Escherichia coli

Biochem J, 1994 Jul 1, 301 ( Pt 1), 17 - 20
Assisted refolding of recombinant prochymosin with the aid of protein disulphide isomerase; Tang B et al.; Protein disulphide isomerase (PDI) was shown to be able to accelerate the refolding of unfolded recombinant prochymosin and to enhance the overall yield of active protein . Unlike previous reports in this study PDI was found to be active at pH values as high as 11 . The coincidence of the similar apparent optimum pH values of uncatalysed and PDI-catalysed reactions suggests that conditions favourable to spontaneous refolding of proteins may help PDI to catalyse thiol/disulphide interchange . Under the conditions described here no exogenously added dithiothreitol was required for PDI-catalysed renaturation, implying that the disulphide form of PDI was reduced to its active form by the free thiol groups in prochymosin molecules.

J Pharmacol Exp Ther, 1994 Jul, 270(1), 356 - 61
Enhanced elimination of biotinylated antibodies by avidin-based hemoperfusion in rats; Burkhart KK et al.; We designed a hemoperfusion system using immobilized avidin for selective removal of biotinylated therapeutic antibodies from rats . Two prototype therapeutic antibodies, ovine antidigoxin Fab fragment and murine monoclonal 8A1 against Escherichia coli J5 lipopolysaccharide endotoxin, were biotinylated using biotin-long-chain-N-hydroxysuccinimide ester . Biotinylated antibodies were administered i.v . to anesthetized rats which were instrumented for measurement of cardiovascular parameters and connected to avidin-hemoperfusion devices . By using several different protocols of antibody administration and hemoperfusion, we found that the half-lives and areas under the time vs . concentration curves of biotinylated antibodies were reduced significantly after passage over immobilized avidin . Avidin hemoperfusion was not associated with adverse changes in blood pressure, heart rate or excess hemolysis . These data suggest that avidin hemoperfusion affords a means for selective removal of biotinylated ligands from serum, and that other therapeutic and potentially toxic compounds could be removed in this manner.

Eur J Biochem, 1994 Jul 1, 223(1), 69 - 77
Expression of phytochrome apoprotein from Avena sativa in Escherichia coli and formation of photoactive chromoproteins by assembly with phycocyanobilin; Hill C et al.; Phytochrome DNAs from oat (Avena sativa L.) encoding the full-length 124-kDa polypeptide, a 118-kDa fragment lacking the first 65 amino acids, and two N-terminal fragments of 65 kDa and 45 kDa were subcloned and expressed in Escherichia coli . Reducing the temperature to 25 degrees C during cell growth and the coexpression of chaperones improved the folding into a functional conformation for most of the polypeptides, and in one case the yield of polypeptides was also enhanced . A maximum yield of reconstitutable apoprotein was obtained by expressing the 65-kDa fragment consisting of 595 amino acids . The apoproteins could be assembled in the dark with phycocyanobilin into photoreversible chromoproteins . The yield of photoreversible pigment could be further increased by far-red/red irradiation cycles, indicating that the presence of the chromophore promotes the correct folding of the binding site . The chromoproteins with an intact N-terminal domain exhibit Pr and Pfr absorption bands, which are blue-shifted relative to the corresponding bands of native phytochrome due to the particular phycocyanobilin structure . The 118-kDa fragment, only lacking the 6-kDa N-terminus, exhibits a strong Pr band, but only a weak Pfr absorbance . This indicates an essential role of the front 6-kDa region of the protein in the formation of the far-red absorbing chromophore-protein complex . Otherwise, the C-terminal region seems to be less important for photoreversibility as indicated by the function of the shorter fragments.

Eur J Biochem, 1994 Jul 1, 223(1), 285 - 92
Isolation and refolding of a mutant methionine-free interleukin-2-receptor alpha chain synthesized as a fusion protein in Escherichia coli; Seipelt I et al.; A soluble domain of the interleukin(IL)-2 receptor, the alpha chain synthesized in Escherichia coli, was employed to study expression and refolding of the protein . The results showed that it is possible to obtain biologically active synthetic methionine-free IL-2 receptor alpha chain (synIL-2R alpha) after BrCN cleavage and renaturation of the crude cleavage material, although the alpha chain is expressed as a deglycosylated, methionine-free protein . The soluble receptor comprises amino acids 1-219 and forms 5 disulfide bonds in its biologically active state . Biological activity has been analysed by affinity chromatography and ELISA with mutant {Ala125}IL-2 and monoclonal antibodies as ligands . Renaturation yield is limited mainly by the high aggregation rate of incorrectly folded protein . Aggregation could be limited by varying the oxidation conditions . The deletion of a non-bridging cysteine at position 192 in the synIL-2R alpha did not affect the renaturation yield of the receptor protein . Additionally a cysteine-free and methionine-free beta-galactosidase derivative was fused to the soluble synIL-2R alpha derivatives to prevent reoxidation of incorrect disulfide bonds in the crude BrCN-cleavage material . It is suggested that cysteine impurities from cyanogen-bromide-cleaved peptides might interfere seriously with the refolding process of the synthetic IL-2 receptor alpha-subunit.

Eur J Biochem, 1994 Jul 1, 223(1), 275 - 83
Purification of ATP synthase from Acetobacterium woodii and identification as a Na(+)-translocating F1F0-type enzyme; Reidlinger J et al.; The ATPase of Acetobacterium woodii was purified after solubilization of membranes with Triton X-100 by poly(ethylene glycol) precipitation and gel filtration . The enzyme consists of at least six subunits of apparent molecular masses of 57, 52, 35, 19, 15 and 4.8 kDa, as determined by SDS/PAGE . The 52-kDa band is immunologically related to the F1F0-ATPase beta subunit of Escherichia coli . The enzyme is not inhibited by vanadate but is inhibited by nitrate, azide and N,N'-dicyclohexylcarbodiimide; the 4.8-kDa subunit specifically reacts with N,N'-dicyclohexyl{14C}carbodiimide, indicating that the enzyme is of the F1F0 type . The enzyme activity is dependent on MgATP (Km = 0.4), has a pH optimum of pH 7-9 and is stimulated by sulfite . ATP hydrolysis is strictly dependent on sodium ions with a Km for Na+ of 0.4 mM . The purified enzyme was reconstituted into liposomes . Upon addition of ATP, primary and electrogenic 22Na+ transport into the lumen of the proteoliposomes was determined . These experiments demonstrate that the ATPase of Acetobacterium woodii is a Na(+)-translocating F1F0-type ATPase.

Carcinogenesis, 1994 Jul, 15(7), 1371 - 5
Synthesis of a 25 base oligonucleotide containing a styrene oxide modification at the O6 position of 2'-deoxyguanosine at a defined site and incorporation studies of the similarly modified 2'-deoxyguanosine-5'-triphosphate; Pongracz K et al.; A diastereomeric mixture of the regioisomers O6-(2-hydroxy-2-phenylethyl)-2'-deoxyguanosine (st6G, beta-isomer) and O6-(2-hydroxy-1-phenylethyl)-2'-deoxyguanosine (alpha-isomer) was site-specifically placed in a 25 base oligonucleotide template 5'-CCGCTAst6GCGGGTACCGAGCTCGAAT-3' using CED phosphoramidite chemistry . Using 32P-post-labeling we found the oligonucleotide to contain 95% of the beta-isomer and 5% of the alpha-isomer of st6G . st6G as the 3'-phosphate was found to be considerably more acid labile than O6-methyl-2'-deoxyguanosine-3'-phosphate, leading to dealkylation during oligonucleotide synthesis . The diastereomeric mixture of O6-(2-hydroxy-2-phenylethyl)-2'-deoxy-guanosine-5'-triphosphate (st6dGTP) was chemically synthesized and used as a substrate for the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I . This study demonstrated that st6dGTP could be incorporated opposite deoxycytidine and did not completely block replication.

Arch Biochem Biophys, 1994 Jul, 312(1), 59 - 66
Expression of modified human cytochrome P450 2E1 in Escherichia coli, purification, and spectral and catalytic properties; Gillam EM et al.; Human cytochrome P450 (P450) 2E1 is of interest because of its role in the oxidation of numerous drugs and carcinogens . The purification of the protein from human liver is difficult, and we report the development of a system for relatively high-level expression in Escherichia coli . A cDNA was prepared from liver cDNA by polymerase chain reaction methods and several variants with modified 5'-termini were constructed . Analysis of seven of these indicated that the highest levels of expression were found when the first 21 codons of the native sequence were deleted and the Trp immediately following the resulting N-terminal Met was changed to Ala (GCT) . Levels of 40-nmol membrane-bound P450 2E1 (liter culture)-1 were routinely recovered . The recombinant P450 2E1 was purified to electrophoretic homogeneity from the bacterial membranes in two ion-exchange steps in > 80% yield . Ferric P450 2E1 was isolated in a mixed spin state . The enzyme was active in chlorzoxazone 6-hydroxylation; the addition of human liver cytochrome b5 lowered the Km for the substrate and increased Vmax . N-Terminal amino acid sequence analysis yielded the expected first 21 residues . The expression system should facilitate the availability of human P450 2E1 and antibodies for studies of the enzyme.

Arch Biochem Biophys, 1994 Jul, 312(1), 210 - 8
High substrate specificity factor ribulose bisphosphate carboxylase/oxygenase from eukaryotic marine algae and properties of recombinant cyanobacterial RubiSCO containing "algal" residue modifications; Read BA et al.; Marine algae play an important role in removing carbon dioxide from the atmosphere . In this investigation, we have determined the substrate specificity factor of ribulose 1,5-bisphosphate carboxylase/oxygenase from several marine chromophytic and rhodophytic algae . The enzymes were purified to homogeneity and all possessed significantly higher substrate specificity factors than the enzymes from terrestrial plants, green algae, or bacteria . There are substantial differences in the sequence in a helix 6 of the large subunit of these enzymes, which is intriguing since residues of this region had been previously shown to influence the ability of ribulose bisphosphate carboxylase to discriminate between CO2 and O2, presumably by influencing the adjacent flexible loop 6 region . Sequence divergence at this and other key regions might contribute to the substantial differences in the substrate specificity factor of the chromophyte/rhodophyte enzyme . Initial studies on probing the basis for the high substrate specificity factor employed single amino acid substitutions in the recombinant cyanobacterial ribulose bisphosphate carboxylase . Residues in the vicinity of loop 6 were changed to reflect the corresponding residues in the chromophyte/rhodophyte large subunit . Some changes in the substrate specificity factor were noted, as were alterations in other important kinetic parameters . Since marine algae show little evidence of photorespiratory metabolism, the high substrate specificity of ribulose bisphosphate carboxylase is consistent with the physiology of these organisms . The results of this study provide further evidence that the properties of this enzyme may evolve or change according to the environment in which the host organism is found.

Arch Biochem Biophys, 1994 Jul, 312(1), 121 - 4
Identification of the N-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system produced by proteolytic digestion; Lee BR et al.; The phosphoenolpyruvate:sugar phosphotransferase system of bacteria plays an important role in the concomitant uptake and phosphorylation of numerous sugars . The first protein in the pathway of phosphotransfer of the phosphoenolpyruvate:sugar phosphotransferase system is Enzyme I . It has been shown that a stable N-terminal domain can be produced by treatment of the purified protein with various proteolytic enzymes . We show here that the region from glutamate-252 to leucine-264 is accessible to proteolysis resulting in N-terminal cores ranging from M(r) 27521 to 28799.

Am J Obstet Gynecol, 1994 Jul, 171(1), 223 - 30
Phosphorylation of 17 beta-hydroxysteroid dehydrogenase in BeWo choriocarcinoma cells; Barbieri RL et al.; OBJECTIVE: The purpose of this study was to test whether 17 beta-hydroxysteroid dehydrogenase might exist in a phosphorylated form . STUDY DESIGN: Phosphorylation of 17 beta-hydroxysteroid dehydrogenase was evaluated in BeWo choriocarcinoma cells . The phosphorylation of 17 beta-hydroxysteroid dehydrogenase expressed in Escherichia coli as a glutathione transferase fusion protein was also studied . RESULTS: Human BeWo choriocarcinoma cells were metabolically labeled with phosphorus 32 orthophosphate . Immunoprecipitates were prepared with rabbit anti-17 beta-hydroxysteroid dehydrogenase antiserum from the labeled cells and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . A phosphorylated protein with a molecular size of 35 kd was obtained from anti-17 beta-hydroxysteroid dehydrogenase immunoprecipitates, which suggested that 17 beta-hydroxysteroid dehydrogenase was phosphorylated in BeWo cells . The predominant phosphoamino acid was phosphoserine . 17 beta-Hydroxysteroid dehydrogenase expressed in E . coli as a glutathione transferase fusion protein was a substrate of protein kinase A in vitro . Protein kinase A phosphorylated the recombinant 17 beta-hydroxysteroid dehydrogenase exclusively on serine . Incubation of BeWo cell lysates with bacterial alkaline phosphatase led to a decrease in the oxidative activity of 17 beta-hydroxysteroid dehydrogenase . Incubation of the alkaline phosphatase inhibitor levamisole with BeWo cell lysates resulted in a higher estradiol-to-estrone conversion rate, compared with cell lysates without any treatment . CONCLUSION: Our data suggest that 17 beta-hydroxysteroid dehydrogenase may exist in phosphorylated forms and that phosphorylation may regulate the activity of 17 beta-hydroxysteroid dehydrogenase in vivo.

J Trauma, 1994 Jul, 37(1), 82 - 9; discussion 89-90
Pre-exposure to hypoxia or septic stimuli differentially regulates endotoxin release of tumor necrosis factor, interleukin-6, interleukin-1, prostaglandin E2, nitric oxide, and superoxide by macrophages; West MA et al.; Shock states with resulting inadequate cellular oxygen delivery may contribute to macrophage (M phi) activation or dysregulation . In this study we compared the effects of transient anoxia and endotoxin pretreatment (LPS1) on M phi mediator release with a second endotoxin stimulus (LPS2) . METHODS: In vitro cultures of murine peritoneal exudate M phi were exposed to 2 hours of hypoxic or normoxic conditions, then incubated 22 hours under identical normoxic conditions +/- 10 ng/mL of LPS1 pretreatment . During the final 24 hours all M phis were exposed to a range of LPS2 concentrations . The M phi supernatants were assayed for tumor necrosis factor (TNF), interleukin 1 (IL-1), interleukin 6 (IL-6), prostaglandin E2 (PGE2), nitric oxide (NO), and superoxide release . RESULTS: LPS1 markedly inhibited M phi TNF release by LPS2, but hypoxia had no effect on LPS2-triggered TNF release . Hypoxia increased M phi IL-6 production in the absence of LPS1, but inhibited the LPS1 augmentation seen under normoxic conditions . Pretreatment with LPS1 increased NO production from LPS2 under normoxic conditions, but hypoxia inhibited this effect . Superoxide production increased by LPS1 under normoxic conditions, but hypoxia significantly inhibited superoxide release . CONCLUSIONS: The effects of transient anoxic exposure on LPS2-triggered M phi function are markedly different from the effects of pretreatment with septic stimuli (LPS1).

J Invest Dermatol, 1994 Jul, 103(1), 7 - 12
A potential laboratory test for dysplastic nevus syndrome: ultraviolet hypermutability of a shuttle vector plasmid; Moriwaki S et al.; The diagnosis of the melanoma-prone disorder dysplastic nevus syndrome (DNS) is based currently on a combination of clinical and histopathologic examinations of patients . To develop a potential laboratory test for DNS, we utilized the observation that an ultraviolet light (UV)-treated mutagenesis plasmid shuttle vector has an abnormally increased frequency of mutations after transfection into lymphoblastoid cells from a patient with familial DNS . pSP189 (containing the bacterial suppressor tRNA gene supF as a marker for mutations and a gene for ampicillin resistance for selection) was treated with UV and transfected into familial DNS, xeroderma pigmentosum complementation group A (XP-A), and normal lymphoblastoid cells by electroporation or diethylaminoethyl (DEAE) dextran . Untreated plasmid pZ189K (containing a gene for kanamycin resistance) was co-transfected as an internal standard to reduce the variability of plasmid survival measurements . After 2 d, plasmids were extracted, used to transform an indicator strain of Escherichia coli, and assayed on plates containing ampicillin or kanamycin . Counting light blue or white colonies (containing mutated supF in the plasmid) and blue colonies (with wild type supF) permitted measurement of the plasmid survival and mutation frequency . Transfection by electroporation or DEAE dextran resulted in abnormally reduced survival of UV-treated plasmid after passage through the XP-A but normal survival in the three DNS lines . Transfection of UV-treated plasmid by DEAE dextran yielded a greater hypermutability with the familial DNS lines than by electroporation . These results suggest that pSP189 UV hypermutability with normal UV survival using DEAE dextran transfection may form the basis of a potential laboratory assay for familial DNS.

J Allergy Clin Immunol, 1994 Jul, 94(1), 88 - 94
IgE-binding capacity of recombinant timothy grass (Phleum pratense) pollen allergens; Laffer S et al.; A panel of 60 cDNA clones coding for IgE-binding proteins from timothy grass pollen was immunocharacterized with sera from 30 patients allergic to grass pollen and antibodies raised against natural grass pollen allergens . In the cases of five representative patients in whom the IgE reactivity pattern with the recombinant allergens had been determined, IgE immunoadsorption experiments were performed . Recombinant Phl p I, Phl p V, and Phl p II and recombinant timothy grass profilin were used for immunoadsorption of the sera, and the percentage of remaining grass pollen-specific IgE was estimated . Although most of the patients showed IgE reactivity to a number of different natural and recombinant timothy grass pollen allergens, up to 66% of IgE directed against blotted total natural grass pollen allergens could be immunoadsorbed from the sera with recombinant Phl p V and Phl p I . The data point to the usefulness of recombinant allergens not only to determine IgE specificities of allergic patients but also to estimate the percentage of specific IgE that individuals produce against certain allergens . The fact that only a limited number of recombinant timothy grass pollen allergens account for a high percentage of grass pollen-specific IgE points to the possible usefulness of recombinant allergens not only for in vitro diagnosis but probably also for specific immunotherapy.

Crit Care Med, 1994 Jul, 22(7), S3 - 7
Biology of proinflammatory cytokines and their antagonists; Moldawer LL; OBJECTIVES: To review the role of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) in the proinflammatory cascade, with particular emphasis on the association between increased concentrations of these cytokines and the sepsis-associated shock syndrome . Data that support the role of these cytokines as proinflammatory mediators provide a rationale for specific anticytokine therapies . DATA SOURCES: Information presented at the 22nd Educational and Scientific Symposium of the Society of Critical Care Medicine on June 9-13, 1993 in New York City was reviewed, along with supportive documentation from the English language literature . STUDY SELECTION: Controlled animal studies that elucidate the relationship between TNF-alpha and IL-1, and endotoxin-induced shock were selected for review . DATA EXTRACTION: This review focused on those data that described the roles of IL-1 and TNF-alpha in the induction of the inflammatory cascade . DATA SYNTHESIS: Information concerning the many aspects of attenuating the systemic inflammatory response was integrated into a description of emerging therapies for septic shock . CONCLUSIONS: Induction of inflammation during sepsis is a complex biological cascade that may be effectively attenuated by novel anticytokine biotherapies.

J Toxicol Environ Health, 1994 Jul, 42(3), 275 - 88
Measurement of environmental formylmethionyl-peptides; Siegel PD et al.; Formylmethionyl-peptides are naturally occurring, biologically active ligands produced by bacteria . They produce a variety of biological effects including neutrophil chemotaxis, cellular degranulation, oxygen-free radical production, and smooth muscle contraction . Our studies have demonstrated that oxidized and reduced forms of formylmethionyl-leucyl-phenylalanine (fMLP) can be detected in bulk environmental organic dust samples . Organic dust fMLP content may not reflect total formylmethionyl-peptide content and pathological sequelae . Attempts to develop a total formylmethionyl-peptide assay that would reflect its pathological potential have thus far been unsuccessful . Information has been derived concerning the biology of formylmethionyl-peptides from these studies . Chromatographic, radioenzymatic, and radioreceptor-ligand binding studies were performed . High-performance liquid chromatography (HPLC) analysis of synthetic and environmental fMLP demonstrated that fMLP is labile, forming three oxidation products . HPLC is limited by inadequate sensitivity for air sample analysis and the probability of the presence of multiple formylmethionyl-peptides . Deformylases were isolated from Escherichia coli, but their usefulness in a competitive assay to detect formylmethionyl-peptides was limited by specificity differences from that for biological receptors . Receptor binding studies were conducted in an attempt to replace the deformylase with a biological receptor . The receptor binding patterns noted were consistent with the existence of three distinct formylmethionyl-peptide receptor subsets in neutrophils and alveolar macrophages . The plurality of fMLP receptor subtypes interfered with formylmethionyl-peptide measurement in a competitive assay . Formylmethionyl-peptides may contribute to organic dust-induced disease, but better techniques for the assessment of exposure to these agents are needed to properly assess their health impact.

J Mol Biol, 1994 Jul 1, 240(1), 87 - 91
Crystals of glutamine-binding protein in various conformational states; Hsiao CD et al.; Crystals of glutamine-binding protein (GlnBP) in various conformational states have been obtained . Crystals of the ligand-free "open" state (denoted form B) have unit cell dimensions a = 86.3 A, b = 86.3 A, c = 81.5 A, alpha = beta = gamma = 90 degrees and diffract to about 2.3 A resolution . An analysis of the intensity data using an Requiv plot indicates that the crystal system is orthorhombic, space group P2(1)2(1)2(1) . Crystals of the ligand-bound "open" state (form B*) are obtained by soaking form B crystals with glutamine (Gln) and diffract to about 1.9 A . Crystals of the GlnBP-Gln complex in a ligand-bound "closed" state (form C) belong to space group P2(1)2(1)2(1) with a = 62.0 A, b = 65.7 A and c = 121.8 A and diffract to about 2.3 A . Crystals of a selenomethionyl GlnBP (form B') are isomorphous to form B crystals and diffract to about 2.1 A resolution.

J Mol Biol, 1994 Jul 1, 240(1), 78 - 86
Structural model of the 50 S subunit of Escherichia coli ribosomes from solution scattering . II . Neutron scattering study; Svergun DI et al.; The X-ray and neutron contrast variation data of the 50 S ribosomal subunit of Escherichia coli in solution are interpreted in the frame of a two-phase model described by the shapes of the 50 S subunit and its RNA-rich core taking into account density fluctuations inside the RNA and the protein moiety . The shape of the envelope of the 50 S subunit and of the RNA-rich core are evaluated with a resolution of about 4 nm . The shape of the envelope is in good agreement with the models of the 50 S subunit obtained from electron microscopy on isolated particles . The shape of the RNA-rich core correlates well with the model of the entire particle determined by the image reconstruction from ordered sheets indicating that the latter model which is based on the subjective contouring of density maps is heavily biased towards the RNA.

J Mol Biol, 1994 Jul 1, 240(1), 66 - 77
Structural model of the 50 S subunit of Escherichia coli ribosomes from solution scattering . I . X-ray synchrotron radiation study; Svergun DI et al.; The application of new methods of small-angle scattering data interpretation to a contrast variation study of the 50 S ribosomal subunit of Escherichia coli in solution is described . The experimental X-ray data from contrast variation with sucrose are analysed in terms of the basic functions in real space and the scattering curves from the volume inaccessible to sucrose and from the regions inside this volume occupied mainly by RNA and by proteins are obtained . From these curves models of the shape of the 50 S subunit and its RNA-rich core are evaluated . These two shapes are positioned so that their difference, which approximates the volume occupied by the proteins, produces a scattering curve which is in good agreement with the scattering from the protein moiety.

J Mol Biol, 1994 Jul 1, 240(1), 52 - 65
Genetic evidence for IS1 transposition regulated by InsA and the delta InsA-B'-InsB species, which is generated by translation from two alternative internal initiation sites and frameshifting; Matsutani S; Insertion sequence IS1 contains two reading frames, insA and B'-insB, which are responsible for its transposition, and was previously shown to express two proteins . The first, InsA, is the product of insA . The second, InsA-B'-InsB is a fusion of InsA with the product of B'-insB . Synthesis of this protein occurs by a -1 frameshift from the 3' region of the insA frame to the open reading frame B', extending from the 5' end of the insB frame . Here, I have shown genetically that IS1 encodes the third species delta InsA-B'-InsB: delta InsA-B'-InsB uses two alternative initiation codons in the middle of the insA frame, and is produced by a frameshift mechanism similar to that used in InsA-B'-InsB expression . Deletion of the small region preceding these initiation codons resulted in decreased expression of delta InsA-B'-InsB, suggesting that the small region play some role in the translation initiation . Surprisingly, it was found that delta InsA-B'-InsB has a transposase-like function and InsA can stimulate the transposition promoted by delta InsA-B'-InsB, while delta InsA-B'-InsB seemed to bind to the left terminal inverted repeat (IRL) of IS1 and inhibit transposition when it was present in excess, as well as InsA represses transposition . It is likely that IS1 transposition activity depends on the ratio of InsA to delta InsA-B'-InsB . A double missense mutation of the internal initiation codons resulted in decreased cointegration activity, showing that delta InsA-B'-InsB is responsible for transposition but InsA-B'-InsB is probably not . Some IS elements, which also contain two tandem, out-of-phase, overlapping genes, appear to express deleted fusion proteins like delta InsA-B'-InsB, but the functions are unknown . The complex phenomena of transposition and its control found in IS1 may be more general in the other mobile DNAs.

J Mol Biol, 1994 Jul 1, 240(1), 1 - 7
Suppression of yeast RNA polymerase III mutations by the URP2 gene encoding a protein homologous to the mammalian ribosomal protein S20; Hermann-Le Denmat S et al.; URP2 was cloned as a multicopy suppressor of several temperature-sensitive mutations defective in RNA polymerase III-dependent transcription, but without effect on mutations affecting RNA polymerase I or II . This single-copy gene encodes a hydrophilic polypeptide of 121 amino acid residues with a predicted molecular mass of 13.9 kDa and a basic isoelectric point of 9.7 . URP2 is a highly expressed gene, judging from its abundant messenger RNA and strong codon bias . The Urp2p protein is essential for cell growth, as shown by the lethal phenotype of the urp2::HIS3 null allele . Given its striking similarity to the S20 ribosomal polypeptide of rat (55% identical residues), Urp2p is in all likelihood the yeast form of this polypeptide . Both proteins are significantly related to S10, a component of the small ribosomal subunit of Escherichia coli that is known to operate as a transcriptional elongation factor . The latter observation suggests that the suppressor effect of URP2 may be due to a direct involvement of Urp2p in RNA polymerase III-dependent transcription . Alternatively, the overexpression of Urp2p could bypass a partial preribosomal RNA processing defect associated with RNA polymerase III mutants . URP2 was assigned to the left arm of chromosome VIII, and maps between DUR3 and YLF1 . The latter gene product has homology to the E . coli gtp1 gene product, and may define a new family of putative GTP-binding proteins.

J Gen Virol, 1994 Jul, 75 ( Pt 7), 1525 - 33
Partial characterization of the lettuce infectious yellows virus genomic RNAs, identification of the coat protein gene and comparison of its amino acid sequence with those of other filamentous RNA plant viruses; Klaassen VA et al.; Purified virions of lettuce infectious yellows virus (LIYV), a tentative member of the closterovirus group, contained two RNAs of approximately 8500 and 7300 nucleotides (RNAs 1 and 2 respectively) and a single coat protein species with M(r) of approximately 28,000 . LIYV-infected plants contained multiple dsRNAs . The two largest were the correct size for the replicative forms of LIYV virion RNAs 1 and 2 . To assess the relationships between LIYV RNAs 1 and 2, cDNAs corresponding to the virion RNAs were cloned . Northern blot hybridization analysis showed no detectable sequence homology between these RNAs . A partial amino acid sequence obtained from purified LIYV coat protein was found to align in the most upstream of four complete open reading frames (ORFs) identified in a LIYV RNA 2 cDNA clone . The identity of this ORF was confirmed as the LIYV coat protein gene by immunological analysis of the gene product expressed in vitro and in Escherichia coli . Computer analysis of the LIYV coat protein amino acid sequence indicated that it belongs to a large family of proteins forming filamentous capsids of RNA plant viruses . The LIYV coat protein appears to be most closely related to the coat proteins of two closteroviruses, beet yellows virus and citrus tristeza virus.

J Biol Chem, 1994 Jul 1, 269(26), 17723 - 9
The N-terminal alpha-helix of fragment B of diphtheria toxin promotes translocation of fragment A into the cytoplasm of eukaryotic cells; Madshus IH; Diphtheria toxin consists of two parts, fragments A and B . Fragment A has enzymatic activity inhibiting protein synthesis . Fragment B binds to cellular receptors and, upon exposure to low pH, inserts into the membrane, forms cation-selective channels, and facilitates translocation of fragment A . Previous data have suggested that the N-terminal part of fragment B, including the amphipathic alpha-helix TH1, plays an active role during translocation of fragment A (Madshus, I . H., Wiedlocha, A., and Sandvig, K . (1994) J . Biol . Chem . 269, 4648-4652) . When replacing charged residues in TH1 with uncharged amino acids, translocation of fragment A was strongly inhibited, virtually without affecting binding of the toxin or channel activity . These data suggest that TH1 may act as a targeting/anchoring sequence . In a mutant with eight positive charges and one negative charge in TH1, increased specific binding was observed, even if TH1 was outside the toxin's binding domain . This suggests that TH1 could be important in binding to parts of the translocation machinery . Fragment A associated with this mutant fragment B was translocated 10-fold more efficiently than wild-type toxin . The fact that this mutant TH1 efficiently promoted translocation, while, a hydrophobic TH1 did not, suggests that TH1 does not interact with the hydrophobic part of the membrane phospholipids.

J Biol Chem, 1994 Jul 1, 269(26), 17663 - 9
DNA-binding domain and recognition sequence of the yeast BAS1 protein, a divergent member of the Myb family of transcription factors; Hovring I et al.; The yeast BAS1 protein is a transcriptional activator with an amino-terminal domain homologous to the DNA-binding domain of the oncoprotein Myb containing three imperfect tryptophan-rich repeats . In contrast to Myb-related transcription factors from higher eukaryotes, where the second and third repeat constitutes a minimal independent DNA-binding domain, all three repeats of BAS1 were found to be necessary for sequence-specific DNA binding . Moreover, an active DNA-binding subdomain was obtained only if the first repeat was enlarged in the amino-terminal direction to include 3 tryptophans and a 23-amino acid insertion and if 55 amino acids carboxyl-terminal to the third repeat were included . The BAS1 DNA-binding site was analyzed in detail and found to cover 8-9 base pairs with no similarity to the Myb recognition element . The binding site included a conserved hexameric TGACTC motif, the methylation of which abolished BAS1 binding, as well as a 3-base pair extension that seemed to have a modulatory effect on BAS1 affinity and where binding was less affected by methylation.

J Biol Chem, 1994 Jul 1, 269(26), 17642 - 8
Breakpoint cluster region gene product-related domain of n-chimaerin . Discrimination between Rac-binding and GTPase-activating residues by mutational analysis; Ahmed S et al.; The breakpoint cluster region gene product (Bcr) is a GTPase-activating protein (GAP) for members of the Rho family, Cdc42Hs, and Rac1, as is the brain protein n-chimaerin . At least 15 proteins have sequence identity to the GAP domain (150 amino acid residues) of Bcr . The widespread occurrence of proteins that possess sequence identity to the Bcr-related GAP domain makes it especially important to understand its structure/function relationships . Amino acid sequence alignment of these proteins reveals three blocks of conservation in the GAP domain . Here, we present a mutational analysis of this domain using n-chimaerin sequences . Ten mutations were constructed (at least two in each of the blocks of conservation), expressed as glutathione S-transferase fusion proteins in Escherichia coli, and purified . Seven of the mutants, including deletions, still possessed GAP activity for Rac1 . Three of the mutants had no Rac1-GAP activity but were still able to bind Rac1 . IC50 values obtained from competition experiments suggest that n-chimaerin and the mutants with no GAP activity bound Rac1 with similar apparent binding constants . Thus, this mutant analysis allows discrimination between Rac1-binding and Rac1 GTPase- activating residues.

J Biol Chem, 1994 Jul 1, 269(26), 17626 - 34
Purification, cloning, and expression of a murine phosphoprotein that binds the kappa B motif in vitro identifies it as the homolog of the human heterogeneous nuclear ribonucleoprotein K protein . Description of a novel DNA-dependent phosphorylation process; Ostrowski J et al.; The kappa B enhancer element regulates expression of many genes involved in immune responses and other processes . kappa B motif binds a number of proteins, some but not all, are related to the NF-kappa B family of transcription factors . We have previously identified a 65-kDa phosphoprotein that is specifically recognized by the kappa B motif (Ostrowski, J., Sims, J . E., Sibley, C . H., Valentine, M . A., Dower, S . K., Meier, K . E., and Bomsztyk, K . (1991) J . Biol . Chem . 266, 12722-12733) . This protein is closely associated with a serine/threonine kinase that is responsive to treatment of cells with interleukin-1 and other agents . We report here purification, cloning, and expression of this kappa B motif-binding phosphoprotein . The primary structure deduced from the isolated murine cDNA, identifies the protein as the homolog of the human heterogeneous nuclear ribonucleoprotein K protein . Antipeptide antibodies and expression of the cloned cDNA in Escherichia coli, demonstrated that the K protein is the authentic phosphoprotein that binds the kappa B motif in vitro . We also demonstrate that the in vitro phosphorylation of the natural and the recombinant K proteins by the associated kinase is stimulated by the kappa B motif.

J Biol Chem, 1994 Jul 1, 269(26), 17464 - 8
The 86-kDa subunit of autoantigen Ku is a somatostatin receptor regulating protein phosphatase-2A activity; Le Romancer M et al.; We previously reported the immunopurification of a somatostatin receptor from the human tumoral gastric cell HGT1 using the monoclonal antibody 30F3 (Reyl-Desmars, F., Le Roux, S., Linard, C., Benkouka, F., and Lewin, M . J . M . (1989) J . Biol . Chem . 264, 18789-18795) . Screening of a lambda gt11 HGT1-cDNA library with 30F3 led us to isolate a cDNA encoding an 86-kDa polypeptide displaying 100% structural identity with the 86-kDa subunit (p86-Ku) of the Ku autoantigen . Recombinant p86 expressed in Escherichia coli cross-reacted with 30F3 and specifically bound {125I-Tyr11}somatstatin-14 . Binding was totally displaced by somatostatin-14, somatostatin-28, and SMS 201-995, with IC50 values of 0.7, 1.0, and 1.2 nM, respectively . In a search for a biological effect associated with binding, we purified a 36-kDa, okadaic acid-sensitive phosphatase (protein phosphatase-2A (PP2A)) from rat gastric cytosol . PP2A catalyzed 32P release from p34cdc2-phosphorylated histone H1 . However, PP2A-induced 32P release was concentration dependently inhibited by recombinant p86-Ku, with a decrease in maximal velocity without a change in Km . Steric exclusion high pressure chromatography indicated that the inhibition resulted from direct interaction of the enzyme with p86-Ku . Furthermore, it was antagonized by increased concentrations of somatostatin-14 and prevented by preincubating p86-Ku with 30F3 . Given the key role played by PP2A in cell cycle regulation, the current findings suggest that p86-Ku could be a physiological target of somatostatin antiproliferative action.

J Biol Chem, 1994 Jul 1, 269(26), 17454 - 7
Expression in Escherichia coli of the two subunits of the isozyme form of wheat germ protein synthesis initiation factor 4F . Purification of the subunits and formation of an enzymatically active complex; van Heerden A et al.; The subunits (p28 and p86) of the isoenzyme form of eukaryotic initiation factor 4F (eIF-(iso)4F) from wheat were expressed separately in Escherichia coli . The subunits were purified by affinity chromatography (p28) and ion-exchange chromatography (p86) . The purified subunits alone did not support polypeptide synthesis in an eIF-(iso)4F and eIF-4F-deficient translation system from wheat germ . However, when the two subunits were mixed together, activity equal to that of the native form of eIF-(iso)4F was obtained . These results show that subunits expressed separately are able to associate and form an enzymatically active complex.

J Biol Chem, 1994 Jul 1, 269(26), 17394 - 6
Ribonuclease A can be transformed into a dimeric ribonuclease with antitumor activity; Di Donato A et al.; A cDNA coding for bovine pancreatic RNase A was mutagenized to insert a proline, a leucine, and 2 cysteine residues, i.e . the residues present at corresponding positions in the subunit of seminal RNase, the only dimeric RNase of the pancreatic-type superfamily . The mutant, expressed in Escherichia coli, eventually aggregated into catalytically active dimers . Like naturally dimeric seminal RNase, at equilibrium the mutant dimeric RNase A adopted two quaternary structures (one with an exchange of the N-terminal segments between partner subunits, the other with no exchange) and displayed a selective toxicity for malignant cells, absent in the monomeric, parent protein.

J Bacteriol, 1994 Jul, 176(14), 4455 - 8
Requirements for mobilization of plasmids RSF1010 and ColE1 by the IncW plasmid R388: trwB and RP4 traG are interchangeable; Cabezon E et al.; Mobilization of plasmid RSF1010 by the IncW plasmid R388 requires the genes involved in W pilus synthesis plus trwB . traG of the IncP plasmid RP4 can substitute for trwB in RSF1010 mobilization by R388 but not in self-transfer of R388 . This result suggests a dual specificity of TrwB-like proteins in conjugation . The same genetic requirements were found for R388 to mobilize the unrelated plasmid ColE1.

J Bacteriol, 1994 Jul, 176(14), 4444 - 7
ssaD1, a suppressor of secA51(Ts) that renders growth of Escherichia coli cold sensitive, is an early amber mutation in the transcription factor gene nusB; Rajapandi T et al.; Complementation analysis of the ssaD1 mutation, isolated as a suppressor of the secA51(Ts) mutation that renders growth of Escherichia coli cold sensitive, was used to show that ssaD corresponds to nusB, a gene known to be important in transcription antitermination . DNA sequence analysis of the ssaD1 allele showed that it creates an amber mutation in the 15th codon of nusB . Analysis of the effect of different levels of NusB protein on secA transcription and translation suggested that NusB plays little or no role in the control of secA expression . Accordingly, mechanisms by which nusB inactivation can lead to suppression of secA51(Ts) and secY24(Ts) mutations without affecting secA expression need to be considered.

J Bacteriol, 1994 Jul, 176(14), 4424 - 9
Genetic determination of the meso-diaminopimelate biosynthetic pathway of mycobacteria; Cirillo JD et al.; The increasing incidence of multiple-drug-resistant mycobacterial infections indicates that the development of new methods for treatment of mycobacterial diseases should be a high priority . meso-Diaminopimelic acid (DAP), a key component of a highly immunogenic subunit of the mycobacterial peptidoglycan layer, has been implicated as a potential virulence factor . The mycobacterial DAP biosynthetic pathway could serve as a target for design of new antimycobacterial agents as well as the construction of in vivo selection systems . We have isolated the asd, dapA, dapB, dapD, and dapE genes involved in the DAP biosynthetic pathway of Mycobacterium bovis BCG . These genes were isolated by complementation of Escherichia coli mutations with an expression library of BCG DNA . Our analysis of these genes suggests that BCG may use more than one pathway for biosynthesis of DAP . The nucleotide sequence of the BCG dapB gene was determined . The activity of the product of this gene in Escherichia coli provided evidence that the gene may encode a novel bifunctional dihydrodipicolinate reductase and DAP dehydrogenase.

J Bacteriol, 1994 Jul, 176(14), 4416 - 23
Cloning, sequencing, and mutational analysis of the hyb operon encoding Escherichia coli hydrogenase 2; Menon NK et al.; The genes encoding the two structural subunits of Escherichia coli hydrogenase 2 (HYD2) have been cloned and sequenced . They occur in an operon (hyb) which contains seven open reading frames . An hyb deletion mutant (strain AP3) failed to grown on dihydrogen-fumarate medium and also produced very low levels of HYD1 . All seven open reading frames are required for restoration of wild-type levels of active HYD2 in AP3 . The hyb operon was mapped at 65 min on the E . coli chromosome.

J Bacteriol, 1994 Jul, 176(14), 4321 - 7
Replacement of diaminopimelic acid by cystathionine or lanthionine in the peptidoglycan of Escherichia coli; Mengin-Lecreulx D et al.; In Escherichia coli, auxotrophy for diaminopimelic acid (A2pm) can be suppressed by growth with exogenous cystathionine or lanthionine . The incorporation of cystathionine into peptidoglycan metabolism was examined with a dapA metC mutant, whereas for lanthionine, a dapA metA mutant strain was used . Analysis of peptidoglycan precursors and sacculi isolated from cells grown with epimeric cystathionine or lanthionine showed that meso-A2pm was totally replaced in the same position by either sulfur-containing amino acid . Moreover, mainly L-allo-cystathionine (95%) or meso-lanthionine (93%) was incorporated into the precursors and sacculi . For this purpose, a new, efficient high-pressure liquid chromatography (HPLC) technique for analysis of the cystathionine isomers was developed . The formation of the UDP-MurNAc tripeptide appeared to be a critical step, since the MurE synthetase accepted meso-lanthionine or D-allo- or L-allo-cystathionine in vitro as good substrates, although with higher Km values . Presumably, the 10-fold-higher UDP-MurNAc-L-Ala-D-Glu pool of cells grown with cystathionine or lanthionine ensured a normal rate of synthesis . The kinetic parameters of the MurF synthetase catalyzing the addition of D-alanyl-D-alanine were very similar for the meso-A2pm-,L-allo-cystathionine-, and meso-lanthionine-containing UDP-MurNAc tripeptides . HPLC analysis of the soluble fragments resulting from 95% digestion by Chalaropsis N-acetylmuramidase of the peptidoglycan material in isolated sacculi revealed that the proportion of the main dimer was far lower in cystathionine and lanthionine sacculi.

J Bacteriol, 1994 Jul, 176(14), 4311 - 5
Physiological role of the chaA gene in sodium and calcium circulations at a high pH in Escherichia coli; Ohyama T et al.; Ohyama et al . previously isolated Escherichia coli mutant RS1, which had a negligible activity for sodium ion extrusion at alkaline pH (T . Ohyama, R . Imaizumi, K . Igarashi, and H . Kobayashi, J . Bacteriol . 174:7743-7749, 1992) . Our present study showed that the mutation of RS1 was compensated for by a cloned chaA gene . It has been proposed that sodium ion extrusion by ChaA is prevented under physiological conditions (D . M . Ivey, A . A . Guffanti, J . Zemsky, E . Pinner, R . Karpel, E . Padan, S . Schuldiner, and T . A . Krulwich, J . Biol . Chem . 268:11296-11303, 1993) . In order to clarify the physiological role of chaA in sodium ion circulation at alkaline pH, we constructed a delta chaA mutant . The resultant mutant, TO112, deficient in both nhaA and chaA, was unable to grow at pH 8.5 in medium containing 0.1 M sodium chloride and had negligible sodium ion extrusion activity . However, TO112 grew at pH 7.0 in medium containing 0.4 M sodium chloride . Sodium ions were extruded from TO112 cells at neutral pH . The extrusion activity at pH 7.5 was greatly reduced by the deletion of nhaB . These data demonstrate that the activity of nhaB is low at high pH and that ChaA extrudes sodium ions at alkaline pH . The uptake of calcium ions by everted membrane vesicles prepared from the delta chaA mutant TO110 was 60% of the activity observed in the vesicles of the wild-type strain at pH 8.5, but the activity at neutral pH was not reduced by the deletion of chaA . Therefore, it was also suggested that ChaA plays a role in calcium ion circulation at alkaline pH.

J Bacteriol, 1994 Jul, 176(14), 4306 - 10
A statistical analysis of the formation of plasmid-free cells in populations of Escherichia coli; Tolker-Nielsen T et al.; By methods analogous to those used in the classical statistical analysis of bacterial mutation, we have analyzed the formation of plasmid-free cells in populations of Escherichia coli harboring pBR322-derived plasmids . Application of fluctuation tests and papilla analysis suggested that there is a high variance in the probability that a plasmid-containing cell will produce a plasmid-free daughter cell . Apparently a subpopulation of plasmid-containing cells gives rise to progeny that produces plasmid-free cells with a high and unpredictable rate . This finding raises the question of whether plasmid maintenance can be adequately described by the conventional mathematical models.

J Bacteriol, 1994 Jul, 176(14), 4296 - 305
Membrane insertion defects caused by positive charges in the early mature region of protein pIII of filamentous phage fd can be corrected by prlA suppressors; Peters EA et al.; The filamentous phage coat protein pIII has been used to display a variety of peptides and proteins to allow easy screening for desirable binding properties . We have examined the biological constraints that restrict the expression of short peptides located in the early mature region of pIII, adjacent to the signal sequence cleavage site . Many functionally defective pIII fusion proteins contained several positively charged amino acids in this region . These residues appear to inhibit proper insertion of pIII into the Escherichia coli inner membrane, blocking the assembly and extrusion of phage particles . Suppressor mutations in the prlA (secY) component of the protein export apparatus dramatically alleviate the phage growth defect caused by the positively charged residues . We conclude that insertion of pIII fusion proteins into the inner membrane can occur by a sec gene-dependent mechanism . The suppressor strains should be useful for increasing the diversity of peptides displayed on pIII in phage libraries.

J Bacteriol, 1994 Jul, 176(14), 4285 - 95
Essential motifs of relaxase (TraI) and TraG proteins involved in conjugative transfer of plasmid RP4; Balzer D et al.; Two essential transfer genes of the conjugative plasmid RP4 were altered by site-directed mutagenesis: traG of the primase operon and traI of the relaxase operon . To evaluate effects on the transfer phenotype of the point mutations, we have reconstituted the RP4 transfer system by fusion of the transfer regions Tra1 and Tra2 to the small multicopy replicon ColD . Deletions in traG or traI served to determine the Tra phenotype of mutant plasmids by trans complementation . Two motifs of TraG which are highly conserved among TraG-like proteins in several other conjugative DNA transfer systems were found to be essential for TraG function . One of the motifs resembles that of a nucleotide binding fold of type B . The relaxase (TraI) catalyzes the specific cleaving-joining reaction at the transfer origin needed to initiate and terminate conjugative DNA transfer (W . Pansegrau, W . Schroder, and E . Lanka, Proc . Natl . Acad . Sci . USA 90:2925-2929, 1993) . Phenotypes of mutations in three motifs that belong to the active center of the relaxase confirmed previously obtained biochemical evidence for the contributions of the motifs to the catalytic activity of TraI . Expression of the relaxase operon is greatly increased in the absence of an intact TraI protein . This finding suggests that the relaxosome which assembles only in the presence of the TraI in addition to its enzymatic activity plays a role in gene regulation.

J Bacteriol, 1994 Jul, 176(14), 4197 - 203
In vivo studies of the role of SecA during protein export in Escherichia coli; Chun SY et al.; SecA is found in Escherichia coli both tightly associated with the cytoplasmic membrane where it functions as a translocation ATPase during protein export and free in the cytosol (R . J . Cabelli, K . M . Dolan, L . Qian, and D . B . Oliver, J . Biol . Chem . 266:24420-24427, 1991; D . B . Oliver and J . Beckwith, Cell 30:311-319, 1982; W . Wickner, A . J . M . Driessen, and F.-U . Hartl, Annu . Rev . Biochem . 60:101-124, 1991) . Here we show that SecA can be immunoprecipitated from the cytosol in complex with both fully elongated and nascent species of the precursor of maltose-binding protein, an exported, periplasmic protein . In addition, under conditions in which the distribution of SecA between the cytosolic and membrane-bound states changes from that normally observed, the distribution of precursor maltose-binding protein changes in parallel . These results support the idea that cytosolic SecA plays a role in export . With the aim of determining the roles of the multiple binding sites for ATP on SecA, we compared the export defect in a culture of E . coli expressing a temperature-sensitive allele of secA with the defect in a culture treated with sodium azide . The results indicate that the mutational change and treatment with sodium azide inhibit export by affecting different steps in the cycle of ATP binding and hydrolysis by SecA.

J Bacteriol, 1994 Jul, 176(13), 4177 - 81
Purification and characterization of the cytochrome bd complex from Azotobacter vinelandii: comparison to the complex from Escherichia coli; Kolonay JF Jr et al.; Partial purification of a cytochrome bd complex from Azotobacter vinelandii grown under high aeration was achieved by isolating respiratory particles enriched in this hemoprotein via differential centrifugation and detergent extraction . The cytochrome bd complex was subsequently solubilized from the inner membrane with dodecyl maltoside and purified to near homogeneity via DEAE-Sepharose chromatography . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the complex consisted of two subunits, with sizes in good agreement with those predicted from the cloned cyd locus (59.7 and 42 kDa) . Spectral analysis of the purified complex indicated that the heme components present were cytochromes b560, b595, and d; CO difference spectral studies identified cytochrome d as a CO-reactive component . The complex had a Km for ubiquinol-1 approximately seven times larger than that for the analogous bd complex from Escherichia coli, and O2 consumption curves revealed a Km value for O2 three times greater than that which we determined for the E . coli bd complex.

J Bacteriol, 1994 Jul, 176(13), 4165 - 7
Flanking sequences affect replication arrest at the Escherichia coli terminator TerB in vivo; Bierne H et al.; We have analyzed the effect of flanking sequences on Tus-induced replication arrest . pBR322 plasmid derivatives which carry the Escherichia coli replication terminator TerB at different locations were used . Efficiency of the replication arrest was estimated from the plasmid copy number and transformation frequency of tus+ cells . We found that flanking sequences do affect replication arrest efficiency, a weak arrest being correlated with the presence of an AT-rich region which is replicated just before TerB . Some sequences located after the replication terminator can also affect replication termination . We propose that the AT-rich regions might impair binding of the Tus protein to the TerB sequence or facilitate helicase-induced unwinding of DNA and Tus displacement from the TerB site.

J Bacteriol, 1994 Jul, 176(13), 4111 - 6
SecF stabilizes SecD and SecY, components of the protein translocation machinery of the Escherichia coli cytoplasmic membrane; Sagara K et al.; The effect of the overproduction of SecF encoded by the tac-secF gene on a plasmid on the synthesis of other Sec proteins was studied in Escherichia coli . SecF overproduction resulted in the simultaneous overproduction of SecD encoded by the tac-secD gene on a plasmid . Deletion of the orf6 gene, located downstream of the secF gene, had no effect on SecD overproduction . A pulse-chase experiment revealed that the overproduction was due to stabilization of SecD with SecF . SecF overproduction also resulted in the overproduction of SecY encoded by the tac-secY gene on a plasmid as well . SecF overproduction also enhanced the level of SecY expressed by the chromosomal secY gene . This SecF effect was not due to its effect on SecD or SecE, since SecF overproduction did not affect the levels of SecD and SecE expressed by the chromosomal secD and secE genes, respectively . SecE-dependent overproduction of SecY has already been demonstrated . It is suggested that SecF interacts with both SecD and SecY . SecE-SecY interaction has been demonstrated . It is likely, therefore, that all Sec proteins in the cytoplasmic membrane interact with each other.

J Bacteriol, 1994 Jul, 176(13), 4081 - 91
Autoregulation of hip, an operon that affects lethality due to inhibition of peptidoglycan or DNA synthesis; Black DS et al.; The hip locus of Escherichia coli affects the frequency of persistence to the lethal consequences of selective inhibition of either DNA or peptidoglycan synthesis . Regulation of the hip operon, which consists of a regulatory region and two genes, hipB and hipA, was examined with strains containing a hip-lac transcriptional fusion placed in single copy at the lambda att site . Disruption of the hip locus increased activity from the fusion 16-fold . Repression was restored by supplying HipB in trans . HipB was overexpressed and purified . On the basis of gel filtration and cross-linking studies, HipB is a dimer in solution . Sequence analysis revealed that HipB is a Cro-like DNA-binding protein . The interaction of HipB with the hip regulatory region was examined by gel retardation, DNase I protection, and methylation protection studies . HipB binds with a Kapp (K apparent) of 40 pM to four operator sites with the conserved sequence TATCCN8GGATA (N represents any nucleotide) . Binding to the operators is nearly simultaneous and appears to be cooperative . Analysis of the role of HipA in the regulation of the hip operon is complicated by the toxicity of HipA in the absence of HipB . Strains disrupted in hipB but not in hipA could not be recovered . Moreover, hipA-containing plasmids cannot be replicated in strains defective in or lacking hipB . HipA is found exclusively in a tight complex with HipB . Although disruption of hipA slightly increased expression from the hip-lac fusion, in vitro studies suggest that HipA does not bind to the hip regulatory region directly but indirectly via HipB.

J Bacteriol, 1994 Jul, 176(13), 4011 - 6
Mutational analysis of sequences in the recF gene of Escherichia coli K-12 that affect expression; Sandler SJ et al.; The level of translation of recF-lacZ fusions is reduced 20-fold by nucleotides 49 to 146 of recF . In this region of recF, we found a previously described ribosome-interactive sequence called epsilon and a hexapyrimidine tract located just upstream of the epsilon sequence . Mutational studies indicate that the hexapyrimidine sequence is involved in at least some of the reduced translation . When the hexapyrimidine sequence is mutant, mutating epsilon increases the level of translation maximally . We ruled out the possibility that ribosome frameshifting explains most of the effect of these two sequences on expression and suspect that multiple mechanisms may be responsible . In a separate report, we show that mutations in the hexapyrimidine tract and epsilon increase expression of the full-sized recF gene.

J Bacteriol, 1994 Jul, 176(13), 3944 - 55
Promoter and operator determinants for fur-mediated iron regulation in the bidirectional fepA-fes control region of the Escherichia coli enterobactin gene system; Hunt MD et al.; The fepA-entD and fes-entF operons in the enterobactin synthesis and transport system are divergently transcribed from overlapping promoters, and both are inhibited by the Fur repressor protein under iron-replete conditions . A plasmid harboring divergent fepA'-phoA and fes-entF'-'lacZ fusions, both under the control of this bidirectional regulatory region, was constructed for the purpose of monitoring changes in expression of the two operons simultaneously . Deletion analysis, site-directed mutagenesis, and primer extension were employed to define both a single promoter governing the expression of fes-entF and two tandemly arranged promoters giving rise to the opposing fepA-entD transcript . A single Fur-binding site that coordinately regulates the expression of all transcripts emanating from this control region was identified by in vitro protection from DNase I nicking . The substitution of one base pair in the Fur recognition sequence relieved Fur repression but did not change the in vitro affinity of Fur for its binding site . Additional mutations in a limited region outside of the promoter determinants for either transcript inhibited expression of both fes and fepA . These observations suggest a mechanism of Fur-mediated regulation in this compact control region that may involve other regulatory components.

J Bacteriol, 1994 Jul, 176(13), 3928 - 35
Characterization of the sigma 38-dependent expression of a core Escherichia coli starvation gene, pexB; Lomovskaya OL et al.; A reverse genetics approach was used to clone a pex starvation gene that codes for an 18-kDa polypeptide, designated PexB . Single-copy pexB-lacZ operon fusions were constructed to study transcriptional regulation and the promoter region of this gene . The induction by carbon starvation or osmotic stress was transcriptional and controlled by sigma 38 but was independent of this sigma factor by the oxidative stress; presumably, it was sigma 70 mediated under the latter stress . During nitrogen starvation, the induction was controlled at the posttranscriptional level . The pexB upstream region contained 245 nucleotides within which sequences approximating the consensus for cyclic AMP receptor protein and integration host factor binding sites were discernible . Deletion of 164 bp of the upstream region, which included these consensus sequences, did not affect starvation-or osmotic stress-mediated induction of pexB but abolished its induction by oxidative stress . The same start site was used in transcription during carbon starvation, osmotic stress, or oxidative stress, suggesting that the pexB promoter can be recognized in vivo by both sigma 38 and sigma 70, depending, presumably, on the presence of appropriate transcriptional factors . The -10 and -35 regions of pexB resembled those of some but not all genes known to be controlled by sigma 38.

Exp Cell Res, 1994 Jul, 213(1), 253 - 65
Adhesion activity in fibronectin's alternatively spliced domain EDa (EIIIA) and its neighboring type III repeats: oncogene-dependent regulation; Xia P et al.; EDa (EIIIA) is one of two alternatively spliced type III repeats in cellular fibronectin (cFN) lacking in plasma fibronectin (pFN) . Previous studies using proteolytic fragments of cFN suggested that EDa may harbor adhesion activity for various Balb/c 3T3 cell derivatives . This putative adhesion activity has now been analyzed more directly . EDa and neighboring type III repeats III11 and III12 from human cFN cDNA were subcloned in various permutations and recombinant minigenes expressed in Escherichia coli . Purified recombinant polypeptide corresponding to EDa type III repeat alone is capable of promoting 3T3 cell attachment and limited cytoplasmic spreading, as are neighboring repeats III11 and III12 when tested as single repeats . While EDa alone exhibited 40-60% of the attachment activity of human pFN depending upon cell type, EDa with both neighboring repeats displayed 70-90% of pFN activity; furthermore, cell spreading was more extensive with the three-repeat molecule . Two experimental approaches indicated that cell surface proteoglycans do not participate in these adhesion processes . Finally, the effects of various oncogenes upon transformation of Balb/c 3T3 cells were investigated . Adhesion activity to all three repeats is completely abrogated by two different ras oncogenes, unaffected by the sis oncogene, and elevated by the src oncogene . Furthermore, ras- revertants of ras-transformed cells had reacquired adhesion activity for EDa and its neighboring repeats . Comparison of individual repeats confirmed oncogene-dependent regulation of receptor activity to these sequences--for 3T3 cells, EDa > III11 = III12, but for src-transformed cells III12 >> EDa > III11 . These studies reveal a new adhesion-promoting activity in alternatively spliced EDa and in neighboring repeats III11 and III12; this receptor activity is regulated either positively or negatively subsequent to transformation by specific oncogenes.

Exp Cell Res, 1994 Jul, 213(1), 128 - 42
Structural elements of the amino-terminal head domain of vimentin essential for intermediate filament formation in vivo and in vitro; Beuttenmuller M et al.; The biological functions of the non-alpha-helical, N- and C-terminal head and tail domains of intermediate filament (IF) proteins are still ill-defined . Previously, it has been shown that the basic, N-terminal head piece of the type III IF protein vimentin is essential for regular IF assembly and that arginine residues within the N-terminus may be involved . In order to identify particular regions within this domain essential for filament formation and stabilization, N-terminally truncated and arginine substitution forms of vimentin were constructed via site-directed in vitro mutagenesis of murine vimentin cDNA . The de novo filament assembly properties of these modified forms were compared with those of wild-type vimentin after transient expression in vimentin-free, cultured cells . In order to investigate their filament assembly competence in vitro, they were also produced in an E . coli expression system . It could be demonstrated that deletion of the first 10, 13, 17, and 32 amino acid residues, respectively, from the N-terminus of vimentin has an increasingly deleterious effect on filament assembly in vitro and network formation in vivo and that, thus, the highly conserved sequence motif, SSYRRXFGG, located in the N-terminus of various IF proteins and partially or totally removed by the above deletions plays a particularly important role in both activities . These results were confirmed and extended by arginine point mutations in the N-terminal head region, which showed that only one of the two adjacent arginine residues located within the conserved sequence motif is essential for filament assembly and stability in vitro as well as network formation in vivo . The neighboring arginine residues could be replaced by lysine residues without severe effects on the assembly properties of the respective mutant proteins . Distinction between the assembly-promoting potentials of the two arginine residues of the N-terminal doublet was considerably facilitated by a Val389-->Asp substitution toward the carboxy-end of the 2B segment of the vimentin rod domain . The synergistic effect of point mutations in this and the N-terminal region of the vimentin molecule implies the interaction of both protein domains in the process of filament assembly . Mutant vimentin proteins that were characterized by distinct incompetence to assemble into IFs caused a massive collapse of the endogenous vimentin filament system when expressed in mouse skin fibroblasts.

Exp Cell Res, 1994 Jul, 213(1), 107 - 12
Minisatellite DNA-binding proteins in mouse brain, liver, and kidney; Shinder GA et al.; Minisatellites are tandemly repeated DNA sequences that are found in most higher eukaryotes . They are genetically unstable and often gain or lose repeat units . Minisatellite repeats contain a "core" sequence which is highly conserved among a family of minisatellites . The core sequence resembles the chi sequence of Escherichia coli which is a binding site for recombination proteins . It has therefore been suggested that minisatellite core sequences may also be binding sites for proteins involved in recombination . In this paper, we report several proteins in mouse brain, liver, and kidney nuclear extracts which bind to various minisatellite sequences . We have found several proteins which have not been previously reported and, in addition, have noted that brain has a different profile of minisatellite-binding proteins than liver and kidney . Moreover, we have also observed probe-specificity in the binding of some of these proteins, suggesting that different "families" of minisatellites may have qualitatively different functions.

Cell, 1994 Jul 1, 77(7), 1093 - 100
Differential evolution of substrates for an RNA enzyme in the presence and absence of its protein cofactor; Liu F et al.; Selection of substrates for an RNA enzyme, the catalytic subunit of RNAase P from E . coli, has been carried out by simulation of evolution in vitro in the presence and absence of the protein cofactor of the enzyme . In the presence of the protein, substrates resembling precursor tRNAs, which were readily cleaved by the catalytic RNA, were selected in addition to others, with different sequences and structures (one of which resembled the precursor to 4.5S RNA) that were not readily cleaved by the catalytic RNA alone . The ribonucleoprotein enzyme is more versatile than the RNA enzyme, and our results suggest that it and 4.5S RNA may have evolved after ancestral tRNAs.

Proc Soc Exp Biol Med, 1994 Jul, 206(3), 273 - 9
Interaction of lactogenic hormones with the soluble extracellular domain of prolactin receptors; Gertler A et al.; Two variants of rabbit prolactin receptor extracellular domain (rbPRLR-ECD) were prepared using insect/baculovirus (amino acids 1-198) and E . coli (amino acids 4-210) expression systems . Bovine PRLR-ECD (bPRPL-ECD amino acids 1-210) and human growth hormone receptor ECD (hGHR-ECD amino acids 1-246) were also prepared using E . coli expression system . All four proteins were purified as monomers with > 95% homogeneity . Their affinity for various lactogenic and somatogenic hormones was determined by binding assays . The stoichiometry of complex formation with these hormones was studied by gel filtration on a Superdex 75 column, and bioactivity was determined by in vitro bioassays . The results summarized in this paper indicate that, in contrast to hGHR-ECD, in which the ability to form a 2:1 complex with hGH is indicative of the biological activity of the hormone, the ability or inability of prolactin and placental lactogen to form 2:1 complexes with rb or bPRLR-ECD cannot predict their biological activity . This conclusion does not preclude however, hormone- or antibody-induced dimerization of the membrane-embedded receptor.

J Histochem Cytochem, 1994 Jul, 42(7), 953 - 6
Development of endogenous beta-galactosidase and autofluorescence in rat brain microvessels: implications for cell tracking and gene transfer studies; Lal B et al.; Cell transplantation is commonly used in studies of CNS development, tumor biology, and gene therapy . Fluorescent dyes and the E . coli lacZ reporter gene are used to identify transplanted cells in host tissues . The usefulness of these methods depends on host autofluorescence and beta-galactosidase (beta-Gal) activity . Our interest in the CNS vasculature led us to examine vascular autofluorescence and beta-Gal activity in postnatal and adult rat brains . Brains were perfusion-fixed (3.7% paraformaldehyde), cryoprotected, and cryostat-sectioned (12 microns) . Autofluorescent vessel profiles were quantitated in sections using rhodamine filter sets and beta-Gal-positive vessels were quantitated under bright-field after incubation of sections with X-Gal chromogenic substrate for 1-18 hr at 37 degrees C . Multifocal vessel autofluorescence appeared in postnatal Day (PND) 18 Lewis rats (0.6 +/- 0.4 vessels/field) and increased tenfold in adults (6.8 +/- 0.3/field) . The numbers of beta-Gal-positive vessels in PND 18 and adult sections incubated with X-Gal for 18 hr were 21.1 +/- 1.7 and 119 +/- 17, respectively . Host beta-Gal staining was similar to that produced by implanted endothelial cells expressing the bacterial lacZ reporter gene . Reducing incubation times in X-Gal to less than 4 hr eliminated endogenous staining and retained lacZ-specific staining . The presence of vascular autofluorescence and endogenous beta-Gal activity must be considered when either fluorescence- or lacZ-dependent cell markers are used in rat brain.

Cancer Res, 1994 Jul 1, 54(13), 3511 - 5
Human monoclonal antibody identified an immunoreactive tetrapeptide sequence (Lys-Tyr-Gln-Ile) in M(r) 43,000 protein of human melanoma; Oka T et al.; The human monoclonal antibody (HuMAb) L92 reacts to an M(r) 43,000 protein associated with human melanoma . To identify the gene encoding its antigenic epitope, a complementary DNA expression library constructed from the human melanoma cell line UCLASO M14 was screened with HuMAb L92 . DNA sequence analysis of the isolated clone revealed that the immunoreactive peptide was composed of 10 amino acids (QDLT-MKYQIF) . The peptide was expressed in Escherichia coli with beta-galactosidase as a fused protein . There is no homology between the cloned sequence and other reported DNA sequences . Western blot analysis showed that the fused protein had specific binding to HuMAb L92 . An antigen-encoding peptide with 10 amino acids was synthesized and tested for its immunoreactivity in vitro . HuMAb L92 reacted specifically to the 10-amino acid peptide in both an antibody-binding inhibition to the M(r) 43,000 protein and a solid-phase enzyme-linked immunosorbent assay . Using several truncated fusion proteins, we found the minimum number of amino acids required for the antibody binding to be 4 (KYQI) . These results suggest that the identified peptide sequence encodes the antigenic epitope of the M(r) 43,000 protein.

Anesth Analg, 1994 Jul, 79(1), 40 - 5
Serum but not plasma produces injury in the perfused rabbit lung; Mouchawar AM et al.; Serum contains proteins that may produce lung injury directly by affecting endothelial cells and indirectly by modulating the effects of endotoxin (lipopolysaccharide) . We studied the effects of 10% serum, 10% plasma, and 10% plasma plus 2 micrograms/mL lipopolysaccharide on pulmonary hypertension and vascular permeability in the isolated perfused rabbit lung . Control lungs perfused with Krebs-Henseleit buffer containing 3% albumin for 2 h had stable pulmonary vascular pressures and permeability (measured by the capillary filtration coefficient) . Serum produced pulmonary hypertension and increased pulmonary vascular permeability . In contrast, plasma, with and without lipopolysaccharide, did not alter pulmonary vascular pressures or permeability . Pretreatment with the cyclooxygenase inhibitor indomethacin prior to the addition of serum prevented the serum-induced increase in pulmonary vascular pressures and permeability . We conclude that the deleterious effects of serum are not due to plasma proteins per se, but instead are related to activation of the coagulation cascade during preparation of the serum . The deleterious effects of serum appear to be mediated by cyclooxygenase metabolites of arachidonic acid . Finally, endotoxin, even with the addition of plasma, does not directly produce lung injury.

Mol Cell Biol, 1994 Jul, 14(7), 4958 - 74
Novel interactions between human T-cell leukemia virus type I Tax and activating transcription factor 3 at a cyclic AMP-responsive element; Low KG et al.; Human proenkephalin gene transcription is transactivated by human T-cell leukemia virus type I (HTLV-I) Tax in human Jurkat T lymphocytes . This transactivation was further enhanced in Jurkat cells treated with concanavalin A, cyclic AMP, or 12-O-tetradecanoylphorbol-13-acetate . Deletion and cis-element transfer analyses of the human proenkephalin promoter identified a cyclic AMP-responsive AP-1 element (-92 to -86) as both necessary and sufficient to confer Tax-dependent transactivation . Different AP-1 or cyclic AMP-responsive element-binding protein (CREB)/activating transcription factor (ATF) proteins which bind this element were expressed in murine teratocarcinoma F9 cells to identify those capable of mediating Tax-dependent transactivation of human proenkephalin gene transcription . Although CREB, c-Fos, c-Jun, and JunD did not have significant effects, JunB inhibited the Tax-dependent transactivation . In contrast, ATF3 dramatically induced Tax-dependent transactivation, which was further enhanced by protein kinase A . Electrophoretic mobility shift assays with recombinant fusion proteins expressed and purified from bacteria indicate that the DNA-binding activity of ATF3 is also dramatically enhanced by Tax . Chimeric fusion proteins consisting of the DNA-binding domain of the yeast transcription factor Gal4 and the amino-terminal domain (residues 1 to 66) of ATF3 were able to mediate Tax-dependent transactivation of a Gal4-responsive promoter, which suggests a direct involvement of this region of ATF3 . Recombinant fusion proteins of glutathione S-transferase with either the amino- or carboxy-terminal (residues 139 to 181) domain of ATF3 were able to specifically interact with Tax . Furthermore, specific antisera directed against Tax coimmunoprecipitated ATF3 only in the presence of Tax.

Mol Cell Biol, 1994 Jul, 14(7), 4712 - 21
Repression of basal transcription by HMG2 is counteracted by TFIIH-associated factors in an ATP-dependent process; Stelzer G et al.; A basal repressor of class II gene transcription was identified, purified, and found to be identical to nonhistone chromosomal protein HMG2 . HMG2 was shown to inhibit basal transcription under conditions in which transcription templates form soluble complexes with HMG2 . Order-of-addition experiments clearly revealed that HMG2 acted after assembly of a TBP-TFIIA-promoter complex and before formation of the fourth phosphodiester bond by RNA polymerase II . Subsequently, an activity that efficiently counteracted repression of transcription by HMG2 in both TBP- and TFIID-containing transcription systems was isolated . Several lines of evidence suggested that antirepression was mediated by a TFIIH-associated factor . The antirepressor first coeluted with TFIIH, was depleted from this fraction by antibodies directed against the TFIIH subunit p62, was dependent on either ATP or dATP, and then was inhibited by the ATP analogs AMP-PNP and ATP gamma S . Relief of HMG2-mediated repression as well as basal promoter function of TFIIH may involve a helicase that coelutes with TFIIH and displays similar nucleotide specificities . Taken together, these data suggest novel consequences of chromatin-associated HMG proteins and they provide direct evidence for a role of TFIIH-associated enzymes in ATP-dependent antirepression of nonhistone chromosomal proteins.

Mol Cell Biol, 1994 Jul, 14(7), 4653 - 61
Transcriptional control of the yeast PDR5 gene by the PDR3 gene product; Katzmann DJ et al.; Saccharomyces cerevisiae cells possess the ability to simultaneously acquire resistance to an array of drugs with different cytotoxic activities . The genes involved in this acquisition are referred to as pleiotropic drug resistant (PDR) genes . Several semidominant, drug resistance-encoding PDR mutations have been found that map near the centromere on chromosome II, including PDR3-1 and PDR4-1 . DNA sequencing of chromosome II identified a potential open reading frame, designated YBL03-23, that has the potential to encode a protein with strong sequence similarity to the product of the PDR1 gene, a zinc finger-containing transcription factor . Here we show that YBL03-23 is allelic with PDR3 . The presence of a functional copy of either PDR1 or PDR3 is essential for drug resistance and expression of a putative membrane transporter-encoding gene, PDR5 . Deletion mapping of the PDR5 promoter identified a region from -360 to -112 that is essential for expression of this gene . DNase I footprinting analysis using bacterially expressed Pdr3p showed specific recognition by this protein of at least one site in the -360/-112 interval in the PDR5 promoter . A high-copy-number plasmid carrying the PDR3 gene elevated resistance to both oligomycin and cycloheximide . Increasing the number of PDR3 gene copies in a delta pdr5 strain increased oligomycin resistance but was not able to correct the cycloheximide hypersensitivity that results from loss of PDR5 . These data are consistent with the notion that PDR3 acts to increase cycloheximide resistance by elevating the level of PDR5 transcription, while PDR3-mediated oligomycin resistance acts through some other target gene.

Mol Cell Biol, 1994 Jul, 14(7), 4522 - 31
CCR4 is a glucose-regulated transcription factor whose leucine-rich repeat binds several proteins important for placing CCR4 in its proper promoter context; Draper MP et al.; The yeast CCR4 protein is required for the expression of a number of genes involved in nonfermentative growth, including glucose-repressible ADH2, and is the only known suppressor of mutations in the SPT6 and SPT10 genes, two genes which are believed to be involved in chromatin maintenance . We show here that although CCR4 did not bind DNA under the conditions tested, it was able to activate transcription when fused to a heterologous DNA-binding domain . The transcriptional activation ability of CCR4, in contrast to that of many other activators, was glucose regulated . Two activation domains one of which was glucose responsive and encompassed a glutamine-proline-rich region similar to that found in other eukaryotic transcriptional factors were identified . The two transactivation regions, when separated from the leucine-rich repeat and the C terminus of CCR4, were unable to complement a defective ccr4 allele, suggesting that the leucine-rich repeat and the C terminus make contacts that link the activation regions to the proper gene context . Native immunoprecipitation of CCR4 revealed that CCR4 was complexed with at least four other proteins . The leucine-rich repeat of CCR4 was both necessary and sufficient for interaction with at least two of these factors . We propose that the leucine-rich repeat links CCR4 through its associated factors to its promoter context at ADH2 and other loci where it is required for proper transcriptional regulation.

J Am Coll Cardiol, 1994 Jul, 24(1), 55 - 60
Dose finding with a novel recombinant plasminogen activator (BM 06.022) in patients with acute myocardial infarction: results of the German Recombinant Plasminogen Activator Study . A study of the Arbeitsgemeinschaft Leitender Kardiologischer Krankenhausärzte (ALKK); Neuhaus KL et al.; OBJECTIVES . The aim of this study was to determine the appropriate dose of a novel recombinant tissue-type plasminogen activator (BM 06.022) for thrombolysis in patients with acute myocardial infarction . BACKGROUND . BM 06.022 is a mutant of tissue-type plasminogen activator expressed in Escherichia coli that can be given as a single bolus because of a prolonged half-life, which might obviate the need for complicated regimens . METHODS . BM 06.022 given as a single bolus was investigated in 142 patients in a multicenter sequential dose-finding study . Efficacy of the drug was assessed from infarct-related artery patency by coronary angiography . RESULTS . With the first dose of 10 MU of BM 06.022, the predefined minimal 90-min patency of 70% was not achieved, as indicated by the sequential probability ratio test after treatment of 42 patients (group A) . The second dose of 15 MU of BM 06.022 was given subsequently in the preset maximum of 100 patients (group B) . Angiography 30, 60 and 90 min after the bolus injection of BM 06.022 revealed a patent infarct-related artery (Thrombolysis in Myocardial Infarction trial {TIMI} grade 2 or 3) in 65% and 66%, 73% and 74% and 66% and 75% of patients in groups A and B, respectively . Very early reocclusion up to the 90-min angiogram occurred in 17% and 13%, late reocclusion until predischarge angiography occurred in 7% and 5%, and rescue percutaneous transluminal coronary angioplasty after the 90-min angiogram was performed in 6 and 14 patients in groups A and B, respectively . Plasma fibrinogen decreased from 2.79 g/liter (range 0.94 to 4.75) to 1.69 g/liter (range 0.0 to 3.95) in group A and from 2.54 g/liter (range 0.0 to 5.02) to 0.92 g/liter (range 0.0 to 2.68) in group B . Two bleeding complications requiring transfusion or surgical intervention and one nonfatal intracranial hemorrhage were encountered . Eight patients had a reinfarction, and five patients died, all of cardiac causes . CONCLUSIONS . With BM 06.022 given as a single bolus, a high early patency rate of the infarct-related coronary artery can be achieved . The speed of thrombolysis seems to be superior to standard thrombolytic drugs . The compound warrants further evaluation with respect to safety and efficacy by clinical end points.

Infect Immun, 1994 Jul, 62(7), 3038 - 40
The eaeB gene of enteropathogenic Escherichia coli is necessary for signal transduction in epithelial cells; Foubister V et al.; An enteropathogenic Escherichia coli mutant carrying an internal deletion in the eaeB gene (UMD864) was unable to activate epithelial cell signals, including tyrosine phosphorylation, cytoskeletal rearrangements, and the release of inositol phosphates, indicating that the eaeB locus encodes a product that is involved in stimulating signals in epithelial cells.

Infect Immun, 1994 Jul, 62(7), 2917 - 29
Actin accumulation associated with clustered and localized adherence in Escherichia coli isolated from patients with diarrhea; Yamamoto T et al.; Escherichia coli D2 (serotype 07:H-) that was isolated from a child with diarrhea hybridized with an F1845 DNA probe used to detect diffuse adherence . Strain D2 adhered to tissue culture cells (HeLa and HEp-2 cells) in a clustered pattern but did not autoagglutinate on the cell surface and induced the elongation of microvilli after 3 h of incubation . After 6 h of incubation, the infected cells were positive for fluorescent-actin staining at the site of clustered adherence . When analyzed with a confocal laser scanning microscope, each D2 cell was surrounded by accumulated actin in a capsule-like formation . Capsule-like, accumulated actin was also observed with enteropathogenic E . coli (EPEC), although in this case, actin accumulation was associated with EPEC microcolonies in a localized pattern . Four other strains of F1845 DNA probe-positive, diffusely adhering E . coli were negative for actin accumulation . Strain D2 did not hybridize with EPEC attaching and effacing DNA or EPEC adherence factor DNA probes . In addition, clustered D2 cells were found inside tissue culture cells . The data suggest a novel infectious mechanism as well as genetic heterogeneity of F1845 DNA probe-positive E . coli . Capsule-like, accumulated actin may protect the bacteria from host defense mechanisms.

Infect Immun, 1994 Jul, 62(7), 2653 - 61
A 9.0-kilobase-pair circular plasmid of Borrelia burgdorferi encodes an exported protein: evidence for expression only during infection; Champion CI et al.; In this study, we report the cloning, sequencing, and molecular analysis of a gene located on a 9.0-kbp circular plasmid of virulent Borrelia burgdorferi B31 designated eppA (exported plasmid protein A) . This gene encodes a precursor protein of 174 amino acids including a signal peptide of 20 amino acids and a type I signal peptidase cleavage site . The mature EppA protein of 154 amino acids has a calculated molecular weight of 17,972 . Several lines of evidence suggest that eppA is not expressed by B . burgdorferi B31 during in vitro cultivation . Immunoblot analysis using hyperimmune rabbit antiserum to recombinant EppA (rEppA) did not detect the presence of EppA in B . burgdorferi B31 cultivated in vitro . Northern blot analysis using total RNA isolated from in vitro-cultivated virulent B . burgdorferi B31 failed to detect an eppA transcript . EppA was not detected in culture supernatants of virulent B . burgdorferi B31 in a sensitive antigen-capture enzyme-linked immunosorbent assay . In contrast, evidence for expression of eppA during infection was based on the observation that patients with Lyme disease as well as rabbits experimentally infected with B . burgdorferi B31 produced antibodies that recognized rEppA . Because the cellular location of EppA in B . burgdorferi cannot be determined in vivo because of very small numbers of organisms present in vertebrate infection, we examined the cellular location of rEppA expressed in Escherichia coli . In E . coli, rEppA is targeted to the outer membrane . In addition, purified E . coli outer membranes containing rEppA treated with chaotrophic agents did not result in rEppA release . These findings are consistent with the idea that EppA is not peripherally associated with the outer membrane of E . coli but rather has an integral outer membrane association.

Immunol Lett, 1994 Jul, 41(2-3), 217 - 23
Immunization of mice with Escherichia coli containing an antigen of the malaria parasite Plasmodium chabaudi chabaudi expressed in lambda gt11 induces high antibody titres; Hartz D et al.; Escherichia coli containing a recombinant malarial protein expressed in lambda gt11 have been evaluated as an antigen delivery system in vivo . They were generated by infecting non-suppressing E . coli cells with a clone from a cDNA library of Plasmodium chabaudi chabaudi in lambda gt11 . This clone (termed clone 6) expresses part of a 93 kDa blood-stage antigen of P . c . chabaudi as beta-galactosidase fusion protein . Immunization of C57B1/6 mice with these infected E . coli cells resulted in an antibody response to the malarial part of the fusion protein comparable to that obtained with purified fusion protein preparations . This method, therefore represents a rapid secondary screening of clones from lambda gt11 expression libraries for immunogenic and potentially protective components . In addition, the administration of whole infected E . coli obviates the need for an adjuvant.

Immunol Lett, 1994 Jul, 41(2-3), 155 - 61
Detection of adult T-cell leukemia-derived factor/human thioredoxin in human serum; Kitaoka Y et al.; Adult T-cell leukemia (ATL)-derived factor (ADF), originally defined as an inducer of interleukin-2 receptor/alpha-chain (IL-2R/p55) of human T-lymphotropic virus type I (HTLV-I) positive T cells, is a human homologue of redox-active coenzyme thioredoxin (Trx) of Escherichia coli . In this study, an enzymatic assay system based on the dithiol-dependent insulin-reducing activity of ADF/Trx was established (insulin-reducing assay) to determine the amount of ADF/Trx in human serum using NADPH and Trx reductase purified from human placenta . Insulin-reducing activity was detected in all of the serum samples from healthy volunteers (n = 30) screened by this assay, with a mean +/- SD of 10.9 +/- 2.4 U/l . This mean value corresponds with the concentration of 223 ng recombinant ADF/Trx (rADF/Trx)/ml . Human serum is known to contain several redox-active proteins with ADF/Trx motifs . To differentiate the contribution of these proteins and ADF/Trx to the insulin-reducing activity, the anti-rADF/Trx monoclonal antibody (mAb)-conjugated affinity column-depleted sera obtained from an identical source was used for analysis . The affinity column-depleted sera demonstrated a loss of over 99% of the original activity, while control column depleted sera lost less than 4% . Furthermore, the amount of affinity-purified ADF/Trx molecules eluted from the same column almost corresponded with the amount estimated by the insulin-reducing activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Biol Pharm Bull, 1994 Jul, 17(7), 980 - 2
Mitogenic activity and the induction of tumor necrosis factor by lipopeptide analogs of the N-terminal part of lipoprotein in the outer membrane of Escherichia coli; Shimizu T et al.; Mitogenicity and the production of tumor necrosis factor (TNF) by a chemically synthesized lipotetrapeptide analog (KAB-8), S-{2,3-bis(palmitoyloxy)-2R-propyl}-N-{(2,2,2)-trichloro- ethoxycarbonyl: Troc group}-(R)-cysteinyl-(S)-seryl-(S)-seryl-(S)-asparagine, the amino acid sequence of which corresponds to that of the lipopeptide part of lipoprotein in Escherichia coli, and several derivatives (KAB-30-41), which possessed the altered glycerocysteine moiety, were examined . A 1-cysteinyl glycerol skeleton-type compound (KAB-8), a propane-type compound (KAB-31), a homoglycerol-type (KAB-39 and 40) and a 2-cysteinyl glycerol-type (KAB-41) exhibited mitogenic activity on splenocytes from C3H/He mice at various concentrations ranging from 0.4 to 100 micrograms/ml . However, propane-type compounds, except KAB-31, and ethane-type compounds showed lower mitogenic activity than other types of compounds . Compounds KAB-8, 31, 40 and 41 induced the production of TNF in peritoneal exudated macrophages from C3H/He mice at concentrations of 25 and 50 micrograms/ml . The results indicate that the structural differences of the glycerol moiety in the synthetic lipopeptides affect the potency of its biological activities.

Bioorg Khim, 1994 Jul, 20(7), 751 - 8
{Cloning transport protein genes for two strains of tobacco mosaic virus and their expression in Escherichia coli cells}; Ivanov KI et al.; Recombinant constructs expressing fusion (His)6 movement proteins of two strains (Cruciferous and Vulgare) of tobacco mosaic virus were generated by cloning the PCR-amplified fragments into the pQE-9 vector . The movement proteins containing N-terminal (His)6 affinity tags were purified on a metal chelate adsorbent.

Zh Mikrobiol Epidemiol Immunobiol, 1994 Jul-Aug, (4), 64 - 6
{Coxiella burnetii antigens--inducers of tumor necrosis factor and interleukin-1}; Tokarevich NK et al.; The cultivation of mouse peritoneal macrophages in the presence of antigenic preparations obtained from C.burnetii was accompanied by the appearance of phagocytes and considerable amounts of tumor necrosis factor and interleukin 1 in the culture medium . The production of cytokines depended on the doses of preparations used as inducers . The special treatment of C.burnetii antigen, selectively removing its phospholipid components, led to a sharp drop in its capacity for stimulating the production of cytokines.

Plasmid, 1994 Jul, 32(1), 32 - 40
A set of beta-galactosidase gene fusion cassettes demonstrates usefulness in expressing HIV-1 genes in Escherichia coli; Valverde V et al.; Heterologous expression in Escherichia coli is often limited by yield and solubility of the foreign protein in the bacterial cytoplasm . In many cases, overexpression also results in growth inhibition . In order to produce retroviral proteins that are especially difficult to overexpress in E . coli, we designed a set of beta-galactosidase fusion cassettes . Fusions with beta-galactosidase increase significantly both yield and solubility of the foreign proteins, thus making purification much easier . These cassettes allow for N- or C-terminal fusions, and the retroviral proteins can be released from the fusion by automaturation in vivo for the HIV-1 protease or cleavage by thrombine for Tat . More generally, any synthetic sequence coding for a given cleavage site can be introduced 5' or 3' to the lacZ gene through a convenient set of unique restriction sites, making these fusion cassettes highly versatile.

Plasmid, 1994 Jul, 32(1), 19 - 31
TrfA-dependent, inner-membrane-associated plasmid RK2 DNA synthesis in Escherichia coli maxicells; Michaels K et al.; Previous results of experiments in which plasmid-encoded proteins were selectively labeled in ultraviolet sensitive "maxicell" mutants suggested that the essential initiation proteins of RK2 (33 and 43 kDa) were bound to the inner membrane of Escherichia coli (D . Kostyal et al., 1989, Plasmid 21, 226-237) . However, in the present studies using a specific polyclonal antibody against the TrfA initiation proteins, significant levels of these proteins were also detected for the first time in the outer membrane fraction as well as the inner membrane fraction . Only in the cytosol fraction were the initiation proteins relatively absent . In order to determine whether initiation and replication were also associated with either or both of the membrane fractions, it was necessary to develop a replicating system more active than the one previously extracted from minicell membranes, which did not separate the membrane into its component parts (J . A . Kornacki and W . Firshein, 1986, J . Bacteriol . 167, 319-326) . In addition, it was also necessary to devise an extraction procedure that did not degrade the supercoil DNA template during the separation of the inner from the outer membrane fraction . Both criteria were met, first by the use of maxicells containing a miniplasmid derivative of RK2 and second by disrupting cell envelopes in the French pressure cell using low pressure . Under these conditions, not only were the two major membrane fractions separated successfully from the cytosol fraction, but supercoil DNA template was also preserved in both fractions, detergents were avoided, and replication was significantly higher than that described in the earlier experiments . TrfA-dependent initiation of DNA replication was associated primarily with the inner membrane fraction.

Mol Biol (Mosk), 1994 Jul-Aug, 28(4), 926 - 31
{Study of the poly(U)-dependent interaction of tRNA(Phe) with the P-site of Escherichia coli ribosomes by chemical modification with nitrosoethylurea}; Nekhai SA et al.; The accessibility of phosphates in E . coli tRNA(Phe) bound to E . coli ribosomes and 30S subunits for attack by an alkylating agent ethylnitrosourea was studied . The experimental technique allowed us to investigate a part of the molecule between N-11 and N-72 . Being bound to the poly(U)-programmed P site of 30S subunit, tRNA(Phe) reveals protection in a discrete region including phosphates 23-44 . This region corresponds to the hairpin formed from the anticodon arm and the adjoining 3' strand of the D stem and the variable loop . On the P site of 70S-poly(U), additional protection was observed in a region between N-45 and N-63 . This region corresponds to the extra loop and T stem, and reveals a fine structure of protection: protected phosphates in positions 45-49, 51, 54-55, 58-61, and 63 alternate with unprotected ones in positions 50, 52-53, 56-57 and 62 . We conclude that ethylnitrosourea can be used for detailed study of tRNA-ribosome contacts.

Mol Biochem Parasitol, 1994 Jul, 66(1), 59 - 69
The Plasmodium falciparum protein RESA interacts with the erythrocyte cytoskeleton and modifies erythrocyte thermal stability; Da Silva E et al.; The ring-infected erythrocyte surface antigen (RESA) associates with spectrin in the erythrocyte membrane (Foley, M., Tilley, L., Sawyer, W . H . and Anders, R . F . (1991) Mol . Biochem . Parasitol., 46, 137-148) . A fragment of the RESA protein, which was expressed in Escherichia coli, was found to bind to inside-out vesicles of erythrocyte membranes in an apparently saturable manner . Upon extraction of inside-out vesicles with Triton X-100, the RESA fragment remained associated with the erythrocyte cytoskeleton . Using the technique of steady-state fluorescence polarisation, we have studied the thermal denaturation of fluorescein-labelled spectrin in the presence of recombinant RESA . We found that the RESA fragment partially protected spectrin against heat-induced conformational changes . Furthermore, erythrocytes infected with a RESA (-) laboratory strain (FCR3) were shown to be more susceptible to heat-induced fragmentation than erythrocytes infected with a RESA (+) strain of the parasite . RESA does not, however, appear to play an essential role in the invasion process per se as erythrocytes resealed to contain anti-RESA antibodies were efficiently invaded.

Mol Biochem Parasitol, 1994 Jul, 66(1), 111 - 8
Expression and characterization of a rac family protein kinase of Entamoeba histolytica; Que X et al.; We previously reported the isolation from Entamoeba histolytica of a novel rac family protein kinase gene, termed Ehrac1, for "related to cAMP-dependent protein kinases and protein kinase Cs" . To study the function and properties of this kinase gene further, we fused the full-length coding region and the truncated catalytic domain of the Ehrac1 gene in frame with the gene encoding glutathione S-transferase in the pGEX-KG vector and expressed the fusion in Escherichia coli . The thrombin-cleaved and uncleaved fusion proteins, GST-Ehrac1 and GST-Ehrac1-c (catalytic domain), were purified and found to exhibit similar protein kinase activities . The Ehrac1 fusion kinase was found to phosphorylate serine/threonine residues exclusively in vitro . The preferred substrate for the enzyme was histone H1 with a Km of approx . 14 microM . Histone H3 and kemptide were phosphorylated at about half the rate of histone H1 . Protamine, enolase, bovine serum albumin, and poly (Glu:Tyr) were not substrates for the enzyme . The protein kinase activity was higher in the presence of Mn2+ than Mg2+ . Neither cAMP, Ca2+, nor Ca2+/calmodulin stimulated enzyme activity . The pH optimum of the enzyme was 7.5 . The Ehrac1 kinase can utilize GTP as well as ATP as a phosphate donor with an apparent Km of 80 microM . Enzyme activity was inhibited 30-40% by a crude cAMP-dependent protein kinase inhibitor from rabbit and by thiol reagents . The expression and purification of enzymatically active Ehrac1 protein kinase should allow further analysis of the regulation and signal transduction pathways of E . histolytica.

Mol Microbiol, 1994 Jul, 13(2), 337 - 48
Identification and characterization of hitherto unknown Mycoplasma pneumoniae proteins; Proft T et al.; Eleven hitherto unknown Mycoplasma pneumoniae proteins were identified and characterized with respect to their size and subcellular location . This was carried out through the construction of in vitro gene fusions between a modified mouse dehydrofolate reductase (dhfr) gene and selected regions (cosmid clones) of the M . pneumoniae genome and expressing them in Escherichia coli . Positive clones were identified using antibodies against specific fractions of M . pneumoniae . The deduced protein sequences of 11 out of 30 clones did not show significant homologies to known proteins in protein data-bank searches . Monospecific antibodies against these 11 fusion proteins were used to determine the size and cellular location of the corresponding M . pneumoniae proteins by immunoscreening Western blots of SDS-acrylamide gels from M . pneumoniae cell extracts.

Mol Microbiol, 1994 Jul, 13(2), 301 - 12
Cloning, sequencing, and characterization of multicopy suppressors of a mukB mutation in Escherichia coli; Yamanaka K et al.; The mukB gene codes for a 177 kDa protein, which might be a candidate for a force-generating enzyme in chromosome positioning in Escherichia coli . The mukB106 mutant produces normal-sized, anucleate cells and shows a temperature-sensitive colony formation . To identify proteins interacting with the MukB protein, we isolated three multicopy suppressors (msmA, msmB, and msmC) to the temperature-sensitive colony formation of the mukB106 mutation . The msmA gene, which could not suppress the production of anucleate cells, was found to be identical to the dksA gene . The msmB and msmC genes suppressed the production of anucleate cells as well as the temperature-sensitive colony formation . However, none of them could suppress both phenotypes in a mukB null mutation . DNA sequencing revealed that the msmB gene was identical to the cspC gene and that the msmC gene had not been described before . A homology search revealed that the amino acid sequences of both MsmB and MsmC possessed high similarity to proteins containing the cold-shock domain, such as CspA of E . coli and the Y-box binding proteins of eukaryotes; this suggests that MsmB and MsmC might be DNA-binding proteins that recognize the CCAAT sequence . Hence, the msmB and msmC genes were renamed cspC and cspE, respectively . Possible mechanisms for suppression of the mukB106 mutation are discussed.

Mol Microbiol, 1994 Jul, 13(2), 265 - 72
The dps promoter is activated by OxyR during growth and by IHF and sigma S in stationary phase; Altuvia S et al.; Dps is a non-specific DNA-binding protein abundant in starved Escherichia coli cells and is important for the defence against hydrogen peroxide . We found that dps mRNA levels are controlled by rpoS-encoded sigma S, the transcriptional activator OxyR and the histone-like IHF protein . In exponentially growing cells, dps is induced by treatment with hydrogen peroxide in an OxyR-dependent manner . This OxyR-dependent induction occurs only during log phase, although the OxyR protein is present in stationary phase . In the stationary phase cells, dps is expressed in a sigma S- and IHF-dependent manner . The purified OxyR and IHF proteins are also shown to bind upstream of the dps promoter . Our results suggest that the dps promoter is recognized by both sigma 70-holoenzyme and sigma S-holoenzyme, since OxyR acts through sigma 70 and the starts of the OxyR- and sigma S-dependent transcripts are identical.

Mol Microbiol, 1994 Jul, 13(2), 243 - 51
Cloning and molecular characterization of a Legionella pneumophila gene induced by intracellular infection and by various in vitro stress conditions; Abu Kwaik Y et al.; The synthesis of a global stress protein (GspA) of Legionella pneumophila is induced in the intracellular environment of the phagocytic cell and by various in vitro stress stimuli . We used techniques of reverse genetics to isolate the gspA gene from a genomic library of L . pneumophila . Sequence analysis of approximately 1700 bp of a representative clone (pBSP1) showed the presence of two open reading frames (ORFs) . ORF1 encoded for a polypeptide with an inferred molecular mass of 19 kDa and an isoelectric point of 6.1 . These predictions correlated with the migration of the GspA protein on two-dimensional SDS-polyacrylamide gels . The predicted amino acid sequence of the GspA protein was identical to 22/23 residues of the N-terminal amino acid sequence derived by Edman degradation of the purified protein . The GspA protein was 41.3% and 36.5% identical to the 16 kDa IbpA and IbpB heat-shock proteins, respectively, of Escherichia coli . Primer extension from mRNA isolated from L . pneumophila showed that transcription of the gspA gene was controlled by two overlapping promoters . One of the promoters was a sigma 70 promoter, while the other was a heat-shock promoter and was regulated by the sigma 32 transcription factor in E . coli . Northern blot analysis showed that the level of gspA mRNA was elevated 3.4-, 5.0-, and 6.7-fold after exposure of L . pneumophila to heat shock, oxidative stress and osmotic shock, respectively . The gspA gene was conserved among 13 serogroups of L . pneumophila . Our data showed that the gspA gene of L . pneumophila, which is induced by intracellular infection and by various stress stimuli, is controlled transcriptionally by two overlapping and separately regulated promoters.

J Cell Sci, 1994 Jul, 107 ( Pt 7), 1949 - 57
Dephosphorylation of the largest neurofilament subunit protein influences the structure of crossbridges in reassembled neurofilaments; Gotow T et al.; Phosphorylation-dependent change in electrophoretic mobility is the most unique characteristic of NF-H, the largest molecular mass subunit of the neurofilament . We dephosphorylated NF-H using Escherichia coli alkaline phosphatase, then reassembled it into neurofilaments with NF-M and NF-L, and into NF-H filaments with NF-H alone . We compared these dephosphorylated filaments with control: projections by low-angle rotary-shadow, crossbridges by quick-freeze deep-etch, and core filament packing density by thin-section electron microscopy . Projections in the dephosphorylated filaments were basically similar in structure to those in control, although there was a tendency for them to be wider and less dense, especially in NF-H filaments . Dephosphorylated filaments were still able to form crossbridges between core filaments, but their crossbridges were significantly wider, less dense, more branched and more irregular than crossbridges in control, and core filaments were more densely packed . These structural differences may be brought about by the removal of phosphate groups from NF-H tail and consequent reduction of electrostatic repulsion between adjacent crossbridges extending from the same core filament . The results indicate that phosphorylation of NF-H is necessary for forming well developed crossbridges, straight and at constant intervals, like those of in vivo axonal neurofilaments.

J Cell Sci, 1994 Jul, 107 ( Pt 7), 1935 - 48
Identification of two N-terminal non-alpha-helical domain motifs important in the assembly of glial fibrillary acidic protein; Ralton JE et al.; The non-alpha-helical N-terminal domain of intermediate filament proteins plays a key role in filament assembly . Previous studies have identified a nonapeptide motif, SSYRRIFGG, in the non-alpha-helical N-terminal domain of vimentin that is required for assembly . This motif is also found in desmin, peripherin and the type IV intermediate filament proteins . GFAP is the only type III intermediate filament protein in which this motif is not readily identified . This study has identified two motifs in the non-alpha-helical N-terminal domain of mouse GFAP that play important roles in GFAP assembly . One motif is located at the very N terminus and has the consensus sequence, MERRRITS-ARRSY . It has some characteristics in common with the vimentin nonapeptide motif, SSYRRIFGG, including its location in the non-alpha-helical N-terminal domain and a concentration of arginine residues . Unlike the vimentin motif in which even conserved sequence changes affect filament assembly, the GFAP consensus sequence, MERRRITS-ARRSY, can be replaced by a completely unrelated sequence; namely, the heptapeptide, MVRANKR, derived from the lambda cII protein . When fused to GFAP sequences with sequential deletions of the N-terminal domain, the lambda cII heptapeptide was used to help identify a second motif, termed the RP-box, which is located just upstream of the GFAP alpha-helical rod domain . This RP-box affected the efficiency of filament assembly as well as protein-protein interactions in the filament, as shown by sedimentation assays and electron microscopy . These results are supported by previous data, which showed that the dramatic reorganization of GFAP within cells was due to phosphorylation-dephosphorylation of a site located in this RP-box . The results in this study suggest the RP-box motif to be a key modulator in the mechanism of GFAP assembly, and support a role for this motif in both the nucleation and elongation phases of filament assembly . The RP-box motif in GFAP has the consensus sequence, RLSL-RM-PP . Sequences similar to the GFAP RP-box motif are also to be found in vimentin, desmin and peripherin . Like GFAP, these include phosphorylation and proteolysis sites and are adjacent to the start of the central alpha-helical rod domain, suggesting that this motif of general importance to type III intermediate filament protein assembly.

Antimicrob Agents Chemother, 1994 Jul, 38(7), 1555 - 60
L-651,392, a potent leukotriene inhibitor, controls inflammatory process in Escherichia coli pyelonephritis; Tardif M et al.; In this study, the relationship between leukotrienes, peritubular cell infiltration with polymorphonuclear cells (PMNs) and renal tubular damage was investigated in a rat model of acute ascending pyelonephritis . Infection was induced by the injection of 10(5) CFU of Escherichia coli into the bladder and occlusion of the left ureter for 24 h . Treatment of infected animals was started 24 h after the induction of pyelonephritis with either hydrocortisone (25 mg/kg of body weight per day), the leukotriene inhibitor L-651,392 (10 mg/kg/day), or the vehicle of L-651,392 and was maintained for 5 days . At the end of treatment, the animals were killed, serum was collected, and both kidneys were removed for colony counts and histopathology . Renal function was evaluated by the measurement of blood urea nitrogen levels and creatinine clearance . The numbers of PMNs and mononuclear cells (MNs) in the cortex and medulla were recorded for all groups on plastic sections done from the left kidney . Infection alone (vehicle of L-651,392) resulted in intensive interstitial infiltration and a severe tubular destruction in the cortex . Treatment with hydrocortisone did not prevent PMN migration and tissue damage . By contrast, treatment with L-651,392 resulted in a significant reduction in PMNs (P < 0.001 in comparisons with all other groups) and greater preservation of the tubular structure despite identical bacterial counts than in the group receiving hydrocortisone . We conclude that L-651,392 prevents inflammatory cells from reaching the site of infection and protects the kidney from tubular damage associated with inflammation during pyelonephritis . Inhibitors of leukotrienes should be further investigated for their potential benefit as adjuvants to antibiotherapy in the treatment of pyelonephritis.

Am J Vet Res, 1994 Jul, 55(7), 991 - 9
Pathogenicity of a bovine attaching effacing Escherichia coli isolate lacking Shiga-like toxins; Fischer J et al.; Escherichia coli isolate 7996-90, obtained from a calf with diarrhea, had negative results of tests for K-88, K-99, 987P, F41, CS31A, F1845, F165 or E . coli adherence factor adhesins and had negative results of tests for the toxins heat-labile, heat-stable A, heat-stable B, Shiga-like toxin (SLT)-I or SLT-II . Strain 7996-90 had localized adherence to HEp-2 cells, caused actin rearrangement in host cells to which it adhered, hybridized with the eaeA probe, and produced the 94-kd outer membrane protein associated with attaching effacing lesions . This isolate caused attaching effacing lesions in Caco-2 cell polar monolayers, rabbit intestinal loops, and the intestines of gnotobiotic pigs . The isolate belongs to serotype O26: NM and is considered a class-II attaching effacing enteropathogenic E coli . Until recent addition of more sensitive assays at veterinary diagnostic laboratories, isolates such as 7996-90 were not readily recognized as pathogens because they failed to fit into the enterohemorrhagic E coli group, members of which, be definition, produce SLT . The assays described can facilitate diagnosis of attaching effacing E coli infection when histologic evaluation is hampered by autolysis.

Anal Biochem, 1994 Jul, 220(1), 82 - 91
Characterization and use of crude alpha-subunit preparations for quantitative immunoblotting of G proteins; Gettys TW et al.; G proteins are heterotrimeric membrane-associated proteins that couple a large number of receptors to a variety of effector systems within the cell . Characterization of G proteins expressed in a particular cell type represents an important first step in defining the potential candidates to which a receptor might couple . A difficulty often encountered using G protein antisera from various commercial and private sources is relating the intensity of bands on a Western blot to the relative amount of G protein present in a membrane preparation . This problem is especially noteworthy when comparing across G protein subtypes due to differences in titer, affinity, and specificity among various antisera . Conventional approaches to obtaining G protein standards of sufficient purity to address these issues in a quantitative manner are time-consuming and difficult, but the procedures outlined herein demonstrate a method for using DEAE fractions from Escherichia coli expressing individual alpha-subunits . The key features of the present approach are to estimate saturable GTP gamma S binding in each alpha-subunit preparation and calculate the moles of alpha-subunit present in the respective preparations based on the known stoichiometry of GTP gamma S binding (1:1) . The extent of correspondence between GTP gamma S binding and immunoreactivity is then determined by trypsin protection assays, which estimate the proportion of immunodetectable G protein which can bind GTP gamma S . After characterization in this manner, DEAE fractions from bacteria transformed with the respective cDNA for Gi alpha-1, G1 alpha-2, and G1 alpha-3 were used to construct standard curves on Western blots and estimate endogenous G protein concentrations in cell lines (CHO and HeLa) and across species (rat and mouse) in isolated adipocyte preparations . Plasma membranes from CHO cells contained Gi alpha-2 (4.8 +/- 0.3 pmol/mg protein) and Gi alpha-3 (0.6 +/- 0.1 pmol/mg protein), but not Gi alpha-1, while HeLa cell membranes contained Gi alpha-1 (0.11 +/- 0.01 pmol/mg protein) and Gi alpha-3 (1.3 +/- 0.1 pmol/mg protein), but not Gi alpha-2 . In contrast, rat and mouse adipocyte membranes contained Gi alpha-1 (48 +/- 2 vs 36 +/- 2 pmol/mg protein), Gi alpha-2 (77 +/- 1.5 vs 25 +/- 1.4 pmol/mg protein), and Gi alpha-3 (26 +/- 1.2 vs 15 +/- 1 pmol/mg protein) . The method described herein provides an innovative solution to the technically difficult problem of obtaining pure standards for the assay of G protein alpha-subunits and does so using simple biochemical and immunological techniques.

Anal Biochem, 1994 Jul, 220(1), 137 - 41
A continuous, anaerobic spectrophotometric assay for chorismate synthase activity that utilizes photoreduced flavin mononucleotide; Ramjee MK et al.; A sensitive, continuous, spectrophotometric assay for chorismate synthase has been developed utilizing photoreduced flavin mononucleotide (FMNH2) as a cofactor under anaerobic conditions . The assay monitors directly the formation of chorismate from 5-enolpyruvylshikimate-3-phosphate (EPSP) at 275 nm with a precision of +/- 2 microM product . The assay conditions have been optimized with respect to FMNH2 (cofactor), EPSP (substrate) and enzyme concentrations, buffer type, and pH . The potential of the assay for detailed steady-state kinetic studies to elucidate the mechanism of action of this commercially important enzyme is also demonstrated.

Anal Biochem, 1994 Jul, 220(1), 115 - 21
An assay for myristoyl-CoA: protein N-myristoyltransferase activity based on ion-exchange exclusion of {3H}myristoyl peptide; French SA et al.; We developed an assay to test for inhibition of myristoyl-CoA:protein N-myristoyltransferase (NMT) activity since this enzyme is important in viral replication and cellular biochemistry . Saccharomyces cerevisae NMT was harvested from Escherichia coli carrying a plasmid vector containing the yeast NMT cDNA . Following the enzyme-catalyzed reaction of {3H}myristoyl-CoA and an octapeptide substrate (GlyAsnAla4Arg2-NH2), the assay mixture was loaded on AG1-8X anion-exchange resin which bound negatively charged reactants and by-products and left a doubly positively charged and nonbinding {3H}myristoyl-peptide product in the supernatant . Optimum conditions for separating reactants and by-products from myristoyl-peptide in a 100-pmol reaction were 450 mg resin and 25% methanol at pH 5.8 . Under these conditions 97% of myristic acid and 98% of myristoyl-CoA bound to the resin, whereas 99% of myristoyl peptide remained in the supernatant . The potent inhibitor S-(2-oxopentadecyl)-CoA was tested in our assay system . In addition, high-specific-activity {3H}myristoyl-CoA, synthesized using acyl-CoA synthetase, was purified on a 200-microCi scale (60 nmol) using a reverse-phase C-18 silica gel cartridge . Impurities, including free CoA, were washed from the column using 10% acetonitrile in 10 mM potassium phosphate buffer, pH 7.5, while purified (95% by radiochemical scan) myristoyl-CoA was eluted from the column using 1:1 acetonitrile:phosphate buffer.

Mutagenesis, 1994 Jul, 9(4), 367 - 75
A preliminary evaluation of the performance of the Muta Mouse (lacZ) and Big Blue (lacI) transgenic mouse mutation assays; Morrison V et al.; All of the published mutagenicity data generated using the Muta Mouse (lacZ) and the Big Blue (lacI) transgenic mouse mutation assays have been re-presented in a standard format . To date, 26 chemicals have been evaluated in one or other of the assays, and eight tissues have been sampled . The present analysis should expedite the further validation of these assays . In particular, it is suggested that data on the present 26 chemicals should be consolidated rather than the generation of partial data sets on additional chemicals.

Brain Res Mol Brain Res, 1994 Jul, 24(1-4), 77 - 88
Cloning of a DNA binding protein that is a tyrosine kinase substrate and recognizes an upstream initiator-like sequence in the promoter of the preprodynorphin gene; Gu J et al.; A 90 bp fragment prepared from the promoter region of the rat preprodynorphin gene formed a complex with rat brain nuclear extracts as assessed by gel mobility shift assays . An 8 base pair sequence, CACTCTCC, termed upstream regulatory element (URE), was identified within this fragment as a binding site by DNase 1 footprint analysis and gel mobility shift assays with synthetic oligonucleotides . The URE is a consensus sequence for a transcription initiator (Inr) element although in the preprodynorphin promoter it is located upstream at -208 and overlaps a region conserved between rat and human promoters . A unique 310 amino acid protein (UreB1) that specifically bound the URE was cloned from a rat brain cDNA library using the URE-containing oligonucleotide . Recombinantly expressed, affinity purified UreB1 protein retains specific binding to the URE oligonucleotide . UreB1 contains a tyrosine kinase phosphorylation consensus and binding is enhanced following phosphorylation with the p43v-abl tyrosine kinase . The UreB1 tyrosine phosphoprotein increases transcription in vitro, consistent with a positive transcriptional regulatory function . UreB1 transcripts are well expressed in subsets of neurons in multiple brain areas suggesting that, in addition to regulation of the preprodynorphin gene, it may have a more generalized role in gene transcription.

Klin Padiatr, 1994 Jul-Aug, 206(4), 342 - 5
{Venous thrombosis of cranial sinuses in asparaginase therapy . A case report}; Schulz U et al.; Changes in the coagulation system due to steroids and asparaginase during treatment for acute lymphoblastic leukemia (ALL) are well known side effects and may cause bleeding or thrombosis . We report the case history of a 7-year old girl who developed thrombosis of the sinus sagittalis superior during ALL-treatment . Diagnosis was made by computed tomography and magnetic resonance imaging after the child became symptomatic with seizure . Until this event the girl had been treated already for two weeks with prednisone and E . coli-asparaginase (4 infusions) . This medication caused distinct hypofibrinogenemia (Fibrinogen 53 mg/dl), prothrombin time expressed as percent of normal values of 58% was also pathological, activated partial thromboplastin time of 35 sec, antithrombin III 120% and thrombocyte count 178 G/l were in normal range . We were not successful in the attempt to adjust the imbalance in the coagulation system by transfusion of fresh frozen plasma (FFP)--seizure happened during FFP-infusion, fibrinogen blood level could be elevated only slightly . Our patient stayed consequently asymptomatic, the clinical recovery was confirmed radiologically.

J Lipid Res, 1994 Jul, 35(7), 1222 - 31
Expression and purification of human cholesterol 7 alpha-hydroxylase in Escherichia coli; Karam WG et al.; Cholesterol 7 alpha-hydroxylase (P450c7) is the first and rate-limiting enzyme in bile acid biosynthesis and is the product of a cytochrome P450 gene, CYP7 . We have previously reported the cloning of a full-length human cholesterol 7 alpha-hydroxylase cDNA (Karam, W . G., and J . Y . L . Chiang . 1992 . Biochem . Biophys . Res . Commun . 185: 588-595) . Using this clone in a polymerase chain reaction, we have generated a cDNA (H7 alpha 1.5) in which the codons for the N-terminal 24 amino acid residues were deleted . The translational product of this cDNA would be a truncated protein, P450c7(delta 2-24) with a hydrophilic NH2-terminal sequence, Met-Ala-Arg-Arg-Arg-Gln.. . This cDNA was cloned into the expression vector pJL and the construct pJL/H7 alpha 1.5 was transformed into E . coli strain TOPP3 . We have also ligated a truncated rat cholesterol 7 alpha-hydroxylase cDNA obtained previously (Li, Y . C., and J . Y . L . Chiang . 1991 . J . Biol . Chem . 266: 19186-19191) into the pJL vector and have transformed this construct (pJL/R7 alpha 1.5) into E . coli strain MV1304 . Both of these systems expressed functional cholesterol 7 alpha-hydroxylase in E . coli . A fivefold improvement in the expression of rat enzyme over the previous expression system was obtained . About 70-80% of the truncated human P450 in the clear lysate was localized in the cytosol . The truncated human and rat P450c7(delta 2-24) were purified to homogeneity . Reconstitution of cholesterol 7 alpha-hydroxylase activity using purified rat or human P450c7(delta 2-24) showed a similar Km of 6 and 7 microM for cholesterol, a Vmax of 0.13 and 0.14 nmol/min, and a turnover number of 1.3 and 1.5 per min, respectively . Immunoblotting experiment revealed that a polyclonal antibody raised against rat microsomal cholesterol 7 alpha-hydroxylase recognized both rat and human P450c7(delta 2-24) . This expression system provides a method for isolation of a large quantity of purified and catalytically active cholesterol 7 alpha-hydroxylase for the study of structure and function of this important enzyme in bile acid synthesis and cholesterol homeostasis.

J Hepatol, 1994 Jul, 21(1), 103 - 9
Hepatitis B virus X gene product acts as a transactivator in vivo; Balsano C et al.; It has previously been shown that the hepatitis B virus X gene product, pX, transactivates homologous and heterologous transcriptional regulatory sequences of viruses and various cellular genes in vitro . However, there is no evidence about the reproducibility and the relevance of this phenomenon in vivo . In this study we crossbred transgenic mice expressing the X gene under the control of the human antithrombin III (ATIII) gene regulatory sequences with transgenics carrying either the chloramphenicol acetyl-transferase or the LacZ bacterial reporter genes driven by the HIV1-LTR, which is known to be activated in trans by pX . Expression of pX in the liver stimulates the HIV1-LTR driven expression of both chloramphenicol acetyl-transferase and beta-galactosidase reporter genes in double transgenic mice . No detectable increase in chloramphenicol acetyl-transferase expression was observed in tissues, such as the spleen, brain and heart, that do not express pX . Our results confirm the transactivating properties of pX in vivo for the first time and support the hypothesis that pX might indeed modify gene expression in HBV-infected hepatocytes and influence viral pathogenesis.

J Comp Pathol, 1994 Jul, 111(1), 33 - 42
Effect of mixed live vaccine (Newcastle disease and infectious bronchitis) and Mycoplasma gallisepticum on the chicken respiratory tract and on Escherichia coli infection; Nakamura K et al.; Interaction between mixed live vaccine (Newcastle disease and infectious bronchitis), Mycoplasma gallisepticum (MG) and Escherichia coli (EC) was studied in specific-pathogen-free chickens, aged 7 days, inoculated intranasally . In the tracheas of chickens inoculated with vaccine, MG and EC, profuse multiplication of EC occurred together with severe and persisent histological lesions, and some birds died from EC infection . Similar though less dramatic effects occurred in birds that received vaccine and EC . The tracheas of chickens inoculated with the vaccine alone, or with MG and EC, or with MG alone, showed comparatively mild effects . There were no histological lesions in the tracheas of chickens inoculated with EC alone . This study suggests that the field use of mixed live vaccine in flocks infected with MG may induce EC septicaemia.

J Cell Biochem, 1994 Jul, 55(3), 389 - 97
Purification and characterization of an activated form of the protein tyrosine kinase Lck from an Escherichia coli expression system; Flint NA et al.; The lymphocyte-specific, nonreceptor protein tyrosine kinase Lck has been purified from an Escherichia coli expression system using a monoclonal antibody column followed by dye-affinity chromatography . Polyacrylamide gel electrophoretic analysis of purified protein revealed a single 56 kDa band, indicating that recombinant Lck was purified to near-homogeneity . The purified enzyme displayed tyrosine kinase activity as measured by both autophosphorylation and phosphorylation of exogenous substrates . Biochemical properties including protein phosphorylation and kinetic characteristics of the enzyme have been assessed . Peptide map analysis revealed that bacterially expressed Lck is phosphorylated predominantly on the autophosphorylation site (tyrosine-394), which is characteristic for activated protein tyrosine kinases . Indeed, we found that the recombinant enzyme is approximately fivefold more active than Lck from resting T cells, which is extensively phosphorylated at the regulatory carboxy-terminal tyrosine residue (tyrosine-505) . Thus, we have overproduced recombinant human Lck in E . coli and developed a simple two-step purification procedure which yields highly active enzyme . This will enable the identification and characterization of potential regulators and targets of Lck and thereby greatly facilitate studies which will clarify its role in T cell signal transduction.

Glia, 1994 Jul, 11(3), 211 - 26
Divergent lineages for oligodendrocytes and astrocytes originating in the neonatal forebrain subventricular zone; Luskin MB et al.; Although previous studies have revealed that the prenatal rat ventricular zone contains separate progenitor cells for neurons, astrocytes, and oligodendrocytes during the development of the cerebral cortex as early as the beginning of neurogenesis (Luskin et al., 1993; Grove et al., 1993), it is still unclear whether there are bipotential progenitor cells in the neonatal telencephalic subventricular zone which give rise to both astrocytes and oligodendrocytes during the peak of gliogenesis . To investigate this possibility, discrete groups of clonally related cells, generated by infecting progenitor cells of the neonatal subventricular zone with a retroviral lineage tracer, were analyzed ultrastructurally . An intracerebral injection of retrovirus encoding the reporter gene E . coli beta-galactosidase (lacZ) was made into the subventricular zone of newborn rats . Two weeks later their brains were perfused, sectioned, and histochemically reacted with X-Gal to identify at the light microscopic level clones of lacZ-positive cells . The sections were processed for electron microscopy to enable the identity of clonally related cells to be assessed at the ultrastructural level . All of the clones analyzed contained cells of the same phenotype and could be divided into four distinct types: immature cell clones situated in the subependymal zone surrounding the lateral ventricle, oligodendrocytes clones, and white or gray matter astrocyte clones . Not all of the cells in every clone displayed ultrastructural features of a mature cell . Rather, in some glial clones the lacZ-positive cells appeared to be at different stages of differentiation . However, we never encountered clones which contained both macroglial subtypes or clones containing neurons . Although the existence of bipotential progenitor cells cannot be completely dismissed, our results indicate the absence of progenitor cells in vivo in the neonatal subventricular zone which divide and generate astrocytes and oligodendrocytes.

Immunology, 1994 Jul, 82(3), 389 - 96
A 14,000 MW lipoprotein and a glycolipid-like structure of Borrelia burgdorferi induce proliferation and immunoglobulin production in mouse B cells at high frequencies; Honarvar N et al.; Sonicated preparations of Borrelia burgdorferi are able to stimulate unselected resting BALB/c spleen cells to proliferate and to produce immunoglobulin in vitro . FACS analysis of target cells prestained with an integrated cell-surface marker as well as cell-depletion experiments demonstrate that the majority of responding lymphocytes are B cells . Limiting dilution analyses of resting B cells revealed high frequencies of cells producing IgM (F 1/11-1/62) or IgG (F 1/5-1/163) in response to B . burgdorferi sonicate (B.b . sonicate) . These numbers were similar to those obtained with lipopolysaccharide (LPS) (IgM: F 1/20-1/84; IgG: F 1/14-1/85) or a synthetic lipopeptide of Braun's Escherichia coli lipoprotein (IgM: F 1/15, 1/19; IgG: F 1/148, 1/34) . The mitogenic structure(s) expressed by B . burgdorferi is distinct from LPS, as similar proliferative responses were obtained with B cells from LPS-resistant (C57BL/10ScCr and C3H/HeJ) and LPS-susceptible (C57BL/10ScSn, C3H/HeN) mice . Furthermore, B-cell mitogenic properties were also found in two distinct fractions of a phenol-chloroform-petroleum ether extract of B . burgdorferi: they consisted of a lipoprotein distinct from the outer surface proteins (Osp) A and B and glycolipid-like structures, respectively . These data suggest that spirochetes express a multitude of distinct structures with mitogenic activity for B cells including various lipoproteins as well as glycolipid(s).

Genetika, 1994 Jul, 30(7), 886 - 97
{Use of a plasmid with integrative function of phage phiC31 for transfer of cloned genes into Streptomyces strains}; Kudriavtseva EA et al.; Opportunities for application of integrative vectors carrying the attP site and the int gene of the temperate actinophage phi C31 for cloning genes in Streptomyces strains were demonstrated . The behavior of the integrative vectors pZAT22 and pTO1 in the model strain S . lividans TK64 and in the bialaphos-producing strain S . hygroscopicus, respectively, was characterized . Restriction maps of the S . lividans and S . hygroscopicus chromosomal regions containing attB sites were constructed . The bar gene of resistance to bialaphos was incorporated into the chromosome of the model strain S . lividans via the integrative pZAT22 vector, the level of expression of this gene within the chromosome of a heterologous host was determined . The possibility of amplification of the bar gene in the chromosome of the strain S . lividans was shown . The conjugative integrative vector pTO1, which carried the cloned bar gene, was introduced into the bialaphos-producing strain by intergeneric matings of Escherichia coli and S . hygroscopicus . The effect of an additional copy of the gene in the chromosome of S . hygroscopicus on strain resistance and productivity was studied.

Eur J Clin Invest, 1994 Jul, 24(7), 454 - 9
Adhesion of Helicobacter pylori and Escherichia coli to human and bovine surface mucus cells in vitro; Nilius M et al.; Helicobacter pylori shows in vivo a specific affinity for epithelial surface mucus cells (SMC) of the human stomach . We studied the in vitro adhesion of five different H . pylori strains and one non-pathogenic Escherichia coli-strain to (a) human antral SMC, obtained during gastroscopy; (b) human tumour SMC, from a carcinoma cell line (CRL 1739 AGS); and (c) bovine SMC, obtained from the abomasum . SMC of different origin were characterized by means of electron microscopy and immunohistochemistry, and showed similar main features: all cells showed intra-cellular structures like zymogens and PAS-positive mucin granules . HSMC were antibody-positive against epithelial cell markers . All five H . pylori strains adhered to human SMC (HSMC) and tumour SMC (TSMC) . Only one strain additionally adhered to bovine SMC (BSMC) . No adhesion to any of these cells was observed with E . coli . Adhesion in vitro is characterized by a close membrane-to-membrane association between H . pylori and the target cells . This phenomenon suggests a specific receptor-ligand interaction.

Curr Genet, 1994 Jul, 26(1), 87 - 90
A versatile shuttle cosmid vector for the efficient construction of genomic libraries and for the cloning of fungal genes; Osiewacz HD; A shuttle cosmid vector, pANsCos1, has been constructed for Escherichia coli and filamentous fungi . This vector contains two cos sequences separated by a single XbaI restriction site . pANsCos1 allows the efficient construction of representative genomic libraries from as little as 15-20 micrograms of genomic DNA . Due to the presence of a functional hygromycin B phosphotransferase gene (hph) transformation of fungal protoplasts with pAN-sCos1, or derivatives of it, results in the formation of hygromycin B-resistant transformants . The T7 and T3 RNA polymerase promoter sequences flanking the cloning site, in combination with two adjacent NotI sites facilitate genomic walking and the rapid construction of restriction maps of cloned inserts.

Curr Genet, 1994 Jul, 26(1), 38 - 44
Multiple-copy integration in the yeast Yarrowia lipolytica; Le Dall MT et al.; Using an EcoRI-BglII fragment of the G unit of the rDNA of Y . lipolytica and a set of 11 deletions in the URA3 promoter, we have constructed several plasmids to test gene amplification in the rDNA . These plasmids contain the rDNA fragment for integration, defective versions of the URA3 gene, the XPR2 gene encoding alkaline extracellular protease (AEP) as a reporter gene, and part of the pBR322 plasmid for selection and replication in E . coli . Among these plasmids, one corresponds to a deletion which allows multiple integration into the rDNA (plasmid pINA773) . Two other plasmids (pINA767 and pINA772) give multiple integration only with a mutated URA3 gene . Transformants carrying these three plasmids were tested for copy number, stability, chromosomal localization and AEP secretion . Transformants containing plasmids pINA767, 772 and 773 displayed an average copy number of 5, 12 and 25-60 copies respectively of the plasmid, as estimated by PCR and DNA hybridization . Integrations occurred in only one chromosome except for transformants containing 60 copies where copies were observed at least in two different chromosomes . Multiple integrations were found both as tandem repeats and as dispersed copies . Plasmid copy number was stable, in both minimum and rich media, for strains containing less than ten copies per cells . However, for higher copy number, multiple integrations were stable only when AEP synthesis was not induced, while in inducing medium stability of the multiple integrations was dramatically affected.

Br Poult Sci, 1994 Jul, 35(3), 427 - 32
Plasma alpha 1-acid glycoprotein concentration in broilers: influence of age, sex and injection of Escherichia coli lipopolysaccharide; Takahashi K et al.; 1 . The influence of age, sex and injection of Escherichia coli lipopolysaccharide (LPS) on the plasma concentration of alpha-1 acid glycoprotein (AGP) was determined in broilers using the single radial immunodiffusion method . 2 . Plasma AGP concentration increased in the 3 d after hatching, and then stabilised at 240 +/- 33 micrograms/ml up to 14 d of age . 3 . No sex-related differences in plasma AGP concentration were observed up to 6 weeks of age . 4 . A single injection of 900 micrograms LPS per chick resulted in a 5-fold increase in AGP concentration compared with that in saline-injected chicks . Multiple injections of LPS (200 micrograms/chick every 2 d for 14 d) caused only a 50% increase in AGP concentration.

J Vet Diagn Invest, 1994 Jul, 6(3), 293 - 6
Cloning, expression, and sequence analysis of the ORF4 gene of the porcine reproductive and respiratory syndrome virus MN-1b; Kwang J et al.; Porcine reproductive and respiratory syndrome virus (PRRSV) MN-1b strain open reading frame 4 (ORF4) has been cloned, sequenced, and expressed in Escherichia coli . The homologies of nucleotide and amino acid sequences between MN-1b (US isolate) and LV (European isolate) are 69% and 64%, respectively . The data also showed that ORF4 of MN-1b is 36 bases shorter than that of LV . Western blot analysis of expressed recombinant ORF4 protein reacted with 65% (26/40) of PRRSV-infected pig sera tested . These results demonstrated that ORF4 of PRRSV may not be a well-conserved region.

Bioconjug Chem, 1994 Jul-Aug, 5(4), 283 - 6
Stabilization of L-asparaginase modified with comb-shaped poly(ethylene glycol) derivatives, in vivo and in vitro; Kodera Y et al.; L-Asparaginase from Escherichia coli was coupled with two types of comb-shaped copolymer of poly-(ethylene glycol) derivative and maleic anhydride (activated PM), having molecular weights of 13,000 and 100,000 (activated PM13 and PM100, respectively) with multivalent reaction sites . After single intravenous injections of PM100-asparaginase and nonmodified asparaginase into rats, the enzymic activity of PM100-asparaginase in serum was well retained for at least 11 days, and the serum L-asparagine concentration remained undetectable for 27 days . The half-lives of PM100-asparaginase and nonmodified asparaginase were 50 and 1.5 h, respectively . Stabilization of L-asparaginase toward heat, urea, and acidity was caused by modifying the enzyme with activated PM13 and PM100 . Especially, PM100-asparaginase retained high enzymic activity toward heat and urea, compared with PM13-asparaginase . It was suggested that these modifiers with a comb-shaped form and with multivalent reactive sites cover the whole surface of the asparaginase molecule and stabilize its conformation possibly through multiple covalent bindings and through various noncovalent interactions.

Int Immunol, 1994 Jul, 6(7), 963 - 71
Demonstration of the target molecule of a protective IgE antibody in secretory glands of Schistosoma japonicum larvae; Nara T et al.; We have demonstrated that a mouse monoclonal IgE antibody, SJ18 epsilon.1, recognizes a 97 kDa surface molecule (Sj97) of Schistosoma japonicum larvae and that the antibody induces partial but significant protection against the skin to lung-stage of S . japonicum infection . The antibody stimulates eosinophil- and macrophage-mediated killing of schistosomula in vitro . In the present study, we isolated the putative full-length cDNA of Sj97 by screening a lambda gt11 cDNA library from S . japonicum adult worms with SJ18 epsilon.1 . The predicted amino acid sequence of the cDNA showed highly significant homology to that of S . mansoni paramyosin, a potential vaccine candidate for schistosomiasis . The deletion mutants of S . japonicum paramyosin were expressed in Escherichia coli and the translation product of 443 amino acid residues of paramyosin was found to be recognized by the antibody . Moreover, we observed by immunoelectron microscopy the presence of paramyosin in the post-acetabular gland as well as in the tegument and muscle layers of the larvae . These results suggest that paramyosin is a secretory protein which may be incorporated into the tegument during the development of schistosomula, thus becoming a target for protective immunity during the migratory phase of the parasite.

Int Immunol, 1994 Jul, 6(7), 1017 - 25
Recombination hotspot associated factors specifically recognize novel target sequences at the site of interchromosomal rearrangements in T-ALL patients with t(8;14)(q24;q11) and t(1;14)(p32;q11); Kasai M et al.; A DNA binding protein was identified which binds to two novel target-like sequences: (i) at the 5' flanking site of the breakpoint junction of chromosome 8 in a patient with T-acute lymphoblastic leukemia (ALL) carrying the t(8;14)(q24;q11) rearrangement and (ii) on chromosome 1 in three of five T-ALL patients with the t(1;14)(p32;q11) rearrangement . This protein {provisionally called recombination hotspot associated factor (ReHF-1)} was also found to bind to a similar target sequence that is present immediately at the 3' end of the human V delta 3 gene segment . In a small number of lines tested, the ReHF-1 protein was expressed in gamma delta lineage T cells and in a number of B cell precursor ALLs whose TCR delta locus has been rearranged . The molecular weight of ReHF-1 protein was determined to be approximately 30 kDa by UV cross-linking analysis . Gel filtration chromatography and sedimentation velocity centrifugation analyses indicate that the ReHF-1 protein exists as a multimeric protein in its native form . These data might suggest a possible role for this protein in the rearrangement of the TCR delta locus . Furthermore, another protein, ReHF-2, that appears to have strict sequence specificity was found to bind only to the complementary single strand of the target sequence . The interaction of these proteins with a conserved target sequence at the chromosomal breakpoint junction might suggest that they are involved in a novel enzymatic mechanism reminiscent of the general features of DNA recombination or replication events in Escherichia coli or Saccharomyces cerevisiae.

Biotechniques, 1994 Jul, 17(1), 130 - 5, 137
Restriction site-independent formation of chimeras from homologous neurotransmitter-transporter cDNAs; Moore KR et al.; To evaluate structure/function relationships among neurotransmitter transporters, chimeric cDNAs were formed between antidepressant- and cocaine-sensitive norepinephrine and serotonin transporters . To eliminate experimenter bias in the positioning of chimeric junctions, we utilized a novel method that involves the in situ formation of chimeras in transformed Escherichia coli from linearized plasmids that bear a single copy of both parental transporter cDNAs . Colonies recovered after growth on selective media frequently contain plasmids bearing chimeric inserts . Coupled with the vaccinia-T7 expression system, this method allowed us to rapidly generate and evaluate multiple transporter chimeras without the need to introduce compatible restriction sites or the time and expense involved in formation of individual chimeric cDNAs . Transporter chimeras with switch points proximal but not distal to the middle of putative transmembrane domain 1 retain serotonin or norepinephrine transport and high-affinity antagonist recognition . Loss of substrate and antagonist recognition despite normal levels of transporter protein by distal chimeras suggests important and divergent interactions between multiple transmembrane domains in forming ligand binding sites . The method of chimera synthesis applied in these studies is likely to be of generic utility particularly when the formation of a library of chimeric cDNAs is desired.

Virus Res, 1994 Jul, 33(1), 55 - 66
In vitro expression of UL56 gene of herpes simplex virus type 1; detection of UL56 gene product in infected cells and in virions; Kehm R et al.; In order to investigate the functional properties of the UL56 gene of herpes simplex virus type 1 (HSV-1), it was necessary to express the UL56 protein in vitro . The DNA sequences corresponding to the open reading frame of the UL56 gene of HSV-1 strain F were amplified from genomic viral DNA by PCR using primers corresponding to the translational start and termination regions of the UL56 ORF . The PCR product (705 bp) was inserted into the EcoRI/XbaI recognition sites of the bacterial expression vector pMal-c2 . This procedure allowed the expression of the viral UL56 gene fused to the maltose-binding protein (MBP) of Escherichia coli, and subsequent cleavage of the fusion protein with the specific protease factor Xa . The induced fusion protein was purified by affinity chromatography using amylose columns . The apparent molecular weight of the fusion protein was about 70 kDa . Factor Xa cleaves the fusion protein into two subfragments of 42 kDa (MBP) and 30 kDa (UL56) . Rabbit antisera induced against recombinant UL56 protein were used for detection of the UL56 gene product during the infection cycles of HSV-1 . The presence of the UL56 protein was detected in infected cells and in HSV-1 virions by Western blot experiments and by immunofluorescence assays . A strong and increasing cytoplasmic fluorescence was observed in RC-37 cells infected with HSV-1 strain F between 6 and 16 h post-infection . In addition it was found that human HSV-1 IgM/IgG positive convalescent sera recognized the recombinant UL56 protein.

Proteins, 1994 Jul, 19(3), 244 - 55
Energy minimization method using automata network for sequence and side-chain conformation prediction from given backbone geometry; Kono H et al.; Globular proteins have high packing densities as a result of residue side chains in the core achieving a tight, complementary packing . The internal packing is considered the main determinant of native protein structure . From that point of view, we present here a method of energy minimization using an automata network to predict a set of amino acid sequences and their side-chain conformations from a desired backbone geometry for de novo design of proteins . Using discrete side-chain conformations, that is, rotamers, the sequence generation problem from a given backbone geometry becomes one of combinatorial problems . We focused on the residues composing the interior core region and predicted a set of amino acid sequences and their side-chain conformations only from a given backbone geometry . The kinds of residues were restricted to six hydrophobic amino acids (Ala, Ile, Met, Leu, Phe, and Val) because the core regions are almost always composed of hydrophobic residues . The obtained sequences were well packed as was the native sequence . The method can be used for automated sequence generation in the de novo design of proteins.

J Photochem Photobiol B, 1994 Jul, 24(2), 129 - 39
Biophysical and biological properties of newly synthesized dioxinocoumarin derivatives . II . Dark and photoinduced effects on T7 phage, yeast and HeLa cells; Csik G et al.; The dioxinocoumarin derivatives 5H-{2}benzopyrano-{3,4-g}{1,4}benzodioxin-5-one (I), 5H-{2}benzopyrano-{3,4-g}{2,3}-dihydro-{1,4}benzodioxin-5-on e II, 6H-{2}benzopyrano{3,4-f}-1,4-benzodioxin-6-one (III) and 6H-{2}benzopyrano{3,4-f}-2,3-dihydro-1,4-benzodioxin-6-one (IV) were synthesized . Their biological effect was studied in the presence and absence of UVA radiation, and compared with that of 8-methoxypsoralen (8-MOP) and angelicin derivatives on T7 phage, diploid yeast (Saccharomyces cerevisiae) and HeLa cells . The photobiological activities of compounds I and III were stronger than that of 8-MOP in phage inactivation and DNA synthesis inhibition in HeLa cells, whereas compounds II and IV, with a saturated dioxin ring, showed very poor activity . The photosensitizing activity of dioxinocoumarins on phage inactivation decreased by a factor of two to three in the absence of oxygen . Treatments with compound I and UVA in the presence of oxygen modified the helical structure and stability of phage DNA and proteins . Compounds I and II were more active than IV for photoinduced cell killing in yeast, although always less active than 8-MOP . At comparable photocytotoxic levels, compounds I and III were as strong inducers of cytoplasmic "petite" mutants in yeast as angelicin, suggesting a possible monofunctional mode of action with cellular DNA.

J Photochem Photobiol B, 1994 Jul, 24(2), 123 - 8
Synergistic action of near-UV and phenylalanine, tyrosine or tryptophan on the inactivation of phage T7: role of superoxide radicals and hydrogen peroxide; Craggs J et al.; Near ultraviolet (NUV) light can cause a variety of damage to biological systems . The effects of NUV are significantly enhanced in the presence of sensitizers . One of the most important targets of such synergistic effects is DNA . Cellular DNA exposed to NUV plus sensitizers is damaged in a variety of ways, DNA strand breaks and interstrand cross-links being the most common effects . In this study, phenylalanine, tyrosine and tryptophan are shown to act as sensitizers for NUV action of phage T7; superoxide anions are produced . The reactive species probably interacts with phage DNA causing damage responsible for phage inactivation . Superoxide dismutase reverses the synergistic activities of phenylalanine and tyrosine on NUV-induced phage inactivation, but catalase is additionally required to reverse the effect of tryptophan . Therefore, it is probable that NUV photolysis of tryptophan causes the production of superoxide ions and hydrogen peroxide, both of which contribute to phage inactivation . The ubiquitous nature of NUV in our environment and the presence of amino acids in skin cells suggests that an important mechanism for the induction of skin cancer in humans by solar exposure is amino acid photolysis by NUV.

J Clin Microbiol, 1994 Jul, 32(7), 1733 - 8
Use of recombinant OspC from Borrelia burgdorferi for serodiagnosis of early Lyme disease; Padula SJ et al.; Infection with Borrelia burgdorferi, the etiologic agent of Lyme disease, is associated with an early and dominant humoral response to the spirochete's 23-kDa outer surface protein C (OspC) . We have cloned and expressed OspC as a fusion protein in Escherichia coli and have shown that patient serum samples react with it in an enzyme-linked immunosorbent assay (ELISA) (S . J . Padula, A . Sampieri, F . Dias, A . Szczepanski, and R . W . Ryan, Infect . Immun . 61:5097-5105, 1993) . Now we have compared the detection of B . burgdorferi-specific immunoglobulin M antibodies in 74 individuals with culture-positive erythema migrans by a whole-cell ELISA, immunoblot, and the recombinant OspC (rOspC) ELISA . Seventy-six negative controls were also studied . With all of the tests, there was a statistically significant association between the duration of disease and the frequency of a positive result . With the rOspC ELISA, the predictive value of a positive test was 100% and the predictive value of a negative test was 74% . Similar results were obtained with the whole-cell ELISA and with the immunoblot using as the source of test antigen a strain of B . burgdorferi which expresses abundant levels of OspC . We conclude that the use of rOspC in an ELISA is a convenient, readily automated, and easily standardized test for the serodiagnosis of early Lyme disease.

J Appl Bacteriol, 1994 Jul, 77(1), 105 - 12
Visible light damage to Escherichia coli in seawater: oxidative stress hypothesis; Gourmelon M et al.; The effect of visible light on Escherichia coli H10407 in seawater microcosms was investigated . Light damage was estimated by loss of colony-forming ability . Illumination of E . coli suspended in oligotrophic seawater with visible light at an intensity of about 40 klux caused a drastic decrease of culturable bacteria which turned to a viable but non-culturable state . In seawater E . coli exhibited weak metabolic activity as estimated by 3H methyl-thymidine incorporation in the cell . Visible light did not significantly alter this metabolic activity and did not involve detectable oxidation of lipid membranes as evaluated by gas chromatography analysis of fatty acids . The involvement of oxygen and reactive oxygen species in phototoxicity was studied . A decrease of the toxic effect was observed when E . coli was exposed to visible light under anaerobic conditions . Scavengers of reactive oxygen species exhibited variable protective effects . beta-Carotene, a singlet oxygen scavenger, and superoxide dismutase were equally ineffective . On the other hand, catalase, which eliminates hydrogen peroxide and thiourea, a hydroxyl radical scavenger, showed a net protection . In addition desferrioxamine B, an iron chelator, was also effective in reducing phototoxicity, probably by preventing hydroxyl radical generation by decomposition of hydrogen peroxide in the presence of iron (Fenton reaction) . Therefore, hydrogen peroxide and hydroxyl radical seem to be reactive intermediates of oxygen-dependent (type II) photosensitized reactions.

Chem Pharm Bull (Tokyo), 1994 Jul, 42(7), 1455 - 8
Acceleration of DNA cleavage by benzidine derivatives under weakly acidic conditions; Yamashita RI et al.; Benzidine derivatives, especially 2,7-diaminofluorene and benzidine, accelerated the cleavage of a plasmid DNA (form I) under weakly acidic conditions at 37 degrees C . The acceleration was not inhibited by EDTA, superoxide dismutase, catalase, or hydroxyl radical scavengers . The rate of the cleavage was influenced by the pH value of the reaction buffer . The decrease in form I DNA in this reaction obeyed first-order kinetics . It was confirmed that the cleavage was enhanced by the amines themselves, and not by their oxidized derivatives . 2,7-Diaminofluorene also cleaved a 3'-end-labeled 40-mer DNA with A and G base specificity . These results indicate that the acceleration is caused by depurination followed by breakage of the DNA sugar backbone.

Plant J, 1994 Jul, 6(1), 87 - 97
A cis-acting element and a trans-acting factor involved in the wound-induced expression of a horseradish peroxidase gene; Kawaoka A et al.; The mechanisms that control the wound-induced expression of the prxC2 gene for horseradish peroxidase (HRP) have been investigated . Analysis of the regulatory properties of 5'-deleted promoters showed that a positive element involved in the response to wounding was located between -307 and -99 bp from the site of initiation of translation . In in vitro binding assays of tobacco nuclear proteins and DNA fragments of prxC2 promoter, the binding site was the Box 1 from -296 to -283 containing the CACGTG motif . To identify the functional role of Box 1, the prxC2 promoter that has been digested from the 5' end to -289 with a disrupted Box 1 was fused to a reporter gene for beta-glucuronidase (GUS) . No induction of GUS activity was observed in transgenic tobacco plants with the prxC2 (-289)/GUS construct . These data indicated that the expression of prxC2 in response to wounding required the Box 1 sequence from -296 to -283 . Furthermore, a tobacco cDNA expression library was screened and a cDNA clone for a protein, designated TFHP-1, that bound specifically to the Box 1 sequence was identified . The putative TFHP-1 protein contains a basic region and leucine zipper (bZip) motif and a helix-loop-helix (HLH) motif . The mRNA for TFHP-1 was abundant in roots and stems, and it was not induced by wounding in leaves . In tobacco protoplasts, antisense TFHP-1 suppressed the expression of prxC2(-529)/GUS.

Plant J, 1994 Jul, 6(1), 55 - 66
Transposition of a Ds element from a plasmid into the plant genome in Nicotiana plumbaginifolia protoplast-derived cells; Houba-Herin N et al.; Nicotiana plumbaginifolia haploid protoplasts were co-transformed with two plasmids, one with a NPT-II/Ds element and one with a gene encoding an amino-terminal truncated Ac transposase . It is shown that Ds can efficiently transpose from extrachromosomal DNA to N . plumbaginifolia chromosomes when the Ac transposase gene is present in trans . Ds has been shown to have transposed into the plant genome in a limited number of copies (1.9 copies per genome), for 21/32 transgenic lines tested . The flanking sequences present in the original plasmid are missing in these 21 plants . In only two of 21 plants was part of the transposase construct integrated . By segregation analysis of transgenic progeny, Ds was shown to be present in the heterozygous state in 10 lines even though haploid protoplasts had been originally transformed . This observation could indicate that integration occurred after or during DNA replication that leads to protoplast diploidization.

Plant J, 1994 Jul, 6(1), 113 - 21
Molecular characterization of Arabidopsis thaliana cDNAs encoding three purine biosynthetic enzymes; Schnorr KM et al.; Glycinamide ribonucleotide (GAR) synthetase, GAR transformylase and aminoimidazole ribonucleotide (AIR) synthetase are the second, third and fifth enzymes in the 10-step de novo purine biosynthetic pathway . From a cDNA library of Arabidopsis thaliana, cDNAs encoding the above three enzymes were cloned by functional complementation of corresponding Escherichia coli mutants . Each of the cDNAs encode peptides comprising the complete enzymatic domain of either GAR synthetase, GAR transformylase or AIR synthetase . Comparisons of the three Arabidopsis purine biosynthetic enzymes with corresponding enzymes/polypeptide-fragments from procaryotic and eucaryotic sources indicate a high degree of conserved homology at the amino acid level, in particular with procaryotic enzymes . Assays from extracts of E . coli expressing the complementing clones verified the specific enzymatic activity of Arabidopsis GAR synthetase and GAR transformylase . Sequence analysis, as well as Northern blot analysis indicate that Arabidopsis has single and monofunctional enzymes . In this respect the organization of these three plant purine biosynthesis genes is fundamentally different from the multifunctional purine biosynthesis enzymes characteristic of other eucaryotes and instead resembles the one gene, one enzyme relationship found in procaryotes.

Plant J, 1994 Jul, 6(1), 105 - 12
Isolation and characterization of two cDNA clones encoding ATP-sulfurylases from potato by complementation of a yeast mutant; Klonus D et al.; Sulfur plays an important role in plants, being used for the biosynthesis of amino acids, sulfolipids and secondary metabolites . After uptake sulfate is activated and subsequently reduced to sulfide or serves as donor for sulfurylation reactions . The first step in the activation of sulfate in all cases studied so far is catalyzed by the enzyme ATP-sulfurylase (E.C . 2.7.7.4.) which catalyzes the formation of adenosine-5'-phosphosulfate (APS) . Two cDNA clones from potato encoding ATP-sulfurylases were identified following transformation of a Saccharomyces cerevisiae mutant deficient in ATP-sulfurylase activity with a cDNA library from potato source leaf poly(A)+ RNA cloned in a yeast expression vector . Several transformants were able to grow on a medium with sulfate as the only sulfur source, this ability being strictly linked to the presence of two classes of cDNAs . The clones StMet3-1 and StMet3-2 were further analyzed . DNA analysis revealed an open reading frame encoding a protein with a molecular mass of 48 kDa in the case of StMet3-1 and 52 kDa for StMet3-2 . The deduced polypeptides are 88% identical at the amino acid level . The clone StMet3-2 has a 48 amino acid N-terminal extension which shows common features of a chloroplast transit peptide . Sequence comparison of the ATP-sulfurylase Met3 from Saccharomyces cerevisiae with the cDNA StMet3-1 (StMet3-2) reveals 31% (30%) identity at the amino acid level . Protein extracts from the yeast mutant transformed with the clone StMet3-1 displayed ATP-sulfurylase activity . RNA blot analysis demonstrated the expression of both genes in potato leaves, root and stem, but not in tubers.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Sci, 1994 Jul, 3(7), 1128 - 30
Structural similarity between binding sites in influenza sialidase and isocitrate dehydrogenase: implications for an alternative approach to rational drug design; Poirrette AR et al.; Using searching techniques based on algorithms derived from graph theory, we have established a similarity between a 3-dimensional cluster of side chains implicated in drug binding in influenza sialidase and side chains involved in isocitrate binding in Escherichia coli isocitrate dehydrogenase . The possible implications of the use of such comparative methods in drug design are discussed.

Protein Sci, 1994 Jul, 3(7), 1125 - 7
Crystallization and preliminary X-ray crystallographic studies of UDP-N-acetylenolpyruvylglucosamine reductase; Benson TE et al.; The overexpression and purification of the second enzyme in Escherichia coli peptidoglycan biosynthesis, UDP-N-acetylenolpyruvylglucosamine reductase (MurB), provided sufficient protein to undertake crystallization and X-ray crystallographic studies of the enzyme . MurB crystallizes in 14-20% PEG 8000, 100 mM sodium cacodylate, pH 8.0, and 200 mM calcium acetate in the presence of its substrate UDP-N-acetylglucosamine enolpyruvate . Crystals of MurB belong to the tetragonal space group P4(1)2(1)2 with a = b = 49.6 A, c = 263.2 A, and alpha = beta = gamma = 90 degrees at -160 degrees C and diffract to at least 2.5 A . Screening for heavy atom derivatives has yielded a single site that is reactive with both methylmercury nitrate and Thimerosal.

Protein Sci, 1994 Jul, 3(7), 1089 - 97
Isolation of Escherichia coli synthesized recombinant eukaryotic proteins that contain epsilon-N-acetyllysine; Violand BN et al.; Recombinant porcine (rpST) and bovine somatotropins (rbST) synthesized in Escherichia coli contain the amino acid, epsilon-N-acetyllysine . This amino acid was initially discovered in place of the normal lysine144 in a modified reversed-phase HPLC (RP-HPLC) species of rpST . Mass spectrometry and amino acid sequencing of a tryptic peptide isolated from this RP-HPLC purified protein were used to identify this altered residue as epsilon-N-acetyllysine . Ion-exchange chromatography was utilized to prepare low isoelectric point (pI) forms of rpST and rbST, which are enriched in epsilon-N-acetyllysine . Electrospray mass spectrometry demonstrated that the majority of the protein in these low pI fractions contained species 42 Da larger than normal . Immobilized pH gradient electrophoresis (IPG) of the ion-exchange purified low pI proteins was used to isolate several monoacetylated species of rpST and rbST . The location of the acetylated lysine in each IPG-purified protein was determined by tryptic peptide mapping and amino acid sequencing of the altered tryptic peptides . Amino acid analyses of enzymatic digests of rpST and rbST were also used to confirm the presence of epsilon-N-acetyllysine in these recombinant proteins . These data demonstrate that a significant portion of rpST and rbST produced in E . coli contain this unusual amino acid.

Protein Sci, 1994 Jul, 3(7), 1058 - 68
The tryptophan residues of mitochondrial creatine kinase: roles of Trp-223, Trp-206, and Trp-264 in active-site and quaternary structure formation; Gross M et al.; The 5 tryptophan residues of chicken sarcomeric mitochondrial creatine kinase (Mib-CK) were individually replaced by phenylalanine or cysteine using site-directed mutagenesis . The mutant proteins were analyzed by enzyme kinetics, fluorescence spectroscopy, circular dichroism, and conformational stability studies . In the present work, Trp-223 is identified as an active-site residue whose replacement even by phenylalanine resulted in > or = 96% inactivation of the enzyme . Trp-223 is responsible for a strong (18-21%) fluorescence quenching effect occurring upon formation of a transition state-analogue complex (TSAC;Mib-CK.creatine.MgADP.NO3-), and Trp-223 is probably required for the conformational change leading to the TSAC-induced octamer dissociation of Mib-CK . Replacement of Trp-206 by cysteine led to a destabilization of the active-site structure, solvent exposure of Trp-223, and to the dissociation of the Mib-CK dimers into monomers . However, this dimer dissociation was counteracted by TSAC formation or the presence of ADP alone . Trp-264 is shown to be located at the dimer-dimer interfaces within the Mib-CK octamer, being the origin of another strong (25%) fluorescence quenching effect, which was observed upon the TSAC-induced octamer dissociation . Substitution of Trp-264 by cysteine drastically accelerated the TSAC-induced dissociation and destabilized the octameric structure by one-fourth of the total free interaction energy, probably by weakening hydrophobic contacts . The roles of the other 2 tryptophan residues, Trp-213 and Trp-268, could be less well assigned.

Protein Sci, 1994 Jul, 3(7), 1052 - 7
A conformational change in the lactose permease of Escherichia coli is induced by ligand binding or membrane potential; Jung H et al.; Lactose transport in membrane vesicles containing lactose permease with a single Cys residue in place of Val 315 is inactivated by N-ethylmaleimide in a manner that is stimulated by substrate or by a H+ electrochemical gradient (delta microH+; Sahin-Toth M, Kaback HR, 1993, Protein Sci 2:1024-1033) . The findings are confirmed and extended in this communication . Purified, reconstituted Val 315-->Cys permease reacts with N-ethylmaleimide or hydrophobic fluorescent maleimides but not with a membrane impermeant thiol reagent, and beta-galactosides specifically stimulate the rate of labeling . Furthermore, the reactivity of purified Val 315-->Cys permease is enhanced by imposition of a membrane potential (delta psi, interior negative) . The results indicate that either ligand binding or delta psi induces a conformational change in the permease that brings the N-terminus of helix X into an environment that is more accessible from the lipid phase.

Mol Reprod Dev, 1994 Jul, 38(3), 268 - 74
Liposome-mediated DNA transfer into chicken primordial germ cells in vivo; Watanabe M et al.; In embryogenesis, avian primordial germ cells (PGCs) circulate temporarily in the blood vessels at stages 10-15 (Hamburger and Hamilton, 1951), before reaching the gonads . In an attempt to transfer cloned genes into PGCs, liposome consisting of reporter plasmid DNA and N-{1-(2,3-Dioleoyloxy)propyl}-N,N,N-trimethylammoniummethylsulf ate was injected into the marginal veins of embryos at stages 11-15 . As reporter plasmids, pRSVZ and pAcZ harboring the Escherichia coli lacZ gene driven, respectively, by the Rous sarcoma virus (RSV) promoter and the chicken beta-actin gene promoter were used . First, 55 embryos were injected with liposome containing pRSVZ and stained for the bacterial beta-galactosidase activity 24 hr after injection . In all the embryos, cells positive for beta-galactosidase activity were observed among the blood cells, endothelial cells, and endocardium cells of the heart, suggesting that transfection took place within the circulatory system . Then, embryos were injected with liposome containing pRSVZ or pAcZ, and stained 2 or 3 d after injection . PGCs positive for beta-galactosidase activity were observed in the gonads in four out of 44 embryos injected with pRSVZ, and 29 out of 71 embryos injected with pAcZ, indicating that the plasmid DNA was transferred into PGCs developing normally . The average number of positive PGCs per embryo was 0.2 and 2.1, respectively, when pRSVZ and pAcZ were introduced . The difference in the number of positive PGCs detected after introduction of the two plasmids suggests that the actin promoter has a higher level of transcriptional activity in PGCs than does the RSV promoter.

Invest Ophthalmol Vis Sci, 1994 Jul, 35(8), 3251 - 9
Sparing of the ipsilateral retina after anterior chamber inoculation of HSV-1: requirement for either CD4+ or CD8+ T cells; Azumi A et al.; PURPOSE . To determine whether CD4+ T cells, CD8+ T cells, or both CD4+ and CD8+ T cells are required for preservation of the ipsilateral retina after uniocular anterior chamber inoculation of herpes simplex virus type 1 (HSV-1) . METHODS . Adult-thymectomized BALB/c mice were T cell depleted by administration of anti-CD4 monoclonal antibody (mAb), anti-CD8 mAb, or anti-CD4 mAb and anti-CD8 mAb together . Control mice were thymectomized but were not T cell depleted . HSV-1 (KOS) was inoculated in one anterior chamber . At intervals after inoculation, the injected eyes were examined histopathologically or homogenized to determine the kinetics of infectious virus recovery . Additional groups of in vivo depleted mice were injected with wild type KOS and RH116 (a mutant of KOS containing the Escherichia coli beta-galactosidase gene) to determine whether viral genes were expressed in the retina in any of the mice . RESULTS . In the inoculated eyes of mice depleted of both CD4+ and CD8+ T cells, there was a significantly higher incidence of acute destructive retinitis at days 9 and 14 postinoculation (PI), and the titer of virus recovered at day 14 PI was significantly higher . Viral gene expression in the retina and the optic nerve was observed after day 7 PI only in the group of mice depleted of both CD4+ and CD8+ cells . In contrast, acute destructive retinitis was not observed in nondepleted mice or in mice depleted of either CD4+ or CD8+ T cells alone, and virus recovery was not significantly different among these three groups of mice . No virus-infected cells were observed in the optic nerve or the sensory retina of nondepleted mice, of mice depleted of only CD4+ cells, or of mice depleted of only CD8+ cells . CONCLUSION . The results of these studies suggest that either CD4+ or CD8+ T cells can spare the retina of the injected eye after uniocular anterior chamber inoculation of HSV-1 . Because virus appeared after day 7 PI in the ipsilateral optic nerve and retina only in mice depleted of both CD4+ and CD8+ T cells, these results suggest that spread of virus to the ipsilateral retina occurs via the optic nerve and that either CD4+ or CD8+ T cells can prevent spread of virus to the inoculated eye resulting in sparing of the ipsilateral retina.

Eur J Biochem, 1994 Jul 1, 223(1), 179 - 87
Expression of functional proliferating-cell nuclear antigen from rice (Oryza sativa) in Escherichia coli . Activity in association with human DNA polymerase delta; Matsumoto T et al.; Proliferating-cell nuclear antigen (PCNA), the auxiliary protein for DNA polymerase delta, is one of the key factors for both PCNA-dependent DNA synthesis and cell-cycle progression . Plant PCNA genes have previously been cloned from rice, carrot, tobacco, and soybean cells by screening the cDNA libraries using similarity to the human or rat PCNA genes . We subcloned the relevant gene from the rice PCNA cDNA into an Escherichia coli expression vector pMAL, and the PCNA protein was expressed in the bacteria in the form of a fusion protein (70 kDa) with maltose-binding protein (MBP) . Monoclonal antibody against human PCNA reacted with both purified fusion protein and a 32-kDa fragment, resulting from restriction protease (factor Xa) digestion of the fusion protein . The N-terminal amino acid sequence of the 32-kDa fragment was identical to that of rice PCNA sequence . Rice PCNA fusion protein was found to stimulate DNA synthesis catalyzed by DNA polymerase delta from human cells (although much less effectively), while having no effect on DNA polymerase alpha activity . The results indicate that plant PCNA functions as one of the cofactors of DNA synthesis as is the case with other eukaryotes.

J Bacteriol, 1994 Jul, 176(14), 4235 - 42
Effects of reduced levels of GroE chaperones on protein metabolism: enhanced synthesis of heat shock proteins during steady-state growth of Escherichia coli; Kanemori M et al.; The GroE heat shock proteins (GroEL and GroES) of Escherichia coli represent major molecular chaperones that participate in folding (and assembly) of a variety of proteins and are essential for cell growth at all temperatures . We have examined the effects of reducing the cellular content of GroE on the synthesis and stability of proteins during steady-state growth with near-normal rates . The GroE protein level was manipulated by placing groE under the control of lacUV5 promoter on a multicopy plasmid in a strain lacking the chromosomal groE operon . When this strain was grown with a limited concentration (40 microM) of inducer (IPTG {isopropyl-beta-D-thiogalactopyranoside}) at 37 degrees C, the GroE level and growth rate were comparable to those of the wild type . When cells were depleted of IPTG, they continued to grow at or below 37 degrees C albeit at reduced rates, despite the much-reduced GroE level (ca . 25% of that of wild type) . Under these conditions, the cellular contents of at least 13 polypeptides were affected . Among the most striking effects was the enhanced synthesis of a set of heat shock proteins which resulted from the increased level of sigma 32 which is required for transcription of heat shock genes . This increase in the sigma 32 level was brought about by both stabilization and increased synthesis of sigma 32 . Other proteins affected by the reduced GroE level included two proteins (enzymes of the Entner-Doudoroff pathway) encoded by the edd-eda operon and the ribosomal protein S6, suggesting that the GroE chaperones are involved in regulating expression of genes for carbohydrate metabolism and in modulating biogenesis or function of the ribosome.

Mol Cell Biol, 1994 Jul, 14(7), 4532 - 45
Hox proteins have different affinities for a consensus DNA site that correlate with the positions of their genes on the hox cluster; Pellerin I et al.; The hox genes, members of a family of essential developmental regulators, have the intriguing property that their expression in the developing murine embryo is colinear with their chromosomal organization . Members of the hox gene family share a conserved DNA binding domain, termed the homeodomain, which mediates interactions of Hox proteins with DNA regulatory elements in the transcriptional control regions of target genes . In this study, we characterized the DNA binding properties of five representative members of the Hox family: HoxA5, HoxB4, HoxA7, HoxC8, and HoxB1 . To facilitate a comparative analysis of their DNA binding properties, we produced the homeodomain regions of these Hox proteins in Escherichia coli and obtained highly purified polypeptides . We showed that these Hox proteins interact in vitro with a common consensus DNA site that contains the motif (C/G)TAATTG . We further showed that the Hox proteins recognize the consensus DNA site in vivo, as determined by their ability to activate transcription through this site in transient transfection assays . Although they interact optimally with the consensus DNA site, the Hox proteins exhibit subtle, but distinct, preferences for DNA sites that contain variations of the nucleotides within the consensus motif . In addition to their modest differences in DNA binding specificities, the Hox proteins also vary in their relative affinities for DNA . Intriguingly, their relative affinities correlate with the positions of their respective genes on the hox cluster . These findings suggest that subtle differences in DNA binding specificity combined with differences in DNA binding affinity constitute features of the "Hox code" that contribute to the selective functions of Hox proteins during murine embryogenesis.

Biochem Cell Biol, 1994 Jul-Aug, 72(7-8), 333 - 42
Characterization of the growth inhibition phenotype of the kilAtelAB operon from IncP alpha plasmid RK2Ter; Turner RJ et al.; The cryptic tellurite resistance (Ter) determinant from RK2Ter has been previously cloned into a pUC8 plasmid (pDT1558) . The Ter.determinant was identified as the kilA locus and comprises an operon of three genes: kilA, telA, and telB (also referred to as klaA, klaB, and klaC on RK2(Tes)) . Cultures harboring the plasmid pDT1558 displayed an extended lag phase in rpoS- hosts, but grew normally in rpoS+ strains . Each of the genes from RK2Ter was subcloned into the expression vector pJF118EH behind an inducible tac promoter using polymerase chain reaction (PCR) . The PCR primers were used to engineer an efficient ribosome-binding site and an adjacent sequence to improve protein expression . Expression plasmids were modified by inclusion of different resistance markers for selection during complementation . The killing phenotype of the kilAtelAB operon was studied with the overexpressing plasmids . Each individual gene specified growth inhibition of Escherichia coli cells, but with different combinations of the genes giving rise to differing degrees of the inhibition . Additionally, the bacteriostatic and bacteriocidal effects of the genes were found to be different depending on whether or not the cultures were grown in minimal salts medium with glucose or in Luria-Bertani medium . Cells harboring the kilA gene alone or the three genes of the kilAtelAB operon expressed either in trans or cis behind the tac promoter were found to form nonseptated filaments up to 10-30 times the length of control cells . The effect of filamentation was greater for cells grown in minimal salts medium with glucose as the carbon source . This study demonstrates that each of the gene products from kilA, telA, and telB, when expressed at high levels either alone or together with another gene in the operon, exhibit some degree of growth inhibition of host cells . This growth inhibition is considered paramount in the stable maintenance of the plasmid within its host.

Biochem Cell Biol, 1994 Jul-Aug, 72(7-8), 313 - 9
Substitutions for Gly-794 show that binding interactions are important determinants of the catalytic action of beta-galactosidase (Escherichia coli); Martinez-Bilbao M et al.; Substitutions of Gly-794 (beta-galactosidase) with Asp, Asn, Glu, and Lys caused decreased binding of substrates and inhibition by substrate analogs, while inhibition by planar and positively charged galactose analogs increased relative to the binding of substrates and the inhibition by substrate analogs . There was a correlation of the relative inhibition with the size of the substituted residue but no relationship to the presence or absence of a negative charge, and as the relative inhibition by the planar and positively charged galactose analogs increased, k3 (hydrolysis; degalactosylation) and kcat/Km (catalytic efficiency) values decreased . The k2 values (glycolytic cleavage; galactosylation) mainly increased for poor substrates (p-nitrophenyl beta-galactoside and lactose) but decreased for o-nitrophenyl beta-galactoside (a good substrate) . Enzymes substituted with Asp or Asn were inhibited to a similar extent by planar and positively charged inhibitors and had similar effects on catalysis, while inhibition and catalytic effects on the enzyme substituted by Glu were quite different . If the negative charge was important, the Asp- and Glu-substituted enzymes should have been inhibited to a similar extent, while the Asn-substituted enzyme should have caused a different degree of inhibition . The enzyme substituted with a Lys at position 794 bound substrates and inhibitors very poorly, but the relative inhibition and the catalysis still correlated to size . Alterations of the size of the residue at position 794 cause modifications in the binding interactions and affected activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Microb Pathog, 1994 Jul, 17(1), 23 - 36
Cloning, characterization and construction of htrA and htrA-like mutants of Brucella abortus and their survival in BALB/c mice; Tatum FM et al.; A genomic library of Brucella abortus S2308 was screened for expression of recombinant proteins recognized by sera from mice and from cattle infected with B . abortus . A positive clone, BA1, expressing a 50 kDa peptide was recognized by both sera . Plasmid pBA1, isolated from BA1, was shown by restriction enzyme digestion to possess a 1.9 kb insert . The nucleotide sequence of the pBA1 insert revealed an open reading frame with of 1539 bases with a coding capacity of 513 amino acids and a predicted molecular weight of 50,992 . The predicted amino acid sequence showed 37% identity to E . coli HtrA, a temperature inducible serine protease . A second B . abortus htrA gene, designated htrA-like, was identified on a different cloned fragment that also encoded B . abortus recA . The nucleotide sequence of the htrA-like gene revealed an open reading frame of 1422 nucleotides with a coding capacity of 474 amino acids and a predicted molecular weight of 50,155 . The deduced amino acid sequence of the htrA-like gene showed 42% and 36% identity with B . abortus and E . coli HtrAs respectively . Western blotting of E . coli lysate containing the htrA-like gene was not recognized by sera from B . abortus-infected cattle or mice . B . abortus htrA but not htrA-like relieved the temperature sensitive phenotype and permitted growth of an E . coli htrA mutant at 42 degrees C . B . abortus htrA and htrA-like mutants were constructed and their survival and growth in BALB/c mice was compared to the parental strain S2308.(ABSTRACT TRUNCATED AT 250 WORDS)

Bioorg Med Chem, 1994 Jul, 2(7), 631 - 7
Enzymatic synthesis of isotopically labeled isoprenoid diphosphates; Christensen DJ et al.; Recombinant yeast isopentenyl diphosphate (IPP) isomerase and avian farnesyl diphosphate (FPP) synthase from overproducing strains of Escherichia coli were used to synthesize FPP from IPP and dimethylallyl diphosphate (DMAPP) . {2,4,5-13C3}IPP and {2,4,5-13C3}DMAPP were synthesized from ethyl {2-13C}bromoacetate and {1,3-13C2}acetone . Thes compounds were used as substrates for enzymatic synthesis of FPP selectivity labeled at the first or third isoprene residue or at all three.

Eur Cytokine Netw, 1994 Jul-Aug, 5(4), 405 - 10
Human interleukin-6 acts as a co-factor for the up-regulation of C3 production by rat liver epithelial cells; Platel D et al.; We investigated the role of human interleukin-6 (IL-6) in the regulation of the third component of complement (C3) biosynthesis by cultured rat liver epithelial cells . A natural human IL-6 (nh IL-6) preparation was shown to up-regulate C3 production, whereas an Escherichia coli-derived recombinant human IL-6 (rh IL-6) displayed no activity on C3 biosynthesis . However, the C3-stimulating activity of the nh IL-6 preparation was only partially reduced when treated with an antihuman IL-6 monoclonal antibody . Binding studies indicated that although it was devoid of any C3 stimulating activity, rh IL-6 specifically bound to hepatic cell receptors (Kd = 0.38 nM) and possessed the same binding affinity as nh IL-6 . Furthermore, the substitution of natural IL-6 molecules for the recombinant IL-6 led to the recovery of the initial C3-stimulating activity . These studies demonstrated that human IL-6 alone does not stimulate rat liver epithelial cell C3 production but is able to accentuate the C3-stimulating activity of unrecognized components which are present in the nh IL-6 preparation . Human IL-6 thus appears to act as a co-factor for the up-regulation of hepatic C3 production.

Avian Dis, 1994 Jul-Sep, 38(3), 589 - 97
In vivo evaluation of the pathogenicity of field isolates of infectious bronchitis virus; Avellaneda GE et al.; The pathogenicity of 13 field isolates of infectious bronchitis virus (IBV) isolated from Georgia broiler farms from 1989 to 1992 was evaluated using the IBV and Escherichia coli mixed-infection model . Based on the clinical signs, mortality, and lesions, the isolates were classified as high, intermediate, and low in pathogenicity . The in vivo classification was compared with the serotype classification results obtained by reverse transcriptase-polymerase chain reaction-restriction fragment length polymorphism analysis . The high-pathogenicity group was composed of five isolates representing three serotypes: Arkansas, Georgia variant (GAV), and Massachusetts . Isolates in the intermediate- and low-pathogenicity groups were all representatives of the Connecticut serotype, except for one isolate, which belonged to the Massachusetts serotype.

Comput Appl Biosci, 1994 Jul, 10(4), 453 - 4
Fast databank searching with a reduced amino-acid alphabet; Landes C et al.; Fast sequence databanks search algorithms generally make use of hash tables and look for exactly matching words . An increased sensitivity--at the expense of a decreased selectivity--can be attained in the case of proteins by using a reduced amino acid alphabet . We propose here an alphabet reduced to 10 symbols, that we used in modified versions of the FASTP and SCAN programs . An application to the aminoacyl-tRNA synthetases shows that this technique may be useful in detecting distant relationships between proteins.

J Biochem (Tokyo), 1994 Jul, 116(1), 95 - 107
X-ray crystallographic study of pyridoxal 5'-phosphate-type aspartate aminotransferases from Escherichia coli in open and closed form; Okamoto A et al.; We determined the three-dimensional structures of aspartate aminotransferase (AspAT) from Escherichia coli and its complex with inhibitor (2-methyl-L-aspartate) at 1.8A resolution . This enzyme reversibly catalyzes the transamination reaction and is a dimer of two identical subunits . Each subunit has 396 amino acid residues and one pyridoxal 5'-phosphate as a cofactor, and is divided into two domains, one large and the other small . Upon binding of the inhibitor, the small domain rotates by 5 degrees toward the large domain to close the active site . This domain movement is caused mainly by small but important main-chain conformational changes in the residues located over the domain interface of the small domain . In chicken mitochondrial AspAT, the domain movement was larger, with a rotational angle of 13 degrees . By comparison of these two structures, the difference in the rotational angles was found to be caused by the larger opening of the domain in the open form of chicken mitochondrial AspAT . Although the overall structures of these two enzymes were almost identical, the surface area of the domain interface in the E . coli enzyme was larger than that in mitochondrial AspAT, suggesting that the structure of the domain interface is responsible for the degree of movement of the small domain.

J Biochem (Tokyo), 1994 Jul, 116(1), 34 - 41
Point mutations at glycine-121 of Escherichia coli dihydrofolate reductase: important roles of a flexible loop in the stability and function; Gekko K et al.; To elucidate the role of a flexible loop in the stability and function of Escherichia coli dihydrofolate reductase, glycine-121 in a flexible loop (residues 117-131), separated by 19 A from active site Asp27, was substituted by site-directed mutagenesis with eight amino acids (Ala, Val, Leu, Asp, Ser, Cys, Tyr, and His) . The free energy change of unfolding decreased in the order of G121A > G121D > G121C > G121S, wild-type > G121H > G121Y > G121L > G121V . The thermal denaturation temperature decreased with all mutations, accompanied by a decrease in the calorimetric enthalpy of denaturation . The steady-state kinetic parameter for the enzyme reaction, Km, was only slightly influenced, but kcat was significantly decreased by the mutations, there being 3- (G121C) to 42-fold (G121L) decreases in kcat/Km compared to that of the wild-type enzyme . The effects of mutations on the stability and enzyme activity were statistically examined as a function of the hydrophobicity and volume of amino acids introduced . The diminished stability and activity with increases in the hydrophobicity and volume of amino acids suggest that the main effect of the mutations would be modification of the flexibility of the loop due to overcrowding of the bulky side chains, overcoming the enhancement of the hydrophobic interaction.

DNA Cell Biol, 1994 Jul, 13(7), 755 - 62
Functional analysis of the cell-specific enhancer in the human proopiomelanocortin gene by beta-galactosidase histochemical staining; Tsukada T et al.; Nucleotide sequences responsible for the cell-specific expression of the human proopiomelanocortin (POMC) gene were analyzed by histochemical staining of beta-galactosidase in culture cells transfected with chimeric genes containing the 5'-flanking regions of the human POMC gene fused to the Escherichia coli lacZ gene . The chimeric genes were stably introduced into various culture cells, including AtT-20 cells, which express the endogenous mouse POMC gene . Whereas the control gene containing the cytomegalovirus enhancer was expressed in all cell lines tested, only AtT-20 cells supported the efficient transcription of the gene containing 2.9 kb of the human POMC 5'-flanking region . These results indicate that the stable transfection-expression system utilizing the histochemical detection of the gene expression is a useful method for the analysis of cell-specific gene expression . These results have also confirmed that the trans-acting factors in mouse AtT-20 cells interact with the human POMC gene promoter region and activate the transcription of the gene . Deletion analysis has demonstrated that the profiles of the transcriptional activity of the various human POMC-lacZ fusion genes are similar to those of the rat POMC gene described previously . Comparison of the human and the rat 5'-flanking sequences revealed close homology in several regions, which might be involved in the efficient transcription of the POMC gene in AtT-20 cells.

Biosci Biotechnol Biochem, 1994 Jul, 58(7), 1231 - 5
Mapping of organic solvent tolerance gene ostA in Escherichia coli K-12; Aono R et al.; The extent of organic solvent tolerance was variable among strains of Escherichia coli K-12 . Genetic analyses of n-hexane-tolerant strains indicated that a number of genes were involved in the solvent-tolerance phenotype . One such gene, designated ostA, was mapped at 1.2 min, close to pdxA . Transduction of ostA from a n-hexane-sensitive strain to a n-hexane-tolerant strain generated n-hexane-sensitive transductants . The sensitive transductant restored n-hexane-tolerance by transduction of ostA from a tolerant strain . Thus, the gene ostA is one of the genes that contributes to deciding the level of organic solvent tolerance in E . coli.

Lett Appl Microbiol, 1994 Jul, 19(1), 44 - 6
Application of the polymerase chain reaction to the identification of Escherichia coli and coliforms in water; Fricker EJ et al.; The polymerase chain reaction (PCR) has been applied to the identification of Escherichia coli and coliforms after overnight growth using two sets of primers described previously . The primer set for E . coli, which was derived from the uid A gene, correctly identified all E . coli strains tested . The sequence was also identified in five non-E . coli coliforms . The coliform primer set correctly identified approximately 70% of the coliforms tested . We conclude that PCR can be used for the rapid identification of E . coli using the primers described here but that further work is required accurately to define a new primer set for the coliform group.

Phytochemistry, 1994 Jul, 36(4), 1027 - 9
Identification of (+)-gallocatechin as a bio-antimutagenic compound in Psidium guava leaves; Matsuo T et al.; From the MeOH-extract of guava leaves, (+)-gallocatechin was isolated as a bio-antimutagenic compound against UV-induced mutation in Escherichia coli . This strengthens the evidence that phenolic compounds require three neighbouring-OH groups in order to possess this activity.

Biotechnol Prog, 1994 Jul-Aug, 10(4), 441 - 6
Rapid quantitation of recombinant retroviruses; Morgan JR et al.; A method for measuring the concentration of recombinant retrovirus encoding the Escherichia coli LacZ gene is described . The assay is based on the quantitative measurement of beta-galactosidase activity in extracts of cells infected with the LacZ-encoding retrovirus . LacZ-encoded beta-galactosidase activity in transduced cells is measured using the colorimetric substrate, o-nitrophenyl beta-D-galactopyranoside (ONPG), and the results are read using an ELISA plate-reader . Because the entire assay is performed in a 96-well plate, large numbers of samples are easily measured, and the assay has the advantages of rapidity, precision, and ease of data collection . The assay was used to determine the optimal concentration of polybrene, a polycation known to enhance the infectivity of retroviruses, and can be used to evaluate other factors that affect infection, as well as the optimal conditions for production of the recombinant retrovirus.

Shock, 1994 Jul, 2(1), 47 - 52
Beneficial effect of H2-agonism and H1-antagonism in rat endotoxic shock; Rixen D et al.; Although histamine release is generally considered harmful in endotoxic shock, several data exist to doubt this view . Own previous studies in rats let us assume a possible beneficial effect only with H1-antagonists, however a detrimental effect on survival with H2-antagonists . Consequently H1- and H2-agonists and antagonists were studied to prove the hypothesis of a beneficial H2-agonistic and H1-antagonistic effect . Two randomized studies were performed in a standardized rat endotoxic shock model (45 mg of Escherichia coli endotoxin/kg body weight (b.w.)) . In both, methylprednisolone (50 mg/kg b.w.) and saline were used as positive and negative controls, respectively . Study I compared the effects of H1- and H2-agonists (betahistine, .1 mg/kg/h, and impromidine, 100 micrograms/kg/h) with H1- and H2-antagonists (astemizole and famotidine both 1 mg/kg b.w.; 20 rats/dose) . Study II was performed to estimate the dose-response relationship of a new, highly potent H2-agonist with additional H1-antagonistic features (BU-E 75: .01, .1, 1.0, 10, and 100 micrograms/kg/h; 20 rats/dose) . Animals receiving impromidine or BU-E 75 all received omeprazole (1 mumol/kg b.w.) to suppress gastric acid secretion . In study I impromidine significantly increased the survival-time and -course compared to famotidine treated animals (p = .01 and p < .05) . Study II showed a positive dose-response relationship of BU-E 75 with an increase in survival rates from 30% (.01 microgram/kg/h) to 70% (100 micrograms/kg/h) . These data strongly support the hypothesis of a beneficial effect of H2-agonism and H1-antagonism on survival parameters in rat endotoxic shock.

Shock, 1994 Jul, 2(1), 34 - 9; discussion 40
Effect of ethanol and sodium arsenite on HSP-72 formation and on survival in a murine endotoxin model; Lappas GD et al.; Recently, investigators reported that prophylactic hyperthermia and induction of heat shock proteins (HSPs) decreased mortality from endotoxin . Although the mechanism by which hyperthermia protects is unknown, two possible etiologies are induction of HSPs and/or production of cytokines, interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor-alpha (TNF-alpha) . The purpose of this study was to determine if in vivo administration of sodium arsenite (NaAsO2) or ethanol, inducers of HSPs in isolated cells, induced HSP-72 production in lung, liver, kidney, and duodenum (organs known to induce HSP-72 by heat) and improved survival from endotoxin . Female ND4 mice were injected intraperitoneally with either NaAsO2 (5.25 mg/kg body weight) or ethanol (4.0 g/kg), immediately, 8 or 18 h prior to Escherichia coli endotoxin injection (20 mg/kg) . Both compounds improved short-term (24 h) survival twofold (p < .01), but failed to improve long-term (7 days) survival . Simultaneous injection of ethanol with endotoxin improved both short-term survival twofold (p < .01), and long-term survival 5-fold (p < .001) . Ethanol induced HSP-72 in kidney, 50% that of the standard (i.e., pooled livers isolated from heat-treated mice); NaAsO2 induced HSP-72 in kidney (approximately 50% of standard) and liver (approximately 21% of standard) . Neither ethanol nor NaAsO2 alone increased circulating concentrations of IL-1 alpha or TNF-alpha . However, ethanol given concurrently with endotoxin produced a significant decrease in TNF-alpha compared to endotoxin alone (p < .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Nat Struct Biol, 1994 Jul, 1(7), 469 - 75
Crystal structure of PvuII endonuclease reveals extensive structural homologies to EcoRV; Athanasiadis A et al.; The crystal structure of the dimeric PvuII restriction endonuclease (R.PvuII) has been determined at a resolution of 2.4A . The protein has a mixed alpha/beta architecture and consists of two subdomains . Despite a lack of sequence homology, extensive structural similarities exist between one R.PvuII subdomain and the DNA-binding subdomain of EcoRV endonuclease (R.EcoRV); the dimerization subdomains are unrelated . Within the similar domains, flexible segments of R.PvuII are topologically equivalent to the DNA-binding turns of R.EcoRV; potential catalytic residues can be deduced from the structural similarities to R.EcoRV . Conformational flexibility is important for the interaction with DNA . A possible classification of endonuclease structures on the basis of the positions of the scissile phosphates is discussed.

Nat Struct Biol, 1994 Jul, 1(7), 453 - 60
Structure of a unique twofold symmetric haem-binding site; Frolow F et al.; Bacterioferritin of Escherichia coli, also known as cytochrome b1, is a hollow, nearly spherical shell made up of 24 identical protein subunits and 12 haems . We have solved this structure in a tetragonal crystal form at 2.9 A resolution . We find that each haem is bound in a pocket formed by the interface between a pair of symmetry-related subunits . The quasi-twofold axis of the haem is closely aligned with the local twofold axis relating these subunits . The axial ligands of the haem are sulphurs of two equivalent methionyl residues (Met 52) from the symmetry-related subunits . A cluster of four water molecules is trapped in the gap between the upper edge of the haem and two extended protein loops which close off the haem from the outer aqueous environment . This is the first structure of a bis-methionine ligated haem-binding site and the first case of a twofold symmetric haem-binding site.

Nat Struct Biol, 1994 Jul, 1(7), 439 - 46
The chaperonin GroEL does not recognize apo-alpha-lactalbumin in the molten globule state; Okazaki A et al.; We investigate here the interaction between GroEL and two kinds of non-native alpha-lactalbumin . alpha-Lactalbumin is a Ca(2+)-binding protein which assumes a molten globule state in the absence of Ca2+ (apo-alpha-lactalbumin) at neutral pH . Our results, obtained by molecular-sieve chromatography and hydrogen-exchange measurements, show that apo-alpha-lactalbumin in this molten globule state is not bound to GroEL either in the absence or in the presence of KCl . On the other hand, we show by molecular-sieve chromatography that alpha-lactalbumin, in which the four disulphide bonds are fully reduced, is bound to GroEL when 50 mM KCl is present . The results demonstrate that the protein state recognized by GroEL is more unfolded and expanded than the typical molten globule state of alpha-lactalbumin.

Gene Ther, 1994 Jul, 1(4), 233 - 8
Tumor cell bystander killing in colonic carcinoma utilizing the Escherichia coli DeoD gene to generate toxic purines; Sorscher EJ et al.; Inefficiency of gene delivery, together with inadequate bystander killing, represent two major hurdles in the development of a toxin-mediated gene therapy for human malignancy . The product of the Escherischia coli DeoD gene (purine nucleoside phosphorylase, PNP) differs from the mammalian enzyme in its substrate specificity and is capable of catalyzing the conversion of several non-toxic deoxyadenosine analogs to highly toxic adenine analogs . We have found that expression of E . coli PNP in < 1% of a human colonic carcinoma cell line leads to the death of virtually all bystander cells after treatment with 6-methyl-purine-2'-deoxyribonucleoside, a deoxyadenosine analog that is a substrate for E . coli PNP but not human PNP . Minimal toxicity was observed in non-transfected or E . coli LacZ transfected cells that were treated with this compound . These results establish a rational approach to achieve significant bystander killing, even after gene transfer to only a small fraction of tumor cells.

Plasmid, 1994 Jul, 32(1), 89 - 94
Differential replication of plasmids during stringent and relaxed response of Escherichia coli; Herman A et al.; Stringent control of DNA replication has been demonstrated for a few replicons like oriC, pBR322, and plasmids derived from coliphage lambda . In this study we investigated the replication of other plasmids harboring a well defined origin (orip15A, oripSC101, and oriRK2 = oriV) in amino-acid-starved stringent and relaxed strains of Escherichia coli . We found differential replication of plasmids during stringent and relaxed response . Inhibition of DNA synthesis or amplification of plasmid DNA in amino acid-starved relA+ and relA- cells depends on the kind of replicon and, surprisingly, on the nature of deprived amino acid . We conclude that there are no general rules for stringent control of DNA replication and each replicon must be considered separately . There are, however, possible explanations for the differences shown between replicons in their response to stringent and relaxed conditions.

Mol Microbiol, 1994 Jul, 13(2), 313 - 26
traJ sense RNA initiates at two different promoters in R100-1 and forms two stable hybrids with antisense finP RNA; Dempsey WB; RNase protection experiments show that the sizes of the two R100 finP molecules are 74 and 135 nucleotides . In an RNase III mutant, finP transcripts form stable double-stranded hybrids of 108 bp and 68 bp with traJ transcripts . RNase protection experiments also show that most R100-1 transcripts originating in traM cross the traM-traJ intergenic region and end inside the untranslated leader region of traJ . Some extend into the traJ open reading frame . These findings mean that the antisense finP RNA, thought to regulate traJ translation, must regulate traJ transcripts from both J and M promoters.

J Cell Sci, 1994 Jul, 107 ( Pt 7), 1959 - 72
Function of type I and type II keratin head domains: their role in dimer, tetramer and filament formation; Hatzfeld M et al.; To examine the role of the keratin head region and its subdomains in filament assembly we constructed several deletion mutants of type I and type II keratins and analysed their in vitro IF forming capacity . The delta K8 (1-74) and delta K18 (1-56), mutants formed only soluble oligomers, predominantly tetramers with their heterotypic partners . K8 mutants that retained either the entire (delta K8 (1-64)) or nearly the entire (delta K8 (1-66)) H1 subdomain formed some short and irregular IF-like structures with K18 . However, filaments never reached the normal length and more protofilamentous material was observed . Analysis of the soluble complexes in 2 M guanidine-HCl indicated that tetramer formation was impaired in the truncated molecules . The length of the deletion correlated with the degree of tetramer destabilization . These results suggest that the head domain--specifically the H1 subdomain of type II keratins-plays a direct role in IF assembly . Its functions include a stabilization of the tetramer molecule, suggesting a role in directing the alignment of dimers as well as in elongation . We also analysed whether both head domains are required or if either type I or type II head domains alone are sufficient for IF formation . Hybrid molecules carrying their partner keratins head domains (K18 (8 head) and K8 (18 head)) were combined with their wild-type partners and tested for IF-forming ability . Both combinations formed filaments distinct from normal IF . The effect of the 'replaced' head domains was not compensated when both hybrid molecules were combined . Taken together, the results indicate that complete removal of the head domains of either K8 or K18 arrested IF assembly at the state of soluble oligomers . Replacement of the head domains by head domains of the complementary partner partly compensated for the effect . However, regular IF formation could not take place when either the head domain was missing or it was replaced by the partner's keratin head.

Antimicrob Agents Chemother, 1994 Jul, 38(7), 1658 - 60
Inhibition of protein synthesis occurring on tetracycline-resistant, TetM-protected ribosomes by a novel class of tetracyclines, the glycylcyclines; Rasmussen BA et al.; One of the two major mechanisms of tetracycline resistance is ribosomal protection . Of this resistance type, tet(M) is the best characterized . Although the mechanism of tet(M) resistance has not yet been fully elucidated, it has been demonstrated that ribosomes isolated from a tet(M) strain are resistant to inhibition of protein synthesis by tetracycline . A new generation of tetracycline compounds, the glycylcyclines, that are able to inhibit protein synthesis occurring on tetracycline-resistant, TetM-protected ribosomes, as well as wild-type, tetracycline-sensitive ribosomes, have been identified.

Genes Dev, 1994 Jul 1, 8(13), 1600 - 12
The cellular concentration of the sigma S subunit of RNA polymerase in Escherichia coli is controlled at the levels of transcription, translation, and protein stability; Lange R et al.; The second vegetative sigma factor sigma S (encoded by the rpoS gene) is the master regulator in a complex regulatory network that governs the expression of many stationary phase-induced and osmotically regulated genes in Escherichia coli . Using a combination of gene-fusion technology and quantitative immunoblot, pulse-labeling, and immunoprecipitation analyses, we demonstrate here that rpoS/sigma S expression is not only transcriptionally controlled, but is also extensively regulated at the levels of translation and protein stability . rpoS transcription is inversely correlated with growth rate and is negatively controlled by cAMP-CRP . In complex medium rpoS transcription is stimulated during entry into stationary phase, whereas in minimal media, it is not significantly induced . rpoS translation is stimulated during transition into stationary phase as well as by an increase in medium osmolarity . A model involving mRNA secondary structure is suggested for this novel type of post-transcriptional growth phase-dependent and osmotic regulation . Furthermore, sigma S is a highly unstable protein in exponentially growing cells (with a half-life of 1.4 min), that is stabilized at the onset of starvation . When cells are grown in minimal glucose medium, translational induction and sigma S stabilization occur in a temporal order with the former being stimulated already in late exponential phase and the latter taking place at the onset of starvation . Although sigma S does not control its own transcription, it is apparently indirectly involved in a negative feedback control that operates on the post-transcriptional level . Our analysis also indicates that at least five different signals {cAMP, a growth rate-related signal (ppGpp?), a cell density signal, an osmotic signal, and a starvation signal} are involved in the control of all these processes that regulate rpoS/sigma S expression.

Bioconjug Chem, 1994 Jul-Aug, 5(4), 327 - 32
DNA-linked RNase H for site-selective cleavage of RNA; Uchiyama Y et al.; ADNA-linked RNase H (Hybrid Enz-1) (Kanaya et al . (1992) J . Biol . Chem . 267, 8492-8498), in which dGTCATCTCC was attached to E . coli RNase H via a covalent linker of 21 A, was altered to improve the site-specific RNA cleavage by increasing the linker length . The sizes of the linkers on these hybrid enzymes (Hybrid Enz-2, -3, and -4) differed by 3 A, the axial rise of the DNA/RNA hybrid, to give 18-, 24-, and 27-A lengths . The conjugate with a size of A was able to cleave a synthetic 22mer RNA (5'-rAAGAUGUCUACGGAGAUGACCA-3'), containing the complementary 9mer RNA sequence (underlined), at one position, A16-U17 . The kinetic parameters of Hybrid Enz-1, -2, -3, and -4 were examined using a 9mer RNA target . The results showed that longer linkers produced higher Km, kcat, and kcat/Km values, and the kcat/Km value of the conjugate with the 27-A linker reached 83% of that of the wild-type RNase H . Hybrid Enz-4 was found to be useful as an RNA restriction endonuclease.

J Clin Microbiol, 1994 Jul, 32(7), 1799 - 804
Characterization and presumptive identification of Helicobacter pylori isolates from rhesus monkeys; Drazek ES et al.; We characterized 38 Helicobacter isolates, including 22 from gastric biopsy samples obtained from 14 rhesus monkeys and single isolates from 16 monkeys in a different colony . Biochemical profiles of these isolates were nearly identical to that of Helicobacter pylori ATCC 43504 . Restriction fragment length polymorphism (RFLP) analysis indicated that each infected monkey harbored one to four strains . The 17 RFLP types found among these 22 isolates differed from all seven RFLPs found among the other 16 isolates . Thus, monkeys within a given colony are more likely to be infected by Helicobacter isolates with the same or a similar RFLP than are monkeys from different colonies . A 16S rRNA gene was amplified by PCR and cloned from the Helicobacter isolate from rhesus monkey 85D08 . Ribotyping with this probe demonstrated less diversity among isolates from rhesus monkeys than was reported among isolates of H . pylori from humans, as did RFLP analysis of a PCR fragment of the ureA-ureB gene cluster . The DNA sequence of the cloned 16S rRNA gene was determined and compared with sequences reported for H . pylori and other Helicobacter species . Our analysis of 127 nucleotides (corresponding with residues 1240 to 1366 of the Escherichia coli 16S rRNA gene) indicated that the Helicobacter isolate from monkey 85D08 was 99.2 to 100% homologous to isolates of H . pylori from humans but only 83.5 to 96.9% homologous with other Helicobacter species in this region of the 16S rRNA gene . These data provide strong support for the presumptive identification of these isolates as H . pylori.

Appl Environ Microbiol, 1994 Jul, 60(7), 2456 - 61
Sequence analysis of 16S rRNA from mycoplasmas by direct solid-phase DNA sequencing; Pettersson B et al.; Automated solid-phase DNA sequencing was used for determination of partial 16S ribosomal DNA sequences of mycoplasmas . The sequence information was used to establish phylogenetic relationships of 11 different mycoplasmas whose 16S rRNA sequences had not been determined earlier . A biotinylated fragment corresponding to positions 344 to 939 in the Escherichia coli sequence was generated by PCR . The PCR product was immobilized onto streptavidin-coated paramagnetic beads, and direct sequencing was performed in both directions . One previously unclassified avian mycoplasma was found to belong to the Mycoplasma lipophilum cluster of the hominis group . Microheterogeneities were discovered in the rRNA operons of Mycoplasma mycoides subsp . mycoides (SC type), confirming the existence of two different rRNA operons . The 16S rRNA sequence of M . mycoides subsp . capri was identical to that of M . mycoides subsp . mycoides (type SC), except that no microheterogeneities were revealed . Furthermore, automated solid-phase DNA sequencing was used to identify a mycoplasmal contamination of a cell culture as Mycoplasma hyorhinis, which proved to be very difficult by conventional methods . The results suggest that the direct solid-phase DNA sequencing procedure is a powerful tool for identification of mycoplasmas and is also useful in taxonomic studies.

Appl Microbiol Biotechnol, 1994 Jul, 41(5), 584 - 90
Flow cytometry studies of recombinant Escherichia coli in batch and continuous cultures: DNA and RNA contents; light-scatter parameters; Fouchet P et al.; Flow cytometry has been used to study the contents of macromolecular compounds and light-scatter parameters in batch and continuous cultures of a recombinant Escherichia coli strain that forms protein inclusion bodies . Changes in relative DNA and RNA contents and cell mass as estimated by forward-angle light scatter were detected and tightly correlated in batch culture . In addition, heterogeneity of wide-angle light scatter (WALS), which we related to the presence of cellular inclusion bodies, was observed . In contrast, the relative RNA content and cell mass did not change during continuous culture, and homogeneity of WALS was found . In addition, unexpected changes in relative DNA content were observed after 67 h of culture, indicating a change in bacterial physiology.

EMBO J, 1994 Jul 1, 13(13), 3077 - 82
Mechanism of the down-regulation of cAMP receptor protein by glucose in Escherichia coli: role of autoregulation of the crp gene; Ishizuka H et al.; Glucose causes catabolite repression by lowering the intracellular levels of both cAMP and cAMP receptor protein (CRP) in Escherichia coli . The molecular mechanism underlying the down-regulation of CRP by glucose has been investigated . We show that glucose lowers the level of crp mRNA without affecting its stability . Replacement of the crp promoter with the bla promoter almost completely abolishes the glucose-mediated regulation of crp expression . Only a slight reduction in the crp expression by glucose is observed in cya- or crp- strains, suggesting that a CRP-cAMP complex is needed for this regulation . We previously showed that transcription of the crp gene is regulated both negatively and positively . Positive autoregulation of crp is caused by the binding of CRP-cAMP to the CRP binding site II located upstream of the crp promoter . Here we show that disrupting the CRP binding site II essentially eliminates the down-regulation of crp expression by glucose . We conclude that the autoregulatory circuit of the crp gene plays a key role in the down-regulation of CRP by glucose.

EMBO J, 1994 Jul 1, 13(13), 2985 - 93
The three-dimensional structure of human erythrocyte aquaporin CHIP; Walz T et al.; Water-permeable membranes of several plant and mammalian tissues contain specific water channel proteins, the 'aquaporins' . The best characterized aquaporin is CHIP, a 28 kDa red blood cell channel-forming integral protein . Isolated CHIP and Escherichia coli lipids may be assembled into 2-D crystals for structural analyses . Here we present (i) a structural characterization of the solubilized CHIP oligomers, (ii) projections of CHIP arrays after negative staining or metal-shadowing, and (iii) the 3-D structure at 1.6 nm resolution . Negatively stained CHIP oligomers exhibited a side length of 6.9 nm with four-fold symmetry, and a mass of 202 +/- 3 kDa determined by scanning transmission electron microscopy . Reconstituted into lipid bilayers, CHIP formed 2-D square lattices with unit cell dimensions a = b = 9.6 nm and a p422(1) symmetry . The 3-D map revealed that CHIP tetramers contain central stain-filled depressions about the fourfold axis . These cavities extend from both sides into the transbilayer domain of the molecule leaving only a thin barrier to be penetrated by the water pores . Although CHIP monomers behave as independent pores, we propose that their particular structure requires tetramerization for stable integration into the bilayer.

Arterioscler Thromb, 1994 Jul, 14(7), 1202 - 9
Role of the lipopolysaccharide (LPS)-binding protein/CD14 pathway in LPS induction of tissue factor expression in monocytic cells; Steinemann S et al.; Endotoxic shock is associated with a coagulopathy, organ failure, and death . Tissue factor (TF) expression by monocytes exposed to bacterial endotoxin (lipopolysaccharide {LPS}) may mediate the coagulopathy and contribute to the high mortality of this disease . We examined the role of the LPS-binding protein (LBP)/CD14 receptor pathway in the LPS induction of TF expression in human monocytic THP-1 cells and peripheral blood monocytes . In THP-1 cells, the threshold concentration of LPS required to induce TF activity in serum-free medium was reduced 20-fold by purified LBP, which also enhanced TF mRNA synthesis . Similarly, monocytes cultured in the presence of serum were induced to express TF antigen at LPS concentrations 100 times lower than monocytes cultured in serum-free medium . An anti-LBP monoclonal antibody indicated that this effect was dependent on the presence of LBP in serum . LPS/LBP induction of TF activity and TF antigen expression in these monocytic cells were also inhibited by an anti-CD14 monoclonal antibody, indicating a requirement for the CD14 receptor . Thus, we suggest that low levels of LPS (5 to 100 pg/mL) present during sepsis induce TF expression in monocytes via the LBP/CD14-dependent pathway.

Mutat Res, 1994 Jul, 315(1), 65 - 74
Biological inactivation of pBR322 plasmid DNA by enzyme- and radiation-induced single-strand damage under various conditions; Ventur Y et al.; The influence of three different kinds of single-strand breaks (ssb) on the biological activity of plasmid DNA (pBR322) was studied . The single-strand breaks were produced either by gamma-irradiation (together with base and sugar damage) or by DNase I digestion which introduced ligatable ssb . Non-ligatable ssb--single-strand gaps of three nucleotides in length--were generated in the nicked DNA by exonuclease III treatment . The biological activity (N/N(o)) of this damaged DNA was assessed in vivo by transformation of E . coli (CMK) repair wild-type cells . The activity of the enzymes of E . coli was studied in vitro by incubation in a protein extract of E . coli making use of an in vitro assay introduced earlier, which makes it possible to distinguish between enzymatic degradation (dsb formation) and repair of damaged plasmid DNA . The biological activity (D37) of DNA with non-ligatable ssb, as determined by electrotransformation, was about 56% lower than that of DNA with ligatable ssb . The biological activity of enzymatically damaged DNA is greater in calcium-treated cells than in electroporated cells . It is proposed that this is due to a calcium-dependent inhibition of nucleases . In contrast to the enzymatically damaged DNA, with gamma-radiation-damaged DNA a calcium-dependent increase in survival was not observed . Therefore, calcium-dependent nucleases do not play a role in the repair of damage produced by gamma-irradiation . The enzyme activity data show that the single-strand damages are either converted into dsb or repaired . A comparison of the efficiency of dsb formation in the extract for two of the single-strand damages is presented . The efficiency depends on the kind of damage and on the presence of cofactors, especially ATP and dNTPs.

Mutat Res, 1994 Jul, 315(1), 1 - 9
Involvement of RecF pathway recombination genes in postreplication repair in UV-irradiated Escherichia coli cells; Tseng YC et al.; Mutations affecting the RecF pathway of recombination (recF, recG, recJ, recN, recO, recQ, recR, ruvA, ruvC) were systematically introduced into two sets of strains: (a) uvrA and uvrA recA2020, (b) uvrA recBC sbcBC and uvrA recBC sbcBC recA2020 . We examined: (i) the effect of these mutations on the repair of DNA daughter-strand gaps which are produced in the nascent DNA synthesized after UV irradiation, and (ii) the ability of recA2020 (a suppressor for the recF mutation) to suppress the UV radiation sensitivity caused by these mutations . In the uvrA cells, mutations in recF, recR or recO genes produced a major deficiency in the repair of daughter-strand gaps, whereas mutations in recJ, recG, recN, recQ, ruvA or ruvC genes had no effect on the repair of daughter-strand gaps . In both uvrA and uvrA recBC sbcBC backgrounds, the UV radiation sensitivity caused by recF, recG, recR, recO, ruvA, or ruvC mutations was partially suppressed by recA2020, whereas the UV radiation sensitivity caused by recJ, recN, or recQ mutations was not suppressed by recA2020 . Partial suppression of the UV sensitivity of recG, ruvA and ruvC mutants was not observed with other suppressors for recF, i.e., recA441, recA720 and recA730 . Taken together, these results further support the notion that the recF, recR and recO gene products (abbreviated as RecFOR) function at the same step in recombination repair, possible as a complex . It also suggests that this putative RecFOR complex does not contain proteins encoded by other genes involved in the RecF pathway of recombination.

J Gerontol, 1994 Jul, 49(4), B145 - 56
Production of age-synchronous mass cultures of Caenorhabditis elegans; Fabian TJ et al.; Methods are described for culturing large populations of age-synchronous Caenorhabditis elegans throughout the adult life span . Contamination of adult populations by progeny was prevented by constructing double-mutant strains that produce progeny at a frequency of less than .005 per adult at the nonpermissive temperature (25.5 degrees C) . Of four double-mutant strains that we have characterized, three have wild-type life spans at 25.5 degrees . The other strain contains a mutant allele, age-1(hx542), that results in an increase in life span of 60% over wild type . All four strains produced sufficient numbers of progeny at the permissive temperature (20 degrees C) to generate populations containing 1-5 x 10(6) nematodes within two weeks . Age-synchronous young adult populations were produced using these strains and have been maintained as adults both in liquid culture and on agar medium . Procedures that reduce E . coli contamination by 30-fold in harvested samples of adults are also described.

Mutat Res, 1994 Jul 1, 308(1), 53 - 64
Beta subunit of DNA polymerase III holoenzyme is induced upon ultraviolet irradiation or nalidixic acid treatment of Escherichia coli; Tadmor Y et al.; Exposure of Escherichia coli to UV irradiation or nalidixic acid, which induce both the SOS and heat shock responses, led to a 3-4-fold increase in the amount of the beta subunit of DNA polymerase III holoenzyme, as assayed by Western blot analysis using anti-beta antibodies . Such an induction was observed also in a delta rpoH mutant lacking the heat shock-specific sigma 32 subunit of RNA polymerase, but it was not observed in recA13 or lexA3 mutants, in which the SOS response cannot be induced . Mapping of transcription initiation sites of the dnaN gene, encoding the beta subunit, using the S1 nuclease protection assay showed essentially no induction of transcription upon UV irradiation, indicating that induction is regulated primarily at the post-transcriptional level . Analysis of translational gene fusions of the dnaN gene, encoding the beta subunit, to the lacZ reporter gene showed induction of beta-galactosidase activity upon UV irradiation of cells harboring the fusion plasmids . Elimination of a 5' flanking DNA sequence in which the dnaN promoters P1 and P2 were located, did not affect the UV inducibility of the gene fusions . Thus, element(s) present from P3 downstream were sufficient for the UV induction . The induction of the dnaN-lacZ gene fusions was dependent on the recA and lexA gene products, but not on the rpoH gene product, in agreement with the immunoblot analysis . The dependence of dnaN induction on the SOS regulators was not mediated via classical repression by the LexA repressor, since the dnaN promoter does not contain a sequence homologous to the LexA binding site, and dnaN mRNA was not inducible by UV light . This suggests that SOS control may be imposed indirectly, by a post-transcriptional mechanism . The increased amount of the beta subunit is needed, most likely, for increased replication and repair activities in cells which have been exposed to UV radiation.

Mol Cell Biol, 1994 Jul, 14(7), 4390 - 7
Proliferating cell nuclear antigen (pol30) mutations suppress cdc44 mutations and identify potential regions of interaction between the two encoded proteins; McAlear MA et al.; In addition to its role as a processivity factor in DNA replication, proliferating cell nuclear antigen (PCNA) may function in the regulation of cell cycle progression . We present genetic evidence that PCNA interacts with the gene product of CDC44, an essential nucleotide-binding protein that encodes the large subunit of yeast replication factor C (K . Fien and B . Stillman, personal communication) . Mutations in POL30 (PCNA) suppress cold-sensitive alleles of cdc44 that contain mutations in or near nucleotide-binding consensus domains, but they do not suppress a null allele . Thus, it appears that PCNA interacts with Cdc44p but cannot substitute for its function . pol30 mutations suppress additional phenotypes of cdc44 mutations, including the cold sensitivity that they were selected to suppress . This observation suggests an intimate association between PCNA and Cdc44p . Each of five independent pol30 mutants contains a unique single mutation that maps to a localized region on one face of the predicted three-dimensional structure of PCNA . This face identifies a region likely to be important for functional interaction between the CDC44 and POL30 gene products.

J Exp Med, 1994 Jul 1, 180(1), 191 - 201
CD19 has a potential CD77 (globotriaosyl ceramide)-binding site with sequence similarity to verotoxin B-subunits: implications of molecular mimicry for B cell adhesion and enterohemorrhagic Escherichia coli pathogenesis; Maloney MD et al.; The glycosphingolipid globotriaosyl ceramide (CD77) and other globo-series glycolipids containing terminal galactose (Gal)alpha 1-4Gal residues function as receptors for the verotoxin (Shiga-like toxin) family of Escherichia coli-elaborated toxins . CD77 is also a marker for germinal center B lymphocytes and Burkitt's lymphoma cells . The pan B cell marker CD19 is a 95-kD membrane protein that appears early in B cell differentiation and is only lost upon terminal differentiation to plasma cells . CD19 is involved in signal transduction and has a regulatory role in B cell proliferation and differentiation in response to activation in vitro . However, an endogenous ligand for CD19 has not yet been identified . We report herein that the extracellular domain of CD19 has a potential CD77-binding site with extensive sequence similarity to the verotoxin B-subunits . These B-subunit-like sequences on CD19 are in close proximity following the organization of intervening amino acids into disulfide-linked domains . Cocapping of CD19 and CD77 on Burkitt's lymphoma-derived Daudi cells with anti-CD19 antibodies indicates that CD19 and CD77 are associated on the B cell surface . Cell surface binding of anti-CD19 antibodies is decreased on CD77-deficient mutant Daudi cells, suggesting that CD77 expression influences the surface expression of CD19 . Wild-type Daudi cells, but not the CD19/CD77-deficient mutants, bind to matrices expressing the carbohydrate moiety of CD77 or other Gal alpha 1-4Gal containing glycolipids . This binding can be inhibited by anti-CD77 antibodies, the CD77-binding verotoxin B-subunit or anti-CD19 antibodies . Daudi cells exhibit a degree of spontaneous homotypic adhesion in culture while the CD77/CD19-deficient Daudi mutants grow as single cells . The stronger homotypic adhesion that occurs in B cells after antibody ligation of CD19 and that involves, to some extent, the integrin system, is also dramatically lower in the mutant cells relative to the parent cell line . However, reconstitution of mutant cells with CD77 restores the anti-CD19 mAb-induced adhesion to wild-type Daudi cell levels . These studies represent the first time that CD19-mediated signaling has been reconstituted in a low-responder B cell line . These convergent observations provide compelling evidence that CD19/CD77 interactions function in adhesion and signal transduction at a specific stage in B cell development and suggest that such interactions have a role in B lymphocyte homing and germinal center formation in vivo . By targeting CD77+ B cells, verotoxins may suppress the humoral arm of the immune response during infection.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochim Biophys Acta, 1994 Jun 30, 1222(2), 179 - 86
Expression and kinetic characterization of barley chymotrypsin inhibitors 1a and 1b; Greagg MA et al.; The genes for chymotrypsin inhibitors 1a and 1b (CI-1a and CI-1b) from barley have been expressed in E . coli, and the CI-1a and CI-1b proteins purified . These proteins, although highly homologous, differ in the active site region at P2, P1' and P3' (Schechter and Berger nomenclature), and so might be expected to have differing specificities . Despite this, analysis of the inhibition kinetics showed that each displayed very similar kinetic behaviour when tested against a range of proteinases . The specificity of the CI-1 proteins is different to that of the other main barley inhibitor, CI-2, and Ki values are found to follow the series subtilisin Carlsberg < neutrophil elastase approximately subtilisin BPN' << chymotrypsin . Only very weak inhibition is found of trypsin, and pancreatic elastase is not measurably inhibited . For the proteinases inhibited most strongly, characteristic slow-binding inhibition kinetics were observed, whereas classical inhibition applied to the weaker interactions . The results are consistent with the major determinant of specificity being the P1 residue of the inhibitor, which is the same in both CI-1a and CI-1b . Consistent with this, is the similar spectrum of specificity found for the homologous inhibitor eglin c from leech, which has the same P1 residue . Both the CI-1 proteins are found to be less stable than CI-2, with CI-1a being significantly less stable than CI-1b as measured by guanidinium hydrochloride unfolding experiments . Possible reasons for the reduced stability are discussed in view of the sequence differences between CI-1a, CI-1b and CI-2.

Biochem Biophys Res Commun, 1994 Jun 30, 201(3), 1561 - 6
Ca2+/calmodulin-dependent protein kinase V and I may form a family of isoforms; Ito T et al.; We reported that polyclonal antibody against Ca2+/calmodulin-dependent protein kinase V (CaM kinase V) reacted to two proteins of rat cerebrum with a molecular mass of 40 and 41 kDa . This antibody revealed the immunoreactivity with CaM kinase I expressed in E . coli (recombinant CaM kinase I), of which molecular mass was 40 kDa, whereas 41 kDa mainly with purified CaM kinase V . The immunoreactive bands of recombinant CaM kinase I and CaM kinase V did not shift by phosphorylation or dephosphorylation . These results suggest that CaM kinase V and CaM kinase I may form a family of isoforms.

Biochem Biophys Res Commun, 1994 Jun 30, 201(3), 1079 - 83
Cooperative binding of zinc to an aminoacyl-tRNA synthetase; Wu MX et al.; Zinc binds to tetrameric alanyl-tRNA synthetase from Escherichia coli with a stoichiometry of one g-atom of zinc per enzyme subunit . The nature of this metal-protein interaction is investigated here through a series of equilibrium dialysis and intrinsic fluorescence experiments . The dialysis data show that zinc binds to this synthetase in a cooperative manner, with half-maximal zinc binding at 0.97 microM free zinc and a Hill coefficient of 1.9 . The cooperative feature is also observed in the zinc-induced quenching of the protein intrinsic fluorescence, indicating that zinc binding induces a conformational change . This is the first report of cooperative binding of zinc to an aminoacyl-tRNA synthetase, and the data provide a rationale for the oligomeric structure of the synthetase specific for alanine.

J Biotechnol, 1994 Jun 30, 35(2-3), 229 - 38
Solid-phase cloning to create sublibraries suitable for DNA sequencing; Hultman T et al.; A solid-phase method is described to create subclones, suitable for DNA sequencing, from lambda or cosmid libraries . The purified target DNA is sonicated and two linkers, with one oligonucleotide biotinylated in the 5'-end, are ligated to the ends of the fragments produced by sonication . After size separation, the fragments are immobilised onto a solid support and the non-biotinylated strand of each immobilised fragment is eluted . In this way, a library of single-stranded fragments is obtained . All fragments contain 'universal' flanking sequences of 22 bases introduced by the linker ligation . These flanking sequences can subsequently be used for solid-phase cloning into a single-stranded vector containing the complementary sequences . Thus, cloning can be achieved without the use of ligase or restriction enzymes . The resulting subclones are used for direct solid-phase sequencing and the immobilised strand can be used to selectively remove homologous DNA from the library of single-stranded fragments . Thus, a sublibrary of non-sequenced fragments can be created . Here, we show that a library of clones, suitable for direct solid-phase sequencing, can be obtained starting with lambda DNA . The efficiency of selective hybridisation of homologous and non-homologous fragments was investigated . The possibility of using this approach for automated cloning strategies for large-scale genomic and cDNA sequencing is discussed.

Biochemistry, 1994 Jun 28, 33(25), 7917 - 24
Kinetics of CheA autophosphorylation and dephosphorylation reactions; Tawa P et al.; The protein kinase CheA of Escherichia coli plays a central role in the signal transduction pathway controlling the swimming behavior of the cell in response to extracellular chemical gradients . CheA autophosphorylates at a rate controlled by the ligand binding state of chemotaxis receptor/transducer proteins . CheA directs the activities of CheY and CheB, effector proteins that become phosphorylated as a result of their interaction with phospho-CheA . In this study, we performed a detailed kinetic analysis of CheA's autophosphorylation reaction, and its dephosphorylation by ADP . Our kinetic data are consistent with a three-step mechanism for CheA autophosphorylation/dephosphorylation involving (i) substrate binding, (ii) phospho-transfer, and (iii) product release . We determined the dissociation constant for the kinetically defined CheA.ATP complex to be approximately 300 microM and the limiting rate constant for autophosphorylation to be approximately 0.026 s-1 at saturating ATP concentration . Our results indicate that the apparent dissociation constant of the phospho-CheA.ADP complex is approximately 42 microM and that the limiting rate constant for CheA dephosphorylation is approximately 0.028 s-1 at saturating ADP concentration . We corroborated the kinetically determined Kd values by performing independent ligand binding experiments . In addition, we found that the kinetics of trans-phosphorylation, involving mutant proteins CheA48HQ and CheA470GK, exhibited kinetic properties similar to those observed for autophosphorylation of wild-type CheA, although the limiting rate constant (0.008 s-1) was somewhat slower for this trans-phosphorylation reaction . These results will provide a framework for assessing the effects of various cheA mutations as well as for exploring the nature of CheA regulation by the chemotaxis receptor/transducer proteins.

Biochemistry, 1994 Jun 28, 33(25), 7745 - 52
Chemical shift assignments and folding topology of the Ras-binding domain of human Raf-1 as determined by heteronuclear three-dimensional NMR spectroscopy; Emerson SD et al.; Raf-1 is a 74-kDa serine-threonine kinase which serves as the immediate downstream target of Ras in the cell growth signal transduction pathway . Recent genetic and biochemical experiments have demonstrated that (1) Ras interacts directly with the amino-terminal domain of Raf and (2) residues 51-131 of the Raf sequence are sufficient to mediate this interaction {Vojtek, A . B., Hollenberg, S . M., & Cooper, J . A . (1993) Cell 74, 205-214} . We have expressed a corresponding segment of the human Raf sequence (Raf55-132) in Escherichia coli as a fusion with maltose binding protein . The fusion protein was purified by affinity chromatography and cleaved at a pre-engineered site with factor Xa protease to liberate the 78-residue fragment of Raf . Raf55-132 bound to Ras with high affinity in a competition assay with GAP . An unlabeled version of Raf55-132 was studied by 2D homonuclear NMR, and uniformly 15N- and 13C/15N-labeled versions of Raf55-132 were studied by 2D and 3D heteronuclear NMR . Nearly complete sequence-specific assignments were made for the backbone HN, H alpha, 15N, and 13C alpha resonances . NOEs were used to determine regions of secondary structure and the overall folding topology . Raf55-132 is an independently folded domain composed of a five-stranded beta-sheet, a three-turn alpha-helix, and possibly an additional one-turn helix . Its structure resembles that of ubiquitin, even though there is no more than 11% sequence homology between the two proteins.

FEBS Lett, 1994 Jun 27, 347(2-3), 300 - 3
Purification, crystallization and preliminary X-ray diffraction analysis of recombinant human neutrophil-activating peptide 2 (rhNAP-2); Kungl AJ et al.; The potent activator and chemoattractant for human neutrophils, neutrophil-activating peptide 2 (NAP-2), has been cloned and expressed in Escherichia coli . The protein has been purified to homogeneity (> 98%) by a series of chromatographic techniques, including reversed phase HPLC . The biological activity of recombinant human NAP-2 (rhNAP-2), characterized by the induction of elastase release from human neutrophils, was found to be comparable to natural NAP-2 . rhNAP-2 has been crystallized by the hanging drop vapor diffusion method . The crystals belong to space group P222 with unit cell dimensions of a = 30.8 A, b = 39.5 A and c = 95.3 A . A packing density of 3.8 A3/Da with a solvent content of approximately 68% is obtained when one molecule per asymmetric unit is assumed . The crystals were shown to diffract to beyond 2.0 A on a conventional X-ray source . They are stable to X-rays for several days and are thus suitable for high resolution structure determination.

FEBS Lett, 1994 Jun 27, 347(2-3), 251 - 6
Actophorin preferentially binds monomeric ADP-actin over ATP-bound actin: consequences for cell locomotion; Maciver SK et al.; Actophorin from Acanthamoeba castellanii severs actin filaments and sequesters actin monomers . Here we report that actophorin binds ADP-bound monomers with higher affinity than ATP-bound monomers . Actophorin is therefore much less efficient at severing actin filaments in the presence of ADP compared to ATP, particularly taking account of the higher critical concentration in ADP . Monomer binding is also reduced in the presence of 25 mM inorganic phosphate (which is assumed to form ADP.Pi-actin) . These findings are discussed in the light of observations on the nucleotide specificity of other monomer binding proteins and related to the role of actin in lamellar protrusion and cell locomotion.

FEBS Lett, 1994 Jun 27, 347(2-3), 137 - 42
A factor protecting mammalian {75Se}SeCys-tRNA is different from EF-1 alpha; Yamada K et al.; In Escherichia coli, an elongation factor (EF-Tu-like) specific to SeCys-tRNA, SELB, has been identified; however, a mammalian counterpart of SELB has not been reported to date . We searched for and found this factor in bovine liver extracts using the assay of {75Se}SeCys-tRNA protecting activity against alkaline hydrolysis (SePF activity) . We found SePF activity in the protein extracts of the precipitate (microsomal fraction) collected at 150,000 x g from bovine liver . The proteins were separated by Sephacryl S-300 chromatography, and the SePF and EF-1 alpha activities were found in the same fraction, indicating that SePF and EF-1 alpha have the same molecular mass (approximately 50 kDa) . We then chromatographed this active fraction using CM-Sephadex C-25 columns . The SePF activity was eluted after the peak of EF-1 alpha activity . This result indicated that this SePF activity was not dependent on EF-1 alpha . In addition to performing these two chromatographies, we investigated pure EF-1 alpha from Bombyx mori but could not detect any SePF activity in B . mori EF-1 alpha . Thus we showed that the SePF activity in bovine liver differs from that of EF-1 alpha in eukaryotes . Therefore the factor protecting {75Se}SeCys-tRNA in bovine liver is not EF-1 alpha and must be a SELB-like factor.

FEBS Lett, 1994 Jun 27, 347(2-3), 133 - 6
Energetic aspects of intramolecular coupling between the nucleotide binding site and the distal switch II region of the yeast RAS2 protein; De Vendittis E et al.; We have studied the interaction of the yeast RAS2 protein with guanine nucleotides using energetic parameters for the dissociation of RAS.nucleotide complexes . The results indicated that a Gly-->Ser substitution at position 82 led to an altered interaction with GppNHp and, to a lesser extent, also with GDP . It was also possible to conclude that structural perturbation of Gly82 can stimulate nucleotide release by decreasing the energetic barrier for nucleotide dissociation . This, together with the observation that residues 80 and 81 are involved in the response of RAS to nucleotide exchange factors without affecting GDP binding per se, suggests a potential mechanism for exchange factor-stimulated GDP release.

Nucleic Acids Res, 1994 Jun 25, 22(12), 2392 - 8
In vivo excision and amplification of large segments of the Escherichia coli genome; Posfai G et al.; In vivo excision and amplification of large segments of a genome offer an alternative to heterologous DNA cloning . By obtaining predetermined fragments of the chromosome directly from the original organism, the problems of clone stability and clone identification are alleviated . This approach involves the insertion of two recognition sequences for a site-specific recombinase into the genome at predetermined sites, 50-100 kb apart . The integration of these sequences, together with a conditional replication origin (ori), is targeted by homologous recombination . The strain carrying the insertions is stably maintained until, upon induction of specifically engineered genes, the host cell expresses the site-specific recombinase and an ori-specific replication protein . The recombinase then excises and circularizes the genomic segment flanked by the two insertions . This excised DNA, which contains ori, is amplified with the aid of the replication protein and can be isolated as a large plasmid . The feasibility of such an approach is demonstrated here for E . coli . Using the yeast FLP/FRT site-specific recombination system and the pi/gamma-ori replication initiation of plasmid R6K, we have devised a procedure that should allow the isolation of virtually any segment of the E . coli genome . This was shown by excising, amplifying and isolating the 51-kb lacZ--phoB and the 110-kb dapX--dsdC region of the E . coli MG1655 genome.

Nucleic Acids Res, 1994 Jun 25, 22(12), 2255 - 63
Footprinting RNA-protein complexes following gel retardation assays: application to the R-17-procoat-RNA and tat--TAR interactions; Pearson L et al.; RNA-protein complexes isolated following a gel retardation assay can be footprinted within the gel matrix using the chemical nuclease activities of 4,7-dimethyl-, 5,6-dimethyl-, and 3,4,7,8-tetramethyl-1,10-phenanthroline-copper . These complexes are more reactive than 1,10-phenanthroline-copper but share its reaction preference for bulges and loops . The interaction of the coat protein of R-17 with its viral RNA target and tat- and tat-derived peptides with HIV TAR RNA have been studied . In both cases, the RNA sequence opposite a 2-3 nucleotide bulge are protected . Tat-derived peptides inhibit cleavage at sites which intact tat does not protect . These results are consistent with transcription studies which have suggested that truncation of tat increases nonspecific binding.

Nucleic Acids Res, 1994 Jun 25, 22(12), 2217 - 21
Nuclease resistance of an extraordinarily thermostable mini-hairpin DNA fragment, d(GCGAAGC) and its application to in vitro protein synthesis; Yoshizawa S et al.; The nuclease resistance of a short, thermostable mini-hairpin, d(GCGAAGC), and other related hairpins was examined . Hairpins possessing a purine-rich (GAA) or (GAAA) loop appeared to be more resistant against nucleases than those with a pyrimidine-rich loop or single-stranded oligomers . Among 8 kinds of oligodeoxyribonucleotides examined, the fragment most resistant against nucleases was a hairpin with the sequence of d(CGCGAAGCG) . This hairpin was then utilized for the stabilization of mRNA in an in vitro translation system; the 3'-terminal region of an mRNA was hybridized with an oligodeoxyribonucleotide including the sequence complementary to the 3'-terminus of the mRNA tagged with the nuclease-resistant d(CGCGAAGCG) hairpin sequence . By using this method, dihydrofolate reductase (DHFR) mRNA was stabilized against nucleases contaminating a cell-free translation system of E.coli, with a consequent increase in protein synthesis efficiency of 200%.

Gene, 1994 Jun 24, 144(1), 75 - 80
Production of recombinant avidin in Escherichia coli; Airenne KJ et al.; A recombinant avidin (re-Avd), containing amino acids (aa) 1-123 of the native chicken egg-white Avd, was produced in Escherichia coli . When cells were grown at 37 degrees C production was over 1 microgram/ml, due to altering the codon preference of the first ten codons . The re-Avd was recovered as a soluble protein from cells grown at 25 or 30 degrees C, whereas at 37 degrees C it was mostly insoluble in inclusion bodies . Our results indicated that, despite the potentially harmful biotin-binding activity of Avd, it is possible to produce biologically active Avd in E . coli which then can easily be purified by affinity chromatography on a biotin column in a single step.

Gene, 1994 Jun 24, 144(1), 59 - 62
ColE1-compatible vectors for high-level expression of cloned DNAs from the T7 promoter; Munson M et al.; A new family of T7-based expression plasmids with unique features is described . The plasmid origin of replication (ori), derived from P15A, is compatible with that of ColE1-derived plasmids, which facilitates the co-production of proteins from these vectors and from ColE1-derived T7 expression vectors in the same cell . The plasmids are medium-copy-number and also carry the M13 ori . Consequently, both double- and single-stranded DNA can be easily obtained . The plasmids encode KmR, thus avoiding the potential for plasmid loss associated with ApR-based systems . One of the plasmids carries the lacI gene, to allow for more stringent regulation of the production of potentially toxic proteins . When the plasmids are introduced into an Escherichia coli strain such as BL21(DE3), which contains the T7 polymerase-encoding gene under control of the lacUV5 promoter, addition of IPTG initiates the production of high levels of the recombinant protein.

J Mol Biol, 1994 Jun 24, 239(5), 726 - 30
Purification and crystallization of the catalytic domain of human protein tyrosine phosphatase 1B expressed in Escherichia coli; Barford D et al.; The amino-terminal 321 residues encoding the catalytic domain of human protein tyrosine phosphatase 1B (molecular mass 37 kDa) has been expressed in Escherichia coli, purified to homogeneity and crystallized . The crystals diffract to 2.4 A resolution when exposed to synchrotron radiation and belong to space group P3(1)21 (or its enantiomorph P3(2)21) with a = 88.4 A, b = 88.4 A, c = 104.0 A, alpha = beta = 90.0 degrees, gamma = 120.0 degrees . There is one molecule of protein tyrosine phosphatase 1B per asymmetric unit and the crystal form is suitable for the determination of the atomic structure of the enzyme.

J Mol Biol, 1994 Jun 24, 239(5), 698 - 712
Detecting patterns in protein sequences; Neuwald AF et al.; The detection of conserved sequence patterns (motifs) in related proteins often yields valuable structural and functional insights . We describe a method that utilizes rigorous statistics and a depth-first search procedure to efficiently and exhaustively search a set of proteins for significant patterns up to a specified length . Additional procedures classify related patterns into groups and identify protein segments most likely to share a common motif . The utility of the method was demonstrated on several difficult test problems; detection of motifs among 56 proteins in the acyltransferase family, detection of a dinucleotide-binding fold present within a small subset of a set of 91 distantly related and unrelated proteins, detection of the helix-turn-helix motif in 15 distantly related proteins and detection of subtle internal repeats in a prenyltransferase . In a search of a large set of sequences for internal repeats, the method detected novel ankyrin-like repeats in an Escherichia coli protein.

J Mol Biol, 1994 Jun 24, 239(5), 664 - 88
Analysis of the DNA-binding affinity, sequence specificity and context dependence of the glucocorticoid receptor zinc finger region; La Baer J et al.; The glucocorticoid receptor binds with high affinity to glucocorticoid response elements (GREs), which commonly consist of imperfect DNA palindromes with hexameric core binding motifs separated by three base-pairs; the receptor dimerizes upon binding to these sequences, with one monomer occupying each core motif . To examine quantitatively the receptor-DNA interaction, and to characterize the effects of single base-pair mutations and sequence context on this interaction, we have studied the binding of a purified glucocorticoid receptor fragment, T7X556, to a 30 bp oligonucleotide containing a complex arrangement of potential receptor binding sites (RBS): two consensus core motifs, RBSo and RBSa reside in the same orientation and overlap by one base-pair, and a third site, RBSb, a partial match to the consensus separated by three base-pairs and in opposite orientation from RBSa . Using four different footprinting reagents, we detected a receptor dimer bound to one face of the DNA double helix, making close contacts with two adjacent major grooves . One monomer bound with high affinity to RBSa and the other bound with lower affinity to RBSb; transfection studies revealed that both binding sites were necessary for GRE activity in vivo . We measured the affinity of the receptor interaction with RBSa, RBSb and non-specific sites, and showed that protein binding at RBSb was improved approximately 150-fold by cooperativity with RBSa . Remarkably, T7X556 failed to bind to the consensus RBSo sequence, even when it was mutated to match exactly RBSa; the preference for RBSa over RBSo was due neither to the presence of RBSb, nor to occlusion of RBSo by protein bound at RBSa . To examine the relative contribution of each core nucleotide to the binding reaction, we saturated RBSo and RBSa with point mutations . The results implied that four RBSa nucleotides, (or 5'- x G x ACA-3'), may make specific contacts with the protein, as certain mutations at these positions reduced binding drastically . Consistent with the footprinting and transfection assays, equivalent mutations in RBSo had no effect on protein binding . Thus, these findings indicate that the consensus core motif alone is not sufficient to specify a functional RBS, and that flanking sequences create an appropriate context for protein binding.

J Mol Biol, 1994 Jun 24, 239(5), 608 - 22
Correlation of translation efficiency with the decay of lacZ mRNA in Escherichia coli; McCormick JR et al.; We isolated mutations in the leader of a ribosomal protein (r-protein)/lacZ fusion gene in Escherichia coli that caused the mRNA to be translated at efficiencies between < 1% and 62% of the rate of wild-type message . Using a subset of these mutants with translation efficiencies between 5% and 62%, we studied both physical and functional decay of the mRNA after rifampicin inhibition of transcription initiation . The decay of physically intact transcript was analyzed by gel electrophoresis of hybrid-selected messenger RNA segments . The output from the message was analyzed by measuring the synthesis rate of r-protein/lacZ fusion protein . Decay of physically intact message after rifampicin addition correlated with the translation efficiency, with the more active messengers being more stable . Different segments of the r-protein/lacZ fusion mRNA decayed with the same rate, indicating that there is no hyper-labile region in the transcript . The decay rate was also independent of the length of the segment probed, suggesting that the mRNA is not degraded by random attacks along the entire length of the molecule . Our results are consistent with an overall 5' to 3' degradation pathway . Surprisingly, the rate of fusion protein synthesis did not decrease immediately after rifampicin addition . Rather, a lag preceded the exponential decay phase; the length of this delay correlated with the translation efficiency, such that the lag increased with increasing efficiency of translation . We suggest that these lags indicate that mRNAs are normally competing for ribosomes during exponential growth and, after rifampicin addition, RNA molecules with longer physical half-lives are translated by ribosomes released from fast decaying messengers.

J Biol Chem, 1994 Jun 24, 269(25), 17297 - 304
Molecular cloning and characterization of a ras-related gene of ran/tc4/spi1 subfamily in Giardia lamblia; Chen LM et al.; The significance of Ras-like proteins in the protozoa is relatively unexplored . In this report, a gene encoding a Ras-like nuclear (Ran) protein was identified in Giardia lamblia by a polymerase chain reaction-based cloning strategy . The sequence analyses suggest that the gene was intronless, and had short 5'-untranslated leader sequences in the corresponding mRNA up to -2, -4, or -29 bases upstream of the first initiation codon . The full-length cDNA sequence predicted a protein comprising 226 amino acids, in which the highly conserved functional motifs of the Ras superfamily were all preserved . This protein showed 52% identity to human TC4 and 50% identity to yeast Spi1 proteins, suggesting that it is closely related to the Ran proteins, and it was therefore designated gRan . gRan produced from recombinant Escherichia coli exhibited GTP binding activity by an overlay assay . In good agreement with the predicted size of gRan, a 27-kDa protein was identified in a lysate of G . lamblia by Western blotting using antiserum raised against recombinant gRan . The protein was further localized in both nuclei of G . lamblia by immunofluorescence staining . Recombinant gRan exhibited low affinity for GTP with a Kd value of 16.8 microM . The affinity was enhanced to a Kd value of 2.2 microM in the presence of 10 mM Mg2+ . The intrinsic GTPase activity of gRan was observed only in the presence of 10 mM Mg2+ and had an estimated Km of 5.6 microM and a Kcat of 0.33/h . These observations demonstrate the presence of Ras-like proteins in the most primitive eukaryotic cells, G . lamblia, and infer that the Ran protein may play a functional role in the nuclei of this organism.

J Biol Chem, 1994 Jun 24, 269(25), 17141 - 5
Production and characterization of recombinant Goodpasture antigen in insect cells; Turner N et al.; The Goodpasture antigen is the target of anti-basement membrane autoantibodies in Goodpasture's disease, a severe human autoimmune disease characterized by glomerulonephritis and lung hemorrhage . It has been identified as the NC1 domain of the alpha 3 chain of type IV collagen (alpha 3(IV)NC1), a minority component of glomerular basement membrane (GBM) . Protocols for obtaining pure human antigen are laborious and low yielding and require cadaver kidneys . Recombinant alpha 3(IV)NC1 produced in Escherichia coli has been insoluble and poorly recognized by patients' autoantibodies . We have used the baculovirus expression system to produce the antigen as a soluble product in Sf9 cells . A transfer vector was constructed from cDNAs encoding the leader peptide, NH2 terminus, and 7 S domain of the human alpha 1 chain of type IV collagen and was joined inframe to the NC1 domain and COOH terminus of the human alpha 3 chain under the control of the polyhedrin promoter . It therefore encodes a hybrid "mini"-collagen chain from which the majority of the central triple helical region has been deleted . The recombinant antigen was seen on SDS-polyacrylamide gel electrophoresis and Western blots of supernatants at its predicted molecular size of 41 kDa and as dimers of 82 kDa . It reacted strongly with human autoantibodies by Western blotting and enzyme-linked immunosorbent assay, inhibited binding of autoantibodies to human GBM, and bound two monoclonal antibodies known to recognize human alpha 3(IV)NC1 . A common alternatively spliced variant alpha 3(IV)NC1 mRNA, leading to a truncated NC1 domain of 60 amino acids, was expressed as a fusion protein with the same alpha 1 NH2-terminal sequence . It failed to be exported from the cell and was not recognized by autoantibodies . Other NC1 domains could be expressed in the same way . These recombinant molecules should prove invaluable for the in vitro study of the immunopathogenesis of Goodpasture's disease, and the approach provides a means by which interactions between the different type IV collagen chains found in GBM could be studied in vitro.

J Biol Chem, 1994 Jun 24, 269(25), 17020 - 4
Characterization and crystallization of recombinant pea cytosolic ascorbate peroxidase; Patterson WR et al.; An Escherichia coli expression system has been developed for pea cytosolic ascorbate peroxidase (APX) . The enzyme was expressed as a fusion product with the E . coli maltose-binding protein for rapid, affinity chromatography purification . Recombinant ascorbate peroxidase (rAPX) was purified by tryptic digestion to separate the maltose-binding protein from rAPX followed by three chromatographic steps . The purified rAPX protein demonstrated identical electrophoretic, enzymatic, and spectral properties when compared to native APX isolated from pea shoots . Upon addition of an equal molar amount of H2O2, rAPX exhibits an initial decrease in the Soret maximum, which slowly converts to a stable, red-shifted Soret peak similar to that observed for cytochrome c peroxidase Compound I, indicating that rAPX Compound I consists of an oxyferryl (Fe(4+)-O) center . rAPX has been crystallized in a form suitable for crystal structure determination, and a preliminary set of native data to 2.6 A have been collected.

J Biol Chem, 1994 Jun 24, 269(25), 17009 - 19
8-Anilino-1-naphthalenesulfonate is a fluorescent probe of conformational changes in the D-galactose-H+ symport protein of Escherichia coli; Walmsley AR et al.; The binding of sugars and antibiotics to the overexpressed D-galactose-H+ symport protein (GalP) can be monitored from changes in the fluorescence of 8-anilino-1-naphthalenesulfonate (ANS) equilibrated with inside-out vesicles . Transported sugars, such as D-glucose and D-galactose, cause an enhancement in the ANS fluorescence of up to 13% . Nontransported sugars that have little, if any, affinity for GalP, such as L-galactose and L-glucose, have no effect upon the ANS fluorescence . Cytochalasin B and forskolin, which are potent inhibitors of the transporter, produce little change in the fluorescence, but are capable of reversing the D-galactose/D-glucose enhancement in fluorescence . Sugars that bind to GalP but are not transported, such as methyl-beta-D-glucose, produce only a slight quench in the ANS fluorescence, but again reverse the enhancement in fluorescence induced by transported sugars . A simple interpretation is that the increase in ANS fluorescence is attributable to the sugar-induced reorientation of the transporter from an inward- to an outward-facing conformation . Nontransported sugars and antibiotics, which are thought to bind at the inner membrane face of the transporter, are able to reverse the fluorescence enhancement by binding to the inward-facing conformation . The postulated reorientation process was sufficiently slow to follow its progress by stopped-flow fluorometry . The Kd for the binding of D-galactose to the inward-facing site was 7.22 (+/- 1.49) mM, and the rate constants for outward and inward reorientation of the transporter were 4.06 (+/- 0.16) s-1 and 1.36 (+/- 0.18) s-1, respectively . The overall Kd values for a range of sugars and antibiotics have been determined, and the involvement of each sugar hydroxyl group in the recognition and translocation processes has been assessed.

J Immunol Methods, 1994 Jun 24, 172(2), 179 - 87
Conjugation of recombinant reverse transcriptase of HIV-1 to beta-D-galactosidase from Escherichia coli for ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) of anti-HIV-1 IgG; Hashinaka K et al.; Recombinant reverse transcriptase (RT) of HIV-1 was conjugated to beta-D-galactosidase from Escherichia coli in three different ways . Maleimide groups were introduced into beta-D-galactosidase molecules using N,N'-o-phenylenedimaleimide in the absence (method I) or presence (method II) of N-ethylmaleimide or into beta-D-galactosidase molecules, which had been treated with excess of 4,4'-dithiodipyridine to block thiol groups, using N-succinimidyl-6-maleimidohexanoate (method III) . Subsequently, the maleimide groups were reacted with thiol groups introduced into recombinant RT molecules using N-succinimidyl-S-acetylmercaptoacetate . The conjugates were tested by a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) . The immune complex consisting of 2,4-dinitrophenyl-bovine serum albumin-recombinant RT conjugate, anti-HIV-1 IgG and recombinant RT-beta-D-galactosidase conjugate was captured by polystyrene beads coated with (anti-2,4-dinitrophenyl group) IgG, eluted with N epsilon-2,4-dinitrophenyl-L-lysine and transferred to polystyrene beads with (anti-human IgG gamma chain) IgG . The conjugate prepared by method III, which showed the least polymerization, the least loss of the specific enzyme activity and the lowest nonspecific binding, improved the sensitivity of the enzyme immunoassay for anti-HIV-1 IgG approximately 30-fold compared with RT-horseradish peroxidase conjugate.

Science, 1994 Jun 24, 264(5167), 1891 - 903
Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP; Pelletier H et al.; Two ternary complexes of rat DNA polymerase beta (pol beta), a DNA template-primer, and dideoxycytidine triphosphate (ddCTP) have been determined at 2.9 A and 3.6 A resolution, respectively . ddCTP is the triphosphate of dideoxycytidine (ddC), a nucleoside analog that targets the reverse transcriptase of human immunodeficiency virus (HIV) and is at present used to treat AIDS . Although crystals of the two complexes belong to different space groups, the structures are similar, suggesting that the polymerase-DNA-ddCTP interactions are not affected by crystal packing forces . In the pol beta active site, the attacking 3'-OH of the elongating primer, the ddCTP phosphates, and two Mg2+ ions are all clustered around Asp190, Asp192, and Asp256 . Two of these residues, Asp190 and Asp256, are present in the amino acid sequences of all polymerases so far studied and are also spatially similar in the four polymerases--the Klenow fragment of Escherichia coli DNA polymerase I, HIV-1 reverse transcriptase, T7 RNA polymerase, and rat DNA pol beta--whose crystal structures are now known . A two-metal ion mechanism is described for the nucleotidyl transfer reaction and may apply to all polymerases . In the ternary complex structures analyzed, pol beta binds to the DNA template-primer in a different manner from that recently proposed for other polymerase-DNA models.

J Biol Chem, 1994 Jun 24, 269(25), 17221 - 7
Slippage synthesis at the galP2 promoter of Escherichia coli and its regulation by UTP concentration and cAMP.cAMP receptor protein; Jin DJ; An intriguing mechanism in regulating transcription initiation from the gal operon in Escherichia coli is described . Initiation from galP2, one of the two promoters of the E . coli galactose operon, is shown to be subject to promoter clearance control in responding to changes in UTP concentration . In vitro, RNA polymerase (RNAP) makes a large amount of nonproductive "stuttering" initiation products at the galP2 promoter at high concentrations of UTP and less of the stuttered products at low concentrations of UTP . Conversely, RNAP makes more productive initiation products at low UTP concentration than at high UTP concentration . The transcription factor cAMP.CRP complex which normally inhibits transcription from galP2 also represses the stuttering synthesis from galP2 . When galactose is used as a sole carbon source and the internal UTP pools are adjusted externally, a cya mutant (in which galP2 is mainly responsible for the expression of the gal operon and galP1 activity is minimal) has a slower growth rate and lower expression of the gal operon at high UTP pools than at low UTP pools . Such an apparent correlation between the in vitro and in vivo results allows one to speculate that changes in UTP concentration can modulate the expression of the gal operon . The implication of a gal promoter being controlled by UTP is discussed.

Nature, 1994 Jun 23, 369(6482), 675 - 7
Structure of the pleckstrin homology domain from beta-spectrin; Macias MJ et al.; The 'pleckstrin homology' or PH domain is a 100-residue protein module . It is present in many kinases, different isoforms of phospholipase C, GTPase-activating proteins and nucleotide-exchange factors . Its function is not known, but many proteins that contain a PH domain interact with GTP-binding proteins . The PH domain in beta-adrenergic receptor kinase may be involved in binding to the beta gamma subunits of a trimeric G-protein . We report here the three-dimensional structure of the PH domain of the cytoskeletal protein spectrin using homonuclear nuclear magnetic resonance . The core of the molecule is an antiparallel beta-sheet consisting of seven strands . The C terminus is folded into a long alpha-helix, and another helix is present in one of the surface loops . The molecule is electrostatically polarized and contains a pocket which may be involved in the binding of a ligand . There is a distant relationship to the peptidyl-prolyl-cis-trans-isomerase FKBP in which this pocket is involved in the binding of the macrocyclic compound FK506 (refs 8-11).

Nature, 1994 Jun 23, 369(6482), 672 - 5
Solution structure of a pleckstrin-homology domain; Yoon HS et al.; Pleckstrin, the major protein kinase C substrate of platelets, contains domains of about 100 amino acids at the amino and carboxy termini that have been found in a number of proteins, including serine/threonine kinases, GTPase-activating proteins, phospholipases and cytoskeletal proteins . These conserved sequences, termed pleckstrin-homology (PH) domains, are thought to be involved in signal transduction . But the details of the function and binding partners of the PH domains have not been characterized . Here we report the solution structure of the N-terminal pleckstrin-homology domain of pleckstrin determined using heteronuclear three-dimensional nuclear magnetic resonance spectroscopy . The structure consists of an up-and-down beta-barrel of seven antiparallel beta-strands and a C-terminal amphiphilic alpha-helix that caps one end of the barrel . The overall topology of the domain is similar to that of the retinol-binding protein family of structures.

Biochim Biophys Acta, 1994 Jun 21, 1218(2), 250 - 3
Cyclic AMP-dependent expression of the Escherichia coli serC-aroA operon; Lim CJ et al.; The Escherichia coli serC-aroA operon encodes biosynthetic enzymes for unrelated amino acid biosynthetic pathways leading to the synthesis of serine and the aromatic amino acids . A serC-aroA-lac translational fusion was constructed in the vector pMC1403 . Synthesis of beta-galactosidase from the serC-aroA-lac fusion was found to be enhanced in the presence of lactose as the sole carbon source . This enhancement was not observed in strains containing a cya or crp mutant . However, the exogenous addition of cAMP greatly increased the beta-galactosidase synthesis in the cya mutant strain . The serC-aroA mRNA content, analyzed by a dot blot assay, also appeared to increase in the serC+ aroA+ cells after the exogenous addition of cAMP . These findings unambiguously indicate that the expression of the serC-aroA operon is positively controlled by cAMP.

Biochim Biophys Acta, 1994 Jun 21, 1218(2), 158 - 62
Escherichia coli gpt as a positive and negative selectable marker in embryonal stem cells; Spring KJ et al.; Transfection of HPRT- L fibroblasts with a plasmid containing two linked selectable markers genes, gpt and neo, regulated by the same eukaryotic control elements, yielded a 6-fold higher transfection frequency on selection for neo than for gpt . Transfection of HPRT- embryonal stem (ES) cells with the same plasmid yielded high levels of transfectants when selected for neo expression with G418, but a level of transfection greater than two orders of magnitude lower was observed when HAT supplemented medium was used to select for gpt expression . Selection for gpt expression in ES cells with medium containing mycophenolic acid and xanthine gave slightly higher frequencies of transfection, but still considerably lower than that for neo selection . In addition, mycophenolic acid exhibited a general cytotoxicity to ES cells with the window between toxicity of this compound to gpt- ES cells and gpt+ ES cells being very narrow . Cells selected with mycophenolic acid and xanthine for expression of gpt remained sensitive to HAT selection . Expression of gpt in a representative ES cell line, selected on mycophenolic acid and xanthine, was verified by Northern analysis and sensitivity to 6-thioguanine . While the level of mRNA expression in this ES cell line was insufficient to support growth via purine salvage when exposed to HAT medium, identical levels of gpt expression in HPRT- L cells, as judged by Northern analysis, allowed for normal growth in HAT medium . This suggests that ES cells place a greater demand on purine nucleotide biosynthesis than L cells . These results are discussed in terms of the use of gpt as a positive and negative selectable marker for gene targeting via homologous recombination in ES cells.

Biochim Biophys Acta, 1994 Jun 21, 1218(2), 145 - 52
Cloning and expression in Escherichia coli of the gene coding for phytoene synthase from the cyanobacterium Synechocystis sp . PCC6803; Martinez-Ferez I et al.; The gene coding for the carotenoid biosynthesis enzyme phytoene synthase (pys) has been cloned from the unicellular cyanobacterium Synechocystis sp . PCC6803 . The gene has been functionally expressed in Escherichia coli, where it directs the biosynthesis of phytoene from geranylgeranyl pyrophosphate (GGPP) . Analysis of the sequence of the Synechocystis pys protein deduced from the gene sequence shows that it is highly homologous to the tomato and Synechococcus phytoene synthases and shows conserved domains with other bacterial phytoene synthase enzymes . The pys gene starts 60 nucleotides downstream of the pds gene (which codes for phytoene desaturase) . However, it seems to be transcribed mainly from its own promoters, because insertions that disrupt the pds gene do not affect significantly the expression of the pys gene.

Proc Natl Acad Sci U S A, 1994 Jun 21, 91(13), 6196 - 200
Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver; Engelhardt JF et al.; First-generation recombinant adenoviruses that lack E1 sequences have shown tremendous promise in animal and human models of gene therapy . Important limitations of these vectors are that recombinant gene expression is transient and inflammation occurs at the site of gene transfer . Our hypothesis for generating vectors with increased persistence is that present recombinant adenoviruses express viral proteins that stimulate cellular immune responses leading to destruction of the infected cells and repopulation of the organ with non-transgene-containing cells . This model predicts that further crippling of the virus will improve persistence and diminish pathology . We describe in this report second-generation recombinant adenoviruses harboring a beta-galactosidase-expressing transgene in which a temperature-sensitive mutation has been introduced into the E2A gene of an E1-deleted recombinant . At nonpermissive temperature, this virus fails to express late gene products, even when E1 is expressed in trans . The biology of this recombinant was studied in vivo in the context of mouse liver, a setting that is permissive for adenovirus type 5 replication . Animals that received the second-generation virus expressed the transgene for at least 70 days, whereas expression of the first-generation virus was no longer than 14 days . In addition, the inflammatory response, as measured by infiltration of CD8+ T cells, was blunted and delayed in livers infected with second-generation virus . These studies illustrate that modifications that disrupt structural protein expression in recombinant adenoviruses may be useful in enhancing their utility for gene therapy.

Proc Natl Acad Sci U S A, 1994 Jun 21, 91(13), 6078 - 82
N-arginine dibasic convertase, a metalloendopeptidase as a prototype of a class of processing enzymes; Pierotti AR et al.; N-Arg dibasic convertase is a metalloendopeptidase from rat brain cortex and testis that cleaves peptide substrates on the N terminus of Arg residues in dibasic stretches . By using both an oligonucleotide and antibodies to screen a rat testis cDNA library, a full-length cDNA was isolated . The sequence contains an open reading frame of 1161 codons corresponding to a protein of 133 kDa that exhibits 35% and 48% similarity with Escherichia coli protease III (pitrilysin, EC 3.4.99.44) and rat or human insulinase (EC 3.4.99.45), respectively . Moreover, the presence of the HXXEH amino acid signature (XX = FL) clearly classifies N-Arg dibasic convertase as a member of the pitrilysin family of zinc-metalloendopeptidases . In addition, a Cys residue that may be responsible for the thiol sensitivity of the insulinase and N-Arg dibasic convertase was proposed . The protein sequence contains a distinctive additional feature consisting of a stretch of 71 acidic amino acids . We hypothesize that this metalloendopeptidase may be a member of a distinct class of processing enzymes.

Proc Natl Acad Sci U S A, 1994 Jun 21, 91(13), 6064 - 8
Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells; Rouet P et al.; Double-strand breaks introduced into DNA in vivo have been shown to enhance homologous recombination in a variety of chromosomal and extrachromosomal loci in Saccharomyces cerevisiae . To introduce double-strand breaks in DNA at defined locations in mammalian cells, we have constructed a mammalian expression vector for a modified form of I-Sce I, a yeast mitochondrial intron-encoded endonuclease with an 18-bp recognition sequence . Expression of the modified I-Sce I endonuclease in COS1 cells results in cleavage of model recombination substrates and enhanced extrachromosomal recombination, as assayed by chloramphenicol acetyltransferase activity and Southern blot analysis . Constitutive expression of the endonuclease in mouse 3T3 cells is not lethal, possibly due to either the lack of I-Sce I sites in the genome or sufficient repair of them . Expression of an endonuclease with such a long recognition sequence will provide a powerful approach to studying a number of molecular processes in mammalian cells, including homologous recombination.

Proc Natl Acad Sci U S A, 1994 Jun 21, 91(13), 5972 - 6
Tau-beta-galactosidase, an axon-targeted fusion protein; Callahan CA et al.; The most commonly used enzymatic reporter molecule, Escherichia coli beta-galactosidase (beta-gal; beta-D-galactoside galactohydrolase, EC 3.2.1.23), fails to readily diffuse into axons; consequently, the morphologies of beta-gal-labeled neurons cannot directly be determined . For analysis of neuronal pathfinding and synaptic connectivity, this information is essential . We have constructed an axon-targeted beta-gal reporter by fusing the cDNA encoding the bovine microtubule-binding protein, tau, to lacZ, the E . coli gene encoding beta-gal . This reporter labels cell bodies and axons when expressed by developing and adult Drosophila neurons . It also reveals the entire cellular extent of nonneuronal cells such as muscle fibers and glia . To generate neuronal markers for studies of Drosophila neural development, we constructed a tau-beta-gal enhancer-trap transposon . From 1500 independent lines generated by mobilization of this transposon, we have isolated a set of useful markers for specific subsets of neurons, glia, and muscles . Since the tau cDNA-lacZ reporter utilizes bovine tau, it may also effectively target beta-gal in vertebrate neurons and prove to be a useful reagent for the analysis of vertebrate nervous systems.

Proc Natl Acad Sci U S A, 1994 Jun 21, 91(13), 5873 - 7
Excision of hypoxanthine from DNA containing dIMP residues by the Escherichia coli, yeast, rat, and human alkylpurine DNA glycosylases; Saparbaev M et al.; The deamination of adenine residues in DNA generates hypoxanthine, which is mutagenic since it gives rise to an A.T to G.C transition . Hypoxanthine is removed by hypoxanthine DNA glycosylase activity present in Escherichia coli and mammalian cells . Using polydeoxyribonucleotides or double-stranded synthetic oligonucleotides that contain dIMP residues, we show that this activity in E . coli is associated with the 3-methyladenine DNA glycosylase II coded for by the alkA gene . This conclusion is based on the following facts: (i) the two enzymatic activities have the same chromatographic behavior on various supports and they have the same molecular weight, (ii) both are induced during the adaptive response, (iii) a multicopy plasmid bearing the alkA gene overproduces both activities, (iv) homogeneous preparation of AlkA has both enzymatic activities, (v) the E . coli alkA- mutant does not show any detectable hypoxanthine DNA glycosylase activity . Under the same experimental conditions, but using different substrates, the same amount of AlkA protein liberates 1 pmol of 3-methyladenine from alkylated DNA and 1.2 fmol of hypoxanthine from dIMP-containing DNA . The Km for the latter substrate is 420 x 10(-9) M as compared to 5 x 10(-9) M for alkylated DNA . Hypoxanthine is released as a free base during the reaction . Duplex oligodeoxynucleotides containing hypoxanthine positioned opposite T, G, C, and A were cleaved efficiently . ANPG protein, APDG protein, and MAG protein--the 3-methyladenine DNA glycosylases of human, rat, and yeast origin, respectively--were also able to release hypoxanthine from various DNA substrates containing dIMP residues . The mammalian enzyme is by far the most efficient hypoxanthine DNA glycosylase of all the enzymes tested.

Proc Natl Acad Sci U S A, 1994 Jun 21, 91(13), 5848 - 52
Localization and characterization of the gene encoding release factor RF3 in Escherichia coli; Grentzmann G et al.; Two protein release factors (RFs) showing codon specificity, RF1 and RF2, are known to be required for polypeptide chain termination in Escherichia coli . A third protein component has also been described that stimulates termination in vitro, but it has remained uncertain whether this protein, RF3, participates in termination in vivo or is essential to cell growth . We report (i) the purification and N-terminal sequencing of RF3; (ii) the isolation of transposon insertion mutants similar to miaD, a suppressor of a leaky UAA mutation affecting the gene miaA, leading to enhanced nonsense suppression; (iii) the localization of the affected gene on the physical map of the chromosome; and (iv) the cloning and sequencing of the wild-type gene, providing proof that it encodes the factor RF3 . We designate the gene prfC . Two transposon insertions were shown to interrupt the coding sequence of prfC, at codons 287 and 426 . The enhanced nonsense suppression in the insertion mutants shows that the product participates in termination in vivo . The isolation of such mutants strongly suggests that the gene product is not essential to cell viability, though cell growth is affected . RF3 is a protein with a molecular weight of 59,460 containing 528 amino acids and displays much similarity to elongation factor EF-G, a GTP binding protein necessary for ribosomal translocation, and other GTP binding proteins known or thought to interact with the ribosome.

Proc Natl Acad Sci U S A, 1994 Jun 21, 91(13), 5838 - 42
Specific human granulocyte-macrophage colony-stimulating factor antagonists; Hercus TR et al.; Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic hemopoietic growth factor and activator of mature myeloid cell function . We have previously shown that residue 21 in the first helix of GM-CSF plays a critical role in both biological activity and high-affinity receptor binding . We have now generated analogues of GM-CSF mutated at residue 21, expressed them in Escherichia coli, and examined them for binding, agonistic, and antagonistic activities . Binding experiments showed that GM E21A, E21Q, E21F, E21H, E21R, and E21K bound to the GM-CSF receptor alpha chain with a similar affinity to wild-type GM-CSF and had lost high-affinity binding to the GM-CSF receptor alpha-chain-common beta-chain complex . From these mutants, only the charge reversal mutants E21R and E21K were completely devoid of agonistic activity . Significantly we found that E21R and E21K antagonized the proliferative effect of GM-CSF on the erythroleukemic cell line TF-1 and primary acute myeloid leukemias, as well as GM-CSF-mediated stimulation of neutrophil superoxide production . This antagonism was specific for GM-CSF in that no antagonism of interleukin 3-mediated TF-1 cell proliferation or tumor necrosis factor alpha-mediated stimulation of neutrophil superoxide production was observed . E . coli-derived GM E21R and E21K were effective antagonists of both nonglycosylated and glycosylated wild-type GM-CSF . These results show that low-affinity GM-CSF binding can be dissociated from receptor activation and have potential clinical significance for the management of inflammatory diseases and certain leukemias where GM-CSF plays a pathogenic role.

Proc Natl Acad Sci U S A, 1994 Jun 21, 91(13), 5818 - 22
Radiation inactivation of human gamma-interferon: cellular activation requires two dimers; Langer JA et al.; gamma-Interferon (IFN-gamma) is a 17-kDa broad-spectrum cytokine which exerts its effects on a variety of target cells through its interaction with the IFN-gamma receptor . Although physicochemical studies of Escherichia coli-derived IFN-gamma, as well as its crystal structure, demonstrate that it is a homodimer in solution (M(r) 34,000), previous radiation inactivation studies yielded a functional size for IFN-gamma of 63-73 kDa in an antiviral assay . To understand the relationship between the solution form of IFN-gamma and the moiety that actually binds to the cellular receptor and activates cells, we examined irradiated nonradioactive and 32P-labeled IFN-gamma for its migration in SDS/polyacrylamide gels (to determine its physical integrity), its binding to cells, its reactivity in an ELISA, and its antiviral activity . The functional size of IFN-gamma differed in the assays, being 22 +/- 2 kDa for the physical destruction of IFN-gamma, 56 +/- 2 kDa for the cellular binding assay, 45-50 kDa for reactivity in the ELISA, and 72 +/- 6 kDa for antiviral activity . The results from the binding assays constitute direct evidence that IFN-gamma binds to its cellular receptor as a dimer . However, for antiviral activity, the functional mass is equivalent to a tetramer . This is consistent with models involving ligand-induced receptor dimerization, whereby two dimers acting in concert (equivalent to the target size of a tetramer) are required to activate cells in the antiviral assay.

Proc Natl Acad Sci U S A, 1994 Jun 21, 91(13), 5813 - 7
GTP-dependent polymerization of Escherichia coli FtsZ protein to form tubules; Bramhill D et al.; The FtsZ protein is a GTPase that is essential for cell division in Escherichia coli . During cytokinesis, FtsZ localizes to a ring at the leading edge of septum synthesis . We report the GTP-dependent polymerization of purified FtsZ measured by sedimentation and light scattering . Electron microscopy of polymerized FtsZ revealed structures including tubules 14-20 nm in diameter with longitudinal arrays of protofilaments . FtsZ depolymerized upon removal of GTP and repolymerized after subsequent GTP addition . Mutant FtsZ84 protein polymerized inefficiently, suggesting that polymerization is important for the cellular role of FtsZ in division . The possibility that tubules of FtsZ protein form a cytoskeleton involved in septum synthesis is consistent with our data.

Proc Natl Acad Sci U S A, 1994 Jun 21, 91(13), 5798 - 802
Identification of the prfC gene, which encodes peptide-chain-release factor 3 of Escherichia coli; Mikuni O et al.; The termination of protein synthesis in bacteria requires two codon-specific polypeptide release factors, RF-1 and RF-2 . A third factor, RF-3, which stimulates the RF-1 and RF-2 activities, was originally identified in Escherichia coli, but it has received little attention since the 1970s . To search for the gene encoding RF-3, we selected nonsense-suppressor mutations by random insertion mutagenesis on the assumption that a loss of function of RF-3 would lead to misreading of stop signals . One of these mutations, named tos-1 (for transposon-induced opal suppressor), mapped to the 99.2 min region on the E . coli chromosome and suppressed all three stop codons . Complementation studies and analyses of the DNA and protein sequences revealed that the tos gene encodes a 59,442-Da protein, with sequence homology to elongation factor EF-G, including G-domain motifs, and that the tos-1 insertion eliminated the C-terminal one-fifth of the protein . Extracts containing the overproduced Tos protein markedly increased the formation of ribosomal termination complexes and stimulated the RF-1 or RF-2 activity in the codon-dependent in vitro termination assay . The stimulation was significantly reduced by GTP, GDP, and the beta,gamma-methylene analog of GTP, but not by GMP . These results fit perfectly with those described in the original publications on RF-3, and the tos gene has therefore been designated prfC . A completely null prfC mutation made by reverse genetics affected the cell growth under the limited set of physiological and strain conditions.

Proc Natl Acad Sci U S A, 1994 Jun 21, 91(13), 5784 - 7
The neuroendocrine polypeptide 7B2 is an endogenous inhibitor of prohormone convertase PC2; Martens GJ et al.; The subtilisin-like prohormone convertase PC2 and the polypeptide 7B2 (an intracellularly cleaved protein of unknown function) are both selectively present in the regulated secretory pathway of neurons and endocrine cells . Here we demonstrate that intact recombinant 7B2 is a potent inhibitor of PC2 and prevents proPC2 cleavage in vitro, whereas the 7B2 cleavage product is virtually inactive . The PC2-related proteinase PC1/PC3 is not inhibited by 7B2 . Furthermore, the carboxyl-terminal half of the 7B2 protein sequence is distantly related to the so-called potato inhibitor I family (which includes subtilisin inhibitors) . Our findings indicate that 7B2 is a physiological inhibitor of PC2 and may provide alternative avenues for the manipulation of peptide hormone levels.

Proc Natl Acad Sci U S A, 1994 Jun 21, 91(13), 5779 - 83
Molecular cloning and characterization of the cDNA coding for the biotin-containing subunit of 3-methylcrotonoyl-CoA carboxylase: identification of the biotin carboxylase and biotin-carrier domains; Song J et al.; Soybean genomic clones were isolated based on hybridization to probes that code for the conserved biotinylation domain of biotin-containing enzymes . The corresponding cDNA was isolated and expressed in Escherichia coli through fusion to the bacterial trpE gene . The resulting chimeric protein was biotinylated in E . coli . Antibodies raised against the chimeric protein reacted specifically with an 85-kDa biotin-containing polypeptide from soybean and inhibited 3-methylcrotonoyl-CoA carboxylase (EC 6.4.1.4) activity in cell-free extracts of soybean leaves . Thus, the isolated soybean gene and corresponding cDNA code for the 85-kDa biotin-containing subunit of 3-methylcrotonoyl-CoA carboxylase . The nucleotide sequence of the cDNA and portions of the genomic clones was determined . Comparison of the deduced amino acid sequence of the biotin-containing subunit of 3-methylcrotonoyl-CoA carboxylase with sequences of other biotin enzymes suggests that this subunit contains the functional domains for the first half-reaction catalyzed by all biotin-dependent carboxylases--namely, the carboxylation of biotin . These domains are arranged serially on the polypeptide, with the biotin carboxylase domain at the amino terminus and the biotin-carboxyl carrier domain at the carboxyl terminus.

Biochemistry, 1994 Jun 21, 33(24), 7691 - 700
Prediction and site-specific mutagenesis of residues in transmembrane alpha-helices of proton-pumping nicotinamide nucleotide transhydrogenases from Escherichia coli and bovine heart mitochondria; Holmberg E et al.; Nicotinamide nucleotide transhydrogenase from bovine heart consists of a single polypeptide of 109 kD . The complete gene for this transhydrogenase was constructed, and the protein primary structure was determined from the cDNA . As compared to the previously published sequences of partially overlapping clones, three residues differed: Ala591 (previously Phe), Val777 (previously Glu), and Ala782 (previously Arg) . The Escherichia coli transhydrogenase consists of an alpha subunit of 52 kD and a beta subunit of 48 kD . Alignment of the protein primary structure of the bovine trashydrogenase with that of the transhydrogenase from E . coli showed an identity of 52%, indicating similarly folded structures . Prediction of transmembrane-spanning alpha-helices, obtained by applying several prediction algorithms to the primary structures of the revised bovine heart and E . coli transhydrogenases, yielded a model containing 10 transmembrane alpha-helices in both transhydrogenases . In E . coli transhydrogenase, four predicted alpha-helices were located in the alpha subunit and six alpha-helices were located in the beta subunit . Various conserved amino acid residues of the E . coli transhydrogenase located in or close to predicted transmembrane alpha-helixes were replaced by site-specific mutagenesis . Conserved negatively charged residues in predicted transmembrane alpha-helices possibly participating in proton translocation were identified as beta Glu82 (Asp655 in the bovine enzyme) and beta Asp213 (asp787 in the bovine enzyme) located close to the predicted alpha-helices 7 and 9 of the beta subunit . beta Glu82 was replaced by Lys or Gln and beta Asp213 by Asn or His . However, the catalytic as well as the proton pumping activity was retained . In contrast, mutagenesis of the conserved beta His91 residue (His664 in the bovine enzyme) to Ser, Thr, and Cys gave an essentially inactive enzyme . Mutation of alpha His450 (corresponding to His481 in the bovine enzyme) to Thr greatly lowered catalytic activity without abolishing proton pumping . Since no other conserved acidic or basic residues were predicted in transmembrane alpha-helices regardless of the prediction algorithm used, proton translocation by transhydrogenase was concluded to involve a basic rather than an acidic residue . The only conserved cysteine residue, beta Cys260 (Cys834 in the bovine enzyme), located in the predicted alpha-helix 10 of the E . coli transhydrogenase, previously suggested to function as a redox-active dithiol, proved not to be essential, suggesting that redox-active dithiols do not play a role in the mechanism of transhydrogenase.

Biochemistry, 1994 Jun 21, 33(24), 7560 - 7
Crystal structure of unmodified tRNA(Gln) complexed with glutaminyl-tRNA synthetase and ATP suggests a possible role for pseudo-uridines in stabilization of RNA structure; Arnez JG et al.; tRNA(2Gln) made in vitro by transcription with T7 RNA polymerase does not contain the pseudouridines at positions 38, 39, and 55, the 4-thiouridine at position 8, or any of the methylated bases found in the tRNA(2Gln) made in vivo . Cocrystals of unmodified tRNA(2Gln) complexed with glutaminyl-tRNA synthetase from Escherichia coli are isomorphous with those of the complex with modified tRNA(2Gln) . A difference electron density map between the complexes with modified and unmodified tRNAs calculated at 2.5-A resolution shows no differences in the protein or tRNA structures, except for some very small shifts in atoms contacting the thiol at the 4 position of uridine 8 that are required to accommodate the smaller oxygen in the unmodified tRNA . Perhaps the most functionally significant change in the unmodified tRNA is the absence of the specifically bound water molecules that are observed to cross-link the N5 of the pseudo-uridines to their 5' phosphate . This suggests a possible role for pseudouridinylation in stabilization of the tRNA through water-mediated linking of these modified bases to the backbone, which is consistent with the lower thermal stability of the unmodified tRNA . An identical water-bridging structure is possible at four of the five other psuedo-uridines in known tRNA structures.

Biochemistry, 1994 Jun 21, 33(24), 7547 - 59
Studies of inhibitor binding to Escherichia coli purine nucleoside phosphorylase using the transferred nuclear Overhauser effect and rotating-frame nuclear Overhauser enhancement; Perlman ME et al.; NMR studies of the adenosine analog tubercidin have been carried out in the presence of Escherichia coli purine nucleoside phosphorylase (PNP) in order to characterize the conformation of the enzyme-complexed nucleoside . Although analysis of transferred NOE data at various enzyme/inhibitor ratios indicated a predominantly syn nucleoside conformation in the enzyme-complexed state, the results, particularly the 8(1') and 8(3') NOE interactions, were not quantitatively consistent with any single bound conformation . Dissociation rate constants for the tubercidin-PNP complex were determined based on analysis of chemical shift and line width data as a function of enzyme/inhibitor ratio, Carr-Purcell-Meiboom-Gill measurements of the transverse relaxation rate as a function of pulse rate, and T1 rho experiments as a function of the spin-lock field strength . Dissociation rate constants of 2100 s-1 at 20 degrees C and 1400 s-1 at 10 degrees C were determined using the latter two methods . These rates are sufficiently high to justify the validity of the transferred NOE method for an enzyme as large as PNP . The possible significance of spin diffusion was investigated by the use of the deuterated analog {2'-2H}tubercidin, for which many of the intraligand spin diffusion pathways are eliminated, and by performing a series of transferred ROE experiments . A comparison of data obtained using transferred NOE and ROE measurements provides a basis for separating direct and indirect relaxation pathways . Both approaches indicated that the relatively significant 8(3') NOE interaction was not dominated by spin diffusion . Furthermore, analysis of chemical shift and transverse relaxation data for the tubercidin H-2 resonance gave inconsistent results for the chemical shift of the bound species and was inconsistent with the assumption of a single, bound conformation . These results were interpreted in terms of a 2:1 ratio of a syn, 3'-exo:anti, 3'-endo geometry for bound tubercidin . Ligand competition experiments using 9-deazainosine show that all of the tubercidin TRNOE effects are reversed by addition of the second nucleoside, suggesting that the TRNOE data for tubercidin arise due to interactions at the active sites of PNP rather than as a consequence of nonspecific binding to the enzyme.

Biochemistry, 1994 Jun 21, 33(24), 7536 - 46
Protein conformational changes induced by 1,1'-bis(4-anilino-5-naphthalenesulfonic acid): preferential binding to the molten globule of DnaK; Shi L et al.; 1,1'-Bis(4-anilino-5-naphthalenesulfonic acid) (bis-ANS), a hydrophobic fluorescent molecular probe which has been shown to bind to compact intermediate states of proteins (molten globules) and also to many nucleotide binding sites, induces a conformational change in DnaK by preferentially binding to its partially folded intermediate state (I) and thus shifting the equilibrium from favoring the native state (N) to favoring the I state . The conformational change was detected by CD, fluorescence emission, size exclusion chromatography, and small-angle X-ray scattering . The presence of bis-ANS significantly decreases the midpoint, Tm, of the initial transition (N-->I) in the thermal unfolding of DnaK, resulting in the apparent destabilization of the native state of DnaK . There is a linear correlation between the apparent free energy (reflected by Tm) of this transition and the concentration of bis-ANS . Bis-ANS does not affect the midpoint of the transition for DnaK from the intermediate to the unfolded state (U) . An additional small transition from I to I*, a more expanded intermediate state, was observed, suggesting that the thermal denaturation of DnaK proceeds via a four-state (N-->I-->I*-->U) unfolding process . The addition of nucleotides, ADP or ATP, to the DnaK-bis-ANS complex causes a decrease in bis-ANS fluorescence emission due to the release of bound bis-ANS from the intermediate state of DnaK . This is due to preferential binding of the nucleotide to the native state of DnaK, resulting in a shift in the equilibrium from the intermediate toward the native state rather than the direct displacement of bis-ANS bound in the nucleotide binding site . Denaturation of DnaK induced by bis-ANS can be minimized by working at a temperature much lower than the Tm of the protein, at low dye concentration, and in the presence of nucleotide . Under these conditions, bis-ANS binds to the native state of DnaK.

Biochemistry, 1994 Jun 21, 33(24), 7510 - 6
Domain structure of Escherichia coli DNA gyrase as revealed by differential scanning calorimetry; Blandamer MJ et al.; The domain structure of DNA gyrase from Escherichia coli has been examined using differential scanning microcalorimetry . The intact enzyme (an A2B2 tetramer) shows at least four transitions with apparent Tm's at 44.8, 53.3, 58.6, and 60.7 degrees C . Comparison with the thermal stabilities of the two separate subunits and genetically-engineered protein fragments has been used to assign these transitions to individual domains within the intact gyrase proteins . The thermal unfolding of DNA gyrase and all individual fragments are irreversible under the conditions of the calorimetric experiment . Further evidence for the assignment of transitions to particular domains has been obtained by studying the effects of tight-binding ligands such as novobiocin on the thermal stabilities of the various protein fragments.

Proc Natl Acad Sci U S A, 1994 Jun 21, 91(13), 5740 - 7
Sorting single molecules: application to diagnostics and evolutionary biotechnology; Eigen M et al.; A method is described that provides for detection and identification of single molecules in solution . The method is based on fluorescence correlation spectroscopy, which records spatio-temporal correlations among fluctuating light signals, coupled with devices for trapping single molecules in an electric field . This technique is applied to studies of molecular evolution, where it allows fast screening of large mutant spectra in which targets are labeled by specific fluorescent ligands . The method expands the horizon in molecular diagnostics by making it possible to monitor concentrations down to (less than) 10(-15) M without any need for amplification.

FEBS Lett, 1994 Jun 20, 347(1), 69 - 72
Cloning and expression of human brain type I inositol 1,4,5-trisphosphate 5-phosphatase . High levels of mRNA in cerebellar Purkinje cells; De Smedt F et al.; In brain and many other tissues, Type I inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase is the major isozyme hydrolysing the calcium-mobilizing second messenger InsP3 . We recently reported the cloning and expression of dog thyroid InsP3 5-phosphatase . During the course of this cloning, screening of a human brain cDNA library allowed us to isolate a cDNA clone D1 with 91% sequence identity with the thyroid sequence . When clone D1 was expressed in Escherichia coli, the fusion protein had InsP3 5-phosphatase activity . M(r) estimates of the recombinant enzyme made by immunodetection, activity assay after SDS/PAGE or silver staining were consistent with the calculated molecular mass . In situ hybridization on human cerebellum sections localised the mRNA for this enzyme to the Purkinje cells.

J Biol Chem, 1994 Jun 17, 269(24), 16766 - 73
Molecular cloning and expression of collagenase-3, a novel human matrix metalloproteinase produced by breast carcinomas; Freije JM et al.; A cDNA coding for a new human matrix metalloproteinase (MMP) has been cloned from a cDNA library derived from a breast tumor . The isolated cDNA contains an open reading frame coding for a polypeptide of 471 amino acids . The predicted protein sequence displays extensive similarity to the previously known MMPs and presents all the structural features characteristic of the members of this protein family, including the well conserved PRCGXPD motif, involved in the latency of the enzyme and the zinc-binding domain (HEXGHXXXXXHS) . In addition, this novel human MMP contains in its amino acid sequence several residues specific to the collagenase subfamily (Tyr-214, Asp-235, and Gly-237) and lacks the 9-residue insertion present in the stromelysins . According to these structural characteristics, the MMP described herein has been tentatively called collagenase-3, since it represents the third member of this subfamily, composed at present of fibroblast and neutrophil collagenases . The collagenase-3 cDNA was expressed in a vaccinia virus system, and the recombinant protein was able to degrade fibrillar collagens, providing support to the hypothesis that the isolated cDNA codes for an authentic collagenase . Northern blot analysis of RNA from normal and pathological tissues demonstrated the existence in breast tumors of three different mRNA species, which seem to be the result of the utilization of different polyadenylation sites present in the 3'-noncoding region of the gene . By contrast, no collagenase-3 mRNA was detected either by Northern blot or RNA polymerase chain reaction analysis with RNA from other human tissues, including normal breast, mammary fibroadenomas, liver, placenta, ovary, uterus, prostate, and parotid gland . On the basis of the increased expression of collagenase-3 in breast carcinomas and the absence of detectable expression in normal tissues, a possible role for this metalloproteinase in the tumoral process is proposed.

J Biol Chem, 1994 Jun 17, 269(24), 16746 - 53
Functional human saposins expressed in Escherichia coli . Evidence for binding and activation properties of saposins C with acid beta-glucosidase; Qi X et al.; Small (80-amino acid) glycoproteins or saposins are important for the in vivo function of several lysosomal hydrolases . Four saposins, A, B, C, and D, are encoded by a single locus termed prosaposin . Saposins C and A are thought to function in vivo as activators of acid beta-glucosidase . The physiologic role of saposin C has been confirmed, whereas that of saposin A role has not . To investigate the effects of saposins C and A on acid beta-glucosidase activity, the coding sequence for the individual saposins was expressed in Escherichia coli and the recombinant proteins purified to homogeneity . Recombinant and natural saposins A and C activated acid beta-glucosidase similarly only in micromolar amounts . Saposin C had specific activation of acid beta-glucosidase activity at < 200 nM . A second phase of activation was achieved at > 1 microM . In comparison, saposin A consistently activated acid beta-glucosidase only at > 1 microM . Two mutant saposins C (Cys382-->Phe and Cys382--Gly) were created and shown to compete with saposin C for a site on acid beta-glucosidase . The mutant saposins did not activate the enzyme . Recombinant saposin A (< 200 nM) competed with saposin C for a site on the enzyme but without activating effects . These studies show that saposin A is not an in vitro activator of acid beta-glucosidase at physiologic concentrations, although binding occurs without activating acid beta-glucosidase . The studies with mutant saposins C indicate that the binding and activation effects of saposins C are distinct events . These results indicate that the saposin C-induced conformational change in the enzyme occurs via highly specific, probably multivalent, interactions between acid beta-glucosidase and saposin C.

J Biol Chem, 1994 Jun 17, 269(24), 16643 - 7
Inhibition of DnaK autophosphorylation by heat shock proteins and polypeptide substrates; Panagiotidis CA et al.; DnaK, the Hsp70 of Escherichia coli, autophosphorylates in vitro . Of the two heat shock proteins that interact with DnaK, GrpE inhibits DnaK phosphorylation, whereas DnaJ has no effect on the reaction . Three synthetic peptides are shown to inhibit DnaK phosphorylation . The potency of a given peptide correlates with its affinity for the DnaK protein . A truncated DnaK that lacks the carboxyl-terminal peptide-binding domain autophosphorylates; this reaction is resistant to the inhibitory peptides . Phosphorylation of the truncated DnaK is still inhibited by GrpE, indicating that the GrpE-binding site resides in the DnaK amino-terminal domain . Thus, DnaK phosphorylation is regulated in vitro, and possibly in vivo, by physiologically relevant substrates and cofactors.

J Biol Chem, 1994 Jun 17, 269(24), 16631 - 7
Null thioredoxin and glutaredoxin Escherichia coli K-12 mutants have no enhanced sensitivity to mutagens due to a new GSH-dependent hydrogen donor and high increases in ribonucleotide reductase activity; Miranda-Vizuete A et al.; This work investigates whether a mutator phenotype is associated to the simultaneous deficiency in thioredoxin and glutaredoxin, the two known hydrogen donors of ribonucleotide reductase . To this end, new Escherichia coli K-12 strains carrying delta trxA and/or grx::kan null mutations were constructed to monitor mutagenesis by selecting forward mutations to L-arabinose resistance . Highly sensitive and specific enzyme-linked immunoassays were developed to confirm that trx-grx- cells lacked thioredoxin and glutaredoxin . A number of remarkable properties were observed in the newly constructed thioredoxin- and glutaredoxin-deficient bacteria compared with the wild type cells . Thus, they (i) grew on minimal medium plates, suggesting that the presence of thioredoxin and glutaredoxin may not be absolutely essential for sulfate reduction; (ii) showed normal mutagenic sensitivities toward a wide variety of DNA-damaging agents, as compared with wild type cells and trx- or grx- single mutants; (iii) displayed 14% of GSH-dependent and 30% of NADPH-dependent ribonucleotide reduction capacity with CDP as substrate in the presence or the absence of exogenous ribonucleotide reductase, respectively; and (iv) showed very high levels of ribonucleotide reductase activity, which was increased from 19- to 23-fold . The existence of a new glutathione-dependent hydrogen donor for ribonucleotide reductase and the high activity levels of this enzyme in trx-grx- defective cells could explain that thioredoxin and the first discovered glutaredoxin are not essential for deoxyribonucleotide synthesis, even under mutagenic stress.

J Biol Chem, 1994 Jun 17, 269(24), 16610 - 7
Induction of heat-stable enterotoxin receptor activity by a human Alu repeat; Almenoff JS et al.; The heat-stable enterotoxins (ST) elaborated by enterotoxigenic Escherichia coli are a family of small cysteine-rich peptides that bind to specific epithelial receptors in the mammalian intestine, causing a secretory diarrhea . The expression of ST receptors is tightly regulated; they are found primarily in intestine, and their expression is developmentally modulated . One receptor for ST has been cloned, and its cDNA encodes a approximately 120-kDa particulate guanylyl cyclase (guanylyl cyclase-C) . Recent studies suggest that there are additional ST receptors that are not homologous to guanylyl cyclase-C . We used an expression cloning strategy to identify intestinal mRNAs that lead to expression of ST receptor activity in transfected cells . Using an ST-specific affinity panning system, we identified a novel 1891-base pair cDNA that does not encode a receptor protein, but instead, consists primarily of untranslated sequence . This cDNA induced receptor activity in both COS and 293 embryonic kidney cells . Northern analysis of the T84 human intestinal cell line, from which this cDNA was cloned, suggests that it is part of a 7.8-kilobase mRNA transcript . This transcript was also identified in human small intestine and colon, as well as in several extra-intestinal tissues . Functional analysis of subcloned fragments reveals that ST binding activity is induced by a 457-base pair human Alu repetitive sequence within the cDNA and that the phenotype is independent of orientation . These findings suggest that a human Alu element induces expression of a unique ST receptor by a transacting mechanism . An unrelated Alu-rich genomic clone did not confer ST binding, suggesting that there may be structural and functional specificity within individual Alu sequences.

J Biol Chem, 1994 Jun 17, 269(24), 16579 - 84
Bimetallic binding motifs in organophosphorus hydrolase are important for catalysis and structural organization; Lai K et al.; Organophosphorus hydrolase is a broad spectrum phosphoric acid hydrolase (EC 3.1.8.1) which appears to contain a binuclear metal center with two metals interactively involved in catalysis and/or structural functions . Site-directed mutagenesis has been employed to evaluate the participation of the various histidine and cysteine residues in metal coordination . The kinetic characteristics and metal binding stoichiometries of the purified site-directed substitutions of each of the histidine and cysteine residues in the catalytic domain of the protein to asparagine and serine residues, respectively, were determined . These data support the hypothesis that the histidines at positions 55, 57, and 201 are coordinated to a metal ion (M1) at the active center of the enzyme and that His254 and His257 are involved in the formation of a second structural metal center (M2) . These and other unidentified amino acids may participate in a co-catalytic center . Although previous solution chemical studies concluded that cysteines are not involved in metal coordination, serine substitutions for Cys59 and Cys227 do affect metal content and catalytic activity . In contrast, substitution of asparagine for His230 does not affect the metal stoichiometry, but does reduce the kcat by 10(-4), indicating that it may be directly involved in the reaction chemistry . The H201N substitution eliminates activity but maintains one molar equivalent of metal and may function as a bridging ligand.

J Biol Chem, 1994 Jun 17, 269(24), 16566 - 73
Repression of AP-1-stimulated transcription by c-Ets-1; Goldberg Y et al.; The transcriptional activities of c-Ets-1 and v-Ets and their functional interaction with the AP-1 factor c-Jun were investigated . Several recombinant Ets proteins were produced and purified either from bacteria or from insect cells . Plasmid DNAs that contained the polyoma virus enhancer Ets/AP-1 element were used as templates for in vitro transcription assays in the presence of HeLa nuclear extract and various combinations of the Jun and Ets proteins . Under these conditions full-length c-Ets-1 on its own does not markedly influence transcription but abolishes the strong transcriptional stimulation normally elicited by Jun . This repression depends on the Ets-binding site and on specific features of c-Ets-1 structure, as both v-Ets and a natural splicing variant c-Ets-1 (delta VII) fail to inhibit Jun activity . These findings suggest that c-Ets may act both as a transcriptional repressor or activator depending on promoter context and splicing pattern.

J Biol Chem, 1994 Jun 17, 269(24), 16549 - 53
Activation and release of enzymatically inactive, full-length rhodanese that is bound to ribosomes as peptidyl-tRNA; Kudlicki W et al.; Synthesis of rhodanese in a cell-free coupled transcription/translation system derived from Escherichia coli leads to an accumulation of full-length rhodanese protein on the ribosomes as well as to enzymatically active protein that is released from the ribosomes into the supernatant fraction . The ribosome-bound protein is enzymatically inactive but can be activated and released from the ribosomes without additional protein synthesis by subsequent incubation in the presence of the added chaperones DnaJ, DnaK, GrpE, GroEL, and GroES plus ATP . Efficient activation requires that all of the chaperones are present together during incubation which yields fully active rhodanese . Incubation in the presence of DnaJ only inhibits release whereas incubation with only GroES or DnaK promotes the release of enzymatically inactive protein . Incubation of the ribosome with puromycin leads to the release of enzymatically inactive protein whereas release and activation in the presence of all of the chaperones is blocked by sparsomycin . The effect of these antibiotics provides very strong evidence that enzymatically inactive, full-length rhodanese is bound to the ribosomes as peptidyl-tRNA and that the peptidyl transferase reaction is required for its release . Considered together, the data indicate that chaperone-mediated late stages of rhodanese folding into the enzymatically active, native conformation are intimately associated with the process of termination and release that occurs as part of the reaction cycle of protein synthesis.

J Mol Biol, 1994 Jun 17, 239(4), 588 - 90
Crystallization of enzyme IIB of the cellobiose-specific phosphotransferase system of Escherichia coli; van Montfort RL et al.; Crystals of enzyme IIB of the cellobiose-specific phosphotransferase system have been obtained from 15% polyethylene glycol 4000 using both streak-seeding and macroseeding techniques at 4 degrees . Crystals were grown with the hanging drop method of vapour diffusion . Addition of 2-propanol and benzamidine/HCl proved essential to obtain single crystals suitable for X-ray analysis . The crystals diffract to 1.8 A resolution and have the monoclinic space group P2(1), with cell dimensions a = 53.6 A, b = 31.7 A, c = 60.0 A and beta = 101.7 degrees . From a self-rotation function it seems likely that there are two molecules in the asymmetric unit related by a non-crystallographic 2-fold axis.

J Mol Biol, 1994 Jun 17, 239(4), 545 - 54
Two-dimensional NMR studies of selenomethionyl calmodulin; Zhang M et al.; Calmodulin (CaM) is a ubiquitous calcium regulatory protein that can interact with almost 30 different target proteins . The majority of the CaM-binding domains of the target proteins are believed to interact with two hydrophobic surfaces on Ca(2+)-CaM; these two regions are very rich in Met residues . To obtain more information about the role of these residues, we have biosynthetically incorporated selenomethionine (SeMet) in place of the nine Met residues of CaM . Amino acid analysis shows that the SeMet-CaM contains 15% Met and 85% SeMet . SeMet-CaM retains many of the properties of the wild-type protein; it activates the enzyme cyclic nucleotide phosphodiesterase, it binds to phenyl-Sepharose and myosin light chain kinase (MLCK) in a calcium-dependent manner, and it experiences a calcium-dependent band shift during SDS-gel electrophoresis . Moreover, by comparing the natural abundance (1H,13C)-heteronuclear multiple quantum coherence (HMQC) spectra of the calcium, apo and target peptide-bound forms of wild-type CaM and SeMet-CaM, we have found that the two proteins have very similar, if not identical, structures . Thus, the substitution of SeMet for Met does not cause a change in the conformation and function of CaM, in agreement with the results obtained for other proteins . The apo, calcium and target peptide-bound forms of SeMet-CaM were subsequently studied by natural abundance (1H,77Se)-heteronuclear multiple bond correlation (HMBC) and (1H,13C)-HMQC NMR . Nine well-resolved 77Se resonances could be observed . Substitution of SeMet for Met gave rise to the same 1H and 13C chemical shift changes for each individual Met residue, this facilitated making the assignments from known 1H,13C assignments of the Met residues . Some of these assignments were confirmed by studying Met-->Leu mutants of CaM . With the exception of Met76, which always remains solvent exposed, all resonances experienced large 77Se chemical shift changes upon the addition of Ca2+ and the MLCK peptide . The large shift changes indicate that the electron distribution in the SeMet side-chain can be adjusted for the different states of CaM, suggesting that the polarizability of sulfur or selenium may be important for the proper functioning of CaM . This study also shows that the natural abundance (1H,77Se)-HMBC experiment provides a sensitive approach for the study of SeMet proteins.

J Mol Biol, 1994 Jun 17, 239(4), 466 - 75
Context-dependent effects of upstream A-tracts . Stimulation or inhibition of Escherichia coli promoter function; Ellinger T et al.; Phased A-tract sequences were inserted in the upstream region of three synthetic promoters known to encompass different rate-limiting steps within the pathway of RNA polymerase-promoter interaction (Ellinger et al., accompanying paper) . Promoter PS1, which is rate-limited in complex formation, was stimulated by A-tracts in vivo . Permanganate probing showed that the stimulation is due to an enhanced ability to compete for limiting RNA polymerase in vivo, leading to the increased formation of open complexes . By contrast, promoters PS2 and PS3, which are rate-limited in steps following open complex formation, were inhibited in vivo by A-tracts . Permanganate probing showed that the inhibition was accompanied by an A-tract-dependent accumulation of stalled initial transcribing complexes . A single A-tract was as effective as three . The phasing of the A-tracts with respect to the core promoter sequence was found to be important for promoter function . The position that caused maximal activation at one promoter caused maximal inhibition at another . These results suggest that the same molecular interaction gives rise to both inhibition and activation . This is likely to be due to facilitated RNA polymerase binding in the presence of A-tracts, which stimulates binding-limited promoters but inhibits promoter function in which polymerase escape and promoter clearance is rate limiting.

J Mol Biol, 1994 Jun 17, 239(4), 455 - 65
Stalling of Escherichia coli RNA polymerase in the +6 to +12 region in vivo is associated with tight binding to consensus promoter elements; Ellinger T et al.; Three synthetic promoters, PS1, PS2 and PS3, which differ in their core promoter elements, were studied in vivo and in vitro . Whereas an increased homology score correlates with higher rates of RNA polymerase binding, it does not correlate with activity in vivo . Permanganate probing in vivo reveals that PS1, which exhibits the lowest homology score, is rate-limited during the early phase of promoter-RNA polymerase interactions . By contrast, PS2 and PS3, with higher homology scores, are limited at a late step involving an open DNA region spanning from +6 to +12, indicating a stalling of RNA polymerase . These complexes disappear upon treatment of cells with rifampicin and are replaced by open complexes covering the start site . Because initiated complexes are selectively insensitive to rifampicin action, this confirms that RNA polymerase stalled at +6 to +12 has initiated RNA synthesis . Kinetic studies indicate that the enzyme is released slowly from this position and that this slow release appears to be responsible for the low promoter activity . For PS3, which exhibits the highest homology score and which binds RNA polymerase most efficiently, the release of the stalled complex is particularly slow . PS3 is found to be the weakest of the three promoters in vivo . These results support models in which promoter activity can be determined by various rate limiting steps, including those following the formation of open complexes and even the initiation of RNA synthesis.

Cell, 1994 Jun 17, 77(6), 943 - 50
Three-dimensional structure of hepatitis B virus core particles determined by electron cryomicroscopy; Crowther RA et al.; Human hepatitis B virus core protein expressed in E . coli assembles into two sizes of particle . We have determined their three-dimensional structures by electron cryomicroscopy and image processing . The large and small particles correspond to triangulation number T = 4 and T = 3 dimer clustered packings, containing 240 and 180 protein subunits, respectively . The local packing of subunits is very similar in the two sizes of particle and shows holes or channels through the shell . The native viral core particle packages RNA and is active in reverse transcription to DNA . The holes we observe may provide access for the necessary small molecules . Shells assembled from the intact core protein contain additional material, probably RNA, which appears as an icosahedrally ordered inner shell in the three-dimensional map.

J Mol Biol, 1994 Jun 17, 239(4), 439 - 54
Nucleolytic inactivation and degradation of the RNase III processed pnp message encoding polynucleotide phosphorylase of Escherichia coli; Hajnsdorf E et al.; The two cleavages made by RNase III in the transcripts of the pnp gene of Escherichia coli, 80 nucleotides upstream of the coding sequence of polynucleotide phosphorylase, were previously demonstrated to trigger the rapid degradation of the pnp messenger . In this paper, we demonstrate that the 5' end of the RNase III processed pnp mRNA is attacked by ribonucleases more efficiently than the rest of the molecule . Several 5' extremities resulting from cleavages occurring in the first 500 nucleotides of the pnp transcript have been identified . Three of them referred to as X, Y and W occur in the wild-type strain at the beginning of the coding sequence of the pnp mRNA . The mRNA appears to be cleaved more efficiently at the X site, proximal to the initiation codon, than at sites Y and W located downstream . In vitro, the maturation at X is catalysed by RNase E but not by RNase III . Accumulation of RNA processed at X in RNase E deficient strains leads us to postulate that X is a high affinity primary site which is slowly cleaved by the residual activity of thermosensitive RNase E at non-permissive temperature and that secondary sites located downstream are processed less efficiently than X . Taken together, our results suggest that in wild-type E . coli the degradation of the RNase III processed mRNA is mediated by RNase E.

Cancer Res, 1994 Jun 15, 54(12), 3196 - 201
Metabolism of bioreductive antitumor compounds by purified rat and human DT-diaphorases; Beall HD et al.; The metabolisms of two standard electron acceptors and a series of bioreductive antitumor compounds by purified rat and human DT-diaphorases (DTD) were compared . DTD was purified from rat liver cytosol and from Escherichia coli in which rat liver or human lung tumor DTD complementary DNA was expressed . Km and kcat values for menadione and 2,6-dichlorophenolindophenol reduction were similar for the three enzyme preparations except that rat E . coli DTD had 2-3-fold higher kcat values for both menadione and 2,6-dichlorophenolindophenol and a 2-3-fold higher Km for menadione than either rat liver or human E . coli DTD . Reduction of the antitumor compounds was 1.9-4.9 times faster with rat E . coli DTD than with human E . coli DTD . The antitumor compounds were reduced in the following order by rat E . coli DTD: 2,5-dimethyl-3,6-diaziridinyl-1,4-benzoquinone > streptonigrin > mitomycin A > diaziquone > mitomycin C (MC) > 5-(aziridin-1-yl)-2,4-dinitrobenzamide . The order was the same for human E . coli DTD with one exception; diaziquone was reduced slightly faster than mitomycin A . Metabolism of doxorubicin could not be detected using rat or human E . coli DTD . MC-induced DNA cross-linking was also more efficient using rat E . coli DTD relative to human E . coli DTD . Metabolism of MC by rat and human E . coli DTD was also compared under aerobic and hypoxic conditions . Rates of reduction of MC and metabolite formation were similar under aerobic and hypoxic conditions, and the toxicity of MC to DTD-rich HT-29 cells was also similar in aerobic and hypoxic conditions . In contrast, the toxicity of MC to DTD-deficient BE cells was potentiated markedly under hypoxia . These data show that although small catalytic differences between rat and human E . coli DTD can be observed, compounds such as 2,5-dimethyl-3,6-diaziridinyl-1,4-benzoquinone and streptonigrin are still excellent substrates for the human enzyme and may be useful in the therapy of tumors high in DTD activity . In addition, metabolism of MC by rat and human E . coli DTD was similar in aerobic and hypoxic conditions; in agreement with these data, cytotoxicity of MC to a DTD-rich cell line was oxygen independent . Increased MC cytotoxicity under hypoxia appears to be mediated by enzymes other than DTD.

J Am Vet Med Assoc, 1994 Jun 15, 204(12), 1949 - 52
Milk production in cows with endotoxin-induced mastitis treated with isotonic or hypertonic sodium chloride solution; Tyler JW et al.; Milk production was monitored in 16 cows for 6 milkings after intramammary infusion of 1 mg of endotoxin in a single forequarter . The cows were randomly assigned to 1 of 2 treatment groups; 8 cows were treated with isotonic saline solution and 8 cows were treated with hypertonic saline solution . Saline solutions were administered IV (5 ml/kg of body weight) 4 hours after infusion of endotoxin . Mean cumulative change in milk yield and interval change in milk yield were greater in cows treated with isotonic saline solution than in cows treated with hypertonic saline solution . Significant differences between treatment groups were not detected.

FEMS Microbiol Lett, 1994 Jun 15, 119(3), 351 - 7
Mutagenesis of a gene encoding a cytochrome o-like terminal oxidase of Azotobacter vinelandii: a cytochrome o mutant is aero-tolerant during nitrogen fixation; Leung D et al.; The amino acid sequence obtained by translating the nucleotide sequence of a 0.55 kb fragment, amplified from Azotobacter vinelandii chromosomal DNA by PCR, was 57% identical to part of the Escherichia coli cyoB gene, encoding subunit I of the cytochrome bo-type quinol oxidase . This fragment was mutated in vitro by insertion of a kanamycin-resistance cassette and introduced into the chromosome of A . vinelandii by homologous recombination . The mutant contained no spectrally detectable cytochrome o . However, in the stationary phase of growth, the level of the alternative oxidase (cytochrome bd) was 11-fold higher than in the wild-type strain . Respiration of the mutant was insensitive to chlorpromazine, an inhibitor thought to act specifically on cytochrome o . Cytochrome o-deficient mutants fixed nitrogen in air, clearly distinguishing the role of this oxidase from that of cytochrome bd, which is required for respiratory protection of oxygen-labile nitrogenase.

Eur J Biochem, 1994 Jun 15, 222(3), 901 - 7
Sequencing and characterization of the sdaC gene and identification of the sdaCB operon in Escherichia coli K12; Shao Z et al.; We describe here the regulatory and coding region, and DNA sequence, for a newly recognized gene, sdaC, which codes for a hydrophobic protein with several predicted membrane-spanning domains . sdaC and sdaB form a single operon, with 57 bp between the end of sdaC and the start of sdaB . Expression of the sdaCB operon is regulated mainly by catabolite repression, but is also slightly sensitive to regulation by leucine-responsive regulatory protein . Cells carrying sdaC on a multicopy plasmid have increased L-serine transport capacity, insensitive to threonine, suggesting that sdaC might code for a recently described highly specific serine transporter {Kayahara, T., Thelen, P., Ogawa, W., Inaba, K., Tsuda, M., Goldberg, E . B . & Tsuchiya, T . (1992) J . Bacteriol . 174, 7482-7485}.

Eur J Biochem, 1994 Jun 15, 222(3), 851 - 9
ATP synthase of yeast mitochondria . Isolation of the F1 delta subunit, sequence and disruption of the structural gene; Giraud MF et al.; The delta-subunit was isolated from the purified yeast F1 . Partial protein sequences were determined by direct methods . From this information, degenerated primers were constructed . A part of the ATP delta gene was amplified by polymerase chain reaction from yeast genomic DNA . From the amplified DNA sequence, a nondegenerated oligonucleotide probe was constructed to isolate a 2.6-kbp BamHI-EcoRI DNA fragment bearing the whole gene . A 1036-bp DraI fragment was sequenced . A 480-bp open reading frame encoding a 160-amino-acid polypeptide is described . The deduced amino acid sequence is 22 amino acids longer than the mature protein, which is 138 amino acids long with a mass of 14,555 Da . The delta-subunit of Saccharomyces cerevisiae is 21%, 35%, 52% identical and 66%, 61% and 92% similar to the epsilon-subunit of Escherichia coli and the delta-subunits of beef heart and Neurospora crassa, respectively . A null mutant was constructed . The mutation was recessive and dramatically affected mitochondrial DNA stability since the transformed cells were 100% cytoplasmic petite . The double mutant (rho-, ATP delta::URA3) displayed low or no ATPase activity with an unstable catalytic sector, since a polyclonal antibody directed against the beta subunit did not coprecipitate the alpha subunit.

Eur J Biochem, 1994 Jun 15, 222(3), 775 - 80
Apparent cooperativity for carbamoylphosphate in Escherichia coli aspartate transcarbamoylase only reflects cooperativity for aspartate; England P et al.; The reaction catalyzed by Escherichia coli aspartate transcarbamoylase (ATCase) proceeds through an ordered mechanism, in which carbamoylphosphate binds first, followed by aspartate; upon binding of this second substrate, the enzyme undergoes a concerted transition from a low-affinity T state to a high-affinity R state . In various studies, conflicting results were obtained concerning the existence of positive cooperativity for the first substrate, carbamoylphosphate . It is shown here that cooperativity for this substrate is only apparent . Indeed, saturation curves for carbamoylphosphate display sigmoidicity only if the aspartate concentration used is high enough to shift ATCase into the R state . Furthermore, it is shown that succinate, an unreactive aspartate analogue which is able to promote the T-->R conformational transition, also induces the appearance of cooperativity for carbamoylphosphate . Similar results were obtained in the course of continuous-flow-dialysis experiments, which show that the binding of carbamoylphosphate is apparently cooperative only in the presence of a concentration of succinate high enough to shift the enzyme into the R state . Taken together, these data show that the apparent cooperativity for carbamoylphosphate is not an intrinsic property of ATCase, as it only reflects the cooperativity for the second substrate, aspartate, as a consequence of the process of ordered substrate binding.

Eur J Biochem, 1994 Jun 15, 222(3), 733 - 41
Cross-reconstitution studies with polypeptides of Escherichia coli and bovine heart mitochondrial F0F1 ATP synthase; Zanotti F et al.; To characterize the role of supernumerary subunits of the mammalian F0F1 ATP synthase, cross-reconstitution of mitochondrial and bacterial F0F1 complexes has been carried out . Escherichia coli F1 (EcF1) can be reconstituted with F1-stripped everted membranes of E . coli (UPEc) and of bovine heart mitochondria (USMP) . Bovine heart mitochondrial F1 (BHF1) can also be reconstituted with both membranes . Both EcF1 and BHF1, when reconstituted with UPEc, exhibited oligomycin-insensitive ATP-hydrolase activity . Subunits of the mammalian F0, in particular F0I-PVP protein, F6 and oligomycin-sensitivity-conferring protein (OSCP) conferred oligomycin sensitivity to the catalytic activity of EcF1 or BHF1 reconstituted with UPEc . Reaction of N,N'-dicyclohexylcarbodiimide and development of inhibition of passive H+ conduction was, in UPEc, considerably slower and exhibited a lower apparent affinity than in USMP . The ATP hydrolase activity of UPEc+EcF1 or UPEc+BHF1 was, also, less sensitive to inhibition by N,N'-dicyclohexylcarbodiimide than USMP+EcF1 or USMP+BHF1 . Addition of mitochondrial F0I-PVP to UPEc enhanced the sensitivity of H+ conduction to oligomycin . F0I-PVP and OSCP added to UPEc, promoted inhibition by N,N'-dicyclohexylcarbodiimide of passive H+ conduction and increased its binding affinity to subunit c of E . coli F0 . The presence of F0I-PVP and OSCP also promoted inhibition by N,N'-dicyclohexylcarbodiimide of the ATP-hydrolase activity of EcF1 or BHF1 reconstituted with UPEc.

EMBO J, 1994 Jun 15, 13(12), 2842 - 8
zeta PKC induces phosphorylation and inactivation of I kappa B-alpha in vitro; Diaz-Meco MT et al.; The zeta isotype of protein kinase C (zeta PKC), a distinct PKC unable to bind phorbol esters, is required during NF-kappa B activation as well as in mitogenic signalling in Xenopus oocytes and mammalian cells . To investigate the mechanism(s) for control of cellular functions by zeta PKC, this enzyme was expressed in Escherichia coli as a fusion protein with maltose binding protein (MBP), to allow immobilization on amylose beads to study signalling proteins in cell extracts that might form complex(es) with zeta PKC . The following evidence for interaction with the NF-kappa B/I kappa B pathway was obtained . MBP-zeta PKC, but not MBP, bound and activated a potentially novel I kappa B kinase of approximately 50 kDa molecular weight able to regulate I kappa B-alpha function . Activation of the I kappa B kinase was dependent on zeta PKC enzymatic activity and ATP, suggesting that zeta PKC controls, directly or indirectly, the activity of a functionally significant I kappa B kinase . Importantly, zeta PKC immunoprecipitates from TNF-alpha-stimulated NIH-3T3 fibroblasts displayed a higher I kappa B phosphorylating activity than untreated controls, indicating the in vivo relevance of these findings . We also show here that zeta PKC associates with and activates MKK-MAPK in vitro, suggesting that one of the mechanisms whereby overexpression of zeta PKC leads to deregulation of cell growth may be accounted for at least in part by activation of the MKK-MAPK complex . However, neither MKK nor MAPK is responsible for the putative I kappa B phosphorylating activity . These data provide a decisive step towards understanding the functions of zeta PKC.

EMBO J, 1994 Jun 15, 13(12), 2764 - 76
Chi sites in combination with RecA protein increase the survival of linear DNA in Escherichia coli by inactivating exoV activity of RecBCD nuclease; Kuzminov A et al.; In Escherichia coli, unprotected linear DNA is degraded by exoV activity of the RecBCD nuclease, a protein that plays a central role in the repair of double-strand breaks . Specific short asymmetric sequences, called chi sites, are hotspots for RecBCD-promoted recombination and are shown in vitro to attenuate exoV activity . To study RecBCD-chi site interactions in vivo we used phage lambda's terminase to introduce a site-specific double-strand break at lambda's cos site inserted into a plasmid . We show that after terminase has cut cos in vivo, nucleases degrade linearized DNA only from the end that does not have a strong terminase binding site . Linearized cosmid DNA containing chi sites in the proper orientation to the unprotected end is degraded more slowly in rec+ E . coli than is chi-less DNA . Increased survival of chi-containing DNA is a result of partial inactivation of exoV activity and is dependent on RecA and SSB proteins . The linearization of chi-containing DNA molecules leads to RecA-dependent formation of branched structures which have been proposed as intermediates in the RecBCD pathway of double-strand break repair.

Biochem J, 1994 Jun 15, 300 ( Pt 3), 765 - 70
The effect of replacing the conserved active-site residues His-264, Asp-312 and Arg-314 on the binding and catalytic properties of Escherichia coli citrate synthase; Man WJ et al.; The first step in the overall catalytic mechanism of citrate synthase is the binding and polarization of oxaloacetate . Active-site residues Arg-314, Asp-312 and His-264 in Escherichia coli citrate synthase, which are involved in oxaloacetate binding, were converted by site-directed mutagenesis to Gln-314, Asn-312 and Asn-264 respectively . The R314Q and D312N mutants expressed negligible overall catalytic activity at pH 8.0, the normal assay pH, but substantial activities for the partial reactions that reflect the cleavage and hydrolysis of the substrate intermediate citryl-CoA . However, when the pH was lowered to 7.0, the overall reaction of the mutants became significant, in contrast to the wild-type enzyme, whereas the two mutants exhibited reduced activities for the partial reactions . This result is consistent with the existence of a rate-limiting step between the two partial reactions for these mutants that is pH-dependent . The Km for oxaloacetate for the two mutants was increased 10-fold and was paralleled by an increase in the Km for citryl-CoA, whereas the Km for acetyl-CoA was increased only 2-fold . Overall, there was a striking parallel between the results obtained for these two mutants, which suggests that they are functionally linked in the E . coli enzyme . The equivalent of these two residues form a salt bridge in the pig heart citrate synthase crystal structure . The H264N mutant, in which the amide nitrogen of asparagine should mimic the delta-nitrogen of histidine, showed negligible activity in terms of both overall and partial catalysis, which may result from a hindrance of conformational change upon oxaloacetate binding . The affinity of this mutant for oxaloacetate appeared to be greatly reduced when investigated using indirect fluorescence and chemical modification techniques.

Biochem J, 1994 Jun 15, 300 ( Pt 3), 757 - 63
Interactions between the Escherichia coli MelR transcription activator protein and operator sequences at the melAB promoter; Williams J et al.; The Escherichia coli MelR protein binds to two sites at the melAB promoter and activates transcription in response to melibiose . The binding of purified MelR to the melAB promoter and to a number of derivatives carrying point mutations has been studied . A melAB promoter fragment that is very weakly activated by the melR gene product has been made by creating multiple symmetrical changes in both MelR-binding sites . A library of random substitutions in MelR was screened to find mutants that were able to activate this mutant promoter . One of these mutants contained a substitution in a proposed helix-turn-helix region that most likely relaxes the sequence-specificity of MelR binding . The other mutants contain substitutions throughout the central part of MelR and appear to alter the conformation of MelR, such that activity is triggered at lower melibiose concentrations and by arabinose.

Biochem J, 1994 Jun 15, 300 ( Pt 3), 717 - 21
Refolding of denatured lactate dehydrogenase by Escherichia coli ribosomes; Chattopadhyay S et al.; Escherichia coli ribosomes were used to refold denatured lactate dehydrogenase from porcine muscle . This activity of ribosomes, unlike most of the chaperons, did not require the presence of ATP . The molar concentration of ribosomes required for this refolding was comparable with that of the enzyme . Restoration of the enzyme activity was demonstrated using assays for both the forward and backward reactions . Binding of the denatured enzyme to ribosomes and its refolding were fairly rapid processes as revealed by the time course of the reaction and inhibition of folding when the denatured enzyme was allowed to refold spontaneously for short times before the addition of ribosomes . This protein-folding activity was detected in 70 S ribosomes as well as its RNA, in 50 S particles and in 23 S rRNA . However, 30 S particles failed to refold the enzyme.

Biochem Biophys Res Commun, 1994 Jun 15, 201(2), 924 - 31
Cytokines induce nitric oxide production in mouse osteoblasts; Damoulis PD et al.; MC3T3-E1 mouse clonal osteogenic cells were incubated with interferon-gamma, interleukin-1 beta, tumor necrosis factor-alpha, and E . coli lipopolysaccharide . TNF alpha, IL-1 beta, and LPS caused a dose- and time-dependent increase of nitrite (NO2-), the stable metabolite of nitric oxide (NO), in conditioned media over 48 hours, while IFN gamma had a minimal effect . Different combinations of the same factors caused a synergistic enhancement of NO2- accumulation, except for IL-1 beta with LPS . The earliest detectable NO2- production was at 6-9 hours, with continued accumulation over 48 hours . NO2- production was inhibited dose-dependently by three arginine analogs known to be specific inhibitors of NO synthase, as well as by actinomycin D, cycloheximide, and dexamethasone; EGTA or indomethacin had a small inhibitory effect . It is concluded that osteoblast-like cells can be induced by proinflammatory cytokines and bacterial endotoxin to produce NO, which can play an important role in bone pathophysiology.

Biochem Biophys Res Commun, 1994 Jun 15, 201(2), 917 - 23
Zinc content of promatrilysin, matrilysin and the stromelysin catalytic domain; Soler D et al.; Promatrilysin expressed in Escherichia coli and Chinese hamster ovary cells contains 2.36 +/- 0.19 and 2.13 +/- 0.39 moles of zinc per mole of protein, respectively, while the activated enzyme contains 2.22 +/- 0.21 . The catalytic domain of stromelysin-1 expressed in E . coli contains 2.22 +/- 0.11 . Thus these matrix metalloproteinases contain two metal binding sites at which zinc is bound firmly and possibly a third site at which it is bound weakly . Promatrilysin and matrilysin do not contain significant amounts of Fe, Cu, Mn, or Ni . All known matrix metalloproteinases have a sequence homologous to the zinc binding site of astacin, HExxHxxGxxH, suggesting that one of the zinc sites is catalytic in agreement with the known inhibition of these enzymes by chelators.

Biochem Biophys Res Commun, 1994 Jun 15, 201(2), 888 - 93
Delivery of plasmid DNA to glial cells using pH-sensitive immunoliposomes; Holmberg EG et al.; Immunoliposomes were constructed with an antibody specific to glial cells . They were used to examine the specificity and efficacy of cell type plasmid transfection . Liposomes contained a beta-galactosidase gene under control of an SV-40 promotor . Two different monoclonal antibodies of a different subclass, IgM and IgG, were examined for their targeting ability using immunoliposomes . Cultured C6 glioma (specific target cell type) and NIH 3T3 (control cell type, fibroblast) cells were transfected using these immunoliposomes . Results indicate a three-fold increase in transfection by the glial specific immunoliposomes, "gliasomes", in glial cell culture over control liposomes . Gliasomes were exposed to NIH 3T3 cells and showed no enhanced transfection over control liposomes . Gliasomes were tested for their specificity by the addition of excess antibody to the cell culture in order to saturate specific receptors on C6 glioma cells . Results indicate a reduced transfection, nearly three-fold, in cells that were saturated with excess antibody prior to exposure to the immunoliposomes.

Biochem Biophys Res Commun, 1994 Jun 15, 201(2), 701 - 8
Genetic tests to reveal TAT homodimer formation and select TAT homodimer inhibitor; Battaglia PA et al.; The HIV Tat protein is essential for productive infection and is a potent activator of viral gene expression . By constructing a genetic fusion between the amino-terminal DNA-binding domain of the lambda repressor (as a reporter for dimerization) and Tat, we show that Tat forms dimers in vivo . By deletion analysis and site-directed mutagenesis, we show that (i) the peptide encoded by exon-1 of Tat is sufficient to promote dimerization and (ii) cys37 is essential for homo-dimerization of Tat protein . Furthermore, by using a new E . coli strain in which the expression of beta-galactosidase is under the negative control of the cl::Tat repressor, we select a protein (CD10/Nep) expressed by human Jurkat T-cells which inhibits Tat dimerization.

Biochem Biophys Res Commun, 1994 Jun 15, 201(2), 635 - 41
TFPACD, a novel bifunctional reagent for reacting with DCCD sites in proteins: studies using Escherichia coli ATP synthase; Phadke AS et al.; A novel cross-linker, 1-{6-(4-azido-2,3,5,6-tetrafluorobenzamido)hexyl}-3-cyclohexylc arbodiimide (TFPACD), has been synthesized and tested by reaction with the Escherichia coli ATP Synthase (ECF1F0) . The reagent has a carbodiimide as one reactive group, which is shown to react with ECF1F0 in a similar way to 1,3-dicyclohexylcarbodiimide (DCCD) and modify the beta subunit of the ECF1 part and the c subunit of the F0 part . Reaction with both the ECF1 and F0 parts of the complex inhibited ATPase activity . The second reactive group in the reagent is the photoactivatable tetrafluorophenylazide moiety . Subsequent UV photolysis of TFPACD--modified ECF1 and ECF1F0 led to generation of cross-linked products in significant yields, one between beta and alpha subunits; the second, dimers of the c subunit of the F0 part.

Biochem Biophys Res Commun, 1994 Jun 15, 201(2), 1021 - 8
cDNA cloning and expression of Cry j II the second major allergen of Japanese cedar pollen; Komiyama N et al.; We have isolated and sequenced a cDNA clone coding for Cry j II, the second major allergen of Japanese cedar (Cryptomeria japonica) pollen . A 1.7-kilobase cDNA clone contained an open reading frame coding for a 514-amino acid protein, including a putative signal peptide of 54 amino acid and three potential glycosylation sites . The deduced amino acid sequence of the Cry j II shows significant identities to those of the polygalacturonases associated with fruit-ripening in tomato (40%) and avocado (43%) and found in pollen of maize (34%) . Cry j II cDNA product expressed in E . coli was recognized by human IgE from patients suffering from cedar pollinosis, suggesting that recombinant Cry j II might be used as an allergen for the clinical diagnosis of cedar pollinosis.

FEMS Microbiol Lett, 1994 Jun 15, 119(3), 309 - 14
Sequences related to the major subunit gene fedA of F107 fimbriae in porcine Escherichia coli strains that express adhesive fimbriae; Imberechts H et al.; Porcine Escherichia coli strains isolated from cases of postweaning diarrhea or edema disease were analysed for the presence of fedA, the major subunit gene of F107 fimbriae . The E . coli isolates were known to contain colonisation factor '8813', or to express F107, 2134P or other fimbriae, different from F4, F5, F6, and F41 . PCR with fedA-specific primers, restriction enzyme digestion of the PCR product, and nucleotide sequence analysis demonstrated that 2134P pili, colonisation factor '8813' and fimbriae identified on Australian strains of the O141 serotype belong to one family of F107 fimbrial antigens.

Biochemistry, 1994 Jun 14, 33(23), 7485 - 94
Crosstalk between domains in the regulatory subunit of cAMP-dependent protein kinase: influence of amino terminus on cAMP binding and holoenzyme formation; Herberg FW et al.; The regulatory (R) subunit of cAMP-dependent protein kinase os an asymmetric multidomain protein with a dimerization domain at the N-terminus, an autoinhibitors site, and two cAMP binding domains at the C-terminus . Activation of the tetrameric holoenzyme is mediated by the cooperative binding of cAMP to the two cAMP binding sites . To better understand how the various domains influence each other, the N-terminus (delta 1-91) up to the autoinhibitor site was deleted . Not only did this monomeric deletion mutant, purified from Escherichia coli, still bind cAMP and the catalytic (C) subunit with high affinity, holoenzyme formation was actually accelerated by at least 50-fold . MgATP also was not required for rapid reassociation of (delta 1-91)R(cAMP)2 and C . The Kd(cAMP) and the Ka(cAMP) were similar to those for holoenzyme formed with full-length R; however, cooperatively was lost . Thus the N-terminus, either by inter- or intraprotomer contacts, not only impedes holoenzyme formation but also influences the cooperative binding of cAMP . The 1-91 deletion also renders the remaining fragment resistant to proteolytic degradation . Finally, unlike full-length R, the mutant protein can migrate freely into the nucleus . Surface plasmon resonance studies for the first time enabled direct measurements of the association and dissociation rate constants both for the intact R and for (delta 1-91)R . Both displayed very fast on-rates (1 x 10(-5) M-1 s-1 and 1.1 x 10(-5) M-1 s-1, respectively) and extremely slow off-rates (2.3 x 10(5) M-1 and 4.3 x 10(5) M-1, respectively) . Thus, unlike the heat-stable protein kinase inhibitor, the region preceding the autoinhibitor site in R does not contribute in a quantitatively significant way to the high-affinity binding of C.

Biochemistry, 1994 Jun 14, 33(23), 7470 - 6
Receptor-binding affinities of human epidermal growth factor variants having unnatural amino acid residues in position 23; Koide H et al.; Ile-23 of human epidermal growth factor (hEGF) has been indicated, by mutagenesis and NMR studies, to be directly recognized by the receptor . In the present study, an unnatural phenylalanine analog, either 2-azaphenylalanine (2aF), 3-azaphenylalanine (3aF), 4-azaphenylalanine (4aF), or 4-fluorophenylalanine (4fF), was incorporated by an in vivo protein synthesis system into position 23 of {Phe23}hEGF, which retains appreciable receptor-binding affinity (about 20% of the wild type) . We compared the receptor-binding affinities of the variants with that of {Phe23}hEGF and found that substitution of Phe-23 with 2aF or 3aF raised the affinity, while substitution with 4aF or 4fF remarkably reduced the affinity . The tertiary structure of {Phe23}hEGF was not significantly affected by the substitution of Phe-23 with 2aF, as shown by the two-dimensional nuclear magnetic resonance analysis . In addition, the substitution of residue 23 with His or Tyr produced an hEGF variant with a slightly higher receptor-binding affinity than that of {Phe23}hEGF . Our results suggest that the receptor has an asymmetric hydrophobic pocket for recognition of the side chain in position 23 of hEGF . Furthermore, on the receptor surface, this pocket seems to be adjacent to a less hydrophobic region with a hydrogen-bond acceptor and donor . Thus, the use of unnatural amino acids in addition to the 20 natural ones allows analyses of the structure-function relationship of a protein at a higher resolution than conventional site-directed substitution by only natural amino acid residues.

Biochemistry, 1994 Jun 14, 33(23), 7398 - 407
Characterization of recombinant horseradish peroxidase C and three site-directed mutants, F41V, F41W, and R38K, by resonance Raman spectroscopy; Smulevich G et al.; Resonance Raman spectra are reported for recombinant horseradish peroxidase C (HRP-C*) and three protein variants prepared by in vitro refolding after Escherichia coli expression . The spectra of their FeII and FeIII forms and of their complexes with benzohydroxamic acid (BHA) were recorded at neutral pH . The residues mutated were on the distal {Phe41-->Trp or Val (F41W, F41V) and Arg38-->Lys (R38K)} side of the heme . The spectra give information on the spin and ligation states via the frequencies of the core size marker bands . No detectable modification in the enzyme structure or in the heme group has been observed in the wild-type recombinant HRP-C* . The FeIII forms of both the recombinant and the plant proteins show the coexistence of a 5-(5-cHS) and a 6-coordinate high-spin (6-cHS) heme, characterized by the anomalous frequencies of certain bands, namely, v3 and v10, which we attribute to a different degree of distortion of the heme planarity with respect to other heme proteins and model compounds, resulting from external forces such as steric contacts within the protein . This effect is partially relieved upon complexation with BHA or as a result of mutation . F41W and F41V are characterized by an increase in a 6-cHS form at the expense of the 5-cHS species, and the R38K by an increase in both the 6-c high-(HS) and low-spin (LS) hemes . The 6-cHS and -LS species are characterized by normal core size marker band frequencies . The FeII-His RR band is at 243 cm-1 in HRP-C*, the high frequency value being due to hydrogen-bonding interactions between the proximal His170 N delta and the carboxylate acceptor group on Asp247 . Mutation at position 38 causes a downshift of 3 cm-1 in the v(Fe-Im) stretching mode, suggesting a weakening of the Fe-Im bond strength . By comparing the results obtained with HRP-C* mutants with those previously reported for the corresponding cytochrome c peroxidase (CCP) mutants, it appears that the distal heme pocket architecture is significantly different in the two peroxidases, although the hydrogen-bonding network coupling the distal and the proximal sides of the heme appears to be conserved . Mutations on the distal side dramatically affect the capability of the protein to bind BHA . F41W and R38K mutants do not bind the substrate, whereas the F41V variant shows a 2-fold increase in affinity.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemistry, 1994 Jun 14, 33(23), 7361 - 7
Subdomain folding of the coiled coil leucine zipper from the bZIP transcriptional activator GCN4; Lumb KJ et al.; One popular model for protein folding, the framework model, postulates initial formation of secondary structure elements, which then assemble into the native conformation . However, short peptides that correspond to secondary structure elements in proteins are often only marginally stable in isolation . A 33-residue peptide (GCN4-p1) corresponding to the GCN4 leucine zipper folds as a parallel, two-stranded coiled coil {O'Shea, E.K., Klemm, J.D., Kim, P.S., & Alber, T.A . (1991) Science 254, 539-544} . Deletion of the first residue (Arg 1) results in local, N-terminal unfolding of the coiled coil, suggesting that a stable subdomain of GCN4-p1 can form . N- and C-terminal deletion studies result in a 23-residue peptide, corresponding to residues 8-30 of GCN4-p1, that folds as a parallel, two-stranded coil with substantial stability (the melting temperature of a 1 mM solution is 43 degrees C at pH 7) . In contrast, a closely related 23-residue peptide (residues 11-33 of GCN4-p1) is predominantly unfolded, even at 0 degrees C, as observed previously for many isolated peptides of similar length . Thus, specific tertiary packing interactions between two short units of secondary structure can be energetically more important in stabilizing folded structure than secondary structure propensities . These results provide strong support for the notion that stable, cooperatively folded subdomains are the important determinants of protein folding.

Biochemistry, 1994 Jun 14, 33(23), 7354 - 60
Kinetics of biotinyl-5'-adenylate synthesis catalyzed by the Escherichia coli repressor of biotin biosynthesis and the stability of the enzyme-product complex; Xu Y et al.; The Escherichia coli repressor of biotin biosynthesis is both a biotin ligase and the repressor of transcriptional initiation at the biotin biosynthetic operon . The small molecule, biotinyl-5'-adenylate (bio-5'-AMP), is the intermediate in the biotin ligation reaction and the positive allosteric effector for sequence-specific DNA binding by BirA . Synthesis of the adenylate from the substrates biotin and ATP is catalyzed by BirA . Although BirA and other biotin holoenzyme synthetases have been the subject of biochemical studies, no direct measurements of the bio-5'-AMP synthesis reaction have been reported . No information relating to the mechanism and kinetic parameters governing adenylate synthesis is available . In addition to this lack of kinetic information, the thermodynamic stability of the BirA-bio-5'-AMP complex is not known . Since the BirA-adenylate complex plays a pivotal role in the biotin regulatory system, both the kinetic and thermodynamic information are essential to a quantitative understanding of the system . We have developed a method for measuring the time course of bio-5'-AMP synthesis . The results of these measurements indicate that the time course is characterized by an initial burst followed by a slow linear phase . The burst corresponds to the rapid synthesis of 1 mol of product per mole of enzyme, and the rate of the slow linear phase is limited by the release of product from the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Jun 14, 33(23), 7309 - 14
The energetic linkage of GTP hydrolysis and the synthesis of activated sulfate; Liu C et al.; ATP sulfurylase, from Escherichia coli K-12, catalyzes both the hydrolysis of GTP and the synthesis of activated sulfate (APS) . This paper describes the energetic linkage of these reactions and the events that couple them . Steady-state and single-turnover experiments suggest that the binding of GTP inhibits APS production and that the hydrolysis of GTP is required to generate the enzyme form(s) that produces APS . It is this progression from the inhibitory, E-GTP, to the productive, E-GDP, complexes in the cycle of APS synthesis that energetically links these two reactions . This model stands in contrast to other GTPase/target systems in which the binding of GTP alone is sufficient to catalyze multiple turnovers of the target reaction . The stoichiometry of GTP hydrolysis to APS synthesis is 1:1, and equilibrium measurements show that -9.1 kcal/mol, produced by the hydrolysis of GTP, is used to thermodynamically drive production of APS and PPi . These findings establish the mechanism of energy transfer in this novel GTPase/target system, and substantially alter our understanding of the energetics of sulfate activation, an essential step in the metabolic assimilation of sulfur.

Biochemistry, 1994 Jun 14, 33(23), 7288 - 93
Properties of the arsenate reductase of plasmid R773; Gladysheva TB et al.; Resistance to toxic oxyanions in Escherichia coli is conferred by the ars operon carried on plasmid R773 . The gene products of this operon catalyze extrusion of antimonials and arsenicals from cells of E . coli, thus providing resistance to those toxic oxyanions . In addition, resistance to arsenate is conferred by the product of the arsC gene . In this report, purified ArsC protein was shown to catalyze reduction of arsenate to arsenite . The enzymatic activity of the ArsC protein required glutaredoxin as a source of reducing equivalents . Other reductants, including glutathione and thioredoxin, were not effective electron donors . A spectrophotometric assay was devised in which arsenate reduction was coupled to NADPH oxidation . The results obtained with the coupled assay corresponded to those found by direct reduction of radioactive arsenate to arsenite . The only substrate of the reaction was arsenate (Km = 8 mM); other oxyanions including phosphate, sulfate, and antimonate were not reduced . Phosphate and sulfate were weak inhibitors, while the product, arsenite, was a stronger inhibitor (Ki = 0.1 mM) . Arsenate reductase activity exhibited a pH optimum of 6.3-6.8 . These results indicate that the ArsC protein is a novel reductase, and elucidation of its enzymatic mechanism should be of interest.

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