J Biol Chem, 1994 Oct 7, 269(40), 24736 - 41 The 50-kDa glucose 6-phosphate-sensitive hexokinase of Schistosoma mansoni; Tielens AG et al.; Hexokinase has been purified from adult Schistosoma mansoni worms and the activity shown to be associated with a single protein species having an M(r) about 50,000 . This protein is recognized on Western blots probed with antisera against rat Type I hexokinase or against a recombinant S . mansoni hexokinase that had been expressed in Escherichia coli using a previously cloned cDNA . An 18-residue N-terminal sequence determined for the purified S . mansoni hexokinase is identical to that deduced from the nucleotide sequence of the cDNA, consistent with the view that the cloned cDNA encodes the hexokinase characterized in the present study . The S . mansoni enzyme has a relatively low Km (approximately 60 microM) for glucose and is sensitive to inhibition (competitive versus ATP, Ki approximately 50 microM) by its product, glucose 6-phosphate (Glc-6-P) . With these kinetic properties and 50 kDa molecular mass, S . mansoni hexokinase resembles the ancestral hexokinase predicted to have given rise, by gene duplication and fusion, to the present day 100-kDa Glc-6-P-sensitive mammalian hexokinases . The schistosomal hexokinase represents the first 50-kDa Glc-6-P-sensitive hexokinase whose sequence has been obtained . The schistosomal hexokinase does not bind to mitochondria, consistent with its lack of a hydrophobic segment at the N terminus which is required for binding of the mammalian Type I and II isoenzymes to mitochondria . The marked Crabtree effect exhibited by S . mansoni cercariae may be at least partly attributed to the expression of rather high levels of a hexokinase having a high affinity for glucose but only a moderate sensitivity to product inhibition by Glc-6-P. J Biol Chem, 1994 Oct 7, 269(40), 24608 - 14 Glutamic acid 86 is important for positioning the 80's loop and arginine 54 at the active site of Escherichia coli aspartate transcarbamoylase and for the structural stabilization of the C1-C2 interface; Baker DP et al.; Glu-86, which interacts with the side chain of Arg-54 across the C1-C2 interface of Escherichia coli aspartate transcarbamoylase, tethers the end of the flexible 80's loop, which moves into the active site during the T to R transition . In order to determine whether this interaction is important for the correct positioning of the 80's loop and Arg-54 at the active site and also for the structural stabilization of the enzyme, a mutant version was created in which Glu-86 was replaced by Gln (Glu-86-->Gln) . Although the mutant holoenzyme exhibits almost normal homotropic cooperativity, both the holoenzyme and catalytic subunit exhibit substantial reductions in activity and affinity for aspartate and carbamyl phosphate . Furthermore, the mutant holoenzyme shows a marked decrease in the activation by ATP and by the bisubstrate analog N-(phosphonoacetyl)-L-aspartate, reduced inhibition by CTP, as well as reduced affinities for these ligands . Results from molecular dynamics simulations of the Glu-86-->Gln and Glu-86-->Ala enzymes suggest that the positions of the 80's loop and Arg-54 are significantly perturbed by the introduction of these mutations . Taken together, these results indicate that the interaction between Glu-86 and Arg-54 is important for the formation of the high affinity, high activity form of the enzyme by stabilizing the correct position of the 80's loop and Arg-54 at the active site . Heat inactivation experiments also demonstrated that Glu-86 plays a significant role in the structural stabilization of the C1-C2 interface, since the temperature required for loss of half of the activity of the Glu-86-->Gln catalytic subunit is reduced by 5 degrees C relative to the wild-type catalytic subunit. J Biol Chem, 1994 Oct 7, 269(40), 24596 - 601 Carboxyl-terminal truncation of recombinant factor XIII A-chains . Characterization of minimum structural requirement for transglutaminase activity; Lai TS et al.; A series of truncation mutants lacking 218, 229, 250, and 269 amino acid residues from the carboxyl terminus of blood coagulation factor XIII A-chains (FXIII A), designated as delta K513, delta A502, delta Y481, and delta K462, respectively, were expressed in Escherichia coli to define the minimum structure required for transglutaminase activity . delta K513 and delta A502 displayed a 3.8-4.7-fold reduction in the Kcat with no change in the Km for the glutamine substrate and a 2-fold increase in the Km of the primary amine substrate . There was no detectable transglutaminase activity for either thrombin-activated delta Y481 or delta K462 . The rate of ammonia release of thrombin-activated delta K513 and delta A502 was reduced 6- and 4-fold, respectively, whereas ammonia release was not detected for the delta Y481 and delta 462 mutants . The Kact for calcium ions of the delta K513 mutant was similar to recombinant FXIIIa, whereas, it was increased by approximately 3-fold for the delta A502 mutant . The rate of fibrin gamma-chain dimer formation for the delta K513 and delta A502 mutants was reduced by approximately 19-fold . delta K462 did not bind to fibrin, while all of the other thrombin-cleaved mutants were bound . In conclusion, these results documented that the carboxyl-terminal calcium binding domain (Asp468-Glu495) was important for FXIIIa to adopt the correct conformation to ensure that efficient catalysis occurred. J Biol Chem, 1994 Oct 7, 269(40), 24586 - 95 Repair of spontaneously deamidated HPr phosphocarrier protein catalyzed by the L-isoaspartate-(D-aspartate) O-methyltransferase; Brennan TV et al.; The non-enzymatic deamidation at residues Asn-12 and Asn-38 of Escherichia coli phosphocarrier protein, HPr, and the repair of the resulting L-isoaspartyl (or beta-aspartyl) derivatives, HPr-1 and HPr-2, by recombinant human S-adenosylmethionine-dependent L-isoaspartate-(D-aspartate) O-methyltransferase (EC 2.1.1.77) were investigated . HPr is a component of the bacterial phosphoenolpyruvate:sugar phosphotransferase system that is involved in the concomitant translocation and phosphorylation of many hexose sugars . The major products of the deamidation reaction, L-isoaspartyl (or beta-aspartyl) residues at positions 12 and 38, were found to be substrates for the L-isoaspartate-(D-aspartate) O-methyltransferase, an enzyme active on a wide variety of peptides and proteins containing these abnormal residues . This enzyme has been shown to catalyze the first step in a process that can convert L-isoaspartyl residues in peptides to normal L-aspartyl residues . The affinity of a recombinant human methyltransferase for HPr-1, a form deamidated at Asn-38, was relatively poor (Km = 3.6 mM), while a greater affinity was found for HPr-2, a form deamidated at both Asn-12 and Asn-38 (Km = 197 microM) . When HPr-2 was incubated with S-adenosylmethionine and the methyltransferase, the bulk of the L-isoaspartyl residues at position 12 was converted to L-aspartyl residues . The major-by-product was the D-isoaspartyl form . The conversion of L-isoaspartyl residues at position 38 to L-aspartyl residues was less complete, reflecting the lower affinity of the methyltransferase for this site . The phosphohydrolysis activity of the repaired form was found to be midway between the form containing only L-aspartyl residues at positions 12 and 38 and the deamidated HPr-2 form. Nature, 1994 Oct 6, 371(6497), 528 - 31 Regulation of retinoid signalling by receptor polarity and allosteric control of ligand binding; Kurokawa R et al.; Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) regulate transcription by binding to response elements in target genes that generally consist of two direct repeat half-sites of consensus sequence AGGTCA (ref . 1) . RAR/RXR heterodimers activate transcription in response to all-trans or 9-cis retinoic acid by binding to direct repeats spaced by five base pairs (DR5 elements), such that RAR occupies the downstream half-site . RXR homodimers activate transcription in response to 9-cis retinoic acid by binding to direct repeats spaced by one base pair (DR1 elements) . Although RXR/RAR heterodimers bind to DR1 elements with higher affinity than RXR homodimers, in most contexts they are unable to activate transcription in response to either all-trans or 9-cis retinoic acid . As a result, RARs inhibit RXR-dependent transcription from these sites . We report that the switching of the RAR from an activator to an inhibitor of retinoid-dependent transcription requires that it be bound to the upstream half-site of DR1 elements and that it allosterically block the binding of ligand to the RXR. Biochim Biophys Acta, 1994 Oct 6, 1214(3), 323 - 32 The effects of phosphoglycerides on Escherichia coli cardiolipin synthase; Ragolia L et al.; Escherichia coli cardiolipin synthase catalyzes the conversion of two phosphatidylglycerol molecules to cardiolipin and glycerol . This enzyme was amplified in strain BL21(DE3) bearing recombinant plasmid pLR3, which was itself constructed by inserting the cls gene downstream from a T7 RNA promoter . Membranes from BL21(DE3)/pLR3 have over 1200 times more cardiolipin synthase activity than do comparable membranes from wild type cells . The enzyme was purified to homogeneity by extraction with Triton X-114 and chromatography on DEAE-cellulose . The purified enzyme migrated as a single band (46 kDa) on SDS-PAGE . This, along with SDS-PAGE analysis of induced protein, supports the notion that cls is the structural gene for cardiolipin synthase . Cardiolipin synthase activity was determined in a mixed micelle assay in which phosphatidyl{2-3H}glycerol was the substrate . The enzyme is inhibited by the product of the reaction, cardiolipin, and by phosphatidate . However, it is not inhibited by two other anionic phosphoglycerides, phosphatidylinositol and bis-phosphatidate . Phosphatidylethanolamine partially offsets inhibition by cardiolipin but not by phosphatidate . Magnesium chloride has the opposite effect . Cardiolipin inhibition of cardiolipin synthase probably plays an important role in regulating cardiolipin synthesis in E . coli. Biochemistry, 1994 Oct 4, 33(39), 11987 - 92 Mechanistic aspects of tagetitoxin inhibition of RNA polymerase from Escherichia coli; Mathews DE et al.; Tagetitoxin inhibits RNA synthesis directed by bacterial RNA polymerase, and the current study explores several mechanistic aspects of this inhibition . Tagetitoxin inhibition of in vitro RNA synthesis directed by Escherichia coli RNA polymerase is independent of the template DNA concentration . The toxin can block Escherichia coli RNA polymerase during elongation of a nascent RNA chain . In abortive initiation assays, the rate of dinucleotide formation is inhibited by tagetitoxin when initiated with ATP or CpA but not when AMP is used to initiate . Formation of longer oligonucleotides is inhibited by the toxin regardless of the initiating nucleotide . These abortive initiation studies indicate that tagetitoxin does not affect nucleotide substrate binding or phosphodiester bond formation and suggest that the toxin may interfere with a subsequent step . It is suggested that tagetitoxin affects the stability of nascent oligonucleotide binding and/or the translocation of the catalytic center with respect to the 3'-OH of nascent oligonucleotides. Biochemistry, 1994 Oct 4, 33(39), 11971 - 9 Melting of a DNA helix terminus within the active site of a DNA polymerase; Hochstrasser RA et al.; Accurate synthesis of DNA by polymerase is due in part to the selective removal of misincorporated nucleotides by a 3'-5' exonuclease activity (proofreading) . Proofreading by an exonuclease domain containing a single-stranded DNA binding site may involve local melting of a duplex DNA substrate . Here we use time-resolved fluorescence spectroscopy to analyze the local melting of a DNA duplex terminus induced by the Klenow fragment of DNA polymerase I . Four oligodeoxynucleotide primer/templates were prepared, each containing the fluorescent adenine analog 2-aminopurine (A*) at the primer 3' terminus, and one of the common DNA bases opposite the A* residue . Fluorescence decays of the duplex DNAs and the single primer oligonucleotide were jointly analyzed using global analysis procedures . Four lifetime components were resolved in the duplex DNAs, representing distinct conformational states of the terminal A* residue: paired A* bases, partially stacked A* bases, and extended A* bases . The variation of the apparent fraction of paired A* bases with temperature was in accord with optical melting data, and the extent of base pairing observed in each duplex was consistent with the base-pairing preferences of A* established in other studies . These results establish that the fluorescence decay characteristics of A* can be used to examine base-pairing interactions at a DNA duplex terminus . Since the fluorescence of A* can be observed without interference from protein amino acid residues, unlike existing methods for monitoring DNA melting transitions, this method was used to examine the extent to which Klenow fragment could induce fraying at each duplex terminus.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Oct 4, 33(39), 11927 - 34 Reactions catalyzed by 5-aminoimidazole ribonucleotide carboxylases from Escherichia coli and Gallus gallus: a case for divergent catalytic mechanisms; Firestine SM et al.; A comparative investigation of the substrate requirements for the enzyme 5-aminoimidazole ribonucleotide (AIR) carboxylase from E . coli and G . gallus has been conducted using in vivo and in vitro studies . In Escherichia coli, two enzymes PurK and PurE are required for the transformation of AIR to 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) . The Gallus gallus PurCE is a bifunctional enzyme containing AIR carboxylase and 4-{(N-succinylamino)carbonyl}-5-aminoimidazole ribonucleotide (SAICAR) synthetase . The E . coli PurE and the C-terminal domain of the G . gallus PurCE protein maintain a significant degree of amino acid sequence identity and also share CAIR as a product of their enzymatic activities . The substrate requirements of AIR carboxylases from E . coli and G . gallus have been compared by a series of in vitro experiments . The carbamic acid, N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) is a substrate for the E . coli PurE (Mueller et al., 1994) but not for the G . gallus AIR carboxylase . In contrast, AIR and CO2 are substrates for the G . gallus AIR carboxylase . The recognition properties of the two proteins were also compared using inhibition studies with 4-nitro-5- aminoimidazole ribonucleotide (NAIR) . NAIR is a tight-binding inhibitor of the G . gallus AIR carboxylase (K(i) = 0.34 nM) but only a steady-state inhibitor (K(i) = 0.5 microM) of the E . coli PurE . These data suggest significant differences in the transition states for the reactions catalyzed by these two evolutionarily related enzymes.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Oct 4, 33(39), 11917 - 26 Carboxylases in de novo purine biosynthesis . Characterization of the Gallus gallus bifunctional enzyme; Firestine SM et al.; Two successive steps in de novo purine biosynthesis are catalyzed by the enzymes 5-aminoimidazole ribonucleotide (AIR) carboxylase and 4-{(N-succinylamino)carbonyl}-5-aminoimidazole ribonucleotide (SAICAR) synthetase . Amino acid sequence alignments of the proteins from various sources suggested that several unusual differences exist within the structure and function of these enzymes . In vertebrates, a bifunctional enzyme (PurCE) catalyzes successive carboxylation and aspartylation steps of AIR to form SAICAR . This is in contrast to the three proteins, PurK, PurE, and PurC, from Escherichia coli which have recently been shown to require 2 equiv of ATP for the AIR to SAICAR conversion in the presence of physiological HCO3- concentrations (Meyer et al., 1992) . A comparative study of these proteins has been initiated using a high-production, heterologous expression system for the Gallus gallus AIR carboxylase-SAICAR synthetase and yields purified enzyme following a two-step procedure . Selective assays have been developed for all the enzymatic activities of the bifunctional protein . The G . gallus AIR carboxylase has no ATP dependence and displays a Km for HCO3- that is 10-fold lower than that for the related PurE protein from E . coli, supporting the hypothesis that the two enzymes require different substrates . No common cofactors or metals are required for catalysis . Each catalytic activity has been shown to be independent by selective inactivation of SAICAR synthetase with the affinity agent 5'-{4-(fluorosulfonyl)benzoyl}-adenosine (FSBA) and inhibition of AIR carboxylase with a tight-binding inhibitor 4-nitro-5-aminoimidazole ribonucleotide (NAIR) . The native protein aggregates, and limited proteolysis indicates that the global structure of the protein involves two independent folding domains, each containing a different catalytic site. Biochemistry, 1994 Oct 4, 33(39), 11868 - 74 Properties of tyrosine 766-->serine mutant of Escherichia coli DNA polymerase I: template-specific effects; Desai SD et al.; In order to determine the role of Tyr 766 of Escherichia coli DNA polymerase I in the catalysis of DNA synthesis, we investigated the properties of a Tyr 766-->Ser (Y766S) mutant of the Klenow fragment of E . coli DNA polymerase I . We found that the rates of incorporation of only dTTP but not the other dNTP substrates were affected in the reactions catalyzed by the mutant enzyme, when homopolymeric template-primers were used . The mutant enzyme exhibited a reduced rate of synthesis only with poly(rA)- or poly(dA)-directed reactions . Examination of the ability of the mutant and the wild-type enzymes to bind to dGTP and dTTP, as judged by UV-mediated cross-linking, indicated nearly identical binding efficiencies of both nucleotides . However, the ability of the mutant enzyme to bind to poly(rA).(dT)15 and poly(dA).(dT)15 was found to be significantly reduced as compared to the binding to heteropolymeric DNA . In order to further define the nature of template-mediated restriction on the catalytic activity of the mutant enzyme, its ability to copy DNA templates containing a stretch of AAAAA and ACACA sequences was compared . The results show that DNA synthesis catalyzed by the mutant enzyme is significantly retarded when it encounters the AAAAA region of the template but not the ACACA region . Product analysis of the reaction directed by the two template-primers showed that the mutant enzyme stalls/terminates synthesis upon encountering an AAAAA sequence in the template.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Oct 4, 33(39), 11767 - 75 Stability of myoglobin: a model for the folding of heme proteins; Hargrove MS et al.; Factors governing the stability of sperm whale, pig, and human metmyoglobin were examined by (1) measuring guanidinium chloride induced unfolding of apoglobins containing 22 replacements at positions 29(B10), 43(CD1), 64(E7), 68(E11), and 107(G8), (2) determining the rates of hemin loss from the recombinant holoproteins, and (3) estimating constitutive expression levels of the corresponding genes in Escherichia coli TB-1 cells . The denaturant titrations were analyzed in terms of a two-step unfolding reaction, N(native apoprotein)-->I(intermediate)-->U(unfolded), in which the intermediate is visualized by an increase in tryptophan fluorescence emission . Two key conclusions were reached . First, high rates of hemin loss are not necessarily correlated with unstable globin structures and vice versa . In general, both rates of hemin loss and the equilibrium constants for apoprotein unfolding must be determined in order to understand the overall stability of heme proteins and to predict the efficiency of their expression . Second, polar residues in the distal pocket cause marked decreases in the overall stability of apomyoglobin . Removal of hemin from V68N and L29N sperm whale myoglobins produces the molten globular I state at pH 7, 25 degrees C, without addition of denaturant . In contrast, the H64L and H64F mutations produce apoproteins which are 10-30 times more stable than wild-type apoglobin . The latter results show that protein stability is sacrificed in order to have the distal histidine (H64) present to increase O2 affinity and inhibit autooxidation. Biochemistry, 1994 Oct 4, 33(39), 11692 - 8 Preparation and kinetic characterization of a fusion protein of yeast mitochondrial citrate synthase and malate dehydrogenase; Lindbladh C et al.; We have expressed the DNA of the fusion of CS1 to MDH1 in Escherichia coli gltA- . The fusion protein (CS1/MDH1) is the C-terminus of CS1 linked in-frame to the N-terminus of MDH1 with a short linker of glycyl-seryl-glycyl . The fusion protein produced was isolated and purified . Gel filtration studies indicated that CS1/MDH1 had a M(r) of approximately 170,000 . Western blotting analysis with SDS gel indicated a M(r) of approximately 90,000-95,000 (theoretical M(r) = 87,000) . This is the expected M(r) for the fusion protein subunit . The kinetics of CS1 and MDH1 activities of the fusion protein were compared to those of the free enzymes . In addition, the effect of AAT reaction, as a competitor for the intermediate OAA of the coupled MDH-CS reaction, was examined . It was observed that AAT was a less effective competitor for OAA when the CS1/MDH1 fusion protein is used than when the separate enzymes are employed . In addition, the transient time for the coupled reaction sequence was less for the fusion protein than for the free enzymes. FEBS Lett, 1994 Oct 3, 352(3), 318 - 22 Purification and spectroscopic characterization of a recombinant amino-terminal polypeptide fragment of mouse epithelial cadherin; Tong KI et al.; Cadherins are a family of Ca(2+)-dependent cell adhesion molecules containing four extracellular tandem repeats each of 110 amino acids . The most amino-terminal repeat is believed to confer the specificity of cell adhesion . A polypeptide containing the amino-terminal repeat of mouse epithelial cadherin has been over-expressed in E . coli and purified to homogeneity . This polypeptide binds Ca2+ with a dissociation constant of 1.6 x 10(-4) M . CD and NMR experiments indicate that the polypeptide adopts a predominantly beta-sheet conformation and that binding of Ca2+ induces only small conformational changes. EMBO J, 1994 Oct 3, 13(19), 4695 - 703 Parental strand recognition of the DNA replication origin by the outer membrane in Escherichia coli; Herrick J et al.; The outer membrane of Escherichia coli binds the origin of DNA replication (oriC) only when it is hemimethylated . We report here the results of a footprinting analysis with the outer membrane which demonstrate that its interaction with oriC occurs mainly at the left moiety of the minimal oriC, where 10 out of 11 Dam methylation sites are concentrated . Two regions, flanking the Integration Host Factor (IHF) sites, are preferentially recognized at the minimum membrane concentration at which oriC plasmid replication is inhibited in vitro . We have identified the putative proteins involved in hemimethylated oriC binding and cloned one of the corresponding genes (hobH) . The purified LacZ-HobH fusion protein specifically binds oriC DNA at the same preferential sites as the membrane . A mutant of the hobH gene reveals partial asynchronous initiation of DNA replication. EMBO J, 1994 Oct 3, 13(19), 4670 - 5 A model of maltodextrin transport through the sugar-specific porin, LamB, based on deletion analysis; Klebba PE et al.; LamB facilitates the uptake of maltose and maltodextrins across the bacterial outer membrane and acts as a general porin for small molecules . Using directed deletion mutagenesis we removed several regions of the LamB polypeptide and identified a polypeptide loop that both constricts the maltoporin channel and binds maltodextrins . In conjunction with a second sugar binding site that we identified at the rim of the channel, these data clarify, for the first time, the mechanism of transport through a substrate-specific porin . Furthermore, unlike the transverse loops of general porins, which originate from a central location in their primary structure, the loop that regulates LamB permeability originates from a C-terminal site . Thus LamB represents a second distinct class of porins in the bacterial outer membrane that is differently organized and separately evolved from OmpF-type, general porins. EMBO J, 1994 Oct 3, 13(19), 4662 - 9 Translocation of N-terminal tails across the plasma membrane; Cao G et al.; Previously we have shown that the first hydrophobic domain of leader peptidase (lep) can function to translocate a short N-terminal 18 residue antigenic peptide from the phage Pf3 coat protein across the plasma membrane of Escherichia coli . We have now examined the mechanism of insertion of N-terminal periplasmic tails and have defined the features needed to translocate these regions . We find that short tails of up to 38 residues are efficiently translocated in a SecA- and SecY-independent manner while longer tails are very poorly inserted . Efficient translocation of a 138 residue tail is restored and is Sec-dependent by the addition of a leader sequence to the N-terminus of the protein . We also find that while there is no amphiphilic helix requirement for N-terminal translocation, there is a charge requirement that is needed within the tail; an arginine and lysine residue can inhibit or completely block translocation when introduced into the tail region . Intriguingly, the membrane potential is required for insertion of a 38 residue tail but not for a 23 residue tail. EMBO J, 1994 Oct 3, 13(19), 4558 - 67 CRP induces the repositioning of MalT at the Escherichia coli malKp promoter primarily through DNA bending; Richet E et al.; Activation of transcription initiation at the Escherichia coli malKp promoter requires the repositioning of MalT, the primary activator, from a set of non-productive sites to a set of productive sites, which is staggered by 3 bp . Occupation of the latter relies on the formation of a higher order structure involving distal MalT sites and the binding of CRP (cAMP receptor protein) to three sites located in the intervening region . We show here that one can successfully replace all of the CRP sites by the binding site of another DNA-bending protein, integration host factor, or by a sequence-directed bend without altering the process of malKp activation . This observation indicates that CRP action at malKp does not involve critical interactions with MalT and that CRP promotes MalT repositioning primarily through DNA bending . This structural role of CRP differs markedly from its role in the activation of the lac promoter. EMBO J, 1994 Oct 3, 13(19), 4549 - 57 The functional subunit of a dimeric transcription activator protein depends on promoter architecture; Zhou Y et al.; In Class I CAP-dependent promoters, the DNA site for CAP is located upstream of the DNA site for RNA polymerase . In Class II CAP-dependent promoters, the DNA site for CAP overlaps the DNA site for RNA polymerase, replacing the -35 site . We have used an 'oriented heterodimers' approach to identify the functional subunit of CAP at two Class I promoters having different distances between the DNA sites for CAP and RNA polymerase {CC(-61.5) and CC(-72.5)} and at one Class II promoter {CC(-41.5)} . Our results indicate that transcription activation at Class I promoters, irrespective of the distance between the DNA sites for CAP and RNA polymerase, requires the activating region of the promoter-proximal subunit of CAP . In striking contrast, our results indicate that transcription activation at Class II promoters requires the activating region of the promoter-distal subunit of CAP. EMBO J, 1994 Oct 3, 13(19), 4536 - 48 Architectural elements in nucleoprotein complexes: interchangeability of specific and non-specific DNA binding proteins; Segall AM et al.; Integration host factor (IHF) is required in lambda site-specific recombination to deform the DNA substrates into conformations active for recombination . HU, a homolog of IHF, can also deform DNA but binds without any apparent sequence specificity . We demonstrate that HU can replace IHF by cooperating with the recombinase protein, integrase, to generate a stable and specific complex with electrophoretic mobility and biochemical activity very close to the complex formed by IHF and integrase . The eukaryotic HMG1 and HMG2 proteins differ entirely in structure from HU but they also bind DNA non-specifically and induce or stabilize deformed DNA . We show that the eukaryotic HMG1 and HMG2 proteins cooperate with integrase at least as well as does HU to make a defined structure . We also find that the eukaryotic core histone dimer H2A-H2B can replace IHF, suggesting that the histone dimer is functional outside the context of a nucleosome . HU and the HMG proteins not only contribute to the formation of stable complexes, but they can at least partially replace IHF for the integrative and excisive recombination reactions . These results, together with our analysis of nucleoprotein complexes made with damaged recombination sites, lead us to conclude that the cooperation between HU and integrase does not depend on protein-protein contacts . Rather, cooperation is manifested through building of higher order structures and depends on the capacity of the non-specific DNA binding proteins to bend DNA . While all these non-specific binding proteins appear to fulfil the same bending function, they do so with different efficiencies . This probably reflects subtle structural differences between the assembled complexes. Virology, 1994 Oct, 204(1), 420 - 4 The protein p30, encoded at the gag-pro junction of mouse mammary tumor virus, is a dUTPase fused with a nucleocapsid protein; Bergman AC et al.; A ribosomal frameshift at the gag-pro junction of mouse mammary tumor virus (MMTV) gives rise to the protein p30 . The protein consists of two domains, the zinc-finger-containing nucleocapsid (NC) protein portion with 95 residues and a C-terminal extension comprising 154 residues . The C-terminal domain shows similarity in sequence with the enzyme dUTPase from other sources . In this paper, we demonstrate that p30 is a functional dUTPase . Overproduction of the NC protein in Escherichia coli, using the native frameshift sequence at the gag stop codon, caused a detectable expression of dUTPase ascribed to a low frequency of readthrough . By a 1-base insertion, eliminating the gag stop codon and fusing the gag and pro reading frames, a plasmid, pET-3d-NCDU, directing overexpression of p30, was constructed . The overproduced protein, purified by phosphocellulose chromatography, shows both zinc-binding and dUTPase activity . Analytical gel filtration and sequence homology to other dUTPases suggest a trimeric assembly of p30 subunits . MMTV thus possesses two different forms of the nucleocapsid protein, the ordinary NC protein and the p30, having the NC protein connected to a domain of dUTPase. Virology, 1994 Oct, 204(1), 376 - 90 Vaccinia virus gene A36R encodes a M(r) 43-50 K protein on the surface of extracellular enveloped virus; Parkinson JE et al.; A characterization of vaccinia virus strain Western Reserve (WR) open reading frame (ORF) A36R is described . This ORF is predicted to encode a 221-amino-acid protein (M(r) 25.1 K) with an amino-terminal hydrophobic sequence, seven potential sites for attachment of N-linked carbohydrate, but no carboxy-terminal transmembrane anchor . It is identical in vaccinia strain Copenhagen and shares 94.6% amino acid identity with the corresponding ORF in variola virus strains Harvey, India-1967, and Bangladesh-1975 . RNA analyses detected a 600-nucleotide, early transcript that initiated 10-13 nucleotides upstream of the A36R ORF, and heterogeneously sized late transcripts running across the ORF . A rabbit antiserum raised against an Escherichia coli glutathione S-transferase fusion protein identified M(r) 43-50 K proteins that accumulated late during vaccinia virus infection and fractionated as integral membrane proteins during Triton X-114 partitioning . Similar polypeptides were expressed by vaccinia virus strains Tian Tan, Tashkent, Lister, Wyeth, Copenhagen, and IHD-J and by rabbitpox virus and cowpox virus (strain Brighton Red) . Immunoblot analysis of purified and protease-digested intracellular mature virus (IMV) and extracellular enveloped virus (EEV) showed that the A36R proteins were present on the surface of EEV with type II membrane topology, but were absent from IMV . A WR deletion mutant lacking the A36R ORF (delta A36R) had a small plaque phenotype on all cell lines tested . IMV formation by delta A36R was unaltered but EEV formation was reduced approximately fivefold compared to wild-type (WT) when measured either by density gradient analysis of isotopically labeled virions or by infectivity assays . Thus the loss of the A36R protein from the EEV surface did not reduce EEV specific infectivity in vitro . Despite this, delta A36R showed striking attenuation compared with WT in a murine intranasal model . Finally, a revertant virus in which the A36R ORF was restored showed WT plaque size, EEV formation, and virulence, demonstrating that all the phenotypic differences of delta A36R were attributable to loss of the A36R gene and not to other mutations acquired during its construction. Virology, 1994 Oct, 204(1), 242 - 50 Identification and characterization of Marek's disease virus genes homologous to ICP27 and glycoprotein K of herpes simplex virus-1; Ren D et al.; We have identified two Marek's Disease Virus (MDV) genes within the EcoRI-B fragment of MDV-GA genomic DNA . EcoRI-B is 11.3-kb long and maps within the long unique region of MDV genomes . A 3.2-kb fragment of EcoRI-B has been sequenced and contains two open reading frames, ORF53 and ORF54 . ORF53 (MDV gK), a homolog to HSV-1 glycoprotein K (gK), is 1062 nucleotides long and encodes 354 amino acids (39.5 kDa) . ORF54, designated MDV ICP27, based on significant similarity to HSV-1 ICP27, is 1419 nucleotides long and encodes 473 amino acids (54.5 kDa) . In Northern blot hybridization, two overlapping transcripts (2.9 and 1.6 kb) were detected in MDV-infected DEF cells treated with cycloheximide, suggesting that both transcripts belong to the immediate-early gene family . Amino acid sequence analysis of MDV gK shows some common glycoprotein features, including a putative N-terminal signal sequence, four N-linked glycosylation sites, and four potential transmembrane domains . Comparison of the predicted amino acid sequence of MDV ICP27 with that of HSV-1 ICP27 and VZV ORF4 shows a high degree of conservation within the C-terminus . The C-terminal region of HSV-1 ICP27 has been demonstrated to be critical to its function . A conserved zinc finger metal-binding motif C(442)-X4-C(447)-X13-H(461)-C(467) was also found in the C-terminus of MDV ICP27 . Furthermore, MDV ICP27 upstream sequences contain four copies of consensus sequence elements similar to the tegument protein target sequence TAATGARAT . TrpE-ICP27 fusion protein was expressed in Escherichia coli, and rabbit antisera were generated using purified fusion protein . A 55-kDa protein has been detected in both MDV-GA- and Md11-infected cells using immunoblot analysis. J Virol, 1994 Oct, 68(10), 6487 - 95 Identification and characterization of a 3C-like protease from rabbit hemorrhagic disease virus, a calicivirus; Boniotti B et al.; Expression studies conducted in vitro and in Escherichia coli led to the identification of a protease from rabbit hemorrhagic disease virus (RHDV) . The gene coding for this protease was found to be located in the central part of the genome preceding the putative RNA polymerase gene . It was demonstrated that the protease specifically cuts RHDV polyprotein substrates both in cis and in trans . Site-directed mutagenesis experiments revealed that the RHDV protease closely resembles the 3C proteases of picornaviruses with respect to the amino acids directly involved in the catalytic activity as well as to the role played by histidine as part of the substrate binding pocket. J Virol, 1994 Oct, 68(10), 6411 - 20 The human papillomavirus type 16 E2 transcription factor binds with low cooperativity to two flanking sites and represses the E6 promoter through displacement of Sp1 and TFIID; Tan SH et al.; The E6 promoters of all genital human papillomaviruses have a characteristic alignment of transcription factor binding sites . Activation of the basic transcription complex at the TATA box depends upon a sequence-aberrant Sp1 site . Repression of E6 promoters is achieved by two binding sites for the viral E2 protein positioned between the Sp1 site and the TATA box . We have purified the human papillomavirus type 16 E2 protein after expression in Escherichia coli and studied its binding and repression properties with oligonucleotides representing the homologous promoter sequences . A Kd value of 3 x 10(-10) M indicated binding properties expected for a native protein . We found low cooperativity in the binding of two E2 dimers to flanking sites, both when these sites were separated by 3 nucleotides, as in the natural promoter, and when they were further apart . E2 protein, bound close to the distal Sp1 site, displaced the Sp1 factor even when the aberrant sequence was replaced by a typical Sp1 core recognition site . The high affinity of E2 protein for its binding site even led to Sp1 displacement at concentrations of E2 protein nearly 2 orders of magnitude lower than those of Sp1 . Functional analyses of mutated E6 promoter sequences showed repression by this distal E2 binding site in the complete absence of binding to the proximal E2 binding site . From our findings and observations published by others, we conclude that each of the E2 binding sites in the E6 promoter of genital human papillomaviruses plays a separate role by displacing the transcription factors Sp1 and TFIID. J Virol, 1994 Oct, 68(10), 6243 - 53 Peripheral blood mononuclear phagocytes mediate dissemination of murine cytomegalovirus; Stoddart CA et al.; Cytomegalovirus is transmitted with blood and organs from seropositive individuals, although the particular leukocyte population harboring latent or persistent virus remains poorly characterized . Murine cytomegalovirus, tagged with the Escherichia coli lacZ gene, was used to identify cells in which virus replicates during acute infection of immunocompetent mice . Recombinant murine cytomegaloviruses, RM461, RM460, and RM427, were constructed to express beta-galactosidase under control of the human cytomegalovirus ie1/ie2 promoter/enhancer . The lacZ gene was inserted between the ie2 and sgg1 genes in RM461 and RM460, disrupting a 0.85-kb late transcript that was found to be dispensable for replication in cultured cells as well as for infection of mice . In BALB/c mice, lacZ-tagged and wild-type viruses exhibited a similar 50% lethal dose and all had the capacity to latently infect the spleen . Peripheral blood mononuclear phagocytes were the major infected leukocyte cell type, as demonstrated by the ability of infected cells to adhere to glass and to phagocytize latex beads; however, these cells did not exhibit typical monocyte markers . Plaque assay for virus and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining of frozen sections of organs from infected mice revealed that the major target organs included the spleen, adrenal glands, liver, and salivary glands, although tissues as diverse as brown fat and lungs were also involved . Individual blue-staining cells were readily identified in all infected tissues . These studies identified a mononuclear phagocyte, possibly a macrophage or dendritic cell precursor, as the vehicle of virus dissemination during acute infection, and demonstrate the utility of using lacZ-tagged murine cytomegalovirus for tropism, pathogenesis, and latency studies. Microbiology, 1994 Oct, 140 ( Pt 10), 2543 - 53 Isolation of the Thiobacillus ferrooxidans ntrBC genes using a T . ferrooxidans nifH-lacZ fusion; Kilkenny CA et al.; An agar plating technique was developed in which the activation of expression of a Thiobacillus ferrooxidans nifH-lacZ gene fusion was used to isolate the ntrBC genes from a T . ferrooxidans gene library . An Escherichia coli ntrC mutant containing the nifH-lacZ fusion was transformed and plated on a low-nitrogen medium so that on flooding with ONPG, the production of yellow colonies indicated the presence of the cloned T . ferrooxidans ntrBC genes . A 4.47 kb region from the T . ferrooxidans chromosome was sequenced . Analysis of the sequence revealed that the ntrB and ntrC genes were closely linked to a third ORF of unknown function . Analysis of the 900 bp region upstream of the T . ferrooxidans ntrBC genes and Southern hybridization experiments confirmed that in T . ferrooxidans ATCC 33020, the glnA and ntrBC genes are unlinked . Expression of the T . ferrooxidans nifH-lacZ fusion in E . coli was activated in the presence of the T . ferrooxidans ntrBC genes and regulated by nitrogen. Microbiology, 1994 Oct, 140 ( Pt 10), 2531 - 41 Two genetically-distinct and differentially-regulated aconitases (AcnA and AcnB) in Escherichia coli; Gruer MJ et al.; An acnA mutant of Escherichia coli was constructed by replacing the chromosomal acnA gene by an internally deleted derivative containing a kanR cassette . Southern and Western blotting confirmed that the acnA gene had been replaced by the disrupted gene and that the aconitase A protein was no longer expressed . However, the mutant failed to exhibit the anticipated glutamate auxotrophy and it retained a residual aconitase activity . This activity was due to an analogous unstable enzyme(s) designated aconitase B . Studies on the regulation of aconitase A synthesis using an acnA-lacZ translational fusion showed that the acnA gene resembles other citric acid cycle genes in being subject to CRP-mediated catabolite repression and ArcA-mediated anaerobic repression . In addition to being activated by the SoxRS oxidative stress regulatory system, the acnA gene appeared to be activated by the ferric uptake regulator (Fur) . It was concluded that the acnA gene belongs to at least four global regulatory networks, crp, arcA, fur and soxRS . In contrast, the aconitase B activity decreased after exposure to oxidative stress and was less affected by anaerobiosis . Comparable studies with the fumarase genes (fumA, B and C) indicated that fumA (encoding the unstable aerobic iron-sulphur-containing fumarase) is activated by the ferric uptake regulator (Fur) and fumC (encoding the stable fumarase) is activated by the SoxRS oxidative stress regulatory system. J Gastroenterol, 1994 Oct, 29(5), 631 - 6 Small duct cholangitis induced by N-formyl L-methionine L-leucine L-tyrosine in rats; Yamada S et al.; Primary sclerosing cholangitis (PSC) frequently accompanies inflammatory bowel diseases . In an attempt to increase our understanding of the pathogenesis of PSC, we studied bile duct changes in rats with colitis which had been given N-formyl L-methionine L-leucine L-tyrosine (fMLT) rectally; fMLT is one of the chemotactic peptides produced by Escherichia coli, and is secreted into the bile by hepatocytes after it enters the portal blood . Transrectal administration of fMLT induced a marked inflammation in the portal triad and mild hepatocyte necrosis on the 4th day . The infiltrating leukocytes in the portal tract were mostly mononuclear cells, which densely infiltrated around the bile ducts . These mononuclear cells appeared to attach to bile duct epithelial cells, and they were more numerous in the smaller bile ducts . Electron microscopy revealed that lymphocytes were in direct contact with bile duct lining cells and that some epithelial cells had degenerated or collapsed . These results suggest that this E . coli-derived peptide may induce cholangitis in the small bile duct through cell-mediated mechanisms . Since these pathologic changes resemble those of the bile duct observed in the early stage of PSC, it can be concluded that bacterial chemotactic peptides may play a role in the pathogenesis of small-duct PSC. Int J Exp Pathol, 1994 Oct, 75(5), 363 - 8 Interferon-gamma and polyunsaturated fatty acids increase the binding of lipopolysaccharide to macrophages; Darmani H et al.; We have previously shown that interferon-gamma (IFN-gamma) increases the polyunsaturated fatty acid content of membrane phospholipids in cells that were sensitive to endotoxin . In this study, IFN-gamma was found to stimulate the binding of endotoxin to the murine macrophage cell line J774.2 and the human monocyte cell line U937 . Interferon-gamma-activated J774.2 cells showed a 66% increase in fluoresceine isothiocyanate (FITC) labelled LPS binding (P < 0.0005 vs control cells) and a 49% increase in tritium labelled LPS binding (P < 0.0001 vs control cells) . Interferon-gamma also induced a 35% increase in binding of FITC-LPS in U937 cells (P < 0.0001 vs control cells) . In contrast, pretreatment of J774.2 cells with interferon-beta (IFN-beta) had no effect on binding of FITC-LPS . Preincubation with exogenously supplied polyunsaturated fatty acids, linoleic and arachidonic acids, resulted in increases of 74% and 69% in FITC-LPS binding, respectively (both P < 0.0005 vs control cells) . On the other hand, pretreatment with the saturated fatty acid, palmitic acid, had no effect on FITC-LPS binding . We propose that IFN-gamma-induced changes in the membrane phospholipid fatty acid composition of macrophage-like cells influence the binding of endotoxin. Liver, 1994 Oct, 14(5), 230 - 3 Endotoxin-induced defenestration of the hepatic sinusoidal endothelium: a factor in the pathogenesis of cirrhosis? Dobbs BR, Rogers GW, Xing HY, Fraser R. Defenestration and capillarisation of the hepatic sinusoidal endothelium occurs early in the pathogenesis of cirrhosis, both in patients suffering from alcohol abuse and in animal models . It is possible also that alcohol abuse promotes the absorption of bacterial endotoxins from the gastrointestinal tract . In this study we have investigated the effects of a single intravenous injection of endotoxin on the hepatic sinusoidal endothelium of rats . Seven days after the dose of endotoxin, the porosity of the sinusoidal endothelium was reduced to 40% of that of controls, due to a decrease in both diameter and number of fenestrae . The livers examined 14 days after dosing exhibited normal porosity . We postulate that bacterial endotoxins play a role in the pathogenesis of cirrhosis by modulating the fenestrated sinusoidal endothelium (liver sieve). Plant Cell, 1994 Oct, 6(10), 1495 - 507 The AAPT1 gene of soybean complements a cholinephosphotransferase-deficient mutant of yeast; Dewey RE et al.; Aminoalcoholphosphotransferases (AAPTases) utilize diacylglycerols and cytidine diphosphate (CDP)-aminoalcohols as substrates in the synthesis of the abundant membrane lipids phosphatidylcholine and phosphatidylethanolamine . A soybean cDNA encoding an AAPTase that demonstrates high levels of CDP-choline:sn-1,2-diacylglycerol cholinephosphotransferase activity was isolated by complementation of a yeast strain deficient in this function and was designated AAPT1 . The deduced amino acid sequence of the soybean cDNA showed nearly equal similarity to each of the two characterized AAPTase sequences from yeast, cholinephosphotransferase and ethanolaminephosphotransferase (CDP-ethanolamine:sn-1,2-diacylglycerol ethanolaminephosphotransferase) . Moreover, assays of soybean AAPT1-encoded enzyme activity in yeast microsomal membranes revealed that the addition of CDP-ethanolamine to the reaction inhibited incorporation of 14C-CDP-choline into phosphatidylcholine in a manner very similar to that observed using unlabeled CDP-choline . Although DNA gel blot analysis suggested that AAPT1-like sequences are represented in soybean as a small multigene family, the same AAPT1 isoform isolated from a young leaf cDNA library was also recovered from a developing seed cDNA library . Expression assays in yeast using soybean AAPT1 cDNAs that differed only in length suggested that sequences in the 5'leader of the transcript were responsible for the negative regulation of gene activity in this heterologous system . The inhibition of translation mediated by a short open reading frame located 124 bp upstream of the AAPT1 reading frame is one model proposed for the observed down-regulation of gene activity. Photochem Photobiol, 1994 Oct, 60(4), 295 - 300 Photosensitized formation of 8-hydroxy-2'-deoxyguanosine and DNA strand breakage by a cationic meso-substituted porphyrin; Nicotera TM et al.; Cationic porphyrins, known to have a high affinity for DNA, are useful tools with which to probe a variety of interactions with DNA . In this study we have examined both DNA strand scission and oxidative DNA base damage, measured by 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation, using a photoactivated cis-dicationic porphyrin . The data demonstrated a dose-dependent formation for each type of DNA damage . Inhibition of strand scission and 8-OHdG formation with the singlet oxygen scavenger 1,3-diphenylisobenzofuran and with MgCl2 and no apparent effect by D2O suggests that a singlet oxygen mechanism generated in close proximity to the DNA may be responsible for the damage . However, a nearly complete inhibition of 8-hydroxy-2'-deoxyguanosine formation in 75% D2O and the substantial enhancement of 8-hydroxy-2'-deoxyguanosine formation in a helium atmosphere by photoactivated porphyrin rules out singlet oxygen as a primary mechanism for this process . These data indicate that distinct mechanisms lead to 8-OHdG formation and strand scission activity. FEMS Microbiol Lett, 1994 Oct 1, 122(3), 297 - 302 Binding protein for Escherichia coli heat-stable enterotoxin II in mouse intestinal membrane; Hitotsubashi S et al.; The protein binding Escherichia coli heat-stable enterotoxin II (STII) was isolated from cell membranes of mouse intestine . The binding of 125I-labeled STII to the proteins was inhibited by unlabeled STII, showing that it is specific . Proteins cross-linked with 125I-STII were purified by column chromatography on hydroxyapatite and TSK gel . Analyses of the purified protein by SDS-polyacrylamide gel electrophoresis and gel filtration showed that the molecular mass was 25 kDa. Appl Environ Microbiol, 1994 Oct, 60(10), 3862 - 3 Immobilization of Escherichia coli expressing the lux genes of Xenorhabdus luminescens; Marincs F et al.; The luxCDABE operon of Xenorhabdus luminescens was cloned into pUC18 to make pLITE27 . Expression of the lux genes from the lac promoter resulted in strong constitutive light emission by Escherichia coli DH5 carrying the recombinant lux plasmid, pLITE27 . When strain DH5(pLITE27) was immobilized with sodium alginate-CaCl2, the embedded cells retained their luminescence up to 2 weeks under appropriate storage conditions. Appl Environ Microbiol, 1994 Oct, 60(10), 3724 - 31 Stoichiometric flux balance models quantitatively predict growth and metabolic by-product secretion in wild-type Escherichia coli W3110; Varma A et al.; Flux balance models of metabolism use stoichiometry of metabolic pathways, metabolic demands of growth, and optimality principles to predict metabolic flux distribution and cellular growth under specified environmental conditions . These models have provided a mechanistic interpretation of systemic metabolic physiology, and they are also useful as a quantitative tool for metabolic pathway design . Quantitative predictions of cell growth and metabolic by-product secretion that are experimentally testable can be obtained from these models . In the present report, we used independent measurements to determine the model parameters for the wild-type Escherichia coli strain W3110 . We experimentally determined the maximum oxygen utilization rate (15 mmol of O2 per g {dry weight} per h), the maximum aerobic glucose utilization rate (10.5 mmol of Glc per g {dry weight} per h), the maximum anaerobic glucose utilization rate (18.5 mmol of Glc per g {dry weight} per h), the non-growth-associated maintenance requirements (7.6 mmol of ATP per g {dry weight} per h), and the growth-associated maintenance requirements (13 mmol of ATP per g of biomass) . The flux balance model specified by these parameters was found to quantitatively predict glucose and oxygen uptake rates as well as acetate secretion rates observed in chemostat experiments . We have formulated a predictive algorithm in order to apply the flux balance model to describe unsteady-state growth and by-product secretion in aerobic batch, fed-batch, and anaerobic batch cultures . In aerobic experiments we observed acetate secretion, accumulation in the culture medium, and reutilization from the culture medium . In fed-batch cultures acetate is cometabolized with glucose during the later part of the culture period.(ABSTRACT TRUNCATED AT 250 WORDS) Appl Environ Microbiol, 1994 Oct, 60(10), 3503 - 7 Role of Na+ in transport of Hg2+ and induction of the Tn21 mer operon; Selifonova OV et al.; The effects of sodium ions on the uptake of Hg2+ and induction of the Tn21 mer operon were studied by using Escherichia coli HMS174 harboring the reporter plasmids pRB28 and pOS14 . Plasmid pRB28 carries merRT', and pOS14 carries merRTPC of the mer operon, both cloned upstream of a promoterless luciferase gene cassette in pUCD615 . The bioluminescent response to 1 microM Hg2+ was significantly inhibited in E . coli HMS174(pRB28) in minimal medium supplemented with sodium ions at 10 to 140 mM . After initial acceleration, light emission declined at 50 nM Hg2+ in the presence of Na+ . The mer-lux assay with resting cells carrying pRB28 and 203Hg2+ uptake experiments showed increased induction and enhanced mercury uptake, respectively, in media supplemented with sodium ions . The presence of Na+ facilitated maintenance of bioluminescence in resting HMS174(pRB28) cells induced with 50 nM Hg2+ . External K+ stimulated bioluminescent response in HMS174(pRB28) and HMS174(pOS14) grown in sodium phosphate minimal medium devoid of potassium ions . Sodium ions appear to facilitate mercury transport . We propose that sodium-coupled transport of mercuric ions can be one of the mechanisms for mercury uptake by E . coli and that the Na+ gradient may energize the transport of Hg2+. Appl Environ Microbiol, 1994 Oct, 60(10), 3485 - 90 Production and release of polyphosphate by a genetically engineered strain of Escherichia coli; Hardoyo et al.; A recombinant strain of Escherichia coli MV1184, which contains plasmid-borne genes encoding the phosphate-specific transport (Pst) system and polyphosphate (polyP) kinase, accumulated high levels of Pi and released polyP into the medium . PolyP could be separated from the culture supernatant by DEAE-Toyopearl 650M chromatography and identified by high-resolution 31P nuclear magnetic resonance spectroscopy . Once E . coli recombinants accumulated high levels of polyP, they released polyP concomitantly with Pi uptake . PolyP release did not accompany the decrease in the cell density, indicating that it is not simply a result of cell lysis . PolyP release ceased when Pi became depleted in the medium and resumed upon addition of Pi to the medium . When Pi uptake was inhibited by 0.1 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP), no polyP release was observed . Furthermore, neither Pi uptake nor polyP release occurred when cells were incubated at 4 degrees C . These findings suggest that the occurrence of polyP release is a possible mechanism that limits a further increase in the cellular polyP concentration in E . coli recombinants . High-resolution 31P nuclear magnetic resonance spectroscopy also detected a surface pool of polyP in intact cells of the E . coli recombinant . The polyP resonance increased when cells were treated with EDTA and broadened upon the addition of a shift reagent, praseodymium . Although the mechanism of surface polyP accumulation is unclear, surface polyP seems to serve as the source for polyP release. Bioessays, 1994 Oct, 16(10), 761 - 6 Endo-exonucleases: enzymes involved in DNA repair and cell death? Fraser MJ. Endo-exonucleases from E . coli to man, although very different proteins, are multifunctional enzymes with similar enzymatic activities . They probably have two common but opposing biological roles . On the one hand, they promote survival of the organism by acting in recombination and recombinational DNA repair to diversify and help preserve the genome intact . On the other hand, they degrade the genomic DNA when it is damaged beyond repair . This ensures elimination of heavily mutagenized cells from the population. J Mol Evol, 1994 Oct, 39(4), 340 - 6 Compositional heterogeneity of the Escherichia coli genome: a role for VSP repair? Gutierrez G, Casadesus J, Oliver JL, Marin A. E . coli genes that contain a high frequency of the tetranucleotide CTAG are also rich in the tetramers CTTG, CCTA, CCAA, TTGG, TAGG, and CAAG (group-I tetramers) . Conversely, E . coli genes lacking CTAG are rich in the tetranucleotides CCTG, CCAG, CTGG, and CAGG (group-II tetramers) . These two gene samples differ also in codon usage, amino acid composition, frequency of Dcm sites, and contrast vocabularies . Group-I tetramers have in common that they are depleted by very-short-patch repair (VSP), while group-II tetramers are favored by VSP activity . The VSP system repairs G:T mismatches to G:C, thereby increasing the overall G+C content of the genome; for this reason the CTAG-rich sample has a lower G+C content than the CTAG-poor sample . This compositional heterogeneity can be tentatively explained by a low level of VSP activity on the CTAG-rich sample . A negative correlation is found between the frequency of group-I tetramers and the level of gene expression, as measured by the Codon Adaptation Index (CAI) . A possible link between the rate of VSP activity and the level of gene expression is considered. Gut, 1994 Oct, 35(10), 1449 - 54 Adhesion of human enterotoxigenic Escherichia coli to human mucus secreting HT-29 cell subpopulations in culture; Kerneis S et al.; Enterotoxigenic Escherichia coli (ETEC) bearing the fimbrial colonisation factor antigens CFA/I, CFA/II, CFA/III, and the non-fimbrial antigen 2230 were tested for their ability to adhere to two cultured human intestinal HT-29 mucus secreting cell subpopulations . These populations are referred to as HT29-MTX and HT29-FU, which differ in the amount of secreted mucins and in their gastric or colonic mucin immunoreactivity respectively . Adherence of radiolabelled bacteria to cell monolayers infected apically was assessed . All ETEC strains adhered to the mucus secreting HT29-FU subpopulation, which secretes mucins of colonic immunoreactivity . Visualisation of bacteria by scanning electron microscopy showed that ETEC bound to the HT29-FU cells possessing a brush border, but not to the mucus and that ETEC binding developed as a function of cell differentiation . The adhesion of ETEC to cells possessing a brush border and to mucus secreting cells was also analysed by indirect immunofluorescence in HT29-MTX cells, which secrete mucins of gastric immunoreactivity . Fluorescein isothiocyanate labelling using specific anti-CFA/I antibody was used to show ETEC; rhodamine isothiocyanate labelling using a monoclonal antibody (designated M1) against purified human gastric mucus was used to detect secreted mucins, and rhodamine isothiocyanate labelling using a monoclonal antibody (designated 4H3) against human dipeptidylpeptidase IV was used to show cells possessing a brush border . Binding of bacteria colocalised with dipeptidylpeptidase IV of enterocytes and not with mucins. Genes Dev, 1994 Oct 1, 8(19), 2363 - 74 Evidence that the cis preference of the Tn5 transposase is caused by nonproductive multimerization; Weinreich MD et al.; The transposase (Tnp) of the bacterial transposon Tn5 acts 50- to 100-fold more efficiently on elements located cis to the site of its synthesis compared with those located in trans . In an effort to understand the basis for this cis preference, we have screened for Tnp mutants that exhibit increased transposition activity in a trans assay . Two mutations in the carboxyl terminus were isolated repeatedly . The EK345 mutation characterized previously increases Tnp activity eightfold both in cis and in trans . The novel LP372 mutation, however, increases Tnp activity 10-fold specifically in trans . Combining both mutations increases Tnp activity 80-fold . Interestingly, the LP372 mutation maps to a region shown previously to be critical for interaction with Inh, an inhibitor of Tn5 transposition, and results in reduced inhibition activity by both Tnp and Inh . Tnp also inhibits Tn5 transposition in trans, and this has been suggested to occur by the formation of inactive Tnp multimers . Because Inh and (presumably) Tnp inhibit Tn5 transposition by forming defective multimers with Tnp, the inhibition defect of the trans-active LP372 mutant suggests that the cis preference of Tnp may also be attributable to nonproductive Tnp-Tnp multimerization . In addition, we show that increasing the synthesis of EK345/LP372 Tnp, but not wild-type Tnp, leads to very high levels of transposition, presumably because this altered Tnp is defective in the inhibitory activity of the wild type protein. Dev Biol, 1994 Oct, 165(2), 639 - 53 Transcriptional and post-transcriptional regulation of maternal and zygotic cytoskeletal tropomyosin mRNA during Drosophila development correlates with specific morphogenic events; Hales KH et al.; We have characterized maternal and zygotic cytoskeletal tropomyosin mRNA expression during Drosophila embryogenesis using in situ hybridization to endogenous cytoskeletal tropomyosin mRNA and a cytoskeletal tropomyosin promoter/beta-galactosidase-encoding fusion gene mRNA in transgenic flies . A 2.0-kb maternal cytoskeletal tropomyosin mRNA is synthesized in the nurse cells and transported into the oocyte during oogenesis . During early embryogenesis, this mRNA becomes localized to the pole cell region and then to the cortex during the cellular blastoderm stage . In later embryos it is localized to the ventral and cephalic furrows and extending germ band . The major zygotic mRNA is 2.4 kb and is first detected at gastrulation . In early embryos, this mRNA is expressed in the invaginating anterior and posterior midgut, the transverse furrows, and the amnioserosa, all regions of the embryo undergoing intense cellular movement, and in later embryos, predominantly in the gut, brain, and epidermis . A transgene construct containing 1.2 kb of 5' cytoskeletal tropomyosin promoter sequences driving expression of Escherichia coli beta-galactosidase and the cytoskeletal tropomyosin 3' untranslated region in transgenic flies has the same distribution of maternal and zygotic transcripts throughout oogenesis and embryonic development as the endogenous transcripts . A transgene containing the 3' untranslated region from either the hsp70 gene or the SV40 early genes, on the other hand, does not express maternal RNA . Furthermore, none of the transgenes expressed in the follicle cells, suggesting that expression in these cells is under different transcriptional control . Our results indicate that maternal and zygotic cytoskeletal tropomyosin mRNAs are localized to specific regions of the developing embryo, particularly in regions of the embryo undergoing cell movement and invagination . Furthermore, the synthesis, transport, and accumulation of this RNA is under transcriptional and post-transcriptional control. Carcinogenesis, 1994 Oct, 15(10), 2143 - 8 An assay for O6-alkylguanine-DNA alkyltransferase based on restriction endonuclease inhibition and magnetic bead separation of products; Wilson BD et al.; We describe an improved, rapid and cost effective assay for measuring O6-alkylguanine-DNA alkyltransferase (AGT) levels in large numbers of small biological samples . The assay is based on the use of a synthetic O6-methylguanine oligonucleotide bound to magnetic beads via a biotin-streptavidin linkage and bound to its complement . A 35S label is incorporated into the free end of the duplex . The basis of the assay lies in the observation that the restriction enzyme PvuII will not cleave the bead-bound duplex oligonucleotide having an O6-methylguanine within the restriction sequence . However, on removal of the methyl group by AGT present in cell extracts prior to incubation with PvuII, the 35S-labelled oligonucleotide is cleaved by the restriction enzyme, releasing a 35S-labelled 8 bp fragment . Due to the stoichiometric nature of the AGT reaction, quantitation of AGT is easily achieved by counting an aliquot of the supernatant after pelleting the unrestricted bead complexes with a magnet . AGT levels measured in certain cell lines and human lymphocytes by the reported assay are comparable to other methods . The assay can be performed in basic laboratories and allows for the rapid processing of many samples simultaneously, which could prove useful in clinical and epidemiological studies. Carcinogenesis, 1994 Oct, 15(10), 2125 - 9 The Fpg protein, a DNA repair enzyme, is inhibited by the biomediator nitric oxide in vitro and in vivo; Wink DA et al.; Nitric oxide has been shown to be a mediator molecule in the regulation of many physiological functions . However, this small diatomic molecule in the presence of O2 generates reactive intermediates which modify DNA bases and inactive enzymes at high concentrations (100 microM) . We report that NO generated by 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO, Et2NN(O)NO-Na+), a compound known to release NO in a predictable manner, caused irreversible damage at physiological concentrations to the zinc finger-containing DNA repair enzyme formamidopyrimidine-DNA glycolyase (Fpg protein) . The inhibition of the enzyme activity was DEA/NO dose and time dependent with IC50s with respect to total NO released from this compound of approximately 110 and approximately 120 mumol/l respectively . This inhibitory effect by P3 was not reversible over time in the presence of reducing agents and/or Zn2+ . Nitrite and diethylamine, the nitrogenous products of the decomposition of DEA/NO, did not inhibit the enzyme . The presence of 500 micrograms/ml bovine serum albumin did not protect the protein from the inhibitory effects of DEA/NO, however, the presence of 10 mM cysteine did dramatically abate the inhibition of the Fpg protein by DEA/NO . Other DNA glycosylases tested were not inhibited by exposure to these concentrations of NO . These results, together with reports of site-directed mutagenesis of this protein, suggest that the cysteine residues contained within the zinc finger motif of the Fpg protein are the primary sites of NO interaction . Our studies were then extended to intact cells . The Fpg protein activity was decreased following treatment in vivo when Escherichia coli MH321 (acr A-) cells were treated with DEA/NO . Furthermore, the Fapy-DNA glycosylase activity in H4 cells, a rat hepatoma line, was decreased when intact cells were incubated with DEA/NO. Plant Mol Biol, 1994 Oct, 26(2), 747 - 56 Vascular expression of the grp1.8 promoter is controlled by three specific regulatory elements and one unspecific activating sequence; Keller B et al.; The bean grp1.8 full-length promoter is specifically active in vascular tissue during normal development of tobacco . Deletion of a negative regulatory element resulted in ectopic activity of the promoter in cortical cells of hypocotyls, roots and stems . A 169 bp fragment (-205 to -36) of the grp1.8 promoter conferred vascular-specific expression to CaMV 35S minimal promoters whereas a 141 bp fragment (-205 to -64) strongly activated these minimal promoters both in vascular and cortical cells . These experiments defined a new regulatory element (VSE) that is essential for vascular-specific expression and is located between -64 and -36 . The 141 bp grp1.8 promoter sequence had enhancer-like properties as it was active in both orientations . A 24 bp sequence (bp -119 to -96, corresponding to the SE1 regulatory element) enhanced expression from several minimal promoters strongly but unspecifically, whereas a 26 bp sequence (-98 to -73, corresponding to the RSE regulatory element) induced vascular-specific expression . Thus, the grp1.8 promoter is regulated by a combinatorial mechanism that can integrate the action of different, non-additively acting regulatory elements into vascular-specific expression. Plant Mol Biol, 1994 Oct, 26(2), 699 - 708 Molecular cloning of P-type ATPases on intracellular membranes of the marine alga Heterosigma akashiwo; Wada M et al.; Two cDNA clones (HAA13 and HAA1) which include conserved regions of genes of P-type ATPases were isolated from the marine alga Heterosigma akashiwo by a method that included the polymerase chain reaction . The longer cDNA (3286 bp), HAA13, consisted of an open reading frame that encoded a 106 kDa polypeptide of 977 amino acids with several possible transmembrane domains and conserved regions of eukaryotic P-type ATPases . One transmembrane domain had a leucine zipper structure . HAA1 was not a full-length gene (2054 bp) and lacked the 5' region, but it also included the conserved regions and putative transmembrane domains . Antibodies against the regions and putative transmembrane domains . Antibodies against the polypeptides encoded by HAA13 and HAA1 that have been expressed in Escherichia coli reacted with 100 kDa and 95 kDa polypeptides, respectively, on intracellular membranes of H . akashiwo cells . Immunostaining of H . akashiwo cells revealed that the HAA13 antigen was distributed on membranes around chloroplasts and the HAA1 antigen was located on small vesicles. Plant Mol Biol, 1994 Oct, 26(2), 679 - 90 Identification of a maize root transcript expressed in the primary response to nitrate: characterization of a cDNA with homology to ferredoxin-NADP+ oxidoreductase; Ritchie SW et al.; To more fully understand the biochemical and molecular events which occur in plants exposed to nitrate, cDNAs whose accumulation was enhanced in nitrate- and cycloheximide-treated maize (Zea mays L . W64A x W182E) roots were isolated . The 340 bp Zmrprn1 (for Zea mays root primary response to nitrate) cDNA also hybridized with a probe enriched for nitrate-induced sequences, and was characterized further . Sequence analysis of a near full-length cDNA (Zmrprn1A) showed strong homology (> 90% amino acid identity) with a root ferredoxin-NADP+ oxidoreductase (FNR) of rice, and 45-50% amino acid identify with leaf FNR genes . When expressed in Escherichia coli, the Zmrprn1A cDNA produced a protein with NADPH: ferricyanide reductase activity, consistent with the enzymatic properties of an FNR . The Zmrprn1 cDNA hybridized with a 1.4 kb transcript which was expressed in the maize root primary response to nitrate . That is, mRNA levels in roots increased rapidly and transiently in response to external nitrate, and low levels of nitrate (10 microM) induced transcript accumulation . The accumulation of the Zmrprn1 transcript was not prevented by cycloheximide, indicating that the cellular factor(s) required for expression were constitutively present in maize roots . The Zmrprn1 mRNA accumulated specifically in response to nitrate, since neither K+ nor NH4+ treatment of roots caused transcript accumulation . Maize leaves had about 5% of the transcript level found in roots, indicating a strong preference for expression of Zmrprn1 in roots . Analysis of maize genomic DNA indicated the presence of only a single gene or very small gene family for the Zmrprn1 . Together, the data indicate that Zmrprn1A encodes a nitrate regulated maize root FNR. Plant Mol Biol, 1994 Oct, 26(1), 423 - 34 Molecular cloning of cucumber phosphoenolpyruvate carboxykinase and developmental regulation of gene expression; Kim DJ et al.; A cDNA library from RNA of senescing cucumber cotyledons was screened for sequences also expressed in cotyledons during post-germinative growth . One clone encodes ATP-dependent phosphoenolpyruvate carboxykinase (PCK; EC 4.1.1.49), an enzyme of the gluconeogenic pathway . The sequence of a full-length cDNA predicts a polypeptide of 74,397 Da which is 43%, 49% and 57% identical to bacterial, trypanosome and yeast enzymes, respectively . The cDNA was expressed in Escherichia coli and antibodies raised against the resultant protein . The antibody recognises a single polypeptide of ca . 74 kDa, in extracts of cotyledons, leaves and roots . The cucumber genome contains a single pck gene . In the seven-day period after seed imbibition, PCK mRNA and protein steady-state levels increase in amount in cotyledons, peaking at days 2 and 3 respectively, and then decrease . Both accumulate again to a low level in senescing cotyledons . This pattern of gene expression is similar to that of isocitrate lyase (ICL) and malate synthase (MS) . When green cotyledons are detached from seedlings and incubated in the dark, ICL and MS mRNAs increase rapidly in amount but PCK mRNA does not . Therefore it seems unlikely that the glyoxylate cycle serves primarily a gluconeogenic role in starved (detached) cotyledons, in contrast to post-germinative and senescing cotyledons where PCK, ICL and MS are coordinately synthesised . While exogenous sucrose greatly represses expression of icl and ms genes in dark-incubated cotyledons, it has a smaller effect on the level of PCK mRNA. Plant Mol Biol, 1994 Oct, 26(1), 353 - 62 Arabidopsis heat shock factor: isolation and characterization of the gene and the recombinant protein; Hubel A et al.; We have isolated the Arabidopsis heat shock factor gene Athsf1 as genomic and corresponding cDNA sequences via cross-hybridization with tomato clones . Sequence analysis indicates only a partial homology with the HSFs from tomato and other organisms which is confined to the DNA-binding and the oligomerization domains . The gene is constitutively expressed but the level of mRNA for Athsf1 increases two-fold upon heat shock . However, the putative promoter region lacks the canonical heat shock elements . After expression in Escherichia coli the recombinant Athsf1 protein binds specifically to a synthetic oligonucleotide containing five heat shock elements . The native size of recombinant ATHSF1 in vitro is consistent with a trimer as demonstrated by chemical cross-linking and pore exclusion limit analysis. Plant Mol Biol, 1994 Oct, 26(1), 225 - 34 Purification and characterization of pea thioredoxin f expressed in Escherichia coli; Hodges M et al.; The recently cloned cDNA for pea chloroplast thioredoxin f was used to produce, by PCR, a fragment coding for a protein lacking the transit peptide . This cDNA fragment was subcloned into a pET expression vector and used to transform E . coli cells . After induction with IPTG the transformed cells produce the protein, mainly in the soluble fraction of the broken cells . The recombinant thioredoxin f has been purified and used to raise antibodies and analysed for activity . The antibodies appear to be specific towards thioredoxin f and do not recognize other types of thioredoxin . The recombinant protein could activate two chloroplastic enzymes, namely NADP-dependent malate dehydrogenase (NADP-MDH) and fructose 1,6-bisphosphatase (FBPase), both using dithiothreitol as a chemical reductant and in a light-reconstituted/thylakoid assay . Recombinant pea thioredoxin f turned out to be an excellent catalyst for NADP-MDH activation, being the more efficient than a recombinant m-type thioredoxin of Chlamydomonas reinhardtii and the thioredoxin of E . coli . At the concentrations of thioredoxin used in the target enzyme activation assays only the recombinant thioredoxin f activated the FBPase. Plant Mol Biol, 1994 Oct, 26(1), 211 - 23 Isolation and characterisation of a maize cDNA that complements a 1-acyl sn-glycerol-3-phosphate acyltransferase mutant of Escherichia coli and encodes a protein which has similarities to other acyltransferases; Brown AP et al.; We selected cDNA plasmid clones that corrected the temperature-sensitive phenotype of Escherichia coli strain JC201, which is deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity . A plasmid-based maize endosperm cDNA library was used for complementation and a plasmid that enabled the cells to grow at 44 degrees C on ampicillin was isolated . Addition of this plasmid (pMAT1) to JC201 restored 1-acyl-sn-glycerol-3-phosphate acyltransferase activity to the cells . Total phospholipid labelling showed that the substrate for the enzyme, lysophosphatidic acid, accumulated in JC201 and was further metabolised to phosphatidylethanolamine in complemented cells . Membranes isolated from such cells were able to convert lysophosphatidic acid to phosphatidic acid in acyltransferase assays . The cDNA insert of pMAT1 contains one long open reading frame of 374 amino acids which encodes a protein of relative molecular weight 42,543 . The sequence of this protein is most similar to SLC1, which is thought to be able to acylate glycerol at the sn-2 position during synthesis of inositol-containing lipids . Homologies between the SLC1 protein, the 1-acyl-sn-glycerol-3-phosphate acyltransferase of E . coli (PlsC) and the maize ORF were found with blocks of conserved amino acids, whose spacing was conserved between the three proteins, identifiable. Plant Mol Biol, 1994 Oct, 26(1), 117 - 30 Reconstitution of the spinach oxygen-evolving complex with recombinant Arabidopsis manganese-stabilizing protein; Betts SD et al.; The psbO gene of cyanobacteria, green algae and higher plants encodes the precursor of the 33 kDa manganese-stabilizing protein (MSP), a water-soluble subunit of photosystem II (PSII) . Using a pET-T7 cloning/expression system, we have expressed in Escherichia coli a full-length cDNA clone of psbO from Arabidopsis thaliana . Upon induction, high levels of the precursor protein accumulated in cells grown with vigorous aeration . In cells grown under weak aeration, the mature protein accumulated upon induction . In cells grown with moderate aeration, the ratio of precursor to mature MSP decreased as the optical density at induction increased . Both forms of the protein accumulated as inclusion bodies from which the mature protein could be released under mildly denaturing conditions that did not release the precursor . Renatured Arabidopsis MSP was 87% as effective as isolated spinach MSP in restoring O2 evolution activity to MSP-depleted PSII membranes from spinach; however, the heterologous protein binds to spinach PSIIs with about half the affinity of the native protein . We also report a correction to the previously published DNA sequence of Arabidopsis psbO (Ko et al., Plant Mol Biol 14 (1990) 217-227). Biopolymers, 1994 Oct, 34(10), 1339 - 47 Detection of monodisperse aggregates of a recombinant amelogenin by dynamic light scattering; Moradian-Oldak J et al.; Recombinant murine amelogenins M179 and M166 were expressed in Escherichia coli and purified . The aggregation properties of these amelogenins have been investigated in aqueous solutions as well as acetonitrile-containing solutions using dynamic light scattering . Dynamic light scattering provides direct measurement of the translational diffusion coefficient and hydrodynamic radius, and of an estimate of the molecular weight . Polydispersity and statistical parameters of how to interpret the analysis are also provided . Amelogenin aggregation was examined in solutions of a range of pH, ionic strengths, and protein concentrations . It was shown that at pH 7.8-8 and ionic strength of 0.02-0.05M the M179 molecules form monodispersed aggregates with hydrodynamic radii ranging from 15 to 19 nm . Analysis of hydrodynamic radii and size distribution of M179 aggregates in acetonitrile-containing solvents compared to that in aqueous solutions indicated a primary role for hydrophobic interactions in the association process of amelogenin molecules to form aggregates . Comparison between the aggregates formed by M179 and M166, which lacks the hydrophilic carboxy-terminal 13 residue sequence of M179, suggested that the self-assembly of amelogenin molecules to form stable and monodisperse aggregates requires the presence of the hydrophilic carboxy-terminal sequence of M179. Arthritis Rheum, 1994 Oct, 37(10), 1445 - 52 Analysis of the specificity of anti-PM-Scl autoantibodies; Ge Q et al.; OBJECTIVE . To compare the specificity of anti-PM-Scl autoantibodies in serum samples from 43 patients with myositis, scleroderma, or both . METHODS . Anti-PM-Scl immunoprecipitates from HeLa cell extract were used as antigen for immunoblot analyses to determine the antigenic components . A series of complementary DNA fragments was expressed in Escherichia coli for immunoblot examination of the reaction with the 100-kd protein . RESULTS . The immunoblot against immunoprecipitates was sensitive and specific for detecting reactions with components of the PM-Scl antigen: 42 of 43 sera (97.7%) reacted with the 100-kd, 27 of 43 (62.8%) with the 70-kd, and 5 of 43 (11.6%) with the 37-kd protein (not previously recognized as antigenic) . Forty-one sera reacted with N-terminal protein S1 (amino acids 11-437), 39 with central protein S2 (amino acids 439-749), and 24 with C-terminal protein S3 (amino acids 750-882) . Of 42 sera tested, 28 (66.7%) reacted most strongly with S1, and 6 (14.3%) reacted most strongly with S2 . Absorption studies implied additional, conformational epitopes not present on the bacterially expressed antigen . CONCLUSION . There was an overall similarity in reactivity to the PM-Scl antigen, but there were differences in the reactivity to the 70-kd and 37-kd proteins, as well as in the relative strength of the reactivity to the S2 protein. Biochem J, 1994 Oct 1, 303 ( Pt 1), 73 - 9 Expression of a biologically active plant cytochrome b5 in Escherichia coli; Smith MA et al.; Cytochrome b5 from tobacco (Nicotiana tabacum) was expressed in Escherichia coli using a T7 polymerase/promoter system as described by Studier, Rosenberg, Dunn and Dubendorff (1990) (Methods Enzymol . 185, 60-89) . Transformed cells were red in colour and accumulated cytochrome b5 to a level of around 30% of the total cell protein . The purified cytochrome had oxidized, reduced and low-temperature absorbance spectra characteristic of plant microsomal cytochrome b5, and exhibited a c.d . spectrum resembling that of a mammalian cytochrome b5 . The recombinant protein appeared to be correctly assembled and biologically active, being reduced by NADH in the presence of microsomal membranes prepared from the developing seeds of sunflower (Helianthus annuus) . Inhibition of haem synthesis in the transformed E . coli cells expressing cytochrome b5, by the use of gabaculin or succinylacetone, prevented the assembly of the cytochrome b5 holoprotein but had little effect on the accumulation of cytochrome apoprotein . The recombinant protein expressed in E . coli therefore has the biochemical features of the higher-plant cytochrome b5 and can be used in studies of plant microsomal oxidation/reduction reactions. Arch Biochem Biophys, 1994 Oct, 314(1), 132 - 41 Purification and characterization of S1 mutants of alpha-lytic protease having altered catalytic properties; Haggett KD et al.; A procedure is described for purifying alpha-lytic protease and its mutants from culture supernatants of recombinant Escherichia coli . The method affords substantial amounts (approx . 80 mg) of homogeneous enzyme . We compared the cleavage preferences of wild-type alpha-lytic protease and of mutants containing the substitutions Ala190 ("parent"), Ala190/Val192/His213/Met218 (mutant 1), Ala190/His213/Leu218 (mutant 9), and Ala190/Thr213/Leu218 (mutant 55), and for each enzyme we found broad agreement between the results obtained with synthetic ester and amide substrates . Kinetic constants were determined for the purified enzymes using selected tetrapeptide p-nitroanilide substrates . Mutant 55 had broad specificity and high activity . In terms of kcat/Km it cleaved at Met and Phe residues two to three times as effectively as the Ala190 enzyme and cleaved at Ala 7 times more effectively than the wild-type protease . The Ala190/His213 enzymes showed a preference for cleavage at His and Met residues . Not only were their kcat values for cleavage at His increased (in relation to the Ala190 enzyme) by an order of magnitude, but they also exhibited large decreases in kcat/Km for cleavage at other residues; for example, the value for cleavage at Phe was 400- to 600-fold lower . Mutant 9 cleaved a recombinant IGF-II fusion protein at a unique His residue and also at a nearby Asn residue. Am J Obstet Gynecol, 1994 Oct, 171(4), 944 - 8 The role of nitric oxide in the pathogenesis of preeclampsia; Seligman SP et al.; OBJECTIVE: Nitric oxide, a potent vasodilator released by endothelial cells, inhibits platelet aggregation and adhesion to vascular endothelial surfaces . Because endothelial cell damage is considered pivotal in the pathogenesis of preeclampsia, this study was initiated to determine whether nitric oxide production is decreased in patients with preeclampsia . STUDY DESIGN: Twenty-six patients with preeclampsia (as defined by a blood pressure > or = 140 mm Hg systolic or 90 mm Hg diastolic plus proteinuria, > or = 300 mg per 24 hours or > or = 2+ by dipstick, both occurring on two occasions > or = 4 hours apart) and 26 normotensive women with singleton gestations in the third trimester were studied . Because nitric oxide is spontaneously oxidized to both nitrite and nitrate, two analytic assays were used serially . Serum nitrite levels were initially determined with the Greiss reagent and subsequently analyzed with Escherichia coli nitrate reductase . RESULTS: With the Greiss reagent alone the mean +/- SEM of serum nitrite level in 26 patients with preeclampsia was significantly decreased compared with 26 normotensive patients (3.46 +/- 1.43 mumol/L vs 4.65 +/- 0.85 mumol/L, p = 0.02) . With the addition of the nitrate reductase enzyme of Escherichia coli the mean +/- SEM of serum nitrite level in 26 preeclamptic patients was again significantly decreased compared with 26 normotensive patients (20.04 +/- 1.25 mumol/L vs 27.38 +/- 2.23 mumol/L, p = 0.02) . One patient with the syndrome of hemolysis, elevated liver enzymes, and low platelets demonstrated a concurrent decrease in serum nitrite over a 2-week period, emphasizing the relationship of nitric oxide to the pathophysiologic features of the syndrome . CONCLUSIONS: Circulating levels of nitrite are decreased in patients with preeclampsia . These data support the concept that diminished nitric oxide synthesis contributes to the pathophysiologic changes seen in preeclampsia. Scand J Immunol, 1994 Oct, 40(4), 457 - 65 Catabolism of the murine IgG1 molecule: evidence that both CH2-CH3 domain interfaces are required for persistence of IgG1 in the circulation of mice; Kim JK et al.; Site-directed mutagenesis of a recombinant Fc-hinge fragment has previously been used to identify a region of the murine IgG1 molecule that controls catabolism, and this site encompasses amino acid residues at the interface of the CH2 and CH3 domains . In the current study the nature of this 'catabolic site' has been further analysed using recombinant techniques . Fc-hinge, CH2-hinge, CH2 and CH3 fragments have been expressed in Escherichia coli, purified and analysed in pharmacokinetic studies in mice . The CH2-hinge has been analysed as both a monomer and dimer, and the dimer has a longer beta phase half-life (61.6 h) than the monomer (29.1 h) . This suggests that two catabolic sites per Fc fragment are required for serum persistence . The need for two functional sites per molecule has been confirmed by the analysis of a hybrid Fc-hinge fragment comprising a heterodimer of one Fc-hinge with the wild type (WT) IgG1 sequence and a mutant Fc-hinge with a defective catabolic site (mutated at His310, Gln311, His433 and Asn434) . This hybrid is cleared with a beta phase half-life of 37.9 h and this is significantly shorter than that of the WT Fc-hinge fragment (82.9 h) . In contrast to the CH2-hinge dimer, the CH3 domain is cleared rapidly (beta phase half-life of 21.3 h) indicating that the region of this domain (His433 and Asn434) previously identified as being involved in the control of catabolism is not sufficient in the absence of the CH2 domain for the serum persistence of an IgG fragment . The data extend our earlier observations concerning a region of the murine IgG1 molecule that is involved in the control of catabolism and have implications for the design of engineered antibodies for therapy. Mol Immunol, 1994 Oct, 31(14), 1047 - 58 Recombinant single-chain Fv fragments carrying C-terminal cysteine residues: production of bivalent and biotinylated miniantibodies; Kipriyanov SM et al.; A murine antibody single-chain Fv (scFv) fragment carrying five C-terminal histidine residues preceded by a cysteine residue and a marker peptide was expressed in Escherichia coli . Its variable heavy (VH) and light (VL) domains are derived from the mouse monoclonal antibody mAb215, which is specific for the largest subunit of RNA polymerase II of Drosophila melanogaster . ScFv' monomers, covalently linked (scFv')2 and non-covalent dimers, as well as aggregated antibody fragments, were isolated from an E . coli cell paste by immobilized metal affinity chromatography in 6 M urea followed by a renaturation procedure that does not use any sulfhydryl agents . In a final step, the components were separated by size exclusion chromatography . All the recombinant antibody fractions demonstrated high antigen-binding activity and specificity as shown by ELISA and Western blot analysis . Affinity measurements carried out by competitive immunoassays showed that covalently linked (scFv')2 have binding constants quite close to those of the parental monoclonal antibodies and four-fold higher than scFv' monomers . ScFv derivatives, specifically biotinylated through the free sulfhydryl group, recognize the corresponding antigen in ELISA and Western blot analysis, thus demonstrating the possibility of using chemically modified scFv antibodies for immunodetection. Mol Cell Biol, 1994 Oct, 14(10), 6879 - 85 A novel mammalian Ras GTPase-activating protein which has phospholipid-binding and Btk homology regions; Maekawa M et al.; We have previously purified a novel GTPase-activating protein (GAP) for Ras which is immunologically distinct from the known Ras GAPs, p120GAP and neurofibromin (M . Maekawa, S . Nakamura, and S . Hattori, J . Biol . Chem . 268:22948-22952, 1993) . On the basis of the partial amino acid sequence, we have obtained a cDNA which encodes the novel Ras GAP . The predicted protein consists of 847 amino acids whose calculated molecular mass, 96,369 Da, is close to the apparent molecular mass of the novel Ras GAP, 100 kDa . The amino acid sequence shows a high degree of similarity to the entire sequence of the Drosophila melanogaster Gap1 gene . When the catalytic domain of the novel GAP was compared with that of Drosophila Gap1, p120GAP, and neurofibromin, the highest degree of similarity was again observed with Gap1 . Thus, we designated this gene Gap1m, a mammalian counterpart of the Drosophila Gap1 gene . Expression of Gap1m was relatively high in brain, placenta, and kidney tissues, and it was expressed at low levels in other tissues . A recombinant protein consisting of glutathione-S-transferase and the GAP-related domain of Gap1m stimulated GTPase of normal Ras but not that of Ras having valine at the 12th residue . Expression of the same region in Saccharomyces cerevisiae suppressed the ira2- phenotype . In addition to the GAP catalytic domain, Gap1m has two domains with sequence closely related to those of the phospholipid-binding domain of synaptotagmin and a region with similarity to the unique domain of Btk tyrosine kinase . These results clearly show that Gap1m is a novel Ras GAP molecule of mammalian cells. Mol Cell Biol, 1994 Oct, 14(10), 6489 - 96 DNA replication from initiation zones of mammalian cells in a model system; Ishimi Y et al.; We reported that DNA replication initiates from the region containing an autonomously replicating sequence from Saccharomyces cerevisiae when negatively supercoiled plasmid DNA is incubated with the proteins required for simian virus 40 DNA replication (Y . Ishimi and K . Matsumoto, Proc . Natl . Acad . Sci . USA 90:5399-5403, 1993) . In this study, the DNAs containing initiation zones from mammalian cells were replicated in this model system . When negatively supercoiled DNA containing an initiation zone (2 kb) upstream of the human c-myc gene was incubated with simian virus 40 T antigen as a DNA helicase, HSSB (also called replication protein A), and DNA polymerase alpha-primase complex isolated from HeLa cells, DNA replication was specifically initiated from the center of the initiation zone, which was elongated bidirectionally in the presence of a DNA swivelase . Without HSSB, the level of DNA synthesis was significantly reduced and the localized initiation could not be detected, indicating that HSSB plays an essential role in the initiation of DNA replication . The digestion of negatively supercoiled template DNA with a single-strand-specific nuclease revealed that HSSB stimulated DNA unwinding in the center of the initiation zone where the DNA duplex is relatively unstable . In contrast, DNA replication started from a broad region of an initiation zone downstream of the dihydrofolate reductase gene from chinese hamster ovary cells, but the center of the region was mapped near the origin of bidirectional DNA replication . These results suggested that this system mimics a fundamental process of initiation of eukaryotic DNA replication . The mechanism of initiation is discussed. J Nucl Med, 1994 Oct, 35(10), 1685 - 90 Synthesis of fluorine-18-labeled biotin derivatives: biodistribution and infection localization; Shoup TM et al.; Recently there has been much interest in the exploitation of the high binding affinity of avidin/biotin as a means of targeting drugs and radionuclides for in vivo applications . We are interested in broadening the application of the avidin/biotin complex to PET . To this end we set out to prepare 18F-labeled biotin analogs . METHODS: Two 18F biotin derivatives, {3aS-(3a alpha,4 beta,6a alpha)}-hexahydro-2-oxo-1H-thieno{3,4- d}imidazole-4-(N-3-(1-{18F}fluoropropyl))pentanamide (1) and {3aS-(3a alpha,4 beta,6a alpha)}-tetrahydro-4-(5-(1-{18F}fluoropentyl)- 1H-thieno{3,4-d}imidazol-2(3H)-(2) were prepared with high specific activity (NCA) and evaluated for their potential in infection localization . RESULTS: Compound 1 binds to avidin and the biodistribution of these derivatives were studied in Escherichia coli infected rats . Half of the infected rats were treated with avidin 24 hr prior to intravenous injection of the 18F-labeled biotin analogs . Biotin 1, without avidin pretreatment, showed a selectivity of 6.08 +/- 1.12 for infection compared to normal muscle . With avidin pretreatment, selectivity increased slightly, giving an infection to normal muscle ratio of 6.39 +/- 0.96 . In contrast, the biodistribution of biotin 2 indicated more binding to normal muscle with an infection to normal muscle ratio of 0.58 +/- 0.07 . This lack of selectivity illustrates the importance of the side-chain amide group in infection localization . There was some defluorination of 1 and 2, as evidenced by increased 18F bone uptake after 60 min: 2.94 +/- 0.37 and 1.17 +/- 0.21 %IG/g +/- s.d., respectively . CONCLUSIONS: Biotin derivatives could be radiofluorinated with high specific activity . Biotin 1, is a potential positron tomography tracer for infection imaging. J Neurosci, 1994 Oct, 14(10), 5844 - 57 Metallothionein III is expressed in neurons that sequester zinc in synaptic vesicles; Masters BA et al.; MT-III, a brain-specific member of the metallothionein gene family, binds zinc and may facilitate the storage of zinc in neurons . The distribution of MT-III mRNA within the adult brain was determined by solution and in situ hybridization and compared to that of MT-I mRNA . MT-III mRNA is particularly abundant within the cerebral cortex, hippocampus, amygdala, and nuclei at base of the cerebellum . Transgenic mice generated using 11.5 kb of the mouse MT-III 5' flanking region fused to the E . coli lacZ gene express beta-galactosidase in many of the same regions identified by in situ hybridization . MT-III mRNA was present in readily identifiable neurons within the olfactory bulb, hippocampus, and cerebellum, and beta-galactosidase activity was localized to neurons throughout the brain, but not to glia, as determined by costaining with X-Gal and neural- and glia-specific antibodies . There is marked correspondence between the neurons that are rich in MT-III mRNA and those neurons that store zinc in their terminal vesicles . MT-III is found complexed with zinc in vivo and its expression in cultured cells leads to the intracellular accumulation of zinc and enhanced histochemical detection of zinc . These results are discussed in light of the possibility that MT-III may participate in the utilization of zinc as a neuromodulator. J Neurol Neurosurg Psychiatry, 1994 Oct, 57(10), 1216 - 20 Mesencephalic and third ventricle cysts: diagnosis and management in four cases; Ramaekers VT et al.; Four infants with obstructive hydrocephalus caused by space occupying third ventricle and mesencephalic cysts are reported . Despite immediate shunt insertion in all patients, there was either lack of clinical improvement or late onset of clinical deterioration . Neuroimaging (CT, MRI, and ventriculography) diagnosed the presence of non-communicating midline outpouchings of the CSF pathways causing obstruction of aqueductal CSF flow and brainstem signs . The cysts were of different origin . In one patient it was caused by a previous thalamic haemorrhage, in another patient by neonatal Escherichia coli meningoventriculitis . In two cases with obstructive hydrocephalus at birth, the aetiology is unclear . Direct puncture and drainage of the cysts led to clinical improvement . The cysts were poorly visualised on CT and could be misinterpreted as an enlarged third ventricle, simulating congenital aqueduct stenosis . Careful neuroradiological investigation is necessary to establish an accurate diagnosis and neurosurgical management . In such cases with hydrocephalus and persisting ventricular enlargement despite shunting, CT ventriculography is a useful tool. J Gen Virol, 1994 Oct, 75 ( Pt 10), 2803 - 6 Nucleotide sequence of Nilaparvata lugens reovirus genome segment S8 coding for the major outer capsid protein; Nakashima N et al.; The complete nucleotide sequence of genome segment 8 (S8) of Nilaparvata lugens reovirus (NLRV) was determined . It consisted of 1802 nucleotides containing a long open reading frame (562 amino acids), which was expressed in Escherichia coli as a fusion protein . The expressed S8 product, a 62K protein, was detected by Western blotting using IgG directed against intact NLRV particles . This result indicates that S8 encodes the major outer capsid protein of NLRV . The protein exhibited 18.6% amino acid sequence identity with the predicted translation product of S10 of rice black-streaked dwarf virus. J Gen Virol, 1994 Oct, 75 ( Pt 10), 2761 - 4 The protease of adenovirus serotype 2 requires cysteine residues for both activation and catalysis; Grierson AW et al.; Sequence analysis and site-directed mutagenesis were used to study the mechanisms of activation and catalysis of the adenovirus type 2 (Ad2) protease . Primary structure alignments of proteases from 12 serotypes and previously elucidated inhibition profiles were used to target residues for mutagenesis . All conserved serine and cysteine residues were mutated separately and following expression in Escherichia coli their activity in a synthetic peptide assay was compared to that of wild-type recombinant protease . Mutants containing altered serine residues were active while mutations to cysteine-104 and cysteine-122 reduced activity by more than 95% . These results taken together with the known inhibition profile of the adenovirus protease confirm that it is a cysteine protease and suggest that one of these residues provides the active site nucleophile while the other is a part of the thiol-disulphide interchange mechanism previously reported to be involved in its activation. J Gen Virol, 1994 Oct, 75 ( Pt 10), 2707 - 17 Comparative studies of the structural proteins and glycoproteins of equine herpesviruses 2 and 5; Agius CT et al.; The structural proteins of equine herpesvirus 2 (EHV-2) and EHV-5, recently shown to be gammaherpesviruses, were identified and compared . Labelled proteins and glycoproteins were separated by SDS-PAGE and although EHV-2 and EHV-5 had similar protein profiles, bands in some positions were virus-specific . Six glycoproteins, with distinct profiles, were identified for both EHV-2 and EHV-5 . Rabbit antisera to EHV-2 and EHV-5 and horse antiserum to EHV-2 were used in radioimmunoprecipitations, Western blot analysis and ELISA to investigate the immunogenicity and cross-reactivity of virus proteins . These analyses revealed that while EHV-2 and EHV-5 proteins share many common epitopes, they also possess type-specific epitopes . A 0.71 kb region of the EHV-2 glycoprotein B (gB) gene was expressed as a fusion protein in Escherichia coli . Antiserum raised in a rabbit to the EHV-2 fusion protein was used to identify a 64K EHV-2 protein as EHV-2 gB . Antiserum to EHV-2 gB was used to identify a 66K EHV-5 protein as EHV-5 gB . These proteins, which may represent subunits of gB rather than the entire molecule, appear the most immunodominant of the structural virion proteins as identified by Western blot. J Gen Virol, 1994 Oct, 75 ( Pt 10), 2567 - 73 Nucleic acid-binding properties of the P1 protein of turnip mosaic potyvirus produced in Escherichia coli; Soumounou Y et al.; The N-terminal P1 protein of turnip mosaic potyvirus (TuMV) polyprotein was overexpressed in Escherichia coli, purified by metal chelation chromatography under denaturing conditions and renatured . U.v . cross-linking experiments indicated that the recombinant protein interacted with RNA, and gel retardation electrophoresis demonstrated that more than one molecule of P1 bound one molecule of RNA . Formation of the protein-RNA complexes was dependent on the conformational state of P1 and was stable at relatively high concentrations of NaCl . P1 had the ability to bind ssRNA and ssDNA, with similar affinity, but was not able to bind to dsDNA . The TuMV protein had the additional characteristic of binding dsRNA with affinity similar to that observed with single-stranded nucleic acids. J Leukoc Biol, 1994 Oct, 56(4), 453 - 7 Endotoxin stimulates the expression of glucose-6-phosphate dehydrogenase in Kupffer and hepatic endothelial cells; Spolarics Z et al.; The aim of the study was to elucidate the effect of lipopolysaccharide (LPS) administration in vivo (Escherichia coli endotoxin, 1 mg/kg body weight) on the expression and cellular activity of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), the rate-limiting enzyme of the hexose monophosphate shunt in hepatic cells . Under basal conditions, Kupffer cells displayed higher activity of G6PDH than endothelial or parenchymal cells . In vivo LPS treatments for 7 and 22 h resulted in 40 and 60% increases, respectively, in the cellular activity of G6PDH in Kupffer cells . G6PDH activity was increased by 140 and 90% after 7- and 22-h LPS treatments in endothelial cells . G6PDH activity in parenchymal cells prepared from animals after 22 h of LPS treatment was decreased by approximately 60% compared with that in cells from saline-injected animals . Total cellular RNA or protein extracts from these cells were analyzed by Northern or Western blots . Under basal conditions, G6PDH mRNA levels relative to total cellular RNA were higher in Kupffer than in endothelial cells and were not detectable in parenchyma cells . LPS injection caused a time-dependent increase in G6PDH mRNA expression in Kupffer and endothelial cells . Western blot analysis of Kupffer cell extracts also showed that LPS treatments caused markedly elevated expression of protein in these cells . These results show that endotoxemia results in marked induction of G6PDH in Kupffer and hepatic endothelial cells but has no such effect in the parenchymal cells . These findings also suggest that the elevated cellular expression of G6PDH is an important regulatory event in the adaptive responses of hepatic nonparenchymal cells to infections . The elevated expression of G6PDH may be important for support of the upregulated NADPH-dependent pathways, such as superoxide anion and nitric oxide production, macromolecular synthesis, or the maintenance of cellular glutathione status. J Infect Dis, 1994 Oct, 170(4), 841 - 7 Reduced virulence of rifampicin-resistant mutants of Francisella tularensis; Bhatnagar N et al.; Rifampicin-resistant mutants of a live vaccine strain (LVS) of Francisella tularensis were produced and screened for virulence in mice; 4 avirulent clones with intraperitoneal (ip) LD50s > 10(6) cfu, compared with 10(2) cfu for LVS, were characterized . Growth characteristics at 37 degrees C, surface envelope proteins, and lipopolysaccharide profiles of resistant mutants were identical to those of LVS . Polymerase activity of the mutants was more resistant than the enzyme from LVS to the inhibitory action of rifampicin . Growth rates for mutants and LVS were similar during the first 5 h at 42 degrees C, but viability of the mutants decreased to < 0.01% at 24 h . LVS and mutants differed in their ability to grow in vitro in host macrophages: LVS increased 580-fold over 72 h; mutants increased 33-fold . After ip inoculation of the organisms into mice, increasing numbers of LVS from peritoneal cells were isolated; mutants decreased over 4 days. J Allergy Clin Immunol, 1994 Oct, 94(4), 689 - 98 Complementary DNA cloning of the major allergen Phl p I from timothy grass (Phleum pratense); recombinant Phl p I inhibits IgE binding to group I allergens from eight different grass species; Laffer S et al.; BACKGROUND: Grass pollens, such as pollen from timothy grass (Phleum pratense), represent a major cause of type I allergy . OBJECTIVE: In this report we attempted to determine how cross-reactive allergenic components of grass pollens from different species can be represented by a minimum number of recombinant allergens . METHODS: We isolated and sequenced a timothy grass pollen cDNA coding for the major allergen Phl p I . A recombinant Phl p I-beta-galactosidase fusion protein, which bound to IgE in 87% of patients with grass pollen allergy, was produced in Escherichia coli . Using recombinant Phl p V and Phl p I, we defined representative patients' sera that bound to group I but not to group V allergens, as well as sera with reactivity against group I and group V allergens . IgE immunoblot inhibition studies were done with nitrocellulose-blotted pollen extracts from eight grass species with different geographic distribution . RESULTS: Preadsorption of patients' sera with recombinant nonfusion Phl p I strongly reduced IgE binding to group I allergens from the eight grasses, showing extensive cross-reactivity between species . CONCLUSION: A single recombinant group I allergen contains many of the IgE epitopes of group I isoallergens from a number of different grass species. J Cell Physiol, 1994 Oct, 161(1), 149 - 59 Interaction of high-molecular-weight basic fibroblast growth factor with endothelium: biological activity and intracellular fate of human recombinant M(r) 24,000 bFGF; Gualandris A et al.; The single-copy gene of human basic fibroblast growth factor (bFGF) encodes four co-expressed isoforms, with an apparent molecular weight (M(r)) of 24 kD, 22.5 kD, 22 kD, and 18 kD, co-translated from a single mRNA . As a tool for the study of the role exerted by the different bFGF isoforms in the biology of endothelial cells, human recombinant 24-kD bFGF was produced and purified from transformed Escherichia coli cells . To this purpose, the novel CUG start codon present in human bFGF cDNA and responsible for the synthesis of 24-kD bFGF was mutagenized to the classic AUG start codon . Transient expression of the mutagenized cDNA in simian COS-1 cells, followed by immunolocalization and subcellular fractionation, resulted in the synthesis of high levels of 24-kD bFGF, which localizes in the cell nucleus as an intact protein . When the same 24-kD bFGF cDNA was expressed in E . coli, the recombinant protein was purified to homogeneity by heparin-Sepharose and ion-exchange chromatography . Recombinant 24-kD bFGF was similar to recombinant 18-kD bFGF in receptor-binding activity and in inducing cell proliferation, plasminogen activator production, and chemotactic movement in cultured endothelial cells . In agreement with the in vitro observations, 24-kD bFGF and 18-kD bFGF exerted a similar angiogenic response when assayed in vivo in the rabbit cornea . Experiments performed with the radiolabeled molecule demonstrated that 24-kD bFGF has an intrinsic ability to bind to high-affinity receptors when added to endothelial GM 7373 cell cultures . Receptor-bound 24-kD bFGF is internalized within the cell and associates with the nucleus with kinetics similar to 18-kD bFGF . Internalized 24-kD bFGF is first processed to the 18-kD form via a chloroquine-insensitive pathway and then to smaller fragments into the lysosomal compartment . At variance with the data obtained in transfected COS-1 cells, only limited amounts of exogenous internalized 24-kD bFGF associates with the nucleus in the intact form, mostly of the nuclear-bound molecule being represented by the processed 18-kD protein and by smaller degradation products . In conclusion, human recombinant 24-kD bFGF exerts a biological response in endothelial cells similar to 18-kD bFGF both in vitro and in vivo . Our data point to a different intracellular behavior of the high-molecular-weight bFGF isoform when added exogenously to cultured cells or when produced endogenously in transfected cells. J Cell Biol, 1994 Oct, 127(2), 467 - 78 Cell cycle analysis of the activity, subcellular localization, and subunit composition of human CAK (CDK-activating kinase); Tassan JP et al.; The activity of cyclin-dependent kinases (cdks) depends on the phosphorylation of a residue corresponding to threonine 161 in human p34cdc2 . One enzyme responsible for phosphorylating this critical residue has recently been purified from Xenopus and starfish . It was termed CAK (for cdk-activating kinase), and it was shown to contain p40MO15 as its catalytic subunit . In view of the cardinal role of cdks in cell cycle control, it is important to learn if and how CAK activity is regulated during the somatic cell cycle . Here, we report a molecular characterization of a human p40MO15 homologue and its associated CAK activity . We have cloned and sequenced a cDNA coding for human p40MO15, and raised specific polyclonal and monoclonal antibodies against the corresponding protein expressed in Escherichia coli . These tools were then used to demonstrate that p40MO15 protein expression and CAK activity are constant throughout the somatic cell cycle . Gel filtration suggests that active CAK is a multiprotein complex, and immunoprecipitation experiments identify two polypeptides of 34 and 32 kD as likely complex partners of p40MO15 . The association of the three proteins is near stoichiometric and invariant throughout the cell cycle . Immunocytochemistry and biochemical enucleation experiments both demonstrate that p40MO15 is nuclear at all stages of the cell cycle (except for mitosis, when the protein redistributes throughout the cell), although the p34cdc2/cyclin B complex, one of the major purported substrates of CAK, occurs in the cytoplasm until shortly before mitosis . The absence of obvious changes in CAK activity in exponentially growing cells constitutes a surprise . It suggests that the phosphorylation state of threonine 161 in p34cdc2 (and the corresponding residue in other cdks) may be regulated primarily by the availability of the cdk/cyclin substrates, and by phosphatase(s). J Bacteriol, 1994 Oct, 176(20), 6404 - 6 Additive effect of tolC and rfa mutations on the hydrophobic barrier of the outer membrane of Escherichia coli K-12; Fralick JA et al.; Studies using tolC mutant derivatives of deep rough (rfa) mutants indicate that tolC and rfa mutations have an additive effect with respect to their sensitivity to hydrophobic agents, suggesting that they are not acting through a mutual mechanism to alter the permeability of the outer membrane. J Bacteriol, 1994 Oct, 176(20), 6392 - 6 Maturation and localization of the TolB protein required