were studied and compared with those of native tryptophan hydroxylases. Int Arch Allergy Immunol, 1994 Dec, 105(4), 381 - 5 Immune response against ovalbumin in rats colonized with an ovalbumin-producing Escherichia coli and the influence of feeding ovalbumin; Dahlman A et al.; The influence of feeding ovalbumin (OA) on the development of IgE/IgG antibodies and delayed-type hypersensitivity (DTH) against OA was studied in rats colonized from birth with an Escherichia coli genetically manipulated to produce OA . At 21 days of age, colonized pups and pups with a normal intestinal flora were weaned onto either an OA-containing or a conventional diet without OA . At 2 months of age the colonized rats showed an increased DTH reaction to OA, but they did not have any anti-OA antibodies in serum . The rats were then immunized intracutaneously with OA in Freund's complete adjuvant . After immunization the colonized rats fed the conventional diet had a significantly higher DTH reaction to OA and significantly higher serum levels of IgE anti-OA antibodies than the uncolonized rats on the same diet . The colonized rats eating the OA-containing diet showed a 73% decrease in the DTH reaction to OA and also significantly lower levels of IgE and IgG antibodies against OA compared with the colonized rats fed conventional diet . The dams colonized as adults by the OA-producing E . coli developed IgE anti-lipopolysaccharide antibodies in serum while the pups colonized via the dams at birth did not . Neonatal colonization with an E . coli strain producing OA resulted in increased DTH reactivity against OA and priming for secondary IgE anti-OA response . Feeding the animals an OA-containing diet from weaning abrogated this intestinally induced hypersensitivity and rendered the animals orally tolerant to OA. Radiat Res, 1994 Dec, 140(3), 356 - 65 Survey of cellular radiosensitivity parameters; Katz R et al.; A model of the formation of particle tracks in emulsion has been extended through the use of biological target theory to formulate a theory of the response of biological cells and molecules of biological importance to irradiation with energetic heavy ions . For this purpose the response to gamma rays is represented by the single-hit, multitarget model with parameters m and D0, while additional parameters kappa (or a0) and sigma 0 are required to represent the size of internal cellular targets and the effective cross-sectional area of the cell nucleus, respectively, for heavy-ion bombardments . For one-or-more-hit detectors, only the first three of these parameters are required and m = 1 . For cells m is typically 2 or more . The model is developed from the concept that response to secondary electrons follows the same functional form for gamma rays and for the gamma rays surrounding an ion's path . Originally applied to dry enzymes and viruses in 1967, the model of the one-hit detector has been extended to emulsions, to other physical and chemical detectors, to single- and double-strand breaks in DNA in EO buffer and to three E . coli strains . The two-hit response has been observed for "track core" effects in radiation chemistry, for supralinearity in thermoluminescent dosimeters and for desensitized nuclear emulsions, where hit numbers up to 6 have been observed . In its extension to biological cells, additional concepts are required relating to the character of the track, namely the grain-count and track-width regimes, and to the ability of multitarget systems to acquire damage from intertrack delta rays (called gamma kill) as well as from intratrack delta rays (called ion kill) . The model has been applied to some 40 sets of radiobiological data obtained from gamma, track-segment heavy-ion and neutron irradiations . Here we elaborate on the meaning of these concepts, tabulate the cellular parameters, and display their systematic behavior and the relationships among them . In particular the parameter kappa, which serves to determine the location in Z*2/beta 2 of the maximum value of the RBE, shows little variation among cell types, while D0, which describes the response to gamma rays and sigma 0, which appears to indicate the target size, varies over many orders of magnitude. J Virol, 1994 Dec, 68(12), 8433 - 6 Molecular dissection of influenza virus nucleoprotein: deletion mapping of the RNA binding domain; Kobayashi M et al.; Influenza virus nucleoprotein (NP) is associated with the genome RNA, forming ribonucleoprotein cores . To identify the amino acid sequence involved in RNA binding, we performed Northwestern blot analysis with a set of N- and C-terminal deletion mutants of NP produced in Escherichia coli . The RNA binding region has been mapped between amino acid residues 91 and 188, a stretch of residues that contains a sequence that is not only highly conserved among NPs from A-, B-, and C-type influenza viruses but also similar to the RNA binding domain of a plant virus movement protein. J Virol, 1994 Dec, 68(12), 8396 - 400 Orientation-specific cis complementation by bulge- and loop-mutated human immunodeficiency virus type 1 TAR RNAs; Braddock M et al.; Tat activates human immunodeficiency type 1 gene expression by binding to TAR RNA . TAR comprises a partially base paired stem and hexanucleotide loop with a tripyrimidine bulge in the upper stem . In vitro, Tat binds to the bulge and upper stem, with no requirement for the loop . However, in vivo, loop sequences are critical for activation, implying that a loop binding cellular factor may be involved in the activation pathway . Given that activation appears to be a two-component system comprising a Tat-bulge interaction and a cellular factor-loop interaction, we considered that it might be possible to spatially separate the two components and retain activation . We have constructed a series of double TAR elements comprising various combinations of mutated TAR structures . Defective TARs with nucleotide substitutions in either the bulge or the loop complemented each other to give wild-type activation . However, the complementation was orientation specific, requiring the intact Tat binding site to reside on the 5'-proximal TAR . These data suggest that provided the wild-type orientation of the bulge and loop elements is retained, there is no requirement for them to coexist on the same TAR structure. J Virol, 1994 Dec, 68(12), 8254 - 64 Inhibition of human immunodeficiency virus type 1 in human T cells by a potent Rev response element decoy consisting of the 13-nucleotide minimal Rev-binding domain; Lee SW et al.; Intracellular immunization is an anti-viral gene therapy strategy based on the introduction of DNA templates into cells to stably express genetic elements which inhibit viral gene expression and replication . We have recently developed an intracellular immunization strategy for human immunodeficiency virus (HIV) infection that uses RNA decoys . RNA decoys are short RNA oligonucleotides corresponding to the HIV trans activation response element (TAR) or Rev response element (RRE) sequences, which function by inhibiting the binding of the HIV regulatory proteins Tat and Rev to the authentic HIV RNA TAR and RRE regions, respectively . In this report we describe the characterization of potent RRE decoys containing the minimal 13-nucleotide primary Rev binding domain of the RRE . Using an improved tRNA cassette to express high levels of RRE transcripts in CEM cells, we found that this new generation of minimal RRE decoys were more potent inhibitors of HIV in isolated cell lines than previously described TAR or RRE decoys . CEM cells expressing RRE decoys exhibited diminished Rev function in cotransfection assays, confirming the specificity of inhibition of HIV by RRE decoys and indicating that the 13-nucleotide minimal Rev binding domain defined by using in vitro binding studies also binds Rev in vivo . Significant differences in the degree of HIV inhibition between individual CEM cell lines transduced with RRE decoy vectors which were not due to sequence alterations in the tRNA-RRE DNA template, differences in RRE decoy expression level, or endogenous variations in the resistance of CEM clonal cell lines to HIV were observed . In order to evaluate the efficacy of RRE decoys in a more realistic fashion than by comparison of individual clonal cell lines, polyclonal populations of transduced CEM cells were infected with HIV . By using a novel flow cytometric method for quantitating intracellular p24 expression, one version of the RRE decoys tested in this study was found to be capable of durably protecting polyclonal populations of CEM cells from HIV. J Virol, 1994 Dec, 68(12), 8239 - 53 Characterization of Marek's disease virus insertion and deletion mutants that lack US1 (ICP22 homolog), US10, and/or US2 and neighboring short-component open reading frames; Parcells MS et al.; We report the characterization of Marek's disease virus (MDV) strains having mutations in various genes that map to the unique short (US) region of the viral genome . A deletion mutant (GA delta 4.8lac) lacks 4.8 kbp of US region DNA, the deleted segment having been replaced by the lacZ gene of Escherichia coli . This deletion results in the loss of the MDV-encoded US1, US10, and US2 homologs of herpes simplex virus type 1, as well as three putative MDV-specific genes, Sorf1, Sorf2, and Sorf3 . Two mutants containing lacZ insertions in the US1 and US10 genes have been constructed, and we have previously reported a US2lac insertion mutant (J . L . Cantello, A . S . Anderson, A . Francesconi, and R . W . Morgan, J . Virol . 65:1584-1588, 1991) . The isolation of these mutants indicates that the relevant genes are not required for growth of MDV in chicken embryo fibroblasts . The mutants had early growth kinetics indistinguishable from those of their parent viruses; however, 5 to 7 days after being plated, the US1 insertion mutant (US1lac) and the GA delta 4.8lac deletion mutant showed a 5- to 10-fold decrease in virus growth . This decrease in virus accumulation correlated with a 30 to 50% decrease in plaquing efficiency when these viruses were plated onto established versus fresh chicken embryo fibroblast monolayers compared with a 10 to 15% decrease seen for the parent viruses and for the US10lac or US2lac insertion mutants . Finally, GA delta 4.8lac could be reisolated from chickens, indicating that the deleted genes are not required for the infection of chickens following intra-abdominal inoculation of an attenuated serotype 1 MDV. J Virol, 1994 Dec, 68(12), 7993 - 8000 Effect of core protein phosphorylation by protein kinase C on encapsidation of RNA within core particles of hepatitis B virus; Kann M et al.; Phosphorylation of core particles derived either from hepatitis B viruses or from livers of hepatitis B-infected individuals has been long recognized, but the nature and function of the phosphorylating enzyme remained unknown . By immunoblotting with a monoclonal antibody, we have now detected protein kinase C within the liver-derived core particles . To study the significance of the encapsidated protein kinase C for the viral life cycle, we established an in vitro assembly system consisting of Escherichia coli-expressed core protein, protein kinase C, and in vitro-synthesized hepatitis B virus RNA . Phosphorylation of the core protein led to a reduced RNA encapsidation capacity of the core particles . Furthermore, RNA and protein kinase C competed for their target sequence, which is the carboxy-terminal arginine-rich domain of the core protein . This finding implies that phosphorylation of the nucleic acid binding site in the core protein occurs within the particles after encapsidation of protein kinase C, pregenomic RNA, and viral polymerase at a later step during viral genome maturation. J Virol, 1994 Dec, 68(12), 7869 - 78 Juxtaposition of two viral DNA ends in a bimolecular disintegration reaction mediated by multimers of human immunodeficiency virus type 1 or murine leukemia virus integrase; Chow SA et al.; Integration of retroviral DNA involves a coordinated joining of the two ends of a viral DNA molecule into precisely spaced sites on target DNA . In this study, we designed an assay that requires two separate oligonucleotides to be brought together via interactions between integrase promoters to form a "crossbones" substrate that mimics the integration intermediate . The crossbones substrate contains two viral DNA ends, each joined to one strand of target DNA and separated by a defined length of target DNA . We showed that purified integrases of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV) could mediate a concerted strand cleavage-ligation between the two half-substrates at one or both viral DNA joining sites (trans disintegration) . Another major product, termed fold-back, resulted from an intramolecular attack on the phosphodiester bond at the viral-target DNA junction by the 3'-OH group of the same DNA molecule (cis disintegration) . The activity of integrase on the crossbones substrate depended on the presence of viral DNA sequences . For trans disintegration, the optimal length of target DNA between the viral DNA joining sites of the crossbones substrate corresponded to the spacing between the staggered joints formed on two opposite strands of target DNA during retroviral DNA integration in vivo . The activity of integrases on crossbones did not require complementary base pairing between the two half-substrates, indicating that the half-substrates were juxtaposed solely through protein-DNA interactions . The crossbones assay, therefore, measures the ability of integrase to juxtapose two viral DNA ends, an activity which heretofore has been difficult to detect by using purified integrase in conventional assays . Certain mutant integrases that were otherwise inactive with the crossbones substrate could complement one another, indicating that no single protomer in the integrase multimer requires a complete set of functional domains either for catalytic activity or for juxtaposition of the two viral DNA ends by the active multimer. J Med Microbiol, 1994 Dec, 41(6), 399 - 404 Detection of enteroadherent Escherichia coli associated with diarrhoea in Italy; Morelli R et al.; One hundred and sixty-eight isolates of Escherichia coli obtained in Italy from 112 children with diarrhoea and 56 age-matched controls were examined by the HEp-2 cell adhesion assay . Sixteen strains showed localised adherence (LA), 29 showed diffuse adherence (DA) and eight strains showed aggregative adherence (AA) . No adhesion pattern was significantly associated with disease . Strains that showed LA or AA were further characterised by serotyping, fluorescent actin staining (FAS) test and hybridisation with the EPEC adherence factor (EAF), E . coli attaching and effacing (eae) and enteroaggregative (EAgg) DNA probes . Strains that showed poor LA were FAS-negative, did not belong to EPEC serotypes and did not hybridise with EPEC probes . Conversely, the two strains that showed a good LA pattern belonged to serotype O128:H2, were FAS positive and hybridised with the eae probe . No isolate hybridised with the EAF probe . Only three of the eight strains with the AA pattern hybridised with the EAgg probe . Probe positivity correlated with the ability to produce clumps at the surface of the liquid culture and to agglutinate rat erythrocytes . In two of these EAgg probe-positive strains, electronmicroscopy revealed the presence of fibrillar bundles which seem to mediate bacterial aggregation. J Med Microbiol, 1994 Dec, 41(6), 393 - 8 Plasmid-coded DNA fragment developed as a specific gene probe for the identification of enteroaggregative Escherichia coli; Debroy C et al.; Enteroaggregative Escherichia coli (EAggEC) is a recently discovered diarrhoeal pathogen implicated as a cause of persistent diarrhoea in children . EAggEC strains exhibit a characteristic pattern of adherence when incubated with HEp-2 cells . Because of the difficulty in identifying this group of bacteria, the epidemiological significance of this pathogen as a diarrhoeal agent has not been fully realised . A gene probe was developed from the 60-MDa plasmid associated with EAggEC strains that encodes the genes for adherence and fimbriae . The sensitivity of the gene probe was 93% and the specificity 98% for detecting EAggEC isolates and is potentially useful for diagnostic and epidemiological studies. J Neurochem, 1994 Dec, 63(6), 2324 - 35 Identification of endogenously phosphorylated KSP sites in the high-molecular-weight rat neurofilament protein; Elhanany E et al.; The high-molecular-weight neurofilament protein (NF-H) is highly phosphorylated in vivo, with estimates as high as 16-51 mol of Pi/mol of protein . Most of the phosphorylation sites are thought to be located on Ser residues in multiple KSP repeats, in the carboxy-terminal tail region of the molecule . Because the extent and site-specific patterns of tail domain phosphorylation are believed to modulate neurofilament structure and function, it becomes essential to identify the endogenous sites of phosphorylation . In this study, we have used selective proteolytic cleavage procedures, Pi determinations, microsequencing, and mass-spectral analysis to determine the endogenously phosphorylated sites in the NF-H tail isolated from rat spinal cord . Twenty Ser residues in NF-H carboxy-terminal tail were analyzed; nine of these, all located in KSP repeats, were phosphorylated . No detectable phosphorylation could be identified in any of the 11 "non-KSP" Ser residues that were examined . KSPXKX, KSPXXX, and KSPXXK motifs were found to be phosphorylated . In addition, a 27-kDa KSP-rich domain, containing 43 virtually uninterrupted KSPXXX repeats, was isolated from the tail domain and found to contain between 30 and 35 mol of Pi/mol of protein . This domain appeared to be highly resistant to endoproteinase Glu-C digestion, although it contains a large number of glutamate residues . It could be proteolyzed, however, after dephosphorylation . This suggests that phosphorylation of the tail domain may contribute to neurofilament stability in vivo . A neuronal-derived protein kinase that specifically phosphorylates only KSPXKX motifs in neurofilaments has been reported . The presence of extensively phosphorylated KSPXXX repeats in NF-H in vivo suggests the existence of yet another, unidentified kinase(s) with specificity for KSPXXX motifs. J Exp Med, 1994 Dec 1, 180(6), 2147 - 53 A genetically detoxified derivative of heat-labile Escherichia coli enterotoxin induces neutralizing antibodies against the A subunit; Pizza M et al.; Escherichia coli enterotoxin (LT) and the homologous cholera toxin (CT) are A-B toxins that cause travelers' diarrhea and cholera, respectively . So far, experimental live and killed vaccines against these diseases have been developed using only the nontoxic B portion of these toxins . The enzymatically active A subunit has not been used because it is responsible for the toxicity and it is reported to induce a negligible titer of toxin neutralizing antibodies . We used