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| Mol Microbiol, 1995 Jul, 17(2), 211 - 20 Comparison of ccd of F, parDE of RP4, and parD of R1 using a novel conditional replication control system of plasmid R1; Jensen RB et al.; A number of plasmid-encoded gene systems are thought to stabilize plasmids by killing plasmid-free cells (also termed post-segregational killing or plasmid addiction) . Here we analyse the mechanisms of plasmid stabilization by ccd of F, parDE of RP4 and parD of R1, and compare them to hok/sok of R1 . To induce synchronous plasmid loss we constructed a novel plasmid replication-arrest system, which possesses the advantage that plasmid replication can be completely arrested by the addition of IPTG, a non-metabolizable inducer . Using isogenic plasmid constructions we have found, for the first time, consistent correlation between the effect on steady-state loss rates and the effect on cell proliferation in the plasmid replication-arrest assay for all three systems . The parDE system had the most pronounced effect both on plasmid stabilization and on plasmid retention after replication arrest . In contrast, ccd and parD both exhibited weaker effects than anticipated from previously published results . Thus, our results indicate that the function and efficiencies of some of the systems should be reconsidered . Our results are consistent with the previously postulated hypothesis that ccd and parDE act by killing plasmid-free segregants, whereas parD seems to act by inhibiting cell division of plasmid-free segregants. Antimicrob Agents Chemother, 1995 Jul, 39(7), 1624 - 8 Analysis of mutations at position 184 in reverse transcriptase of human immunodeficiency virus type 1; Boyer PL et al.; We have analyzed recombinant human immunodeficiency virus type 1 reverse transcriptases that contain mutations at position 184 . These variants retain high levels of RNA-dependent DNA polymerase activity and show resistance to ddITP . However, the mutants varied in their ability to polymerize processively . The variants Met184Ile and Met184Val showed slight reductions in processivity relative to that of the wild-type enzyme; the variants Met184Ala and Met184Leu showed considerable reductions in their processivity. RNA, 1995 Jul, 1(5), 501 - 9 The conformation of 23S rRNA nucleotide A2058 determines its recognition by the ErmE methyltransferase; Vester B et al.; The ErmE methyltransferase confers resistance to MLS antibiotics by specifically dimethylating adenine 2058 (A2058, Escherichia coli numbering) in bacterial 23S rRNA . To define nucleotides in the rRNA that are part of the motif recognized by ErmE, we investigated both in vivo and in vitro the effects of mutations around position A2058 on methylation . Mutagenizing A2058 (to G or U) completely abolishes methylation of 23S rRNA by ErmE . No methylation occurred at other sites in the rRNA, demonstrating the fidelity of ErmE for A2058 . Breaking the neighboring G2057-C2611 Watson-Crick base pair by introducing either an A2057 or a U2611 mutation, greatly reduces the rate of methylation at A2058 . Methylation remains impaired after these mutations have been combined to create a new A2057-U2611 Watson-Crick base interaction . The conformation of this region in 23S rRNA was probed with chemical reagents and it was shown that the A2057 and U2611 mutations alone and in combination alter the reactivity of A2058 and adjacent bases . However, mutagenizing position G-->A2032 in an adjacent loop, which has been implicated to interact with A2058, alters neither the ErmE methylation at A2058 nor the accessibility of this region to the chemical reagents . The data indicate that a less-exposed conformation at A2058 leads to reduction in methylation by ErmE . Nucleotide G2057 and its interaction with C2611 maintain the conformation at A2058, and are thus important in forming the structural motif that is recognized by the ErmE methyltransferase. RNA, 1995 Jul, 1(5), 478 - 90 Reverse splicing of the Tetrahymena IVS: evidence for multiple reaction sites in the 23S rRNA; Roman J et al.; Group I introns in rRNA genes are clustered in highly conserved regions that include tRNA and mRNA binding sites . This pattern is consistent with insertion of group I introns by direct interaction with exposed regions of rRNA . Integration of the Tetrahymena group I intron (or intervening sequence, IVS) into large subunit rRNA via reverse splicing was investigated using E . coli 23S rRNA as a model substrate . The results show that sequences homologous to the splice junction in Tetrahymena are the preferred site of integration, but that many other sequences in the 23S rRNA provide secondary targets . Like the original splice junction, many new reaction sites are in regions of stable secondary structure . Reaction at the natural splice junction is observed in 50S subunits and to a lesser extent in 70S ribosomes . These results support the feasibility of intron transposition to new sites in rRNA genes via reverse splicing. Anat Embryol (Berl), 1995 Jul, 192(1), 77 - 87 Cell death in the olfactory epithelium; Magrassi L et al.; In the nervous system of vertebrates the olfactory epithelium presents unique cytological characteristics . In the olfactory mucosa, olfactory neurons die and are replaced from undifferentiated neuroblasts over the entire life span of the animal . It remains unclear whether these neurons die as a result of a direct insult from the environment or in fulfillment of a physiological program of cell death . We have studied the distribution and the characteristics of cell death in the olfactory epithelium of normal, adult rats . The olfactory epithelium contains pycnotic bodies resembling those described for thymocytes undergoing terminal apoptotic changes . These appear at all levels in the epithelium, under both light and electron microscopes and can also be demonstrated after vital staining with acridine orange . Chromatin condensation into large blocks, often located at the nuclear periphery, is a morphological hallmark of the nuclei of mature olfactory neurons, which also present an increase in electron density of the cytoplasm . After non-radioactive in situ labeling of fragmented DNA, the nuclei of olfactory neurons are positive . Under the same reaction conditions (mild protease digestion), most of the nuclei of the supporting and basal cells are negative . In vivo incorporation of 5-bromouridine, a marker of RNA synthesis, is also lower in olfactory neurons than in basal and supporting cells . These findings suggest that olfactory neurons are committed very early to physiological cell death. Virus Res, 1995 Jul, 37(2), 87 - 97 Adenovirus protease expressed in insect cells cleaves adenovirus proteins, ovalbumin and baculovirus protease in the absence of activating peptide; Keyvani-Amineh H et al.; The adenovirus type 2 protease (EP) was expressed by infecting insect cells with a recombinant baculovirus . Immunoblot and activity analysis showed EP to be present in both the nucleus and cytoplasm . While the insect cell expressed EP was more soluble than the Escherichia coli expressed EP, its activity was one quarter of the latter, suggesting that eukaryotic postsynthetic modifications are not essential for enzyme activity . EP inactivated a cytoplasmic cathepsin-like baculovirus-encoded cysteine protease which carries a single EP cleavage site and which was capable of digesting most adenovirus structural proteins in vitro . In addition to cleavage of the baculovirus protease, the adenovirus EP was also able to cleave ovalbumin and canine adenovirus protein pre-VII, in the absence of activating peptide . EP activation therefore may occur by means of factors other than the specific activating peptide. Plasmid, 1995 Jul, 34(1), 58 - 61 Analysis of a retron EC86 and EC67 insertion site in Escherichia coli; Lim D; The Escherichia coli clinical strain 49 contains the integration sites for the retrons EC86 and EC67 but not the retrons themselves . To compare the chromosomal structures before and after retron integration, the DNA sequence of the integration site in strain 49 was determined and compared to the corresponding sequences in strains containing EC86 and EC67 . The results suggested that when these retrons inserted into the E . coli chromosome they replaced a 3.5-kb fragment of chromosomal DNA . It is proposed that the replacement of preexisting DNA by a retron may be a general mechanism for retron integration, since in the three examples in which the integration sites are known, the retrons appear to have integrated into the chromosome by replacing a preexisting DNA segment. Plasmid, 1995 Jul, 34(1), 1 - 10 Ligation of nonmatching DNA molecule ends; Cimmino C et al.; T4 DNA ligase can promote the in vitro ligation of blunt DNA ends to ends bearing a 2-nucleotide single-stranded protrusion . This was shown by digestion of plasmids pBR322 and pSP71 with the appropriate restriction enzymes followed by recircularization of the plasmids and transformation of Escherichia coli . It could be ruled out that such nonmatching ligations are due to the presence of contaminating nucleases . The efficiency of ligation is of the same order of magnitude as that obtained with blunt end ligations . The interaction of a number of different combinations of blunt and sticky ends, the latter bearing both 3' and 5' protrusions, was investigated . Ligation of nonmatching ends was shown to take place in all cases . Several ligation junctions were sequenced, showing that during the ligation process the 2-nucleotide protrusion is trimmed away . In two instances the ligation event was accompanied by the specific loss of either 3 or 15 nucleotide pairs as well as the protrusion . An intermolecular ligation involving nonmatching ends was also performed, demonstrating that this form of ligation can be usefully employed in molecular cloning experiments. Proteins, 1995 Jul, 22(3), 293 - 7 Crystallization and preliminary X-ray analysis of the cytoplasmic domain of human erythrocyte band 3; Kiyatkin AB et al.; A cytoplasmic domain of the human erythrocyte membrane protein band 3 (M(r) = 42,500), residues 1-379, expressed in and purified from E . coli, has been crystallized by the method of vapor diffusion in sitting drops with subsequent streak-seeding at room temperature . Initial crystals were grown from solutions containing 65-68% saturated ammonium sulfate at pH 4.9 and 2 mg/ml protein . Subsequent streak-seeding into solutions of 50-53% ammonium sulfate at pH 4.9 and 7 mg/ml protein produced single crystals suitable for X-ray analysis, which contained pure protein as revealed by gel electrophoresis . The crystals belong to the monoclinic space group C2 with cell dimensions of a = 178.8 A, b = 90.5 A, c = 122.1 A, and beta = 131.3 degrees and diffract at least to 2.7 A resolution (at 100 K) . A self-rotation function shows the presence of approximate 222 local symmetry. Proteins, 1995 Jul, 22(3), 290 - 2 Crystallization and preliminary X-ray crystallographic studies of nonhistone region of macroH2A.1; Vijay-Kumar S et al.; Histone macroH2A has a novel hybrid structure consisting of a large nonhistone region and a region that closely resembles a full-length histone H2A . One key to understanding macroH2A function is determining the structure and function of its nonhistone region . The nonhistone region of one of the two known macroH2A subtypes was expressed in Escherichia coli and purified using affinity and molecular sieve chromatography . Crystals of the protein suitable for structural studies were grown from polyethylene glycol solutions by vapor equilibration techniques . The crystals belong to the hexagonal space group P6(4) (or its enantiomorph P6(2)) with unit cell parameters: a = b = 106.2 A, c = 125.9 A, alpha = beta = 90 degrees, and gamma = 120 degrees . There are four molecules in the asymmetric unit . Self-rotation function studies revealed three twofold noncrystallographic rotation axes related approximately by 222 symmetry . These crystals have 47% solvent content and diffract to 3.8 A resolution. Ontogenez, 1995 Jul-Aug, 26(4), 300 - 9 {The binding of exogenous DNA pRK31acZ by rabbit spermatozoa, its transfer to oocytes and expression in preimplantation embryos}; Kuznetsov AV et al.; It was shown that 35.3% ejaculated rabbit spermatozoa washed from seminal plasma are capable of interacting with heterogeneous DNA . The major part (85.2%) of bound DNA was located in the post-acrosomal part of the sperm head . After incubation with plasmid pRK31acZ the spermatozoa transferred it in the oocytes during in vivo fertilization, as shown by expression of reporter gene lacZ in 19.3% of preimplantation embryos . Additional treatment of the mobile spermatozoa with DMSO and heat shock raised the efficiency of exogenous DNA incorporation in the spermatozoa, as expressed in the percentage of embryos containing bacterial beta-galactosidase (61.7%) . It is proposed to use the capture of heterogeneous DNA by the spermatozoa and its transfer in the oocytes for production of transgenic animals and in studies of regulation of gene expression at the early stages of embryogenesis. Nucl Med Commun, 1995 Jul, 16(7), 615 - 20 In vitro evaluation of neutrophil viability after exposure to a hypotonic medium; Thorson LM et al.; Separation techniques for radiolabelled leukocytes have inherent problems with contaminants (e.g . platelets and erythrocytes) . Hypotonic lysis methods can eliminate the erythrocytes, but the question of neutrophil viability after an exposure to a hypotonic solution (i.e . sterile water) remains . Ficoll/ hypaque two-density gradient separation was performed on donor whole blood to obtain a pure neutrophil suspension . A timed sequence of water exposure was done for 5-100 s on the neutrophil preparations . The viability of these preparations was evaluated using flow cytometry and chemotaxis . The trypan blue staining method was used to document cell death . With water exposures ranging up to 100 s, 2.04 +/- 1.80% neutrophils exhibited cellular degradation by flow cytometry, and all samples demonstrated viable neutrophils by chemotaxis and trypan blue staining . The hypotonic medium exposure times for leukocyte separations should be less than 30 s for neutrophils to retain their viability by these in vitro techniques. Mol Biol (Mosk), 1995 Jul-Aug, 29(4), 826 - 31 {Cloning an extended palindromic sequence of Tetrahymena pyriformis ribosomal DNA}; Mukha DV et al.; Simple and effective method for isolation of native copies of ribosomal DNA (rDNA) of T . pyriformis by pulse field gel electrophoresis is suggested . Cloning of long palindrome sequence of rDNA from T . pyriformis in plasmid is described. Mol Microbiol, 1995 Jul, 17(1), 25 - 35 The role of cysteine residues in the transport of mercuric ions by the Tn501 MerT and MerP mercury-resistance proteins; Morby AP et al.; Each cysteine residue in the MerT and MerP polypeptides of bacterial transposon Tn501 was replaced by serine, and the mercury-resistance phenotypes of the mutants were determined in Escherichia coli . Cys-24 and Cys-25 in the first transmembrane region of MerT were essential for transport of mercuric ions through the cytoplasmic membrane, and mutations Cys-76-Ser, Cys-82-Ser or Gly-38-Asp in MerT or Cys-36-Ser in MerP all reduced transport and resistance . Deletion of the merP gene slightly reduced mercuric ion resistance and transport, whereas a Cys-33-Ser mutation in MerP appears to block transport of mercuric ions by MerT . The effects of deleting merP on mutations in merT were tested . The 116-amino-acid MerT protein is sufficient for mercuric ion transport across the cytoplasmic membrane. Brain Res Mol Brain Res, 1995 Jul, 31(1-2), 165 - 72 Tyrosine kinase phosphorylation of GABAA receptors; Valenzuela CF et al.; Phosphorylation of purified bovine brain GABAA receptors by the tyrosine kinase, pp60v-src was examined . pp60v-src phosphorylated two bands of 54-62 kDa and 48-51 kDa that migrated to approximately the same position as bands recognized by antisera against the beta 2 and gamma 2 GABAA receptor subunits, respectively . Bacterially expressed proteins containing the putative large cytoplasmic loops of the beta 1 and gamma 2L subunits were phosphorylated by pp60v-src, indicating that the phosphorylation sites are located in these subunit domains . The tyrosine kinase inhibitors, genistein and the tyrphostins B-42 and B-44, inhibited muscimol-stimulated 36Cl- uptake in mouse brain membrane vesicles (microsacs) . magnitude of the tyrphostin B-44-induced inhibition of muscimol-stimulated 36Cl- uptake was significantly reduced in microsacs that were lysed and resealed under conditions that inhibit phosphorylation . GABA-gated Cl- currents were also inhibited by genistein and tyrphostin B-44 in Xenopus oocytes expressing alpha 1 beta 1 and alpha 1 beta 1 gamma 2L subunits . Consequently, protein tyrosine kinase-dependent phosphorylation appears to be another mechanism of regulating the function of GABAA receptors. J Biol Chem, 1995 Jun 30, 270(26), 15908 - 14 Maturation of pre-tRNA(fMet) by Escherichia coli RNase P is specified by a guanosine of the 5'-flanking sequence; Meinnel T et al.; The C+1/A+72 base pair at the top of the acceptor stem of Escherichia coli tRNA(fMet) accounts for several of the specialized roles of this tRNA in translation initiation . According to the rules of RNA substrate recognition by RNase P, the C+1/A+72 pair is likely to disfavor the 5'-maturation of pre-tRNA(fMet) . Indeed, in contrast to other E . coli tRNA species, tRNA(fMet) was not properly matured when overproduced from a multicopy expression vector . Half of the recovered tRNA(fMet) retained an extension at the 5' side . Such a defect of tRNA(fMet) processing could be cured by changing bases C+1 and A+72 by a Watson-Crick base pair or by non-paired bases, provided one of them was a G . It could also be compensated by either (i) over-expression of RNase P or (ii) introduction within the plasmid of one out of the three 5'-flanking sequences naturally occurring in the four E . coli tRNA(fMet) genes . The effect of these flanking sequences on the maturation of tRNA(fMet) could be accounted for by the presence of a G located 2 bases upstream from C+1 . Notably, this G is the only residue that is conserved in the 5'-flanking sequences of all four E . coli tRNA(fMet) genes. J Biol Chem, 1995 Jun 30, 270(26), 15762 - 9 Identification of key charged residues of human interleukin-5 in receptor binding and cellular activation; Graber P et al.; Interleukin-5 (IL-5) is a cytokine that plays a major role in the differentiation and activation of eosinophils . In order to identify which charged residues of human IL-5 are important in binding to its receptor and subsequent cellular activation, we have systematically replaced all of the clusters of charged amino acids with alanine residues . The mutants have been expressed in Escherichia coli, renatured, and purified . They were assayed for ability to cause proliferation of the erythroleukaemic cell line TF-1 and the up-regulation of eosinophil adhesion to ICAM-1 . In addition, we studied receptor binding using either immobilized recombinant IL-5 receptor alpha-chain or the alpha/beta-receptor complex expressed on TF-1 cells . The key charged residue involved in binding to the beta-chain of the receptor is Glu-12 . This residue is in an identical position to those previously identified in IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) involved in binding to the receptor beta-chain . The alpha-chain binding site is shown to involve the side chains Arg-90 and Glu-109, located in the second beta sheet and after the end of the fourth helix, respectively . It is unique to IL-5 and does not occur in IL-3 or GM-CSF . Understanding the topology of the interaction of IL-5 with its receptor chains will help in the search for rationally designed antagonists of IL-5 function. J Biol Chem, 1995 Jun 30, 270(26), 15620 - 7 Domain closure in the catalytic chains of Escherichia coli aspartate transcarbamoylase influences the kinetic mechanism; Lee BH et al.; The closure of the two domains of the catalytic chains of Escherichia coli aspartate transcarbamoylase, which is critical for completion of the T-->R transition, is stabilized by salt-bridges between Glu-50 and both Arg-167 and Arg-234 . Mutation of Glu-50 to Ala shifts the enzyme toward a low activity, low affinity state (Newton, C . J., and Kantrowitz, E . R . (1990) Biochemistry, 29, 1444-1451) . Kinetic isotope effects (KIE) and equilibrium isotope exchange kinetics (EIEK) have been used to probe the dynamic properties of the Glu-50-->Ala enzyme . Unlike the behavior of the wild-type enzyme, the observed kinetic isotope effect for 13C versus 12C at the carbonyl group of carbamoyl phosphate (CP) increased upon the binding of ligands which promote the formation of the R-state (Asp, N-phosphonacetyl-L-aspartate (PALA), or ATP) . The maximum rate for the {14C}Asp<-->Carbamoyl aspartate (CAsp) exchange with the Glu-50-->Ala enzyme was 500-fold slower than for the wild-type enzyme; however, the rate for the {14C}CP<-->CAsp exchange was only 50-fold slower, reversing the relative rates observed with the wild-type enzyme . In addition, upon variation of substrate pairs involving Asp or CAsp, loss of inhibition effects in the CP<-->CAsp exchange indicated that the Glu-50-->Ala substitution caused the kinetic mechanism for the mutant enzyme to shift from ordered to random . Computer simulations of the EIEK data indicate that the Glu-50-->Ala mutation specifically causes strong decreases in the rates of catalysis and association-dissociation for Asp and CAsp, with minimal effects on the CP and Pi on-off rates . With substrates bound, the Glu-50-->Ala enzyme apparently does not attain a full R-state conformation . The PALA-activated Glu-50-->Ala enzyme, however, exhibits substrate affinities comparable to those for the wild-type enzyme, but fails to restore the preferred order substrate binding . Unlike the wild-type enzyme, both the T and R-states of the Glu-50-->Ala enzyme contribute to catalysis . A third state, I, is proposed for the Glu-50-->Ala enzyme, in which random order substrate binding is exhibited, and the catalytic step contributes significantly to overall rate limitation. J Biol Chem, 1995 Jun 30, 270(26), 15545 - 50 Spectroscopic studies of the characterization of recombinant human dihydrolipoamide dehydrogenase and its site-directed mutants; Liu TC et al.; In this paper, we report the overexpression and single-step purification of recombinant wild-type and site-directed mutants of human dihydrolipoamide dehydrogenase in Escherichia coli and detailed spectroscopic studies aimed at understanding the catalytic mechanism of this enzyme . One mutation (K37E) has been identified in a patient lacking dihydrolipoamide dehydrogenase activity and has been reported previously (Liu, T.-C., Kim, H., Arizmendi, C., Kitano, A., and Patel, M . S . (1993) Proc . Natl . Acad . Sci . USA . 90, 5186-5190), while the other two mutations were previously generated specifically to address the role of the active-site base (His-452) and its ion pair (Glu-457) . Circular dichroic and fluorescence spectroscopic data illustrate the role of these amino acids in maintaining the structure and function of human dihydrolipoamide dehydrogenase . While mutant H452Q is severely crippled in catalysis of the physiological reaction, the reverse reaction is affected in the E457Q mutant . The K37E mutant shows very little deviation from the wild-type enzyme. J Biol Chem, 1995 Jun 30, 270(26), 15539 - 44 Identification of an essential second metal ion in the reaction mechanism of Escherichia coli adenylosuccinate synthetase; Kang C et al.; This study reports that two Mg2+ ions are required for Escherichia coli adenylosuccinate synthetase activity . The first metal ion is presumably coordinated with beta- and gamma-phosphoryl groups of GTP to provide an electron sink, and the second one seems to interact with aspartate in the enzyme active site . Regarding the latter metal ion, kinetic studies show that aspartate and the second Mg2+ ion bind to the enzyme active site randomly with a kcat value of 1.47 s-1 and with Km values for aspartate and Mg2+ of 225 and 114 microM, respectively . The dissociation constants for aspartate and Mg2+ of the enzyme.GTP.IMP.(aspartate or Mg2+) complex are 79.2 and 40.0 microM, respectively . However, variable amounts of aspartate or Mg2+ did not show any significant changes in the Km values for GTP and IMP . Kinetic studies using Mn2+ and Ca2+ ions indicate that the kcat values (0.930 and 0.235 s-1, respectively) were slightly decreased compared with the value obtained using Mg2+; however, the Km values for aspartate and GTP in the presence of Mn2+ and Ca2+ were significantly decreased compared with those obtained using Mg2+ ion (4.5 and 4.6 times for Mn2+ ion and 5.6 and 5.8 times for Ca2+ ion, respectively) . On the other hand, the Km values for IMP were not significantly changed (1.9 and 1.8 times for Mn2+ and Ca2+ ions, respectively) . Taken together, these kinetic results imply that aspartate may interact with Mg2+ to form a Mg.aspartate complex in the enzyme active site . An inhibition study of the enzyme with ZnCl2 (its Ki value is 29 nM) also suggested that Zn2+ competes with aspartate as well as Mg2+, implying that Zn2+ might form a complex with aspartate in the active site . On the basis of these results, it is suggested that Mg.aspartate complex formation in the active site of adenylosuccinate synthetase may be important in activation of the protonated amino group of aspartate, enhancement of the enzyme's binding affinity, and its specificity for aspartate. Science, 1995 Jun 30, 268(5219), 1866 - 72 Crystal structure of DNA photolyase from Escherichia coli; Park HW et al.; Photolyase repairs ultraviolet (UV) damage to DNA by splitting the cyclobutane ring of the major UV photoproduct, the cis, syn-cyclobutane pyrimidine dimer (Pyr <> Pyr) . The reaction is initiated by blue light and proceeds through long-range energy transfer, single electron transfer, and enzyme catalysis by a radical mechanism . The three-dimensional crystallographic structure of DNA photolyase from Escherichia coli is presented and the atomic model was refined to an R value of 0.172 at 2.3 A resolution . The polypeptide chain of 471 amino acids is folded into an amino-terminal alpha/beta domain resembling dinucleotide binding domains and a carboxyl-terminal helical domain; a loop of 72 residues connects the domains . The light-harvesting cofactor 5,10-methenyltetrahydrofolylpolyglutamate (MTHF) binds in a cleft between the two domains . Energy transfer from MTHF to the catalytic cofactor flavin adenine dinucleotide (FAD) occurs over a distance of 16.8 A . The FAD adopts a U-shaped conformation between two helix clusters in the center of the helical domain and is accessible through a hole in the surface of this domain . Dimensions and polarity of the hole match those of a Pyr <> Pyr dinucleotide, suggesting that the Pyr <> Pyr "flips out" of the helix to fit into this hole, and that electron transfer between the flavin and the Pyr <> Pyr occurs over van der Waals contact distance. Cell, 1995 Jun 30, 81(7), 1085 - 93 The MIM complex mediates preprotein translocation across the mitochondrial inner membrane and couples it to the mt-Hsp70/ATP driving system; Berthold J et al.; We have identified a complex in mitochondria that functions as a part of the preprotein import machinery of the inner membrane (MIM complex) . Two known components, MIM23 and MIM17, and two novel components, MIM33 and MIM14, were found as constituents of this complex . In the presence of a translocating chain, the outer membrane import machinery (MOM complex) and the MIM complex form translocation contact sites . On the matrix side, the MIM complex is associated with the mt-Hsp70-MIM44 system . We propose a structure of the import machinery in which the MIM complex constitutes a proteinaceous channel that accepts preproteins from the MOM complex, facilitates their reversible transmembrane movement, and mediates unidirectional transport by linkage to the ATP-dependent mt-Hsp70-MIM44 system. J Biol Chem, 1995 Jun 30, 270(26), 15711 - 8 Studies of the functional topography of the catalytic center of Escherichia coli primase; Mustaev AA et al.; The catalytic center of E . coli primase (581 amino acids) was identified by using, in the G4oric single-strand binding protein (SSB) primer RNA (pRNA) synthesis system, ATP and AMP derivatives, which were modified on the 5' side with reactive groups that can be cross-linked to the ATP binding site plus {alpha-32P}GTP . The position of the covalently attached 32P-labeled dinucleotide was mapped by chemical and enzymatic cleavage of labeled wild type and deletion mutants of primase . The catalytic center involves one of the Lys residues Lys-211, Lys-229, and Lys-241 . The ATP binding site is preformed in primase, and the cross-linked ATP residue can be elongated to a 5-nucleotide limit, which implies significant stretching of the catalytic center during pRNA synthesis . His-43 close to the N terminus in a proposed zinc finger and Lys-528 near the C terminus were also cross-linked to ATP residues in the primase ATP binding site, suggesting that these regions are topographically close to the catalytic center during pRNA synthesis . When cross-linking was performed on the preformed primase/SSB/G4oric complex with long arm reagents (12-15 A), SSB was also labeled, indicating a close proximity to the site of pRNA synthesis. Biochem Pharmacol, 1995 Jun 29, 50(1), 39 - 44 Effect of alkyl-N-purine DNA glycosylase overexpression on cellular resistance to bifunctional alkylating agents; Bramson J et al.; Increased activity of alkyl-N-purine DNA glycosylase (ANPG; a.k.a . N3-methyladenine DNA glycosylase) has been correlated with resistance to both chloroethylnitrosoureas and nitrogen mustards . Also, overexpression of the human glycosylase in Escherichia coli results in resistance to alkylating agents . To determine how overexpression of the protein affects resistance to these bifunctional alkylating agents in mammalian cells, wild-type CHO-AA8 cells were transfected with an expression construct containing the human ANPG cDNA . Several clonally isolated lines that expressed increasing levels of glycosylase activity were selected . None of these lines displayed increased resistance to either bis-chloroethylnitrosourea or melphalan . To determine how overexpression of this protein affects cells in the absence of nucleotide excision repair, the mutant CHO-UV20 cell line was transfected with the same expression construct . This cell line lacks functional ERCC-1 protein and displays extreme hypersensitivity to bifunctional alkylating agents . Again, none of the UV20 transfectants displayed increased resistance . The results of these experiments indicate that unlike E . coli, overexpression of the glycosylase alone is not sufficient to confer resistance to bifunctional alkylating agents in this system . Structural differences between mammalian cells and E . coli may explain the interesting result that a mammalian gene can confer drug resistance in E . coli but not in mammalian cells. Nature, 1995 Jun 29, 375(6534), 809 - 12 Powering the flagellar motor of Escherichia coli with an external voltage source; Fung DC et al.; Rotary motors of bacterial flagella are driven by ions that move across the cytoplasmic membrane down an electrochemical gradient . For Escherichia coli, the ions are protons, and the maximum work per unit charge that they can do is the protonmotive force . To test whether motor efficiency is limited by proton leakage or mechanical nonlinearities, we measured torque as a function of protonmotive force . Filamentous cells were drawn into micropipettes and energized with an external voltage source . Torque was proportional to protonmotive force up to -150 mV, twice the span accessible by earlier techniques . This is consistent with a mechanism in which a fixed number of protons, working at unit efficiency, carry the motor through each revolution . We also found that individual torque-generating elements inactivate at low potentials or potentials of reverse sign . When normal potentials are restored, they reactivate sequentially. Biochemistry, 1995 Jun 27, 34(25), 8180 - 9 A single substitution in the motif 1 of Escherichia coli lysyl-tRNA synthetase induces cooperativity toward amino acid binding; Commans S et al.; The constitutive lysyl-tRNA synthetase (LysRS) of the Escherichia coli strain OEL134 differs from the wild-type enzyme by the single substitution of threonine 208 with methionine . In vitro study of the isotopic {32P}PPi-ATP exchange reaction catalyzed by purified T208M LysRS revealed specific features that are not observed with the wild-type LysRS: (i) The steady state of the reaction was reached after a approximately 1-min lag when the addition of the enzyme was used to initiate the reaction . This lag disappeared upon preincubation of the enzyme with lysine and ATP . (ii) The variation of the steady state rate as a function of the lysine concentration in the assay was sigmoidal (Hill coefficient of 1.65), suggesting cooperativity of lysine binding to this dimeric enzyme . The allosteric behavior of the mutant enzyme was further established by showing that, at low concentrations of lysine, low amounts of cadaverine stimulated T208M LysRS activity . T208A LysRS, in which threonine 208 had been changed into alanine by site-directed mutagenesis, displayed the same properties as T208M LysRS . Remarkably, Thr 208 makes part of the first signature motif of class II aminoacyl-tRNA synthetases, a motif likely to be involved in the dimerization of the enzyme subunits . Therefore, the behavior of the Thr 208 mutants of LysRS supports the idea that the dimerization of class II aminoacyl-tRNA synthetases is important for an efficient structuration of their active site. Biochemistry, 1995 Jun 27, 34(25), 8115 - 22 High resistance of Escherichia coli ribonuclease HI variant with quintuple thermostabilizing mutations to thermal denaturation, acid denaturation, and proteolytic degradation; Akasako A et al.; To test whether the combination of multiple thermostabilizing mutations is a useful strategy to generate a hyperstable mutant protein, five mutations, Gly23-->Ala, His62-->Pro, Val74-->Leu, Lys95-->Gly, and Asp134-->His or Asn, were simultaneously introduced into Escherichia coli ribonuclease HI . The enzymatic activities of the resultant quintuple mutant proteins, 5H- and 5N-RNases HI, which have His and Asn at position 134, respectively, were 35 and 55% of that of the wild-type protein . The far-UV and near-UV CD spectra of these mutant proteins were similar to those of the wild-type protein, suggesting that the mutations did not seriously affect the tertiary structure of the protein . The differences in the free energy change of unfolding between the wild-type and mutant proteins, delta delta G, were estimated by analyzing the thermal denaturation of the proteins by CD . The 5H-RNase HI protein, which was slightly more stable than the 5N-RNase HI, was more stable than the wild-type protein by 20.2 degrees C in Tm and 5.6 kcal/mol in delta G at pH 5.5 . In addition, the 5H-RNase HI was highly resistant to proteolysis and acid denaturation . The effects of each mutation on the thermal stability and the susceptibility to chymotryptic digestion were nearly cumulative, and the 5H-RNase HI undergoes chymotryptic digestion at a rate that is 41 times slower than that of the wild-type protein . Good correlation was observed between the thermal stability and the resistance to chymotryptic digestion for all proteins examined.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Jun 27, 34(25), 8110 - 4 Minimum folding unit of dystrophin rod domain; Kahana E et al.; Fragments of the rod domain of dystrophin, which consists of spectrin-like repeating sequences, have been prepared by expression in Escherichia coli . The phasing established earlier for the dystrophin rod, as well as for Drosophila spectrin and smooth muscle alpha-actinin, suggested a length of less than 113 residues for the dystrophin repeat that we have chosen . Fragments with a common N-terminus and lengths between 113 and 119 residues were prepared . The formation of the stable native tertiary fold could be recognized by resistance to proteolysis, the circular dichroism spectrum in the regions of both peptide and aromatic absorption bands and the resolution of the long-wavelength component in the tryptophan absorption spectrum . It was found that the critical length for folding was 117 residues: shortening the chain by 1 further residue resulted in loss of the capacity to form a defined tertiary structure . Residue 117 is a glutamine; replacement of this by a methionine residue did not impair the ability of the chain to enter the folded conformation, implying that it is the length of the C-terminal alpha-helix, rather than any specific side-chain interaction, that is critical in determining the stability of the native structure . The fragment of 119 residues forms a significantly more stable structure than that of 117 . It appears that the minimum unit capable of forming the native fold extends some residues into the adjoining sequence repeat. Biochemistry, 1995 Jun 27, 34(25), 7988 - 95 "Prohormone thiol protease" (PTP) processing of recombinant proenkephalin; Schiller MR et al.; The "prohormone thiol protease" (PTP) from adrenal medullary chromaffin granules has been demonstrated as a novel cysteine protease that converts the model enkephalin precursor, ({35S}Met)-preproenkephalin, to appropriate enkephalin related peptide products {Krieger, T . J., & Hook, V . Y . H . (1991) J . Biol . Chem . 266, 8376-8383; Kreiger, T . J., Mende-Mueller, L., & Hook, V . Y . H . (1992) J . Neurochem . 59, 26-31; Azaryan, A . V., & Hook, V . Y . H . (1994) FEBS Lett . 341, 197-202} . In this report, PTP processing of authentic proenkephalin (PE) was examined with respect to production of appropriate intermediate products, and kinetics of PE processing were assessed . Recombinant PE was obtained by high level expression in Escherichia coli, with the pET3c expression vector; PE was then purified from E . coli by DEAE-Sepharose chromatography, preparative gel electrophoresis, and reverse-phase HPLC . Authentic purified PE was confirmed by amino acid composition analyses and peptide microsequencing . In time course studies, PTP converted PE (12 microM) to intermediates of 22.5, 21.7, 12.5, and 11.0 kDa that represented NH2-terminal fragments of PE, as assessed by peptide microsequencing . Differences in molecular masses of the 22.5, 21.7, 12.5, and 11.0 kDa products reflect PTP processing of PE within the COOH-terminal region of PE, which resembles PE processing in vivo {Liston, D . L., Patey, G., Rossier, J., Verbanck, P., & Vanderhaeghen, J . (1983) Science 225, 734-737; Udenfriend, S., & Kilpatrick, D . L . (1983) Arch . Biochem . Biophys . 221, 309-314} . Products of 12.5, 11.0, and 8.5 kDa were generated by PTP cleavage between Lys-Arg at the COOH-terminus of (Met)enkephalin-Arg6-Gly7-Leu8.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Jun 27, 34(25), 7967 - 72 Interruption of the water chain in the reaction center from Rhodobacter sphaeroides reduces the rates of the proton uptake and of the second electron transfer to QB; Baciou L et al.; A chain of bound water molecules was recently identified in the photosynthetic reaction center (RC) from Rhodobacter sphaeroides by X-ray crystallography {Ermler et al . (1994) Structure 2, 925-936} . The possible role of the chain in proton transfer from the solution to the secondary quinone (QB) was investigated by site-directed mutagenesis and flash-induced absorbance spectroscopy . Pro L209, situated along the water chain about 9 A from QB, was changed into the aromatic residues Phe and Tyr in order to interrupt the chain . In the PL209Y (Pro L209-->Tyr) mutant, the very small changes in the QA-QB<==>QAQB- equilibrium constant (K2) and the first electron-transfer rates (kAB(1)) indicate that the mutation does not lead to large structural changes . In the PL209F (Pro L209-->Phe) mutant, a 7-fold decrease of kAB(1) is observed . It follows a pH dependence parallel to that of the wild type . It is consistent with no modification of the pK of the Glu L212 determined from the pH dependence of K2 . The decreased kAB(1) may reflect some slight structural modification in this mutant and/or rearrangement of the cluster of charged residues close to the L209 position . The major effect of the mutations observed is a concomitant decrease of the rates of the second electron transfer, kAB(2), and of the proton uptake upon the second flash . The relative decrease of the kAB(2) rate values in the mutants is more pronounced above pH 8 . Our results indicate that the mutations have specifically altered the pathway of proton transfer to QB.(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1995 Jun 26, 367(2), 145 - 8 Cloning and expression of the cDNA coding for the erythrocyte isoenzyme of human acylphosphatase; Fiaschi T et al.; Three independent cDNAs coding for the erythrocyte isoform of human acylphosphatase were isolated and characterized . All the clones were incomplete at the 5' end, but Northern blot analysis using the cDNA as a probe showed the presence of an unusually long mRNA 5'-untranslated region . The transcript was present in a variety of human cell lines of different origins, although at different levels . Southern blot analysis on DNA from different individuals revealed a simple hybridization pattern . Large amounts of pure enzyme with kinetic characteristics very similar to those of the native protein were expressed in E . coli. Biochem Biophys Res Commun, 1995 Jun 26, 211(3), 804 - 11 RecA-assisted rapid enrichment of specific clones from model DNA libraries; Teintze M et al.; An approach to library screening is being developed, in which the desired clone is "fished" out of a mixture of all the recombinants in a library with a RecA-coated probe . In the current embodiment of this method, we used as a probe the (+) strand of an M13 phage containing a fragment of the human albumin gene and a (dA)49 stretch . We screened a library of two plasmids, one containing the same albumin fragment as the probe, and one heterologous to the probe in 50-100 fold molar excess . The plasmids were linearized . Probe and library were reacted in the presence of RecA, the mixture was loaded onto an oligo(dT) column, which retained the probe-target complex by base-pairing to the dAs of the probe, the uncaptured plasmids were washed, and the probe-target complex was released from the column, religated and propagated into E . coli . Recovery of the homologous target was 15-28%, and enrichment for the homologous plasmid was 200 to 400-fold . This approach may provide a general method for expedited DNA library screening. Biochem Biophys Res Commun, 1995 Jun 26, 211(3), 754 - 60 Apoc-III-beta-galactosidase hybrid distinguishes between VLDL and LDL phospholipids; Trieu VN et al.; A plasmid directing the expression of preapolipoprotein C-III fused to beta-galactosidase was constructed . Escherichia coli harboring this construct produced the hybrid, with full beta-galactosidase activity, at 0.6% of total cellular protein . Purified preapoC-III-beta-galactosidase hybrid exhibited specific binding to very low density lipoproteins, which was inhibited by purified apoC-III . No binding to low density lipoproteins was observed . Binding was mediated by the phospholipids of very low density lipoproteins, as the hybrid had a specific affinity for phospholipids and no detectable affinity for triglycerides, cholesteryl esters, or cholesterol . The differential binding suggests that there are differences between the phospholipids of very low density lipoproteins and the phospholipids of low density lipoproteins. Mol Gen Genet, 1995 Jun 25, 247(6), 764 - 7 Influence of DNA topology on expression of the tdc operon in Escherichia coli K-12; Wu Y et al.; TdcB activity expressed from the chromosomal gene and LacZ expression from single-copy tdc-lacZ transcriptional and translational fusions were measured in Escherichia coli strains harboring mutations in the genes encoding DNA gyrase, topoisomerase I and the HU protein . The pattern of tdc operon expression in these mutants suggests that relaxation of supercoiled DNA enhances tdc transcription in vivo. Mol Gen Genet, 1995 Jun 25, 247(6), 735 - 41 The spectra of base substitutions induced by the impCAB, mucAB and umuDC error-prone DNA repair operons differ following exposure to methyl methanesulfonate; Doyle N et al.; We have used the lacZ reversion assay to study the mutation spectra induced by the Escherichia coli chromosomal umuDC operon and of its two plasmid-borne analogues impCAB and mucAB following exposure of cells to UV light and methyl methanesulfonate (MMS) . We have shown that the impCAB, mucAB and umuDC operons all produce a similar response to UV light which results almost exclusively in AT-->GC transitions . However, we found that the three operons produced different responses to alkylating agents . We found that with MMS the chromosomal umuDC operon produced almost exclusively AT-->GC transitions, whilst both mucAB and impCAB produced predominantly transversions . In the case of the impCAB operon the mutation spectrum contained more AT-->TA than GC-->TA transversions; this balance was reversed with mucAB . The effect of the copy number of the error-prone DNA repair operons upon the mutagenic spectra was also studied . The results obtained suggest that the copy number of the imp operon does not greatly affect the specificity of base substitutions observed . However, an increase in the copy number of the umuDC operon greatly affected the specificity of base substitution, such that virtually no transitions were produced and the spectrum was dominated by GC/AT-->TA transversions . It appears that the three error-prone DNA repair operons impCAB, mucAB and umuDC, despite showing strong structural and functional homologies, can display major differences in the spectrum of base changes induced during mutagenesis.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Gen Genet, 1995 Jun 25, 247(6), 726 - 34 Studies on the binding of integration host factor (IHF) and TraM to the origin of transfer of the IncFV plasmid pED208; Di Laurenzio L et al.; The origin of transfer (oriT) of the IncFV plasmid pED208 contains a region with three binding sites for both the plasmid-encoded TraM protein and the integration host factor (IHF) of Escherichia coli, a sequence-specific DNA-binding protein . One region, containing overlapping TraM and IHF binding sites, could be interpreted as containing two binding sites for each protein . Using gel retardation assays, an affinity constant for IHF binding to the three main sites was estimated in the presence and absence of 0.1 M potassium glutamate, which increased the avidity of IHF binding to the weaker sites by two orders of magnitude . DNase I protection analyses and electron microscopy were used to determine the affinity of IHF for oriT-containing DNA in the presence and absence of TraM . The binding of IHF and TraM was found to be non-cooperative by the two techniques employed . Electron microscopy also demonstrated that IHF bent the oriT region in a manner consistent with its previously determined mode of action, while TraM had no discernible effect on the appearance of the DNA . This suggested that IHF and TraM interact with a 295 bp sequence in the oriT region and organize it into a higher order structure that may have a role in the initiation of DNA transfer and control of traM expression. Nucleic Acids Res, 1995 Jun 25, 23(12), 2105 - 19 Analysis of the Escherichia coli genome VI: DNA sequence of the region from 92.8 through 100 minutes; Burland V et al.; The 338.5 kb of the Escherichia coli genome described here together with previously described segments bring the total of contiguous finished sequence of this genome to > 1 Mb . Of 319 open reading frames (ORFs) found in this 338.5 kb segment, 147 (46%) are potential new genes . The positions of several genes which had been previously located here by mapping or partial sequencing have been confirmed . Several ORFs have functions suggested by similarities to other characterised genes but cannot be assigned with certainty . Fifteen of the ORFs of unknown function had been previously sequenced . Eight transfer RNAs are encoded in the region and there are two grey holes in which no features were found . The attachment site for phage P4 and three insertion sequences were located . The region was also analysed for chi sites, bend sites, REP elements and other repeats . A computer search identified potential promoters and tentative transcription units were assigned . The occurrence of the rare tetramer CTAG was analysed in 1.6 Mb of contiguous E.coli sequence . Hypotheses addressing the rarity and distribution of CTAG are discussed. J Biol Chem, 1995 Jun 23, 270(25), 15434 - 42 CuZn superoxide dismutase (SOD-1):tetanus toxin fragment C hybrid protein for targeted delivery of SOD-1 to neuronal cells; Francis JW et al.; Increased levels of CuZn superoxide dismutase (SOD-1) are cytoprotective in experimental models of neurological disorders associated with free radical toxicity (e.g . stroke, trauma) . Targeted delivery of SOD-1 to central nervous system neurons may therefore be therapeutic in such diseases . The nontoxic C-fragment of tetanus toxin (TTC) possesses the nerve cell binding/transport properties of tetanus holotoxin and has been used as a vector to enhance the neuronal uptake of proteins including enzymes . We have now produced a recombinant, hybrid protein in Escherichia coli tandemly joining human SOD-1 to TTC . The expressed hybrid protein (SOD:Tet450) has a subunit molecular mass of 68 kDa and is recognized by both anti-SOD-1 and anti-TTC antibodies . Calculated per mol, SOD:Tet450 has approximately 60% of the expected SOD-1 enzymatic activity . Analysis of the hybrid protein's interaction with the neuron-like cell line, N18-RE-105, and cultured hippocampal neurons by enzyme immunoassay for human SOD-1 revealed that SOD:Tet451 association with cells was neuron-specific and dose-dependent . The hybrid protein was also internalized, but there was substantial loss of internalized hybrid protein over the first 24 h . Hybrid protein associated with cells remained enzymatically active . These results suggest that human SOD-1 and TTC retain their respective functional properties when expressed together as a single peptide . SOD:Tet451 may prove to be a useful agent for the targeted delivery of SOD-1 to neurons. J Biol Chem, 1995 Jun 23, 270(25), 15353 - 8 Complex interactions between yeast TFIIIB and TFIIIC; Chaussivert N et al.; Transcription of yeast class III genes requires the sequential assembly of the general transcription factors TFIIIC and TFIIIB, and of RNA polymerase III, into an initiation complex composed of at least 25 polypeptides . The 70-kDa subunit of TFIIIB (TFIIIB70) is central in this network of interactions as it contacts both TATA-binding protein and a subunit of polymerase III . We show here that the TATA-binding protein interacts with the carboxyl-terminal part of TFIIIB70 . TFIIIB70 also contacts TFIIIC (factor tau) via its tau 131 subunit . The protein domains of tau 131 and TFIIIB70 involved in this interaction, either positively or negatively, were mapped using the two-hybrid system . We provide evidence that intramolecular interactions mask functional domains in both polypeptides. J Biol Chem, 1995 Jun 23, 270(25), 15315 - 9 Catalytic activities of glycogenin additional to autocatalytic self-glucosylation; Alonso MD et al.; Glycogenin is the autocatalytic, self-glucosylating protein that initiates glycogen synthesis in muscle and other tissues . We have sequenced the cDNA for rabbit muscle glycogenin and expressed and purified the protein in high yield as well as two mutant proteins in which Phe or Thr replaces Tyr-194, the site of glucosylation . While the wild-type protein can self-glucosylate, the mutants cannot, but all three utilize alternative acceptors by intermolecular glucose transfer for which the mutants have altered specificity . Tyr-194 is therefore not essential for the catalytic activity of glycogenin . All three proteins also hydrolyze UDP-glucose to glucose at rates comparable with the rate of self-glucosylation . The hydrolysis is competitive with glucose transfer to p-nitrophenyl alpha-maltoside . Self-glucosylation, glucosylation of other acceptors, and hydrolysis all appear to be catalyzed by the same active center . In the absence of peptidase inhibitors, the homogenous recombinant proteins of M(r) 37,000 break down to equally active species having M(r) 32,000 . The kinetics of self-glucosylation catalyzed by the wild-type enzyme suggest that the reaction could be intermolecular rather than, as previously reported, intramolecular . The wild-type recombinant enzyme and native muscle glycogenin, which is phosphorylated, are inhibited quite differently by ATP at physiological concentration. J Biol Chem, 1995 Jun 23, 270(25), 15162 - 9 The zinc-binding site of Escherichia coli glutamyl-tRNA synthetase is located in the acceptor-binding domain . Studies by extended x-ray absorption fine structure, molecular modeling, and site-directed mutagenesis; Liu J et al.; The zinc contents of fragments of Escherichia coli glutamyl-tRNA synthetase, as well as the conservation of the CYC sequence only in zinc-containing glutamyl-tRNA synthetases, suggested that the 98CYCX24-CRHSHEHHADDEPC138 includes some or all residues involved in binding its zinc atom (Liu, J., Lin, S.-X., Blochet, J.-E., Pezolet, M., and Lapointe, J . (1993) Biochemistry 32, 11390-11396) . Extended x-ray absorption fine structure (EXAFS) shows that this zinc atom has a four-coordinate non-planar coordination environment with 3 sulfur and 1 nitrogen atoms with bond lengths, respectively, 2.37 +/- 0.02 A and 2.01 +/- 0.02 A, presumably belonging to 3 cysteine residues and 1 histidine residue . Conservative replacement of each histidine and cysteine residue of the 98C-138C segment, respectively, with glutamine (Q) and serine (S), yields variants H129Q, H131Q, H132Q, and C138S (which sustain the growth at 42 degrees C of E . coli JP1449, whose glutamyl-tRNA synthetase is thermosensitive) and C98S, C100S, C125S, and H127Q (which do not) . The amount of this enzyme in these mutants is at least 1 order of magnitude larger than that in a wild type strain; however, no glutamyl-tRNA synthetase activity is detectable in extracts of the variants C100S and C125S, whereas its specific activity in those of C98S and H127Q is about 10-fold lower than in cells overproducing the wild type enzyme or the variants H129Q, H131Q, H132Q, and C138S . These results indicate that the zinc atom present in E . coli glutamyl-tRNA synthetase is bound by the 2 evolutionarily conserved cysteines at positions 98 and 100, and by Cys125 and His127 . Molecular modeling of the N-terminal half of this enzyme, using the known structure of E . coli glutaminyl-tRNA synthetase, supports this conclusion and suggests that the 98C-127H segment does not have the characteristics of the classical zinc fingers. Science, 1995 Jun 23, 268(5218), 1769 - 72 Construction of a soluble adenylyl cyclase activated by Gs alpha and forskolin; Tang WJ et al.; A soluble adenylyl cyclase was constructed by linkage of portions of the cytosolic domains of the mammalian type I and type II enzymes . The soluble enzyme was stimulated by both forskolin and the alpha subunit of the heterotrimeric guanine nucleotide-binding protein (G protein) Gs (Gs alpha) . Expression of the construct complemented the catabolic defect in a strain of Escherichia coli that is deficient in adenylyl cyclase activity . The active, approximately 60-kilodalton enzyme accumulated in the cytoplasmic fraction of E . coli to yield activities in excess of 1 nanomole per minute per milligram of protein . The two sets of transmembrane helices of mammalian adenylyl cyclases are thus not necessary for the catalytic or the most characteristic regulatory activities of the enzyme . This system may be useful for both genetic and biochemical analysis of G protein-regulated adenylyl cyclases. Science, 1995 Jun 23, 268(5218), 1766 - 9 Transcriptional activation by tetracyclines in mammalian cells; Gossen M et al.; A transcriptional transactivator was developed that fuses the VP16 activation domain with a mutant Tet repressor from Escherichia coli . This transactivator requires certain tetracycline (Tc) derivatives for specific DNA binding . Thus, addition of doxycycline to HeLa cells that constitutively synthesized the transactivator and that contained an appropriate, stably integrated reporter unit rapidly induced gene expression more than a thousandfold . The specificity of the Tet repressor-operator-effector interaction and the pharmacological characteristics of Tc's make this regulatory system well suited for the control of gene activities in vivo, such as in transgenic animals and possibly in gene therapy. Science, 1995 Jun 23, 268(5218), 1721 - 7 Crystal structure of lac repressor core tetramer and its implications for DNA looping; Friedman AM et al.; The crystal structure of the tryptic core fragment of the lac repressor of Escherichia coli (LacR) complexed with the inducer isopropyl-beta-D-thiogalactoside was determined at 2.6 A resolution . The quaternary structure consists of two dyad-symmetric dimers that are nearly parallel to each other . This structure places all four DNA binding domains of intact LacR on the same side of the tetramer, and results in a deep, V-shaped cleft between the two dimers . Each monomer contributes a carboxyl-terminal helix to an antiparallel four-helix bundle that functions as a tetramerization domain . Some of the side chains whose mutation reduce DNA binding form clusters on a surface near the amino terminus . Placing the structure of the DNA binding domain complexed with operator previously determined by nuclear magnetic resonance onto this surface results in two operators being adjacent and nearly parallel to each other . Structural considerations suggest that the two dimers of LacR may flexibly alter their relative orientation in order to bind to the known varied spacings between two operators. J Mol Biol, 1995 Jun 23, 249(5), 914 - 22 The action of Escherichia coli endonuclease III on multiply damaged sites in DNA; Chaudhry MA et al.; Energy deposition by ionizing radiation can lead to the formation of clustered DNA damage, i.e . more than one lesion situated within a helical turn of DNA . Among the postulated lesions are those characterized by damaged bases and abasic sites on opposite strands . Enzymatic removal of such lesions may inadvertently lead to the formation of double-strand breaks . To test this hypothesis, we have constructed model substrates containing damaged bases (5,6-dihydrothymine) or abasic sites set one, three, five and seven bases apart on opposite strands, and examined the reactivity of Escherichia coli endonuclease III towards these substrates . Endonuclease III demonstrates two activities; as a glycosylase that removes saturated pyrimidine bases, such as dihydrothymine, and as an AP lyase that cleaves DNA strands at abasic sites . Analysis of endonuclease III-treated dihydrothymidine containing plasmid DNA by agarose gel electrophoresis indicated that the enzyme generated only single-strand breaks when the base damage was set one and three base-pairs apart, and only slowly introduced double-strand breaks in the other substrates . Endonuclease III treatment of the abasic site-containing DNA, however, readily yielded double-strand breaks . Taken together, these results indicate that the glycosylase activity of the enzyme, but not the AP lyase activity, is inhibited by the presence of a closely positioned break in the opposite strand. J Mol Biol, 1995 Jun 23, 249(5), 890 - 902 Nag repressor-operator interactions: protein-DNA contacts cover more than two turns of the DNA helix; Plumbridge J et al.; The NagC repressor binds to two sites in the intergenic nagE-B region overlapping the divergently expressed nagE and nagB promoters . In addition the NagC repressor binds to two sites upstream of the manXYZ operon . Although basically palindromic, there is little sequence consensus between the four operators . To identify the DNA sequence important for NagC recognition, we have taken advantage of the fact that repression of the nagE and nagB genes requires the formation of a loop of DNA between molecules of the repressor bound to the nagE and nagB operators . The nagE operator was systematically mutagenised and the effect of the mutations measured on the level of expression from a nagB-lacZ fusion . These experiments showed that the most important positions for recognition are the two A.T base-pairs at positions-5 and -6 from the centre of symmetry . These are the only absolutely conserved bases in the four operators . Certain changes of residues at position -3 and -4 have fairly strong effects while changes at -7 to -10 have only minor effects . However the presence of a G or C base at positions + 11 or -11 produces a NagC binding site with considerably higher affinity than the wide-type nagE operator both in vitro and in vivo, a "super-operator" . The presence of a super-operator considerably increased the stability of the binary looped NagC-DNA complex in vitro . However in the presence of cAMP/CAP, NagC showed the same apparent binding affinity to wild-type and super-operators indicating that one role of cAMP/CAP in the repression complex is to reduce the need for high affinity sites . These super-operators allow a higher level of repression of the nagE promoter compared to the nagB, presumably due to the existence of linear complexes of NagC bound to BoxE. J Mol Biol, 1995 Jun 23, 249(5), 857 - 68 Phage T4-coded Stp: double-edged effector of coupled DNA and tRNA-restriction systems; Penner M et al.; The optional Escherichia coli prr locus encodes two physically associated restriction systems: the type IC DNA restriction-modification enzyme EcoprrI and the tRNA(Lys)-specific anticodon nuclease, specified by the PrrC polypeptide . Anticodon nuclease is kept latent as a result of this interaction . The activation of anticodon nuclease, upon infection by phage T4, may cause depletion of tRNA(Lys) and, consequently, abolition of T4 protein synthesis . However, this effect is counteracted by the repair of tRNA(Lys) in consecutive reactions catalysed by the phage enzymes polynucleotide kinase and RNA ligase . Stp, a short polypeptide encoded by phage T4, has been implicated with activation of the anticodon nuclease . Here we confirm this notion and also demonstrate a second function of Stp: inhibition of EcoprrI restriction . Both effects depend, in general, on the same residues within the N-proximal 18 residue region of Stp . We propose that Stp alters the conformation of EcoprrI and, consequently, of PrrC, allowing activation of the latent anticodon nuclease . Presumably, Stp evolved to offset a DNA restriction system of the host cell but was turned, eventually, against the phage as an activator of the appended tRNA restriction enzyme. J Chromatogr A, 1995 Jun 23, 705(1), 163 - 9 Detection of neu differentiation factor with a biospecific affinity sensor during chromatography; Lu HS et al.; A technique using a biospecific affinity sensor, BIAcore, was applied to monitor and determine mammalian cell-derived neu differentiation factor (NDF) in column fractions during chromatography . Specific purified polyclonal antibody against Escherichia coli-derived NDF was chemically bound to the surface of BIAcore sensor chips and the derivatized sensor chips were used to detect the specific binding of NDF . The measurement of NDF at very low levels can be assessed by injecting small volumes of the crude media or column fractions into the BIAcore sensor containing antibody-bound sensor chips . This automated procedure performed under computer programming control allows direct measurement of multiple NDF samples in a short period of time and provides excellent quantitative data, which is not possible using other related methods such as Western blotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stimulatory activity assay on receptor autophosphorylation. J Biol Chem, 1995 Jun 23, 270(25), 14951 - 7 T cell-targeted immunofusion proteins from Escherichia coli; Better M et al.; Fusion proteins between cell-targeting domains and cytotoxic proteins should be particularly effective therapeutic reagents . We constructed a family of immunofusion proteins linking humanized Fab, F(ab')2, or single chain antibody forms of the H65 antibody (which recognizes the CD5 antigen on the surface of human T cells) with the plant ribosome-inactivating protein gelonin . We reasoned that such an immunofusion would kill human target cells as efficiently as the previously described chemical conjugates of H65 and gelonin (Better M., Bernhard, S . L., Fishwild, D . M., Nolan, P . A., Bauer, R . J., Kung, A . H . C., and Carroll, S . F . (1994) J . Biol . Chem . 269, 9644-9650) if both the recognition and catalytic domains remained active, and a proper linkage between domains could be found . Immunofusion proteins were produced in Escherichia coli as secreted proteins and were recovered directly from the bacterial culture supernatant in an active form . All of the immunofusion proteins were purified by a common process and were tested for cytotoxicity toward antigen-positive human cells . A 20-60-fold range of cytotoxic activity was seen among the fusion family members, and several fusion proteins were identified which are approximately as active as effective chemical conjugates . Based on these constructs, immunofusion avidity and potency can be controlled by appropriate selection of antibody domains and ribosome-inactivating protein. Nature, 1995 Jun 22, 375(6533), 688 - 91 Tilting of the light-chain region of myosin during step length changes and active force generation in skeletal muscle; Irving M et al.; Force generation and relative sliding between the myosin and actin filaments in muscle are thought to be caused by tilting of the head region of the myosin crossbridges between the filaments . Structural and spectroscopic experiments have demonstrated segmental flexibility of myosin in muscle, but have not shown a direct linkage between tilting of the myosin heads and either force generation or filament sliding . Here we use fluorescence polarization to detect changes in the orientation of the light-chain region of the head, the part most likely to tilt, and synchronized head movements by imposing rapid length steps . We found that the light-chain region of the myosin head tilts both during the imposed filament sliding and during the subsequent quick force recovery that is thought to signal the elementary force-generating event. Biochemistry, 1995 Jun 20, 34(24), 7913 - 22 Stimulation of DNA synthesis by mouse DNA helicase B in a DNA replication system containing eukaryotic replication origins; Matsumoto K et al.; A number of DNA helicases have been isolated from mammalian cells, but their abilities to stimulate DNA replication accompanied with DNA unwinding have not been addressed so far . We constructed a model DNA replication system using the yeast autonomously replicating sequence (ARS) as the replication origin . In this system, SV40 T antigen as a DNA helicase assembles to the replication origin where the DNA duplex is unwound by torsional stress due to the negative supercoiling of template DNA, which leads to bidirectional DNA replication from the origin . We report here that DNA helicase B isolated from mouse FM3A cells can greatly stimulate DNA synthesis in this replication system in place of SV40 T antigen . DNA synthesis was dependent on the presence of single-stranded DNA binding protein (RP-A), DNA polymerase alpha/primase from mouse cells, and Escherichia coli DNA gyrase . DNA gyrase was required not only at elongation as a DNA swivelase but also at initiation to increase negative superhelical density of template DNA with the assistance of RP-A . A mammalian DNA fragment containing a replication initiation zone upstream of the c-myc gene as well as the yeast ARS fragment acted as a cis-element in this system using DNA helicase B . Both DNA helicase B and SV40 T antigen have the ability to extensively unwind the template DNA in the presence of RP-A and DNA gyrase, which may be crucial for stimulation of DNA synthesis in this system. Biochemistry, 1995 Jun 20, 34(24), 7819 - 24 A nontransportable substrate for lactose permease; Seibert C et al.; A substrate for lactose permease of Escherichia coli was synthesized that binds to the protein with a relatively high affinity, but is not transported to any detectable extent . This substrate, 6'-{(N-phenylalanylphenylalanyl)amino}hexyl 1-thio-beta-D-galactoside, is a peptide galactoside composed of a bulky aromatic dipeptide that is linked to galactose via an aminohexyl spacer . Binding of the peptide galactoside to lactose permease in cytoplasmic membranes was determined in a competition assay yielding a dissociation constant of 150 microM . Transport was measured by a counterflow assay using lipid vesicles with reconstituted lactose permease . An upper limit for the rate constant of transport was obtained as 0.02 s-1, 3 orders of magnitude smaller than the value for lactose. Virology, 1995 Jun 20, 210(1), 194 - 201 Expression and purification of a recombinant tobacco etch virus NIa proteinase: biochemical analyses of the full-length and a naturally occurring truncated proteinase form; Parks TD et al.; The tobacco etch virus 27-kDa nuclear inclusion a (NIa) proteinase was expressed in Escherichia coli as a recombinant fusion protein containing a seven-histidine tag at the amino-terminus . Catalytically active and inactive (by virtue of a single amino acid change) forms of the proteinase were purified to homogeneity in a two-column chromatographic procedure . The active form of the proteinase was slowly converted to a lower molecular weight form, while the inactive form was not . This conversion was dilution independent and thought to be intramolecular . Isolation of the approximately 2-kDa peptide cleavage product and determination of its N-terminal amino acid sequence positioned the cleavage site 24 amino acids from the carboxy-terminus of the proteinase . A recombinant NIa proteinase lacking the C-terminal 24 amino acids was shown to possess limited activity . Kinetic analyses of cleavage of a synthetic peptide by the full-length or truncated proteinase were conducted and indicated that the Km of the truncated proteinase was approximately fourfold higher than that of the full-length form . The truncated proteinase was approximately one-twentieth as efficient in proteolysis of the test peptide substrate as the full-length form. Biochim Biophys Acta, 1995 Jun 20, 1267(2-3), 83 - 91 The GTPase activity of the Escherichia coli Ffh protein is important for normal growth; Samuelsson T et al.; The Escherichia coli (E . coli) Ffh protein is homologous to the 54kDa subunit of the eukaryotic signal recognition particle . We have examined an intrinsic GTPase activity of this protein and have created mutations in one sequence motif (GXXXXGK) of the putative GTP binding site . When glycine-112 was changed to valine (Ffh-G112V), Vmax was reduced to only 4% of the wildtype level . On the other hand, when glutamine-109 was altered to glycine (Ffh-Q109G), the major effect was a 50-fold increase in Km . These results show that the residues Q-109 and G-112 are essential for the binding and hydrolysis of GTP and that they are part of a catalytic site structurally related to that of many other GTPase proteins . Expression of the mutant protein Ffh-G112V in E . coli was highly toxic in the presence of the wildtype protein . In contrast, genetic complementation experiments showed that a viable strain could be constructed where the Ffh-Q109G mutant protein replaced wildtype Ffh . However, expression of the mutant protein had a negative effect on growth rate at 30 degrees C and resulted in elongated cells . These results demonstrate that the GTPase activity of the Ffh protein is required for proper function of the protein in vivo. Biochim Biophys Acta, 1995 Jun 20, 1267(2-3), 131 - 3 Evidence for stimulation of the inositol triphosphate-Ca2+ signalling system in rat enterocytes by heat stable enterotoxin of Escherichia coli; Chaudhuri AG et al.; In Escherichia coli heat stable enterotoxin (STa)-treated rat enterocytes, the rise of inositol triphosphate (IP3) preceded the rise of {Ca2+}i . Chelation of extracellular Ca2+ with EGTA and suspension of cells in Ca2+ free buffer both demonstrated the enterotoxin-induced initial rise of {Ca2+}i with a concomitant loss of sustained phase . Furthermore, pretreatment of cells with dantrolene resulted in a decrease of the early response of {Ca2+}i, indicating the initial effect of the rise of {Ca2+}i was mostly due to its mobilization from some IP3-sensitive intracellular stores. Proc Natl Acad Sci U S A, 1995 Jun 20, 92(13), 6102 - 6 Domains of transcription factor Sp1 required for synergistic activation with sterol regulatory element binding protein 1 of low density lipoprotein receptor promoter; Yieh L et al.; Feedback regulation of transcription from the low density lipoprotein (LDL) receptor gene is fundamentally important in the maintenance of intracellular sterol balance . The region of the LDL receptor promoter responsible for normal sterol regulation contains adjacent binding sites for the ubiquitous transcription factor Sp1 and the cholesterol-sensitive sterol regulatory element-binding proteins (SREBPs) . Interestingly, both are essential for normal sterolmediated regulation of the promoter . The cooperation by Sp1 and SREBP-1 occurs at two steps in the activation process . SREBP-1 stimulates the binding of Sp1 to its adjacent recognition site in the promoter followed by enhanced stimulation of transcription after both proteins are bound to DNA . In the present report, we have defined the protein domains of Sp1 that are required for both synergistic DNA binding and transcriptional activation . The major activation domains of Sp1 that have previously been shown to be essential to activation of promoters containing multiple Sp1 sites are required for activation of the LDL receptor promoter . Additionally, the C domain is also crucial . This slightly acidic approximately 120-amino acid region is not required for efficient synergistic activation by multiple Sp1 sites or in combination with other recently characterized transcriptional regulators . We also show that Sp1 domain C is essential for full, enhanced DNA binding by SREBP-1 . Taken together with other recent studies on the role of Sp1 in promoter activation, the current experiments suggest a unique combinatorial mechanism for promoter activation by two distinct transcription factors that are both essential to intracellular cholesterol homeostasis. Proc Natl Acad Sci U S A, 1995 Jun 20, 92(13), 6042 - 6 Dissection of transcription factor TFIIF functional domains required for initiation and elongation; Tan S et al.; TFIIF is unique among the general transcription factors because of its ability to control the activity of RNA polymerase II at both the initiation and elongation stages of transcription . Mammalian TFIIF, a heterodimer of approximately 30-kDa (RAP30) and approximately 70-kDa (RAP74) subunits, assists TFIIB in recruiting RNA polymerase II into the preinitiation complex and activates the overall rate of RNA chain elongation by suppressing transient pausing by polymerase at many sites on DNA templates . A major objective of efforts to understand how TFIIF regulates transcription has been to establish the relationship between its initiation and elongation activities . Here we establish this relationship by demonstrating that TFIIF transcriptional activities are mediated by separable functional domains . To accomplish this, we sought and identified distinct classes of RAP30 mutations that selectively block TFIIF activity in transcription initiation and elongation . We propose that (i) TFIIF initiation activity is mediated at least in part by RAP30 C-terminal sequences that include a cryptic DNA-binding domain similar to conserved region 4 of bacterial sigma factors and (ii) TFIIF elongation activity is mediated in part by RAP30 sequences located immediately upstream of the C terminus in a region proposed to bind RNA polymerase II and by additional sequences located in the RAP30 N terminus. Proc Natl Acad Sci U S A, 1995 Jun 20, 92(13), 5973 - 6 Residues in the pathway through a membrane transporter; Yan RT et al.; The structure of solute transporters is understood largely from analysis of their amino acid sequences, and more direct information is greatly needed . Here we report work that applies cysteine scanning mutagenesis to describe structure-function relations in UhpT, a bacterial membrane transporter . By using an impermeant SH-reactive agent to probe single-cysteine variants, we show that UhpT transmembrane segment 7 spans the membrane as an alpha-helix and that the central portion of this helix is exposed to both membrane surfaces, forming part of the translocation pathway through this transporter. Proc Natl Acad Sci U S A, 1995 Jun 20, 92(13), 5875 - 9 Immortalization of human primary B lymphocytes in vitro with DNA; Kempkes B et al.; Epstein-Barr virus (EBV) is a human DNA tumor virus that efficiently immortalizes human primary B lymphocytes in vitro . Although viral genes that are expressed in latently infected B lymphocytes have been shown to function in cellular growth control, their detailed genetic analysis has been cumbersome for two reasons . The viral genome is too large to permit genetic engineering and human primary B lymphocytes, the only targets for infection by EBV in vitro, are both intractable in culture and recalcitrant to DNA transfection . To overcome these obstacles, we have assembled all the essential genes of EBV on a single recombinant vector molecule in Escherichia coli . We show here that this mini-EBV plasmid can yield immortalized B cells upon transfer of its naked DNA into human primary B lymphocytes . Established cell lines carry recombinant vector DNA and cannot support virus production . Because this DNA can be easily manipulated in E . coli, mutant mini-EBVs as well as foreign genes can now be introduced and studied successfully in recipient B lymphocytes from any human donors . These mini-EBVs therefore are potentially useful for human gene therapy. Proc Natl Acad Sci U S A, 1995 Jun 20, 92(13), 5768 - 72 Transcription in archaea: similarity to that in eucarya; Langer D et al.; We present homologies between archaeal and eucaryal DNA-dependent RNA polymerase (RNAP) subunits and transcription factors . The sequences of the Sulfolobus acidocaldarius subunits D, E, and N and alignments with eucaryal homologs are presented here . The similarities between archaeal transcription factors and their eucaryal homologs TFIIB and TBP have been established in other laboratories . The archaeal RNAP subunits H, K, and N, respectively, show high sequence similarity to ABC27, ABC23, and ABC10 beta (found in all three eucaryal RNAPs); subunit D, to AC40 (common to polymerase II and polymerase III) and B44 (polymerase II); and subunit L, to AC19 and B12.5 . The similarity of subunit D and its eucaryal homologs to bacterial alpha is limited to the "alpha-motif," which is also present in subunit L and its eucaryal homologs . Genes encoding homologs of the related eucaryal RNAP subunits A12.2/B12.6 and also homologs of eucaryal transcription elongation factors of the TFIIS family have been detected in Sulfolobus acidocaldarius and Thermococcus celer . In archaea, the protein is not an RNAP subunit . Together with the sequence similarities between archaeal box A-containing and eucaryal TATA box-containing promoters, this shows that the archaeal and eucaryal transcription systems are truly homologous and that they differ structurally and functionally from the bacterial transcription machinery . In contrast, however, a number of genes for the archaeal transcription apparatus are organized in clusters resembling the clusters of transcription-associated genes in Bacteria. Proc Natl Acad Sci U S A, 1995 Jun 20, 92(13), 5783 - 7 A molecular sensor system based on genetically engineered alkaline phosphatase; Brennan CA et al.; Binding and signaling proteins based on Escherichia coli alkaline phosphatase (AP; EC 3.1.3.1) were designed for the detection of antibodies . Hybrid proteins were constructed by using wild-type AP and point mutants of AP {Asp-101 --> Ser (D101S) and Asp-153 --> Gly (D153G)} . The binding function of the hybrid proteins is provided by a peptide epitope inserted between amino acids 407 and 408 in AP . Binding of anti-epitope antibodies to the hybrid proteins modulates the enzyme activity of the hybrids; upon antibody binding, enzyme activity can increase to as much as 300% of the level of activity in the absence of antibody or can decrease as much as 40%, depending on the presence or absence of the point mutations in AP . The fact that modulation is altered from inhibition to activation by single amino acid changes in the active site of AP suggests that the mechanism for modulation is due to structural alterations upon antibody binding . Modulation is a general phenomenon . The properties of the system are demonstrated by using two epitopes, one from the V3 loop of human immunodeficiency virus type 1 gp120 protein and one from hepatitis C virus core protein, and corresponding monoclonal antibodies . The trend of modulation is consistent for all hybrids; those in wild-type AP are inhibited by antibody, while those in the AP mutants are activated by antibody . This demonstrates that modulation of enzyme activity of the AP-epitope hybrid proteins is not specific to either a particular epitope sequence or a particular antibody-epitope combination. FEBS Lett, 1995 Jun 19, 367(1), 5 - 11 Mutations in the hydrophobic domain of poliovirus protein 3AB abrogate its permeabilizing activity; Lama J et al.; Poliovirus protein 3AB contains a predicted amphipathic helix that could lead to pore formation in membranes . We have introduced various mutations in the hydrophobic domain of the protein and the membrane-modifying properties of the resulting mutants have been analyzed . Expression of wild type 3AB protein in E . coli increases the influx and efflux of different molecules such as nucleosides, lactose analogues and antibiotics . Thus, 3AB expression makes E . coli cells two orders of magnitude more sensitive to hygromycin B, a non-permeant inhibitor of translation, and causes a 15-20-fold enhancement in the efflux of uridine . Changes in membrane permeability take place under conditions where no cellular lysis is detected and when other molecules such as beta-galactosidase or polyribonucleotides are kept inside the cell . These membrane modifications can be blocked to different extents by amino acid substitutions in the membrane-spanning region of the protein . These results suggest that poliovirus protein 3AB could possess an intrinsic ability to form pores in natural membranes, thus allowing the flux of small hydrophylic molecules through them. FEBS Lett, 1995 Jun 19, 367(1), 49 - 52 Production of functional chick liver HMG 2a protein in Escherichia coli; Oka T et al.; An efficient Escherichia coli system for the production of a variant form of high-mobility group-2a protein (HMG 2a), having the additional 5 amino acid residues (Ala-Pro-Thr-Leu-Glu) at the NH2-terminal, has been constructed . cDNA encoding HMG 2a was ligated with the Omp A signal peptide sequence and was inserted into an inducible bacterial expression vector pSH-L . After the plasmid introduced into E . coli was expressed by temperature shift, the recombinant product was purified by trichloacetic acid precipitation followed by Bio-Rex 70 column chromatography . The purified product showed the expected NH2-terminal sequence and the superhelical activity of circular DNA similar to the authentic HMG 2a isolated from chick liver. FEBS Lett, 1995 Jun 19, 367(1), 28 - 32 Cloning and expression of cDNA encoding a new type of ascorbate peroxidase from spinach; Ishikawa T et al.; A cDNA clone (SAP1) encoding a peroxidase was isolated from a spinach cDNA library using monoclonal antibodies raised against Euglena ascorbate peroxidase . The deduced amino acid sequence of SAP1 had higher homology with the cytosolic ascorbate peroxidases from plant sources than with bacterial peroxidases and classical plant peroxidases . The peroxidase activity of recombinant SAP1 protein expressed in E . coli was 1.6-fold higher with ascorbate than with guaiacol, which was similar to those of endogenous cytosolic ascorbate peroxidases . Here we conclude that SAP1 belongs to a new type of ascorbate peroxidase from spinach. FEBS Lett, 1995 Jun 19, 367(1), 33 - 8 Tetrahydropyrimidine derivatives inhibit binding of a Tat-like, arginine-containing peptide, to HIV TAR RNA in vitro; Lapidot A et al.; The ability of a small molecule, 2-methyl,4-carboxy,5-hydroxy-3,4,5,6-tetrahydropyrimidine (THP(A)), which accumulates intracellularly in various streptomyces, to inhibit the interaction of Tat peptide (R52) with TAR RNA is presented . Using gel-shift assay, we found that the inhibition constant Ki of THP(A) is 50-100 nM, which is in the range of the binding constants of Tat peptide and protein . THP(A) is approximately 10(6) times more tightly bound than the free L-arginine . The high binding affinity may be attributed to the special delocalized positive charge on the NCN group and the hydroxyl group at the 5 position of this molecule . A model for THP(A)-TAR interaction, analogous to the arginine guanidinum group-TAR interaction, is presented . The relatively high uptake of THP(A) by mammalian cells warrants in vivo Tat/TAR inhibition studies. J Biol Chem, 1995 Jun 16, 270(24), 14679 - 84 Low affinity binding of phorbol esters to protein kinase C and its recombinant cysteine-rich region in the absence of phospholipids; Kazanietz MG et al.; Binding of phorbol esters to protein kinase C (PKC) has been regarded as dependent on phospholipids, with phosphatidylserine being the most effective for reconstituting binding . By using a purified single cysteine-rich region from PKC delta expressed in Escherichia coli we were able to demonstrate that specific binding of {3H}phorbol 12,13-dibutyrate to the receptor still takes place in the absence of the phospholipid cofactor . However, {3H}phorbol 12,13-dibutyrate bound to the cysteine-rich region with 80-fold lower affinity in the absence than in the presence of 100 micrograms/ml phosphatidylserine . Similar results were observed with the intact recombinant PKC delta isolated from insect cells . When different phorbol derivatives were examined, distinct structure-activity relations for the cysteine-rich region were found in the presence and absence of phospholipid . Our results have potential implications for PKC translocation, for inhibitor design, and for PKC structural determination. J Biol Chem, 1995 Jun 16, 270(24), 14576 - 81 Purification of mu-calpain by a novel affinity chromatography approach . New insights into the mechanism of the interaction of the protease with targets; Molinari M et al.; A calmodulin-binding motif is a common structural feature of a number of calpain substrates (1) . Since a calmodulin-like domain has been identified in both subunits of the calpain molecule, the proposal was made that the domain(s) would recognize the calmodulin-binding motifs of the substrates prior to the enzymatic modification by calpain . In keeping with the proposal, a successful attempt to purify mu-calpain from human erythrocytes was made by using an affinity chromatography approach in which the synthetic peptide C49, containing the calmodulin-binding domain of the plasma membrane Ca(2+)-ATPase, was coupled to a Sepharose matrix . The calmodulin-like domain of the catalytic subunit of human mu-calpain expressed in Escherichia coli was also retained by the C49-Sepharose column . Both mu-calpain and the calmodulin-like domain interacted with C49 in a Ca(2+)-dependent way and were eluted from the column by Ca(2+)-chelating agents . The finding confirmed the interaction between the calmodulin-binding domain of the plasma membrane Ca(2+)-ATPase and the calmodulin-like domain of mu-calpain . Experiments were performed to establish whether irreversibly inactivated mu-calpain or its expressed C-terminal portion containing the calmodulin-like domain could activate the hydrolysis of ATP by the plasma membrane Ca2+ pump, in keeping with evident ATPase stimulation of the same pump by calmodulin . A stimulation was observed, but it was much weaker than that induced by calmodulin. J Biol Chem, 1995 Jun 16, 270(24), 14412 - 9 Structural organization of procaryotic and eucaryotic Hsp90 . Influence of divalent cations on structure and function; Jakob U et al.; Hsp90 is a very abundant molecular chaperone that apparently helps to protect cellular proteins from denaturation upon temperature upshift . The unusual ability of Hsp90 to function under conditions where other proteins unfold prompted us to investigate the stability and structural organization of Hsp90 itself . Both procaryotic and eucaryotic members of the Hsp90 family were found to have very similar physicochemical properties: (i) they are stable against thermal unfolding up to at least 50 degrees C, (ii) they show biphasic, reversible unfolding transitions in guanidinium chloride, and (iii) their oligomerization state is strongly and rapidly affected by millimolar concentrations of divalent cations . In the presence of MnCl2 and MgCl2 defined changes in the quaternary structure of Hsp90 could be observed which resulted in a decrease in thermostability and an increased tendency to form larger aggregates . The addition of divalent cations also almost completely abolished the chaperone function of Hsp90 and induced release of folding intermediates of citrate synthase bound to Hsp90 . These modulating effects of divalent cations on structure and function of Hsp90 in vitro represent a potential mechanism for regulation of Hsp90 chaperone action in vivo. J Biol Chem, 1995 Jun 16, 270(24), 14297 - 304 Mutagenesis studies of the phosphorylation sites of recombinant human pyruvate dehydrogenase . Site-specific regulation; Korotchkina LG et al.; Mammalian pyruvate dehydrogenase (alpha 2 beta 2) (E1) is regulated by phosphorylation-dephosphorylation, catalyzed by the E1-kinase and the phospho-E1-phosphatase . Using site-directed mutagenesis of the three phosphorylation sites (sites 1, 2, and 3) on E1 alpha, several human E1 mutants were made with single, double, and triple mutations by changing Ser to Ala . Mutation at site 1 but not at sites 2 and/or 3 decreased E1 specific activity and also increased Km values for thiamin pyrophosphate and pyruvate . Sites 1, 2, and 3 in the E1 mutants were phosphorylated either individually or in the presence of the other sites by the dihydrolipoamide acetyltransferase-protein X-E1 kinase indicating a site-independent mechanism of phosphorylation . Phosphorylation of each site resulted in complete inactivation of the E1 . However, the rates of phosphorylation and inactivation were site-specific . Sites 1, 2, and 3 were dephosphorylated either individually or in the presence of the other sites by the phospho-E1-phosphatase resulting in complete reactivation of the E1 . The rates of dephosphorylation and reactivation were similar for sites 1, 2, and 3, indicating a random dephosphorylation mechanism. J Biol Chem, 1995 Jun 16, 270(24), 14286 - 91 Characterization of the binding site for cyclothialidine on the B subunit of DNA gyrase; Nakada N et al.; The mechanism of inhibition of DNA gyrase by cyclothialidine, a novel gyrase inhibitor isolated from Streptomyces filipinensis NR0484, has been studied further by using {14C}benzoylcyclothialidine and a reconstituted Escherichia coli gyrase system consisting of the A subunit, the B subunit and relaxed ColE1 DNA . The mechanism of inhibition was also studied with the 43-kDa N-terminal fragment of the B subunit . The {14C}benzoylcyclothialidine could bind to the B subunit alone but not to the A subunit nor to the plasmid DNA alone . Furthermore, the compound also bound to the 43-kDa N-terminal fragment of the B subunit . Scatchard analysis of {14C}benzoylcyclothialidine binding to DNA gyrase showed that the binding affinity of the compound increased, depending on the assembly of the gyrase (A2B2) . DNA complex . This suggests that the binding site of cyclothialidine on the B subunit or its vicinity causes a conformational change during the assembly of the gyrase.DNA complex (increase in affinity: B-->A2B2-->A2B2.DNA) . Furthermore, displacement curves of {14C}benzoylcyclothialidine binding by nonlabeled cyclothialidine, ATP analogues, and coumarin antibiotics indicated that cyclothialidine, coumarins, and ATP share a common (or overlapping) site of action on the B subunit of DNA gyrase; however, the microenvironment of the binding sites may differ. J Mol Biol, 1995 Jun 16, 249(4), 785 - 99 Crystal structure of Escherichia coli QOR quinone oxidoreductase complexed with NADPH; Thorn JM et al.; The crystal structure of the homodimer of quinone oxidoreductase from Escherichia coli has been determined using the multiple isomorphous replacement method at 2.2 A resolution and refined to an R-factor of 14.1% The crystallographic asymmetric unit contains one functional dimer with the two subunits being related by a non-crystallographic 2-fold symmetry axis . The model consists of two polypeptide chains (residues 2 through 327), one NADPH molecule and one sulphate anion per subunit, and 432 water molecules . Each subunit consists of two domains: a catalytic domain and a nucleotide-binding domain with the NADPH co-factor bound in the cleft between domains . Quinone oxidoreductase has an unusual nucleotide-binding fingerprint motif consisting of the sequence AXXGXXG . The overall structure of quinone oxidoreductase shows strong structural homology to that of horse liver alcohol dehydrogenase. Neurosci Lett, 1995 Jun 16, 192(3), 169 - 72 Neurotrophin-3 is expressed in the posterior lobe of mouse cerebellum, but does not affect the cerebellar development; Tojo H et al.; We replaced the neurotrophin-3 (NT-3) gene with Escherichia coli-derived lacZ via homologous recombination in embryonic stem (ES) cells and generated the mutant mice . Here we show the in vivo expression of NT-3 in the cerebellum during the postnatal period . A high level of lacZ expression was found in the granular layer of posterior lobe (lobules VII to X) in the postnatal NT-3(+/-) cerebellum . The expression in these regions was reduced with age . Although the Purkinje cells are considered to be a target of NT-3 and the NT-3(-/-) mice displayed severe moving disorders like ataxia, no histological abnormality was observed in their cerebellum . These findings suggest that the NT-3 expressed in the cerebellum gives some trophic effects primarily to the posterior lobe, however, the deficiency does not affect its development. Biochem J, 1995 Jun 15, 308 ( Pt 3), 901 - 8 Modulation of the activity of human 17 alpha-hydroxylase-17,20-lyase (CYP17) by cytochrome b5: endocrinological and mechanistic implications; Lee-Robichaud P et al.; Using NADPH-cytochrome P-450 reductase as electron donor the homogeneous pig 17 alpha-hydroxylase-17,20-lyase (CYP17) was shown to catalyse the conversion of delta 5, as well as delta 4, steroids (pregnenolone and progesterone respectively) predominantly into the corresponding 17 alpha-hydroxylated products . The latter were then cleaved by the lyase (desmolase) activity of the enzyme into androgens . Cytochrome b5 stimulated both these activities, but its most noticeable effect was on the formation of delta 16-steroids, which compulsorily required the presence of cytochrome b5 . These results on the pig enzyme confirm the original findings {Nakajin, Takahashi, Shinoda and Hall (1985) Biochem . Biophys . Res . Commun . 132, 708-713} . The human CYP17 expressed in Escherichia coli {Imai, Globerman, Gertner, Kagawa and Waterman (1993) J . Biol . Chem . 268, 19681-19689} was also purified to homogeneity and was found to catalyse the hydroxylation of pregnenolone and progesterone without requiring cytochrome b5 . Like the pig CYP17, the human CYP17 also catalysed the cytochrome b5-dependent direct cleavage of pregnenolone into the delta 5,16-steroid, but unlike it the human enzyme did not cleave progesterone at all . 17 alpha-Hydroxypregnenolone was, however, cleaved into the corresponding androgen but only in the presence of cytochrome b5 . 17 alpha-Hydroxyprogesterone was a poor substrate for the human CYP17; although it was converted into androstenedione in the presence of cytochrome b5 its K(m) was 5 times higher and Vmax . 2.6 times lower than those for the hydroxylation of progesterone . The endocrinological and mechanistic implications of these results are discussed. EMBO J, 1995 Jun 15, 14(12), 2958 - 66 Type III restriction endonucleases translocate DNA in a reaction driven by recognition site-specific ATP hydrolysis; Meisel A et al.; Type III restriction/modification systems recognize short non-palindromic sequences, only one strand of which can be methylated . Replication of type III-modified DNA produces completely unmethylated recognition sites which, according to classical mechanisms of restriction, should be signals for restriction . We have shown previously that suicidal restriction by the type III enzyme EcoP15I is prevented if all the unmodified sites are in the same orientation: restriction by EcoP15I requires a pair of unmethylated, inversely oriented recognition sites . We have now addressed the molecular mechanism of site orientation-specific DNA restriction . EcoP15I is demonstrated to possess an intrinsic ATPase activity, the potential driving force of DNA translocation . The ATPase activity is uniquely recognition site-specific, but EcoP15I-modified sites also support the reaction . EcoP15I DNA restriction patterns are shown to be predetermined by the enzyme-to-site ratio, in that site-saturating enzyme levels elicit cleavage exclusively between the closest pair of head-to-head oriented sites . DNA restriction is blocked by Lac repressor bound in the intervening sequence between the two EcoP15I sites . These results rule out DNA looping and strongly suggest that cleavage is triggered by the close proximity of two convergently tracking EcoP15I-DNA complexes. Biochem Biophys Res Commun, 1995 Jun 15, 211(2), 627 - 38 Recombinant expression of hepatitis A virus protein 3A: interaction with membranes; Pisani G et al.; The function of hepatitis A virus (HAV) protein 3A and its structural requirements were studied in vitro and in a bacterial expression system by comparing the polypeptide precursor 3AB derived from a cytopathogenic strain with that of an attenuated strain . Although the precursor polypeptides 3AB of both HAV strains bind to microsomal membranes after translation in vitro they differ in inducing membrane permeability when expression is induced in bacteria . Intake and release of macromolecules was dramatically increased when 3AB of the cytopathogenic strain was expressed . Amino acid sequence alignments suggest that membrane binding might be due to a hydrophobic stretch near the C-terminus of 3A found in all picornaviruses whereas the ability to induce permeability of E . coli membranes is determined by an amphipathic helix formed at the N-terminus of 3A of HAVFG. Biochem Biophys Res Commun, 1995 Jun 15, 211(2), 365 - 9 Deletion and site-directed mutagenesis of EF-hand domain of phospholipase C-delta 1: effects on its activity; Nakashima S et al.; In order to elucidate a role of a putative EF-hand motif (144-172) in phospholipase C-delta 1 (PLC-delta 1), deletion and point mutation of the enzyme were performed and the mutated cDNAs were expressed in CHO cells and E . coli AD202 strain . Deletion of amino acid residues of 141-236 or 173-236 resulted in abolition of PLC activity . However, the decreased PLC activity to 15-20% by deletion of the EF-hand motif (144-172) was still Ca(2+)-dependent . Furthermore, mutants, in which conserved Asp153, Asp157, Glu164 or all these acidic amino acids in the EF-hand motif were replaced with alanine residues, showed nearly the same PLC activity and Ca(2+)-dependency as those of wild-type . These results suggest that the region containing the EF-hand motif may not play a role in regulation of Ca(2+)-sensitivity of PLC-delta 1, but is important for its activity. Nature, 1995 Jun 15, 375(6532), 554 - 60 The 2.2 A crystal structure of the Ras-binding domain of the serine/threonine kinase c-Raf1 in complex with Rap1A and a GTP analogue; Nassar N et al.; The X-ray crystal structure of the complex between the Ras-related protein Rap1A in the GTP-analogue (GppNHp) form and the Ras-binding domain (RBD) of the Ras effector molecule c-Raf1, a Ser/Thr-specific protein kinase, has been solved to a resolution of 2.2 A . It shows that RBD has the ubiquitin superfold and that the structure of Rap1A is very similar to that of Ras . The interaction between the two proteins is mediated by an apparent central antiparallel beta-sheet formed by strands B1-B2 from RBD and strands beta 2-beta 3 from Rap1A . Complex formation is mediated by main-chain and side-chain interactions of the so-called effector residues in the switch I region of Rap1A. Cancer Res, 1995 Jun 15, 55(12), 2503 - 6 Mutational effects on the p16INK4a tumor suppressor protein; Yang R et al.; Several point mutations of p16INK4a were studied by site-specific mutagenesis and functional analysis to assess the effects of these mutations on the function of the protein . These mutations were reported in several malignancies . Three deletional mutants of p16INK4a were also analyzed to reveal the relationship between p16INK4a and p15INK4b and to test the importance of the ankyrin repeats observed in both proteins . We studied the activity of these mutants using the yeast two-hybrid system and an in vitro kinase assay . Our results suggest that point mutations in the conserved ankyrin consensus affect the activity of p16INK4a . However, not all of the point mutations observed in tumors have a detectable effect on the activity . The COOH-terminal region of p16INK4a is not required for the protein to bind and to inhibit CDK4, but the deletion of the 4th ankyrin repeat abolished the activity completely. J Immunol, 1995 Jun 15, 154(12), 6355 - 64 Neutralizing murine monoclonal antibodies to human IL-5 isolated from hybridomas and a filamentous phage Fab display library; Ames RS et al.; Conventional hybridomas and combinatorial Ab libraries were used to develop neutralizing murine mAbs to human IL-5 . Mice were immunized with rIL-5 . Spleens from two mice were used to generate hybridomas . Spleens from an additional three mice were used to construct a combinatorial library . In both instances, Abs were identified and selected by ELISA using 96-well plates coated with rIL-5 . These Abs were tested for the ability to block binding of iodinated rIL-5 to the alpha-chain of the human IL-5 receptor (IL-5R alpha) and to inhibit proliferation of IL-5-dependent cells . By hybridoma technology, 16 mAbs were obtained, 11 of which blocked binding to IL-5R alpha, including three that inhibited proliferation . Quantitative binding assays and sequence analysis revealed that these latter three mAbs were closely related . Combinatorial cloning and selection by phage display was used to isolate 24 bacterial colonies secreting Fabs that bound to 125I-rIL-5 and to rIL-5-coated plates . Sequencing of 10 of the Fabs indicated that four unique Abs were obtained, comprising one predominant VH paired with one of two different VL . The sequence of the Fabs was distinct from the sequences of the neutralizing mAbs . In contrast to the mAbs, none of the Fabs blocked binding of 125I-IL-5 to IL-5R alpha or neutralized the biologic activity of IL-5 . The inability to identify neutralizing Fabs was shown not to result from their monovalency, because a Fab derived from one of the neutralizing mAbs, by cloning and expression of its Fd and kappa light chains, retained neutralizing activity . By chain shuffling, pairing of the Fd fragment of the heavy chain of one of the neutralizing mAbs (2B6), with the light chain library derived from the IL-5-immunized mice, neutralizing Fabs were obtained . These Fabs contained light chain sequences closely related to the original light chain of 2B6 . Hence, chain shuffling allowed detection of a light chain sequence that was not evident upon two-chain combinatorial selection . The results reveal differences in the Abs obtained from a combinatorial library vs hybridomas and demonstrate how these approaches can be used in concert to select mAbs with neutralizing activity. Clin Chim Acta, 1995 Jun 15, 237(1-2), 43 - 58 Recombinant M-, B- and MB-type isozymes of human phosphoglyceric acid mutase: their large-scale production and preparation of polyclonal antibodies specific to M- and B-type isozymes; Uchida K et al.; Human phosphoglyceric acid mutase is a dimer comprising M-, B- and MB-type isozymes composed from the combination of the muscle-specific (M) and non-muscle-specific (B) subunits . Human DNAs coding M and B subunits were, respectively, reconstructed at their 5' regions without changing amino acid sequences, and expressed directly in Escherichia coli under the control of the trp promoter . M- and B-type isozymes were over-produced in the bacterial cytoplasm as soluble, active forms, which have been purified and characterized . MB-type was synthesized in vitro by recombining M- and B-type . All three recombinant isozymes thus obtained showed the same properties as the naturally-occurring ones with respect to the properties tested . Polyclonal IgGs specific to the M-type, B-type and MB-type were prepared from rabbits immunized with M- and B-type, using columns bound with M- and B-type . A method for the immunoassay of MB-type which is specifically present in cardiac muscle, is now under development. FEMS Microbiol Lett, 1995 Jun 15, 129(2-3), 237 - 42 Identification of EaeA protein in the outer membrane of attaching and effacing Escherichia coli O45 from pigs; Zhu C et al.; We have previously reported that the production of attaching and effacing lesions by Escherichia coli O45 isolates from pigs is associated with the eaeA (E . coli attaching and effacing) gene . In the present study, expression of the EaeA protein, the eaeA gene product, among swine O45 E . coli isolates was examined . The majority (20/22) of attaching and effacing positive, eaeA+ E . coli O45 isolates, but none of ten attaching and effacing negative, eaeA- or eaeA+ isolates, expressed a 97-kDa outer membrane protein as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis . Amino-terminal amino acid sequencing demonstrated a high homology between this 97-kDa protein of swine E . coli O45 and the EaeA protein (intimin) of human enteropathogenic E . coli and enterohemorrhagic E . coli . In addition, a serological relationship between the EaeA proteins of swine O45, rabbit (RDEC-1) and human (E2348/69) attaching and effacing E . coli strains was observed . Our results indicate an association between expression of the EaeA protein and attaching and effacing activity among O45 E . coli isolates . The data also suggest an antigenic relatedness of the EaeA proteins of swine, rabbit, and human attaching and effacing E . coli. FEMS Microbiol Lett, 1995 Jun 15, 129(2-3), 175 - 81 Non-reciprocal regulation of Rhodobacter capsulatus and Rhodobacter sphaeroides recA genes expression; Fernandez de Henestrosa AR et al.; The Rhodobacter capsulatus recA gene has been isolated and sequenced . Its deduced amino acid sequence showed the closest identity with the Rhodobacter sphaeroides RecA protein (91% identity) . However, the promoter regions of both R . capsulatus and R . sphaeroides recA genes are only 64% similar . An Escherichia coli-like LexA binding site was not present in the upstream region of the R . capsulatus recA gene . Nevertheless, the R . capsulatus recA gene is inducible by DNA damage in both hetero- and phototrophically growing conditions . The R . capsulatus recA gene is poorly induced when inserted into the chromosome of R . sphaeroides, indicating that the recA gene of both bacteria possess different control sequences despite their phylogenetically close relationship. FEMS Microbiol Lett, 1995 Jun 15, 129(2-3), 157 - 62 A recombinant foot-and-mouth disease virus antigen inhibits DNA replication and triggers the SOS response in Escherichia coli; Benito A et al.; The 3D gene of foot-and-mouth disease virus encodes the viral RNA dependent RNA polymerase, also called virus infection associated (VIA) antigen, which is the most important serological marker of virus infection . This 3D gene from a serotype C1 virus has been cloned and overexpressed in Escherichia coli under the control of the strong lambda lytic promoters . The resulting 51 kDa recombinant protein has been shown to be immunoreactive with sera from infected animals . After induction of gene expression, an immediate and dramatic arrest of cell DNA synthesis occurs, similar to that produced by genotoxic doses of the drug mitomycin C . This effect does not occur during the production of either a truncated VIA antigen or other related and non-related viral proteins . The inhibition of DNA replication results in a subsequent induction of the host SOS DNA-repair response and in an increase of the mutation frequency in the surviving cells. Genes Dev, 1995 Jun 15, 9(12), 1543 - 57 A developmentally regulated chromosomal origin of replication uses essential transcription elements; Marczynski GT et al.; Only one of the two chromosomes in the asymmetric Caulobacter predivisional cell initiates replication in the progeny cells . Transcription from a strong promoter within the origin occurs uniquely from the replication-competent chromosome at the stalked pole of the predivisional cell . This regulated promoter has an unusual sequence organization, and transcription from this promoter is essential for regulated (cell type-specific) replication . Our analysis defines a new class of bacterial origins and suggests a coupling between transcription and replication that is consistent with the phylogenetic relationship of Caulobacter to the ancestral mitochondrion. Eur J Biochem, 1995 Jun 15, 230(3), 958 - 64 Site-directed mutagenesis studies on the lima bean lectin . Altered carbohydrate-binding specificities result from single amino acid substitutions; Jordan ET et al.; The wild-type seed lima bean lectin (LBL), and recombinant LBL expressed in Escherichia coli show specificity for the human blood group A immunodominant trisaccharide GalNAc alpha 1-3{Fuc alpha 1-2}Gal beta 1-R . We have generated four site-specific mutants of LBL, two of which show altered specificity for extended carbohydrate structures . Four mutants, {C127Y}LBL, {H128P}LBL, {H128R}LBL and {W132F}LBL were expressed in E . coli . Two mutants show altered specificity for the substituent at the C2 hydroxy group of the penultimate Gal in the wild-type ligand which is alpha-L-fucose in the A trisaccharide . The mutant {C127Y}LBL showed specificity for the A disaccharide (GalNAc alpha 1-3Gal) and GalNAc alpha 1-4Gal, with free hydroxyl groups at the C2 position of Gal . The mutant {H128P}LBL bound the Forssman disaccharide structure GalNAc alpha 1-3GalNAc, in which the C2 hydroxyl group is substituted with an acetamido group . The third and fourth mutants, {H128R}LBL and {W132F}LBL, exhibited wild-type specificities, both recognizing the A trisaccharide . All of these mutant lectins bound the terminal GalNAc residues exposed on asialoovine submaxillary mucin, thus indicating that the monosaccharide-binding site had not been altered . We also determined that all but one mutant ({C127Y}LBL) retained the high-affinity binding site for N6 derivatives of adenine, indicative of tetramer formation; each mutant also expressed the low-affinity binding site for 8-anilinonaphthalene 1-sulfonate (1/monomer) . Thus, by targeting two residues in LBL, we have identified a region of the protein that is part of the extended carbohydrate-binding site and which is specifically involved in the binding/recognition of substituents at the C2 position of the penultimate Gal of the A disaccharide . We have determined, by site-directed mutagenesis, that an essential Cys residue is involved in the specificity of LBL for the A trisaccharide. Eur J Biochem, 1995 Jun 15, 230(3), 1111 - 7 Site-directed mutagenesis and structure/function studies of casein kinase II correlate stimulation of activity by the beta subunit with changes in conformation and ATP/GTP utilization; Jakobi R et al.; Casein kinase II exists in vivo as an active holoenzyme consisting of catalytic alpha and/or alpha' and regulatory beta subunits, which form a tetrameric structure of alpha 2 beta 2 . Unlike most other protein kinases, casein kinase II uses both ATP and GTP effectively as phosphate donors . Two residues unique to the catalytic subunit of casein kinase II, Val66 and Trp176, were mutated to Ala66 and Phe176, respectively, the amino acids present in more than 95% of the identified protein kinase sequences . Using recombinant alpha subunit expressed in Escherichia coli, the mutations have been previously shown to reduce the utilization of GTP by changing Km values for ATP and GTP and to reduce the approximate fivefold stimulation observed upon addition of the regulatory subunit {Jakobi, R . & Traugh, J . A . (1992) J . Biol . Chem . 267, 23,894-23,902} . In this study, the mutations are shown to affect the catalytic activity of the reconstituted holoenzyme by changing both Km and Vmax values . The Vmax for ATP is reduced by the mutation of Trp176 to phenylalanine, but no change is observed with GTP . The Val66 to alanine and Val66/Trp176 to alanine/phenylalanine mutations reduce the Vmax values for ATP and GTP to levels comparable to those of the catalytic subunits alone, indicating that changes in the stimulation of activity by the beta subunit are due to changes in Vmax . Structural studies using ultraviolet CD spectroscopy show that changes in stimulation of Vmax by the beta subunit are correlated with changes in the secondary structure; the extent of these changes is reduced by both mutations . Correlation of changes in secondary structure and stimulation of activity by the beta subunit indicate that the formation of the wild-type holoenzyme causes conformational changes in the active site, leading to an increased rate of reaction . As shown by the mutations, Val66 and Trp176 are involved both in the conformational changes and in the selectivity of ATP and GTP. Eur J Biochem, 1995 Jun 15, 230(3), 1053 - 8 Vitamin-B12-independent methionine synthase from a higher plant (Catharanthus roseus) . Molecular characterization, regulation, heterologous expression, and enzyme properties; Eichel J et al.; Methionine synthases catalyze the formation of methionine by the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine . This reaction is the last step in L-methionine biosynthesis, and it also serves to regenerate the methyl group of S-adenosylmethionine, a cofactor required for biological methylation reactions . We describe the cloning, expression and characterization of a methionine synthase from the higher plant Catharanthus roseus . cDNAs were identified that encoded a protein of 85 kDa sharing 50% identify with the cobalamin-independent methionine synthase from Escherichia coli (MetE) and 41% identity with a partial sequence of a yeast homolog of MetE . The C . roseus protein was expressed at high levels in E . coli . The enzyme accepts the triglutamate form of methyltetrahydrofolate as a methyl donor but not the monoglutamate form, and it does not require S-adenosylmethionine or cobalamin for activity . The properties indicate that the enzyme is a cobalamin-independent methionine synthase (EC 2.1.1.14) . In contrast to the E . coli MetE, the plant protein does not require phosphate or magnesium ions for activity . Immunoblots of plants extracts showed that the protein was localized in the cytosol, and was present in a variety of plant species . A nutritional downshift of the C . roseus cell culture revealed a strong, transient transcriptional activation, but no significant increment in the total level of the protein . The availability of the protein and the cDNA now provide tools to investigate the complexities of methionine biosynthesis in plants. J Immunol Methods, 1995 Jun 14, 183(1), 95 - 101 Mutagenesis of immunoglobulin-like domains from the extracellular human interferon-gamma receptor alpha chain and their recognition by neutralizing antibodies monitored by surface plasmon resonance technology; Ruegg N et al.; The extracellular portion of the human interferon-gamma receptor (IFN-gamma R) is predicted to adopt two Ig-like domains each with Ig superfamily type C2 topology . Surface plasmon resonance technology has been used here to compare the equilibrium and kinetic constants for binding reactions between these and several mutant domains, and neutralising anti-IFN-gamma R monoclonal antibodies . The biosensor measurements provide a sensitive method for monitoring the effects of mutations on the functional epitopes recognized by the neutralising antibodies . Thus, using recombinant native-like proteins made in E . coli, the ten N-terminal residues of the receptor were found not to be essential for domain folding, nor for recognition by the antibodies A6, D2 and gamma R38 . In a similar way, residues in the interdomain region were found to play an important functional role in the epitope recognized by the antibody gamma R99. Biochemistry, 1995 Jun 13, 34(23), 7694 - 702 Involvement of histidine-91 of the beta subunit in proton translocation by the pyridine nucleotide transhydrogenase of Escherichia coli; Glavas NA et al.; The pyridine nucleotide transhydrogenase (EC 1.6.1.1) carries out transmembrane proton translocation coupled to transfer of a hydride equivalent between NAD+ and NADP+ . Mutations were made in histidine-91 of the beta subunit of the pyridine nucleotide transhydrogenase of Escherichia coli . This amino acid is the only conserved charged residue in the transmembrane domains of this enzyme and thus potentially is involved in proton translocation by the transhydrogenase . The mutant beta H91N retained 80% of the hydride transfer activity while proton translocation was reduced to 7% . This behavior is consistent with a role for beta His91 in the proton translocation pathway . Other mutations at this residue affected the conformation of the enzyme . Thus, the enzyme in mutants beta H91C, beta H91T, and beta H91S was unable to undergo the conformational change that occurred on binding of the substrates NADP+ or NADPH . By contrast, the enzyme in the beta H91K mutant was present in the NADP(H)-induced conformation even in the absence of these substrates . Further evidence for the linkage between beta His91 and the conformation of the beta subunit was obtained by labeling the transmembrane domain of the beta subunit with {14C}N,N'-dicyclohexylcarbodiimide (DCCD) . Labeling occurred most readily with the enzyme of beta H91K . It is concluded that beta His91 is a component of the proton translocation pathway of the transhydrogenase and that its state of protonation is probably linked to conformational changes induced by binding/debinding of substrates during the catalytic cycle of the enzyme. Biochemistry, 1995 Jun 13, 34(23), 7686 - 93 Resonance Raman characterization of the binary and ternary complexes of thymidylate synthase with 5-nitrodeoxyuridylate; Austin JC et al.; Resonance Raman (RR) spectra are reported for the binary complex of Escherichia coli thymidylate synthase (TS) with the substrate analog inhibitor 5-nitrodeoxyuridylate (NDU) . The TS/NDU binary complex RR spectrum shows many similarities to the RR spectra of thiol adducts of NDU or of 5-nitro-1-methyluracil formed in solution, providing strong evidence in support of the formation of a covalent link between Cys146 of TS and C6 of NDU . Spectral differences between the model compounds and the binary complex reflect the consequences of fixing the conformations of the uracil and ribose rings at the enzyme active site . The RR spectra of the ternary complexes of TS/NDU with either tetrahydrofolate (H4-folate) or the cofactor 5,10-methylenetetrahydrofolate (CH2H4-folate) show that a covalent link is not formed between C11 of CH2H4-folate and C5 of NDU . Neither does the methylene bridge of CH2H4-folate remain intact in the ternary complex; either CH2H4-folate is present as the N5 iminium cation species or the methylene group is lost as formaldehyde . A shift in the NO2 symmetric stretching frequency in the ternary complex indicates expulsion of water molecules from the region of the NO2 group by the cofactor. Biochemistry, 1995 Jun 13, 34(23), 7678 - 85 Stereoelectronic activation of methylenetetrahydrofolate by thymidylate synthase: resonance Raman spectroscopic evidence; Austin JC et al.; Resonance Raman (RR) spectra are reported for the ternary complex of Escherichia coli thymidylate synthase with the cofactor 5,10-methylenetetrahydrofolate (CH2-H4-folate) and the inhibitor 5-fluoro-2'-deoxyuridylate, excited at 337 or 356 nm, in resonance with perturbed absorption bands of the p-aminobenzoylglutamate (PABA-Glu) portion of the cofactor . For comparison, RR spectra were obtained with 260 nm excitation for PABA-Glu in various solvents, and for CH2H4-folate and H4-folate in aqueous solution . These reference spectra are assigned to modes of PABA-Glu in its benzenoid form . The ternary complex RR spectra are very different, however, and are assigned, with the aid of isotopic data, to the PABA-Glu in a predominantly quinoid form . Similar spectra were obtained for the ternary complexes of the E58Q and K48Q mutants, indicating that neither Glu58 nor Lys48 are essential for maintaining the quinoid structure, even though their side chains complement the dipolar charge distribution of the quinoid form of PABA-Glu . Since these are the only charged residues in the PABA-Glu vicinity, electrostatic stabilization is not essential to maintenance of the quinoid structure . It is proposed that quinoid formation results from steric forces, probably resulting from the protein conformation change known to accompany cofactor binding, which enforce coplanarity of the PABA-Glu ring and substituents . This stereoelectronic change activates the cofactor by opening the methylene bridge . A second RR spectrum of the ternary complex, previously proposed to reflect an alternate structure, is shown to result instead from irreversible formation of a laser-induced photoproduct. Biochemistry, 1995 Jun 13, 34(23), 7668 - 77 Heteronuclear NMR studies of the interactions of 15N-labeled methionine-specific transfer RNAs with methionyl-tRNA transformylase; Wallis NG et al.; In Escherichia coli the methionylated initiator methionyl-tRNA (tRNAfMet) is formylated on the aminoacyl moiety by the enzyme methionyl-tRNA transformylase . The methionylated elongator methionyl-tRNA (tRNAmMet) is not modified in this way . In order to gain structural information about this specific recognition, solution NMR studies were carried out . To be able to identify changes that were occurring in the tRNA molecule on interaction with the methionyl-tRNA transformylase, the imino protons involved in secondary and tertiary base pairing in the tRNAfMet and tRNAmMet molecules first had to be assigned to specific resonances in the NMR spectra . A combination of 2D NOESY, 2D HMQC, and 3D NOESY--HMQC spectra were used on uniformly 15N-labeled samples . After assignment of the base pairs of the tRNA, the two forms of tRNA were separately mixed with transformylase in a 1:1 molar ratio . The HMQC spectra of both the tRNAmMet and the tRNAfMet showed general broadening, but in the tRNAfMet HMQC spectra a decrease in the intensity of several resonances was also observed . These resonances had been assigned to the acceptor stem of the tRNA, confirming site-directed mutagenesis experiments that it is the acceptor stem of the tRNA which is important in conferring the specificity for the transformylase . The loss of intensity of the acceptor stem resonances suggests that this part of tRNAfMet melts upon binding to the enzyme. Biochemistry, 1995 Jun 13, 34(23), 7622 - 8 Solution structure of the C-terminal single-stranded DNA-binding domain of Escherichia coli topoisomerase I; Yu L et al.; Escherichia coli DNA topoisomerase I catalyzes the interconversion of different topological forms of DNA . In this paper we describe NMR studies of a 14K C-terminal fragment of this enzyme that binds preferentially to single-stranded DNA and enhances the enzyme's ability to relax negatively supercoiled DNA under high salt conditions . The 1H, 13C, and 15N resonances of the protein were assigned from a number of heteronuclear multidimensional NMR experiments, and the three-dimensional structure of the protein was determined from a total of 2188 NMR-derived restraints . The root-mean-square deviation about the mean coordinate positions for residues 13-120 is 0.68 +/- 0.11 A for the backbone atoms and 1.09 +/- 0.09 A for all heavy atoms . The overall fold, which consists of two four-stranded beta-sheets separated by two helices, differs from other DNA- and RNA-binding proteins such as gene 5, cold shock protein, and hnRNP C . From an analysis of the changes in chemical shift upon the addition of single-stranded DNA, the location of the oligonucleotide binding site was determined . The binding site consists of a beta-sheet containing positively charged and aromatic amino acids and, in spite of its different structure, is similar to that found in other proteins that bind single-stranded oligonucleotides. Biochemistry, 1995 Jun 13, 34(23), 7576 - 85 Cyanide and carbon monoxide binding to the reduced form of cytochrome bo from Escherichia coli; Mitchell R et al.; Cyanide binds to fully reduced cytochrome bo and induces a blue shift of the Soret absorption band of the high-spin heme o and a change in the visible region spectrum consistent with the expected conversion to a low-spin state . The dissociation constant, determined by titration of the extent of the binding spectrum, is 7.0 +/- 0.6 mM at pH 7.0 . In contrast, cyanide does not bind significantly in this concentration range to the reduced form of cytochrome bd . The reduced cyanide compound of cytochrome bo can be laser photolyzed . Typically, less than 20% photolysis was attained with conditions that give essentially full photolysis of the carbon monoxide compound . The subsequent monophasic kinetics of recombination of cyanide at varying cyanide concentrations were used to determine kon, koff, and dissociation constant values at pH 7.0 of 572 +/- 43 M-1 s-1, 4.2 +/- 0.7 s-1, and 7.3 +/- 1.3 mM, respectively . The dissociation constant changes very little in the pH range 6-8, indicating that a proton is bound together with the cyanide anion, as predicted by our recent proposal of a requirement for electroneutrality in the binuclear center {Mitchell, R., & Rich, P . R . (1994) Biochim . Biophys . Acta 1186, 19-26} . Competition studies confirm that cyanide and carbon monoxide cannot bind simultaneously, so that their binding sites must overlap . A small fraction of the reduced unliganded enzyme appears to have a distinct photolysis spectrum in the absence of added ligands, and this is transformed into a typical ferrous cyanide compound only at very high cyanide concentrations . Cyanide binding and photolysis were also examined in a number of mutant forms of cytochrome bo, and in a wild-type form which was partially depleted in CuB . Dramatic changes in rate constants and binding constants were found in several cases . Data from several mutants were compared with analogous data on the binding and photolysis of carbon monoxide, and the effects of mutation were quite different with the two ligands . A model is developed to explain the observed effects of point mutations on the recombination kinetics of both carbon monoxide and cyanide . Overall, the results indicate that the CuB site is required for binding of cyanide, but not carbon monoxide, to the reduced enzyme, possibly by providing the site for binding of the associated proton. FEBS Lett, 1995 Jun 12, 366(2-3), 115 - 8 Cloning and controlled overexpression of the gene encoding the 35 kDa soluble lytic transglycosylase from Escherichia coli; Dijkstra AJ et al.; The lytic transglycosylases of Escherichia coli are involved in peptidoglycan metabolism and resemble the lysozymes not only in activity, but in the case of the 70 kDa soluble lytic transglycosylase (Slt70), also structurally . Here we report the cloning of the gene that encodes the 35 kDa soluble lytic transglycosylase (Slt35) of E . coli . Based on the sequence of the full-length gene, Slt35 is very likely to be a proteolytically truncated form of a slightly larger protein . The homology between Slt35 and Slt70, albeit poor, indicates that the active site architecture of both proteins may be alike . Using the T-7 promoter system, Slt35 was overproduced in large quantities and purified to homogeneity for crystallographic purposes. FEBS Lett, 1995 Jun 12, 366(2-3), 109 - 14 The GLUT4 glucose transporter and the alpha 2 subunit of the Na+,K(+)-ATPase do not localize to the same intracellular vesicles in rat skeletal muscle; Lavoie L et al.; The GLUT4 glucose transporter and the alpha 2 subunit of the Na+,K(+)-ATPase of rat skeletal muscle are two proteins which redistribute from intracellular membranes to plasma membranes following in vivo insulin stimulation . Here we show that although both proteins co-segregate after subcellular fractionation of unstimulated rat hindlimb muscles, they do not share the same intracellular residence inside the muscle fibre . By immunogold single- and double-labeling on ultrathin muscle cryosections with specific antibodies, the GLUT4 glucose transporter and the Na+,K(+)-ATPase alpha 2 subunit were observed on different vesicular structures within the cell . GLUT4 was detected on subsarcolemmal and perinuclear membranes, and at the junction between myofibrillar A and I bands where triads are localized . The alpha 2 subunit of the Na+,K(+)-ATPase was observed at the plasma membrane and in distinct subsarcolemmal vesicles and intermyofibrillar membranes . Quantitative analysis of double-labeling of GLUT4 and Na+,K(+)-ATPase alpha 2 subunit revealed that less than 6% of the two proteins co-localize in the same continuous vesicular structures . The differential intracellular localization of the two proteins was further confirmed by immunopurification of GLUT4-containing membranes from muscle homogenates, in which the alpha 2 subunit of the Na+,K(+)-ATPase was found only at the same extent as the alpha 1 subunit of the enzyme, a protein exclusively present at the plasma membrane. Nucleic Acids Res, 1995 Jun 11, 23(11), 2030 - 6 Analysis of the proteins and cis-acting elements regulating the stress-induced phage shock protein operon; Weiner L et al.; The phage shock protein operon (pspABCE) of Escherichia coli is strongly induced by adverse environmental conditions . Expression is controlled principally at the transcriptional level, and transcription is directed by the sigma factor sigma 54 . PspB and PspC are required for high-level psp expression during osmotic shock, ethanol treatment and f1 infection, but heat-induced expression is independent of these proteins . We report here that the promoter region contains an upstream activation sequence (UAS) that is required for psp induction and has the enhancer-like ability to activate at a distance . A DNA-binding activity is detected in crude protein extracts that is dependent on the UAS and induced by heat shock . We further show that integration host factor (IHF) binds in vitro to a site between the UAS and sigma 54 recognition sequence . In bacteria lacking IHF, psp expression is substantially reduced in response to high temperature and ethanol . During osmotic shock in contrast, psp expression is only weakly stimulated by IHF, and IHF mutants can strongly induce the operon . The dependence of psp expression on IHF varies with the inducing condition, but does not correlate with dependence on PspB and PspC, indicating distinct, agent-specific activation mechanisms. Nucleic Acids Res, 1995 Jun 11, 23(11), 1990 - 6 Transformation of Escherichia coli with large DNA molecules by electroporation; Sheng Y et al.; We have examined bacterial electroporation with a specific interest in the transformation of large DNA, i.e . molecules > 100 kb . We have used DNA from bacterial artificial chromosomes (BACs) ranging from 7 to 240 kb, as well as BAC ligation mixes containing a range o different sized molecules . The efficiency of electroporation with large DNA is strongly dependent on the strain of Escherichia coli used; strains which offer comparable efficiencies for 7 kb molecules differ in their uptake of 240 kb DNA by as much as 30-fold . Even with a host strain that transforms relatively well with large DNA, transformation efficiency drops dramatically with increasing size of the DNA . Molecules of 240 kb transform approximately 30-fold less well, on a molar basis, than molecules of 80 kb . Maximum transformation of large DNA occurs with different voltage gradients and with different time constants than are optimal for smaller DNA . This provides the opportunity to increase the yield of transformants which have taken up large DNA relative to the number incorporating smaller molecules . We have demonstrated that conditions may be selected which increase the average size of BAC clones generated by electroporation and compare the overall efficiency of each of the conditions tested. Nucleic Acids Res, 1995 Jun 11, 23(11), 1919 - 22 The T-T pyrimidine (6-4) pyrimidinone UV photoproduct is much less mutagenic in yeast than in Escherichia coli; Gibbs PE et al.; We have examined the mutagenic properties of the T-T pyrimidine (6-4) pyrimidinone UV photoproduct in Saccharomyces cerevisiae, transforming the yeast cells either with single-stranded vectors that carried this adduct at a unique site or with gapped duplex vectors in which the adduct was located within a 28 nt single-stranded region . In an earlier study with SOS-induced Escherichia coli, we found that this photoproduct is highly mutagenic, specifically generating 3' T-->C substitutions in >85% of replicated molecules, and ascribed this specificity to the formation of a stable guanine-pyrimidinone mispair via hydrogen bonds at N-3 and O-2 . In contrast, this adduct is very much less mutagenic in yeast, with 60-70% of molecules being replicated accurately and only 12-20% of them exhibiting 3' T-->C substitutions . The enhanced accuracy may reflect the ability of a yeast DNA polymerase, but not E.coli DNA polymerase III, to trap the adduct in a configuration favorable for the formation of an adenine-pyrimidinone base pair. Nucleic Acids Res, 1995 Jun 11, 23(11), 1894 - 900 The level of the pUB110 replication initiator protein is autoregulated, which provides an additional control for plasmid copy number; Muller AK et al.; Plasmids control their copy number by limiting the amount of the initiator for DNA replication . The plasmid pUB110 initiator protein is termed RepU . Expression of the pUB110 repU gene is controlled by two antisense RNAs that interfere with repU mRNA translation . Genetic evidence suggests that Rep protein levels may be regulated by additional uncharacterized mechanisms . The repU gene product was radiolabeled and purified by monitoring the radioactive label . RepU overproduction was performed in cells containing the plasmid leading strand replication origin (dso), to allow for a putative inactivation of RepU . Polypeptides with apparent molecular masses of 42 (RepU*) and 39 (RepU) kDa were purified, both having the N-terminal sequence expected for the repU gene . The RepU/RepU* protein mixture bound specifically to dso . At low protein concentrations, about six RepU/RepU* protomers bound to the dso region . At higher concentrations, an extended nucleoprotein complex was formed . The promoter for the repU gene was localized downstream of the dso region . The results suggest that the extended RepU/RepU*-dso DNA complex interferes with repU promoter utilization . This provides an additional copy number control by limiting RepU concentration . Our results suggest that during replication the RepU protein might be converted into an inactive RepU-RepU* hetero-oligomer, further limiting the amount of RepU protein available for replication initiation. Mol Gen Genet, 1995 Jun 10, 247(5), 623 - 32 Two enzymes together capable of cysteine biosynthesis are encoded on a cyanobacterial plasmid; Nicholson ML et al.; The cyanobacterium Synechococcus sp . PCC 7942 contains two endogenous, genetically cryptic plasmids of 8.0 and 48.5 kb, which have been designated pANS and pANL, respectively . Characterization of the 3.8 kb Ba6 BamHI fragment of pANL identified three open reading frames which were transcriptionally regulated by sulfur availability and the protein CysR . One of these genes, designated srpG, encodes a protein which exhibits 67% amino acid identity to the Escherichia coli enzyme O-acetyl-L-serine (thio)-lyase A . Overlapping the 3' end of srpG is a second gene, designated srpH, which encodes a protein with similarity to the amino-terminal region of serine acetyltransferase enzymes . DNA hybridization results indicate that there is second copy of srpG in Synechococcus sp . PCC 7942, which is consistent with previous isoenzyme studies on O-acetyl-L-serine (thiol)-lyase in cyanobacteria . The introduction of srpG and srpH into E . coli cysKcysM and cysE mutant strains, respectively, results in the complementation of the lesion in cysteine biosynthesis . Additionally, the E . coli cysK cysM strain containing srpG is able to utilize sulfate more efficiently than thiosulfate, indicating that SrpG is probably a type A O-acetyl-L-serine (thiol)-lyase . The possible function of these genes in the adaptation of cyanobacteria to sulfur stress is discussed. Mol Gen Genet, 1995 Jun 10, 247(5), 529 - 36 Oenothera mitochondrial orf454, a gene involved in cytochrome c biogenesis corresponds to orf169 and orf322 of Marchantia; Gruska I et al.; We have characterized a mitochondrial gene in Oenothera, designated orf454, capable of encoding a component of the cytochrome c biogenesis system . This open reading frame is interrupted by an intron of 941 nucleotides showing high similarity to a group II intron residing in the rpl2 gene . RNA editing, which is observed at 18 cytidine positions within the orf454 reading frame, improves the similarity to protein-coding sequences in bacteria and higher plants and removes the last 16 amino acids . orf454 also shows high sequence similarity to two overlapping reading frames (orf169 and orf322) of Marchantia mitochondria . These ORFs belong to an operon-like cluster of genes in the liverwort that is not conserved in Oenothera mitochondria . However, in bacteria these reading frames are organized like the Marchantia gene cluster . It has been shown by genetical analysis in Rhodobacter capsulatus that these genes are essential for cytochrome c biogenesis . Genes of bacterial operons-ccl1 in Rhodobacter and yejR and nrfE in Escherichia coli - show high sequence similarity to the mitochondrial reading frames orf577 and orf454 of Oenothera . orf454, which we describe here, is homologous to the C-terminal region of these bacterial genes, while the previously described orf577 is homologous to the N-terminal region. Mol Gen Genet, 1995 Jun 10, 247(5), 521 - 8 Regulation of lrp gene expression by H-NS and Lrp proteins in Escherichia coli: dominant negative mutations in lrp; Oshima T et al.; Lrp (leucine-responsive regulatory protein) is a global transcription factor of Escherichia coli and regulates, negatively or positively, many genes including lysU, which encodes lysyl-tRNA synthetase . Dominant negative mutations that derepress lysU expression were isolated in this study . These mutations affected a predicted DNA-binding domain of Lrp and mutants were defective DNA-binding domain of Lrp and mutants were defective both in activation of ilvIH expression and in repression of lysU expression . Consistent with the previous notion that lrp is autoregulated, lrp expression was derepressed by these mutations and repressed by multi-copy plasmids carrying lrp+ . Moreover, we found by gene fusion and Northern blot hybridization that the "histone-like" protein, H-NS, bound specifically to a promoter segment of lrp in vitro, and the level of lrp expression increased in the hns null mutant . These results indicated that the lrp gene is not only feedback regulated by Lrp but is also controlled by H-NS protein. J Mol Biol, 1995 Jun 9, 249(3), 654 - 64 Investigating the structural determinants of the p21-like triphosphate and Mg2+ binding site; Cronet P et al.; Amongst the superfamily of nucleotide binding proteins, the classical mononucleotide binding fold (CMBF), is the one that has been best characterized structurally . The common denominator of all the members is the triphosphate/Mg2+ binding site, whose signature has been recognized as two structurally conserved stretches of residues: the Kinase 1 and 2 motifs that participate in triphosphate and Mg2+ binding, respectively . The Kinase 1 motif is borne by a loop (the P-loop), whose structure is conserved throughout the whole CMBF family . The low sequence similarity between the different members raises questions about which interactions are responsible for the active structure of the P-loop . What are the minimal requirements for the active structure of the P-loop? Why is the P-loop structure conserved despite the diverse environments in which it is found? To address this question, we have engineered the Kinase 1 and 2 motifs into a protein that has the CMBF and no nucleotide binding activity, the chemotactic protein from Escherichia coli, CheY . The mutant does not exhibit any triphosphate/Mg2+ binding activity . The crystal structure of the mutant reveals that the engineered P-loop is in a different conformation than that found in the CMBF . This demonstrates that the native structure of the P-loop requires external interactions with the rest of the protein . On the basis of an analysis of the conserved tertiary contacts of the P-loop in the mononucleotide binding superfamily, we propose a set of residues that could play an important role in the acquisition of the active structure of the P-loop. J Mol Biol, 1995 Jun 9, 249(3), 564 - 75 E-box variants direct formation of distinct complexes with the basic helix-loop-helix protein ALF1; Bonven BJ et al.; The murine transcription factor ALF1 belongs to the class of basic helix-loop-helix proteins specific for the NCAGNTGN-version of the E-box . Binding of homodimeric ALF1 to variants of this motif was studied by a combination of binding site selection technology and DNA modification interference analysis . The results showed that substitutions at the non-conserved positions in the E-box sequence could cause profound alterations in the patterns of specific contacts at the protein-DNA interface . Thus, both the overall extent of the binding region and the backbone phosphate contact pattern differed markedly between closely related E-boxes with similar affinities for ALF1 . The identity of the base at the inner N was an important determinant of contact pattern specification . The E-box variants differed in their ability to mediate ALF1 dependent transcriptional activation in vivo . We discuss the possibility that adaptability in basic helix-loop-helix protein-DNA interactions can result in complexes with different functional properties. J Biol Chem, 1995 Jun 9, 270(23), 14115 - 22 Artificial transmembrane segments . Requirements for stop transfer and polypeptide orientation; Chen H et al.; Transmembrane segments of proteins generally consist of a long stretch of hydrophobic amino acids, which can function to initiate membrane insertion (start-stop sequences), initiate translocation (signal-anchor sequences), or stop further translocation of the following polypeptide chain (stop-transfer sequences) . In this study, we have taken Escherichia coli alkaline phosphatase, a transported and water-soluble protein, and examined the requirements for converting it into a transmembrane protein with particular orientation . Since the wild type enzyme is transported, there is no predisposition against membrane translocation, yet it is not a membrane protein, so it does not possess any intrinsic membrane topogenic preferences . A series of potential transmembrane segments was introduced into an internal position of the enzyme to test the ability of each to initiate translocation, stop translocation, and adopt a particular orientation . For this purpose, cassette mutagenesis was used to incorporate new structural segments composed of polymers of alanines and leucines . The threshold value of hydrophobicity required to function as a stop-transfer sequence was determined . For a transmembrane segment of typical length (21 residues), this value is equivalent to the hydrophobicity of 16 alanines and 5 leucines . Interestingly, much shorter segments will also suffice to stop translocation, but these must be composed of more highly hydrophobic residues (e.g . 11 leucines) . When the wild type amino-terminal signal peptide is deleted or made dysfunctional, sufficiently hydrophobic internal segments can initiate translocation of the following polypeptide and function as a signal anchor . Furthermore, in so doing, the orientation of the protein is changed from N(out)-C(in) to N(in)-C(out). J Biol Chem, 1995 Jun 9, 270(23), 14042 - 6 The ATP synthase gamma subunit . Suppressor mutagenesis reveals three helical regions involved in energy coupling; Nakamoto RK et al.; A role in coupling proton transport to catalysis of ATP synthesis has been demonstrated for the Escherichia coli F0F1 ATP synthase gamma subunit . Previously, functional interactions between the terminal regions that were important for coupling were shown by finding several mutations in the carboxyl-terminal region of the gamma subunit (involving residues at positions 242 and 269-280) that restored efficient coupling to the mutation, gamma Met-23-->Lys (Nakamoto, R . K., Maeda, M., and Futai, M . (1993) J . Biol . Chem . 268, 867-872) . In this study, we used suppressor mutagenesis to establish that the terminal regions can be separated into three interacting segments . Second-site mutations that cause pseudo reversion of the primary mutations, gamma Gln-269-->Glu or gamma Thr-273-->Val, map to an amino-terminal segment with changes at residues 18, 34, and 35, and to a segment near the carboxyl terminus with changes at residues 236, 238, 242, and 246 . Each second-site mutation suppressed the effects of both gamma Gln-269-->Glu and gamma Thr-273-->Val, and restored efficient coupling to enzyme complexes containing either of the primary mutations . Mapping of these residues in the recently reported x-ray crystallographic structure of the F1 complex (Abrahams, J . P., Leslie, A . G., Lutter, R., and Walker, J . E . (1994) Nature 370, 621-628), reveals that the second-site mutations do not directly interact with gamma Gln-269 and gamma Thr-273 and that the effect of suppression occurs at a distance . We propose that the three gamma subunit segments defined by suppressor mutagenesis, residues gamma 18-35, gamma 236-246, and gamma 269-280, constitute a domain that is critical for both catalytic function and energy coupling. J Biol Chem, 1995 Jun 9, 270(23), 14031 - 41 Glutathionylspermidine metabolism in Escherichia coli . Purification, cloning, overproduction, and characterization of a bifunctional glutathionylspermidine synthetase/amidase; Bollinger JM Jr et al.; Glutathionylspermidine (GSP) synthetases of Trypanosomatidae and Escherichia coli couple hydrolysis of ATP (to ADP and Pi) with formation of an amide bond between spermidine (N-(3-aminopropyl)-1,4-diaminobutane) and the glycine carboxylate of glutathione (gamma-Glu-Cys-Gly) . In the pathogenic trypanosomatids, this reaction is the penultimate step in the biosynthesis of the antioxidant metabolite, trypanothione (N1,N8-bis-(glutathionyl)spermidine), and is a target for drug design . In this study, GSP synthetase was purified to near homogeneity from E . coli B, the gene encoding it was isolated and sequenced, the enzyme was overexpressed and purified in quantity, and the recombinant enzyme was characterized . The 70-kDa protein was found to have an unexpected second catalytic activity, glutathionylspermidine amide bond hydrolysis . Thus, the bifunctional GSP synthetase/amidase catalyzes opposing amide bond-forming and -cleaving reactions, with net hydrolysis of ATP . The synthetase activity is selectively abrogated by proteolytic cleavage 81 residues from the C terminus, suggesting that the two activities reside in distinct domains (N-terminal amidase and C-terminal synthetase) . Proteolysis at this site is facile in the absence of substrates, but is inhibited in the presence of ATP, glutathione, and Mg2+ . A series of analogs was used to probe the spermidine-binding site of the synthetase activity . The activity of diaminopropane as a substrate, inactivity of the C4-C8 diaminoalkanes, and greater loss of specificity for analogs modified in the 3-aminopropyl moiety than for those modified in the 4-aminobutyl moiety indicate that the enzyme recognizes predominantly the diaminopropane portion of spermidine and corroborate N-1 (the aminopropyl N) as the site of glutathione linkage (Tabor, H . and Tabor, C . W . (1975) J . Biol . Chem . 250, 2648-2654) . Trends in Km and kcat for a set of difluorosubstituted spermidine derivatives suggest that the enzyme may bind the minor, deprotonated form of the amine nucleophile. J Biol Chem, 1995 Jun 9, 270(23), 13916 - 24 Reduced chaperone-like activity of alpha A(ins)-crystallin, an alternative splicing product containing a large insert peptide; Smulders RH et al.; alpha-Crystallin is a multimeric protein complex which is constitutively expressed at high levels in the vertebrate eye lens, where it serves a structural role, and at low levels in several non-lenticular tissues . Like other members of the small heat shock protein family, alpha-crystallin has a chaperone-like activity in suppressing nonspecific aggregation of denaturing proteins in vitro . Apart from the major alpha A- and alpha B-subunits, alpha-crystallin of rodents contains an additional minor subunit resulting from alternative splicing, alpha A(ins)-crystallin . This polypeptide is identical to normal alpha A-crystallin except for an insert peptide of 23 residues . To explore the structural and functional consequences of this insertion, we have expressed rat alpha A- and alpha A(ins)-crystallin in Escherichia coli . The multimeric particles formed by alpha A(ins) are larger and more disperse than those of alpha A, but they are native-like and display a similar thermostability and morphology, as revealed by gel permeation chromatography, tryptophan fluorescence measurements, and electron microscopy . However, as compared with alpha A, the alpha A(ins)-particles display a diminished chaperone-like activity in the protection of heat-induced aggregation of beta low-crystallin . Our experiments indicate that alpha A(ins)-multimers have a 3-4-fold reduced substrate binding capacity, which might be correlated to their increased particle size and to a shielding of binding sites by the insert peptides . The structure-function relationship of the natural mutant alpha A(ins)-crystallin may shed light on the mechanism of chaperone-like activity displayed by all small heat shock proteins. J Biol Chem, 1995 Jun 9, 270(23), 13748 - 56 Chimeric muscle and brain glycogen phosphorylases define protein domains governing isozyme-specific responses to allosteric activation; Crerar MM et al.; Muscle and brain glycogen phosphorylases differ in their responses to activation by phosphorylation and AMP . The muscle isozyme is potently activated by either phosphorylation or AMP . In contrast, the brain isozyme is poorly activated by phosphorylation and its phosphorylated a form is more sensitive to AMP activation when enzyme activity is measured in substrate concentrations and temperatures encountered in the brain . The nonphosphorylated b form of the brain isozyme also differs from the muscle isozyme b form in its stronger affinity and lack of cooperativity for AMP . To identify the structural determinants involved, six enzyme forms, including four chimeric enzymes containing exchanges in amino acid residues 1-88, 89-499, and 500-842 (C terminus), were constructed from rabbit muscle and human brain phosphorylase cDNAs, expressed in Escherichia coli, and purified . Kinetic analysis of the b forms indicated that the brain isozyme amino acid 1-88 and 89-499 regions each contribute in an additive fashion to the formation of an AMP site with higher intrinsic affinity but weakened cooperativity, while the same regions of the muscle isozyme each contribute to greater allosteric coupling but weaker AMP affinity . Kinetic analysis of the a forms indicated that the amino acid 89-499 region correlated with the reduced response of the brain isozyme to activation by phosphorylation and the resultant increased sensitivity of the a form to activation by saturating levels of AMP . This isozyme-specific response also correlated with the glycogen affinity of the a forms . Enzymes containing the brain isozyme amino acid 89-499 region exhibited markedly reduced glycogen affinities in the absence of AMP compared to enzymes containing the corresponding muscle isozyme region . Additionally, AMP led to greater increases in glycogen affinity of the former set of enzymes . In contrast, phosphate affinities of all a forms were similar in the absence of AMP and increased approximately the same extent in AMP . The potential importance of a number of isozyme-specific substitutions in these sequence regions is discussed. J Biol Chem, 1995 Jun 9, 270(23), 13681 - 7 Metabolic sources of hydrogen peroxide in aerobically growing Escherichia coli; Gonzalez-Flecha B et al.; Exposure of cells to hydrogen peroxide (H2O2) mediates adaptive responses or oxidative damage, depending on the magnitude of the challenge . Determining the threshold for peroxide-mediated oxidative stress thus requires quantitation of the changes in endogenous H2O2 production . The intracellular steady-state concentrations of H2O2 were measured in intact Escherichia coli under different conditions . Compounds that block electron transport at NADH dehydrogenase (rotenone) or between ubiquinone and cytochrome b (antimycin) showed that univalent reduction of O2 can occur at these sites in vivo to form superoxide anion (O2-), in agreement with reports for mammalian mitochondria . Mutational inactivation of different components of the respiratory chain showed that H2O2 production also depended on the energy status of the cell and on the arrangement of respiratory chain components corresponding to particular growth conditions . Production rates for O2- and H2O2 were linearly related to the number of active respiratory chains that reached maximal values during exponential growth . In the strains defective in respiratory chain components, catalase activity was regulated to compensate for changes in the H2O2 production rates, which maintained intracellular H2O2 at 0.1-0.2 microM during aerobic growth over a wide range of cell densities . The expression of a katG'::lacZ fusion (reporting transcriptional control of the catalase-hydroperoxidase I gene) was increased by H2O2 given either as a pulse or as a steady production . This response not only depended on the type and severity of the stimulus but was also strongly influenced by the growth phase of the cells. J Biol Chem, 1995 Jun 9, 270(23), 13620 - 9 Implication of mammalian ribosomal protein S3 in the processing of DNA damage; Kim J et al.; A human apurinic/apyrimidinic endonuclease activity, called AP endonuclease I, is missing from or altered specifically in cells cultured from Xeroderma pigmentosum group-D individuals (XP-D cells) (Kuhnlein, U., Lee, B., Penhoet, E . E., and Linn, S . (1978) Nucleic Acids Res . 5,951-960) . We have now observed that another nuclease activity, UV endonuclease III, is similarly not detected in XP-D cells and is inseparable from the AP endonuclease I activity . This activity preferentially cleaves the phosphodiester backbone of heavily ultraviolet-irradiated DNA at unknown lesions as well as at one of the phosphodiester bonds within a cyclobutane pyrimidine dimer . The nuclease activities have been purified from mouse cells to yield a peptide of M(r) = 32,000, whose sequence indicates identity with ribosomal protein S3 . The nuclease activities all cross-react with immunopurified antibody directed against authentic rat ribosomal protein S3, and, upon expression in Escherichia coli of a cloned rat cDNA for ribosomal protein S3, each of the activities was recovered and was indistinguishable from those of the mammalian UV endonuclease III . Moreover, the protein expressed in E . coli and its activities cross-react with the rat protein antibody . Ribosomal protein S3 contains a potential nuclear localization signal, and the protein isolated as a nuclease also has a glycosylation pattern consistent with a nuclear localization as determined by lectin binding . The unexpected role of a ribosomal protein in DNA damage processing and the unexplained inability to detect the nuclease activities in extracts from XP-D cells are discussed. Gene, 1995 Jun 9, 158(2), 305 - 6 Isolation of cDNA clones encoding the mouse protein V-1; Pennica D et al.; The amino acid (aa) sequence of rat V-1, a developmentally regulated brain protein, was used to isolate clones encoding mouse V-1, a protein of 118 aa, from an embryoid body cDNA library . The aa sequences of rat and mouse V-1 are identical . V-1 shares several properties (including a 23 of 24 aa match of a tryptic peptide) with myotrophin, a protein that induces cardiac myocyte hypertrophy . Attempts to show that V-1 produced in Escherichia coli could induce the cardiac myocyte hypertrophy ascribed to myotrophin were unsuccessful. Biochim Biophys Acta, 1995 Jun 9, 1244(2-3), 377 - 83 Carbohydrate specificity of the Escherichia coli P-pilus papG protein is mediated by its N-terminal part; Hansson L et al.; The adherence of pyelonephritic Escherichia coli isolates to mammalian host cells is mediated by the P-pili structures on the bacterial surface . The protein constituting the distal part of the pili structure, papG, interacts with glycan receptors on the host cell . Variation in specificity for different glycoconjugates between the isolates, that may reflect variation in host tropism, has been correlated to three different classes of papG . Truncated variants of the class I, II and III papG adhesins were produced as fusion protein in E . coli and analysed for carbohydrate binding . The results showed that both carbohydrate binding and specificity of the papG adhesin resided in a linear part of the N-terminus of the protein. Biochim Biophys Acta, 1995 Jun 9, 1244(2-3), 259 - 68 Synthesis and characterisation of fluorescent oligonucleotides . Effect of internal labelling on protein recognition; Hagmar P et al.; Fluorescently labelled 42 base pair DNA duplexes were synthesised to examine the interaction between the TyR repressor protein of Escherichia coli and its DNA recognition sequence . An Fmoc-protected 5-(3-aminoprop-l-yn-l-yl)-2'-deoxyuridine phosphoramidite was synthesised and incorporated into oligonucleotides using standard beta-cyanoethyl phosphoramidite chemistry . Oligonucleotides containing the 3-aminopropynyl nucleotide at internal positions were reacted with fluorescein isothiocyanate to generate fluorescent DNA molecules useful for characterising interactions between DNA and proteins . Short DNA duplexes were investigated with respect to their melting temperatures and their ability to bind TyrR . Oligonucleotides containing a TyrR binding site were labelled in the central region of the recognition sequence or near the 5' edge of the recognition sequence . Fluorescein-labelled oligonucleotides could hybridise to form duplex DNA, and gel retardation experiments showed that the presence of the dye did not alter the binding affinity for the TyrR protein significantly . Fluorescence anisotropy measurements were used to examine the binding equilibrium in low and high salt buffers . A dissociation constant of 200-500 nM was obtained for the interaction of the TyrR dimer with a 42 bp duplex containing a centrally located 22 bp TyrR binding site. Nature, 1995 Jun 8, 375(6531), 503 - 6 Retinoblastoma-protein-dependent cell-cycle inhibition by the tumour suppressor p16; Lukas J et al.; D-type cyclins, in association with the cyclin-dependent kinases Cdk4 or Cdk6, promote progression through the G1 phase of the cell cycle by phosphorylating the retinoblastoma protein (RB) . The activities of Cdk4 and Cdk6 are constrained by inhibitors such as p16, the product of the CDKN2 gene on human chromosome 9p21 (refs 12-14) . The frequent deletion or mutation of CDKN2 in tumour cells suggests that p16 acts as a tumour suppressor . We show that wild-type p16 arrests normal diploid cells in late G1, whereas a tumour-associated mutant of p16 does not . Significantly, the ability of p16 to induce cell-cycle arrest is lost in cells lacking functional RB, including primary fibroblasts from Rb-/- mouse embryos . Thus, loss of p16, overexpression of D-cyclins and loss of RB have similar effects on G1 progression, and may represent a common pathway to tumorigenesis. Biochim Biophys Acta, 1995 Jun 6, 1256(3), 305 - 9 Effect of Escherichia coli lipopolysaccharide on surfactant secretion in primary cultures of rat type II pneumocytes; Romero C et al.; The purpose of this study is to evaluate the effect of the Escherichia coli lipopolysaccharide on the secretion of phosphatidylcholine, the principal component of pulmonary surfactant, in primary cultures of rat alveolar type II pneumocytes . Lipopolysaccharide stimulated phosphatidylcholine secretion in a time- and dose-dependent manner . At a concentration of 200 micrograms/ml, lipopolysaccharide stimulated the release of phosphatidylcholine 4-fold over the basal secretory rate, and the concentration producing half the maximal response was 20 micrograms/ml . The stimulatory effect of lipopolysaccharide on phosphatidylcholine secretion was additive to that of the protein kinase C activator TPA, which is a potent stimulator of surfactant secretion . Lipopolysaccharide did not activate protein kinase C, which suggests that stimulation of phosphatidylcholine secretion by the endotoxin was through a mechanism independent of protein kinase C activation. Biochemistry, 1995 Jun 6, 34(22), 7548 - 62 A heuristic approach to the analysis of enzymic catalysis: reaction of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-alpha-aminobutyrate and delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-allylglycine catalyzed by isopenicillin N synthase isozymes; Blackburn JM et al.; Isopenicillin N synthase (IPNS) catalyzes the oxidative cyclization of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine to isopenicillin N . It is proposed that the multiple products produced from certain substrate analogues result from pathway branching after formation of a ferryl oxene intermediate . We have been interested in ascertaining the reasons for multiple product formation . One possibility is that the products are predisposed toward formation once the beta-lactam ring and the ferryl oxene are produced . Alternately, the products may be persuaded into being by the enzyme restricting conformations such that otherwise less favorable chemistry can take place . With the existing description of the IPNS catalytic cycle, this fundamental question has not been answerable . We describe here the application of a heuristic method to resolve this key issue . It was reasoned that by comparing the ratios of products formed by a set of perturbed IPNS variants it might be possible to generate qualitative information about the relative magnitude of certain activation parameters . If certain product ratios are affected but others are not, then it should be possible to say which steps in the reaction are dictated merely by chemical fundamentals and which steps are directly effected by the enzyme . In this paper we report the high-level expression, purification, and characterization of four IPNS isozymes . Comparison of the product ratios obtained on incubation of unnatural substrate analogues with four IPNS isozymes corresponding to perturbed active site variants shows substantial variation in some cases and little in others . Interpretation of the results obtained with delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-alpha-aminobutyrate (ACAB) allows conclusions to be drawn regarding the role of the enzyme in restricting available conformations of the natural substrate to disfavor certain otherwise chemically favorable pathways and hence products . The results obtained with delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-allylglycine, while rather more complex, substantiate the conclusions drawn from the ACAB data . A major conclusion is that, in the oxidation of ACV, IPNS is a negative catalyst of cepham formation but a positive catalyst of penam formation. Biochemistry, 1995 Jun 6, 34(22), 7542 - 7 Tritium isotope effects in adenosylcobalamin-dependent glutamate mutase: implications for the mechanism; Marsh EN; The transfer of tritium between adenosylcobalamin and substrate in the reaction catalyzed by glutamate mutase was examined to investigate the possibility of a protein-based radical intermediate . There was no evidence that tritium was transferred to the protein during the reaction, as tritium neither became stably bound to the protein nor exchanged with water . The kinetics of tritium transfer from adenosylcobalamin to 3-methylaspartate was investigated . Both the transfer of tritium to product and the exchange of enzyme-bound and free coenzyme contribute to the kinetics of tritium loss from adenosylcobalamin . By varying the experimental conditions, the rates of both coenzyme exchange and tritium transfer could be measured . Exchange of adenosylcobalamin with enzyme is very slow, k off = 0.01 s-1, which may reflect a conformational change in the coenzyme and/or protein involved in forming active holo enzyme . The rate constants for the loss of tritium from adenosylcobalamin and the appearance of tritium in 3-methylaspartate are much faster and very similar, k = 0.67 +/- 0.05 s-1 and k = 0.50 +/- 0.05 s-1, respectively, consistent with the transfer of tritium occurring directly between coenzyme and substrate . The isotope effect, calculated from the rate constants for tritium transfer, and kcat, determined for the overall reaction under the same conditions, are between 13.5 and 18 . These values are typical of primary isotope effects seen for enzymes in which hydrogen transfer is substantially rate limiting . A protein radical, therefore, appears unlikely to feature in the mechanism of this enzyme. Biochemistry, 1995 Jun 6, 34(22), 7533 - 41 Determination of subunit dissociation constants in native and inactivated CTP synthetase by sedimentation equilibrium; Robertson JG; Sedimentation equilibrium was used to correlate changes in aggregation state with active site modification of Escherichia coli CTP synthetase . The native enzyme equilibrated between monomers, dimers, and tetramers in the absence of substrates . At enzyme concentrations above 5 microM, tetramers represented 40% of the species in solution . Inactivation by 6-diazo-5-oxonorleucine (DON) or thiourea dioxide reduced the amount of tetramer to below detectable limits . However, inactivated enzyme still equilibrated between monomers and dimers . Simultaneous analysis of multispeed data at three protein concentrations yielded estimates of the dissociation constants for the monomer-dimer and dimer-tetramer equilibria . For multiple data sets of native enzyme, K1,2 was between 1 and 2 microM, and K2,4 was between 1 and 18 microM . For DON inactivated enzyme, K1,2 was 3-4 microM, and for thiourea dioxide inactivated enzyme, K1,2 was approximately 1 microM . The values for K1,2 are consistent with previously published studies by gel filtration, demonstrating that the enzyme dissociates to monomers in very dilute solution (Anderson, 1983) . However, the sedimentation equilibrium experiments are the first to show that the enzyme forms tetramers in the absence of nucleotides . This result implies the presence of stable conformations in the native enzyme capable of dynamic equilibrium between monomers, dimers, and tetramers . The results presented here illustrate the sensitivity of sedimentation equilibrium for measuring the aggregation state of equilibrating enzyme species and demonstrate that active site modifications disrupt the quaternary structure of CTP synthetase. Biochemistry, 1995 Jun 6, 34(22), 7525 - 32 Mechanism of metal-independent hydroxylation by Chromobacterium violaceum phenylalanine hydroxylase; Carr RT et al.; Phenylalanine hydroxylase converts phenylalanine to tyrosine utilizing a tetrahydrobiopterin cofactor . Several key mechanistic questions have yet to be resolved, specifically the identity of the hydroxylating species and the role of the non-heme iron which is present in all of the mammalian PAHs . Recently, we have demonstrated that a bacterial PAH from Chromobacterium violaceum does not require any redox active metal for activity {Carr, R . T., & Benkovic, S . J . (1993) Biochemistry 32, 14132-14138} . To identify the function of iron in the mammalian PAH's, we have undertaken a series of experiments to compare the mechanisms of this metal-independent PAH with the iron-dependent PAH from rat liver . Using {4-2H}phenylalanine as a substrate gave a kinetic isotope effect on hydroxylation of unity for CVPAH which is in agreement with previous values reported for RLPAH . The {4-2H}phenylalanine underwent an NIH shift upon hydroxylation by CVPAH . The extent of deuterium retention at the 3-position of the tyrosine product was identical within experimental error for both RLPAH and CVPAH using {4-2H}phenylalanine and {2,3,5,6-2H}phenylalanine as substrates . This suggests that PAH from either source probably does not directly mediate the NIH shift mechanism . No uncoupled pterin turnover was observed for CVPAH with either L-tyrosine or p-chloro-L-phenylalanine as substrate or tetrahydropterin as cofactor, each of which causes uncoupled turnover with RLPAH . CVPAH readily accepts 4-methylphenylalanine as a substrate giving 4-(hydroxymethyl)phenylalanine as the major product and 3-methyltyrosine as the only other minor product.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Jun 6, 34(22), 7517 - 24 Substrate determinants of the course of tartrate dehydrogenase-catalyzed reactions; Serfozo P et al.; The substrate specificity of tartrate dehydrogenase has been probed using a series of alternative substrates to identify the molecular interactions which determine whether a particular substrate undergoes enzyme-catalyzed decarboxylation or not . A series of 3-substituted malate analogs, in which F, Cl, Br, I, SH, or NH2 substituents were placed at the 3R- or 3S-position, was prepared, and the product resulting from the action of tartrate dehydrogenase on each compound was identified . All of the halomalates and both diastereomers of aminomalate underwent oxidative decarboxylation; both diastereomers of 3-thiomalate underwent net nonoxidative decarboxylation . The results were interpreted in terms of a model in which decarboxylation is conformationally controlled . The data are not consistent with a model which suggests that substrates assume the conformation that is necessary to avoid steric crowding between the enzyme and the substituent at the 3-position of the substrate . These data are consistent with a model in which the course of the reaction with (+)-tartrate and meso-tartrate is dictated by the coordination of the substrate hydroxyls to the active site Mn2+ . However, the observed reactivities of the 3-methyltartrate diastereomers are not consistent with this model, either: (2R,3R)-3-methyltartrate undergoes oxidative decarboxylation, and (2R,3S)-3-methyltartrate undergoes simple oxidation . These results suggest that for these compounds the conformation is dictated by the positioning of the hydrophobic substituent in a specific binding pocket.(ABSTRACT TRUNCATED AT 250 WORDS) Biochem Biophys Res Commun, 1995 Jun 6, 211(1), 40 - 8 Analysis of translational termination of recombinant human methionyl-neurotrophin 3 in Escherichia coli; Meng SY et al.; A highly efficient UGA stop codon readthrough event during the synthesis of human neurotrophin 3 in E . coli is described . The incorporation of a Trp residue at the UGA stop codon is confirmed combining both the chemical analyses and the molecular and genetic data in this report . The 3' adjacent nuleotide to the UGA stop codon plays a crucial role in determining the readthrough efficiency in the order of A > G > C > U . The replacement of UGA with UAA or UAG totally abolished this readthrough phenomenon and the use of StpR host cells also prevented the occurrence of UGA readthrough . Gene dosage (or plasmid copy number) effect was not indicated in this event; however, the titration of RF-2 by mRNA transcripts under over-expression conditions might explain why tRNAtrp competes so well with RF-2 for UGA . Another apparently less produced readthrough product resulting from a transcript with no stop codon is also recorded, and the addition of a second in-frame stop codon increased the amount of the observed readthrough product. Biochem Biophys Res Commun, 1995 Jun 6, 211(1), 340 - 6 Acute endotoxin tolerance is accompanied by stimulated glucose use in macrophage rich tissues; Spolarics Z et al.; A single injection of E . coli LPS at a dose of 10 mg/kg b.w . ("high dose") is lethal in Sprague Dawley rats . However, animals given a sublethal dose of LPS (0.5 mg/kg bw; "low dose") at time zero, followed by a second high dose injection at 48 h, display endotoxin tolerance with 100% survival . The aim of the present study was to assess the relationship between this observed endotoxin tolerance and the endotoxin-induced glucose metabolic response in selected tissues and nonparenchymal hepatic cells . In each experimental group two injections, the first at time zero, the second at 48 h were given in vivo . Four experimental groups constituted these studies: A) saline followed by saline, B) low dose LPS followed by saline, C) saline followed by high dose LPS, and D) low dose LPS followed by high dose LPS . In vivo glucose use in tissues and cells was measured 3h after the last treatments employing the 2-deoxy-glucose tracer technique . Glucose use by liver, lung, spleen and intestine was not different between saline/saline (group A) and low dose LPS/saline injected (group B) animals . Saline/high dose LPS injection (group C) doubled glucose uptake, while the sequential LPS injections (group D) caused an additional, 2-3 fold increase in the glucose use by these tissues . Hepatic endothelial cells showed a similarly elevated glucose use in vivo in both group C and D . Kupffer cells from group D animals, however, displayed markedly elevated glucose use in vivo as compared to cells from group C . Our data indicate that high dose LPS in endotoxin tolerant animals is accompanied by a more markedly stimulated tissue glucose use than found following lethal LPS treatment alone . This increased peripheral glucose use may support cellular functions responsible for the protection of the host. Proc Natl Acad Sci U S A, 1995 Jun 6, 92(12), 5640 - 4 Rep protein of tomato yellow leaf curl geminivirus has an ATPase activity required for viral DNA replication; Desbiez C et al.; The Rep protein of geminiviruses is the sole viral protein required for their DNA replication . The amino acid sequence of Rep protein contains an NTP binding consensus motif (P-loop) . Here we show that purified Rep protein of tomato yellow leaf curl virus expressed in Escherichia coli exhibits an ATPase activity in vitro . Amino acid exchanges in the P-loop sequence of Rep causes a substantial decrease or loss of the ATPase activity . In vivo, mutant viruses carrying these Rep mutations do not replicate in plant cells . These results show that ATP binding by the Rep protein of geminiviruses is required for its function in viral DNA replication. Proc Natl Acad Sci U S A, 1995 Jun 6, 92(12), 5635 - 9 RuvC protein resolves Holliday junctions via cleavage of the continuous (noncrossover) strands; Bennett RJ et al.; The RuvC protein of Escherichia coli resolves Holliday junctions during genetic recombination and the postreplicational repair of DNA damage . Using synthetic Holliday junctions that are constrained to adopt defined isomeric configurations, we show that resolution occurs by symmetric cleavage of the continuous (noncrossing) pair of DNA strands . This result contrasts with that observed with phage T4 endonuclease VII, which cleaves the pair of crossing strands . In the presence of RuvC, the pair of continuous strands (i.e., the target strands for cleavage) exhibit a hypersensitivity to hydroxyl radicals . These results indicate that the continuous strands are distorted within the RuvC/Holliday junction complex and that RuvC-mediated resolution events require protein-directed structural changes to the four-way junction. Proc Natl Acad Sci U S A, 1995 Jun 6, 92(12), 5487 - 90 Adaptive reversion of an episomal frameshift mutation in Escherichia coli requires conjugal functions but not actual conjugation; Foster PL et al.; Adaptive reversion of a lac- frameshift mutation in Escherichia coli appears to be due to DNA polymerase errors, implying that DNA is being synthesized although the cells are not dividing . Here we report that the production of adaptive lac+ revertants (i) is much higher when the mutational target is on the F' episome than when it is on the bacterial chromosome; (ii) is enhanced by functions required for conjugation; but (iii) does not require conjugation per se . These results suggest that, in static cells, DNA synthesis is initiated from the conjugal origin of transfer . Mutations may arise as polymerase errors during this synthesis or during synthesis stimulated by recombination among the multiple gene copies. Proc Natl Acad Sci U S A, 1995 Jun 6, 92(12), 5416 - 20 Identification of functionally important helical faces in transmembrane segments by scanning mutagenesis; Lee GF et al.; We applied mutational analysis to a protein domain that functions in neither catalysis nor binding but, rather, in transmembrane signaling . The domain is part of chemoreceptor Trg from Escherichia coli . It contains four transmembrane segments, two from each subunit of the homodimer . We used cysteine scanning to investigate the functional importance of each of 54 residues in the two transmembrane segments . Cysteines at some positions resulted in subtle but significant reductions in tactic response . Those positions defined a specific helical face on each segment, implying that the segments function as helices . The functionally important faces corresponded to structural, helical packing faces identified independently by biochemical studies . All functionally impaired receptors exhibited altered signaling properties, either reduced signaling upon stimulation or induced signaling in the absence of stimulation . The distribution of substitutions creating these two phenotypes implied that conformational signaling involves movement between the two transmembrane helices within a subunit and that signaling is optimal when stable interactions are maintained across the interface between subunits. Biochem Biophys Res Commun, 1995 Jun 6, 211(1), 106 - 14 The influence of RNA and DNA template structures during transcript elongation by RNA polymerases; Sastry SS et al.; It was previously thought that elongating Escherichia coli transcription ternary complex consists of an RNA polymerase molecule enclosing 17 +/- 1 melted bases (bubble) of the template DNA and a 12-base-pair RNA-DNA hybrid ("transcription bubble paradigm") . Recent evidence suggests that ternary elongation complexes are heterogeneous and possibly vary in bubble size and length of RNA-DNA hybrid . We used a new type of assay to address the relative contributions of bubble size, secondary structure of RNA and RNA-DNA hybrid length during elongation . Synthetic RNA-DNA bubble duplexes are assembled in vitro . RNA structure 5' to the RNA-DNA hybrid, hybrid length and bubble size are systematically changed . The relative efficiency of E . coli and T7 RNA polymerases to elongate RNA primer is quantitated . RNA elongation was high (approximately 22-30%) when a stable hairpin was present towards the 5' end of the primer . Efficiency of elongation was lower for RNA primers without hairpins . Hairpin RNAs with presumed RNA-DNA hybrids of 3-7 bp were efficiently elongated compared to hairpins that presumably form 10bp hybrids . Preformed bubbles of different sizes (2,5 or 20 bases) were functional in all cases where elongation was moderate or high . We concluded that RNA secondary structure plays a dominant role compared to hybrid length or bubble size in determining efficient elongation by RNA polymerases. FEBS Lett, 1995 Jun 5, 366(1), 61 - 4 Human glucose-6-phosphate dehydrogenase . Lysine 205 is dispensable for substrate binding but essential for catalysis; Bautista JM et al.; By site-directed mutagenesis of the cloned human glucose-6-phosphate dehydrogenase cDNA, lysine 205 (the residue that after reacting with pyridoxal-5'-phosphate renders inactive enzyme) was mutated to threonine (K205T) to remove the amino group, or to arginine (K205R) to displace the position of the amino group, in order to analyze the role of its nucleophilic group in position epsilon . Compared to the wild-type enzyme, the K205T and K205R mutants retain a specific activity of 2.6 and 11.4%, respectively; their catalytic specificity (Kcat/Km) is drastically decreased, whereas the Km values for both substrates are only slightly increased . These findings in the light of the 3D structure of G6PD suggest that the epsilon-amino group of lysine 205 can favour a hydrogen bond within the active pocket essential for catalysis. FEBS Lett, 1995 Jun 5, 366(1), 17 - 20 On the distribution of ligands within the asymmetric chaperonin complex, GroEL14.ADP7.GroES7; Girshovich AS et al.; In the presence of MgATP or MgADP the E . coli chaperonin proteins, GroEL and GroES, form a stable asymmetric complex with a stoichiometry of two GroEL7:one GroES7: seven MgADP . The distribution of the ligands between the two heptameric GroEL rings is crucial to our understanding of the mechanism of chaperonin-assisted folding, being either cis (i.e . {GroEL7.MgADP7.GroES7}-{GroEL7}) or trans (i.e . {GroEL7.MgADP7}-{GroEL7.GroES7} . On the basis of cross-linking experiments with 8-azido-ATP and the heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), it was suggested that GroES and MgADP are bound to the same GroEL ring which resists proteinase K digestion {Nature 366 (1993) 228-233} . However, we find that the SPDP-promoted cross linking of GroES and GroEL occurs in the absence of Mg2+, ADP or ATP, which are required for the formation of the asymmetric complex . Cross-linking is shown to occur only when the SPDP-modified GroES is co-precipitated with GroEL by trichloracetic acid . Furthermore, there are structural grounds for questioning whether SPDP can crosslink, in a physiologically relevant manner, an amino group of GroES with any of the cysteinyl groups of GroEL. FEBS Lett, 1995 Jun 5, 366(1), 1 - 5 Structural analysis and comparison of the C-terminal transport signal domains of hemolysin A and leukotoxin A; Yin Y et al.; NMR spectroscopy was used to study the structure of the C-terminal signal sequences of the bacterial toxins, hemolysin A(HlyA) and leukotoxin A (LktA) . The two signals share little sequence homology; however, both can direct toxin transport with equal efficiency . We report here that in a membrane mimetic environment both peptides form two short non-interacting alpha-helices separated by a short loop . This higher order structure may be a common feature of C-terminal signals and may be required for interaction with the membrane associated transporter complex. J Mol Biol, 1995 Jun 2, 249(2), 478 - 92 Slow ligand-induced transitions in the allosteric phosphofructokinase from Escherichia coli; Auzat I et al.; The fluorescence of the unique tryptophan residue of the allosteric phosphofructokinase from Escherichia coli varies upon binding of any ligand, whether substrate or effector, suggesting that the protein undergoes a conformational change . This fluorescent probe has been exploited to determine the rates of the structural transitions that occur upon ligand binding and that are responsible for the remarkable allosteric behavior of this enzyme . The kinetics of fluorescence changes measured after rapidly mixing phosphofructokinase with one of its ligands show the presence of several allosteric transitions with widely different rates, ranging from a few hundred s-1 to less than 0.1 s-1 . The rate of each conformational change increases with the concentration of the ligand used to trigger it, suggesting that ligands induce a conformational change and do not displace a pre-existing equilibrium . The hypothesis that each ligand stabilizes a different conformational state of the protein is confirmed by the kinetics of displacement of one ligand by another: for instance, the binary complexes between phosphofructokinase and either its substrate, fructose-6-phosphate, or its allosteric activator, ADP, have the same low fluorescence and should be in the same active state, but they show different rates of conformational transition upon binding the inhibitor phosphoenolpyruvate . It appears that phosphofructokinase can exist in more than two states . Some conformational changes between these multiple states are slow enough to play an important role in the kinetics of the reaction catalyzed by phosphofructokinase, and could even explain part of its allosteric behavior . These results show that steady-state measurements are not sufficient to analyze the regulatory properties of E . coli phosphofructokinase. J Mol Biol, 1995 Jun 2, 249(2), 270 - 82 Mechanism of post-segregational killing by hok-homologue pnd of plasmid R483: two translational control elements in the pnd mRNA; Nielsen AK et al.; The pnd system of plasmid R483 mediates plasmid stabilization by killing of plasmid-free cells . The pnd mRNA is very stable and can be translated into PndA protein, a cell toxin which kills the cells from within by damaging the cell membrane . Translation of the pnd mRNA is inhibited by the PndB antisense, a small labile RNA of 63 nt . The rapid decay of the PndB antidote leads to onset of PndA synthesis in plasmid-free segregants or after addition of rifampicin . Surprisingly however, the full-length pnd mRNA was found to be translationally inactive whereas a 3'-end truncated version of it was found to be active . We have therefore suggested previously, that the 3'-end of the full-length pnd mRNA encodes a fold-back inhibitory sequence (fbi), which prevents its translation . Here we present an analysis of the metabolism of the pnd mRNAs . A mutational analysis shows that single point mutations in the fbi motif results in more rapid truncation . The fbi mutations could not be complemented by second-site mutations in either of the pndA or pndC Shine-Dalgarno (SD) elements . Surprisingly, mutations in the pndC SD element also lead to a more rapid truncation . The effect of these latter mutations was, however, complemented by mutations in a proposed anti-SD element upstream of the pndC SD . Mutations in the anti-SD element were lethal . These results show, that the pnd mRNA contains two negative control elements, one located in its very 3'-end (fbi), and one located just upstream of the pndC SD region (the anti-SD element) . These observations add to the complexity of the induction scheme previously proposed to explain activation of pndA expression in plasmid-free cells: In addition to its negative effect of translation, the fbi structure also maintains a reduced processing rate in the 3'-end of the mRNA . This permits the accumulation of a reservoir of pnd mRNA, which can be activated by 3'-end processing in plasmid-free cells . The anti-SD may prevent translation of the pnd mRNA during transcription, thus preventing detrimental synthesis of toxin. J Biol Chem, 1995 Jun 2, 270(22), 13384 - 91 Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle . V . Four different polymerase-clamp complexes on DNA; Stukenberg PT et al.; Several different subassemblies of DNA polymerase III holoenzyme can be purified from Escherichia coli . Toward the goal of understanding the functional significance of these subassemblies, we have used the gamma complex clamp loader and the beta ring to assemble each different polymerase onto DNA . Through use of radioactive labeled proteins, the subunit structure of each resulting processive polymerase has been determined . Use of DNA polymerase III core, the gamma complex, and beta results in a core-beta complex on DNA; the gamma complex is not incorporated into the structure . The addition of tau to the assembly reaction to form either core1-tau 2 or core2-tau 2 results in a more efficient polymerase and more stabile association of core-tau beta on DNA, although the gamma complex still does not remain on DNA . The gamma complex clamp loader was retained on DNA with the other subunits only if it was first assembled into the polymerase (Pol) III* structure . The clamp loader within Pol III* appeared to be capable of loading two beta clamps onto DNA for both core polymerases within Pol III*, consistent with the hypothesis that one replicase can simultaneously replicate both strands of a duplex chromosome . These findings extend those of an earlier study showing that distinctive polymerases can be assembled depending on the presence or absence of tau (Maki, S., and Kornberg, A . (1988) J . Biol . Chem . 263, 6561-6569) . The significance of these distinct polymerases in separate paths of DNA metabolism is discussed. J Biol Chem, 1995 Jun 2, 270(22), 13378 - 83 Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle . IV . ATP-binding site mutants identify the clamp loader; Xiao H et al.; The gamma complex (gamma delta delta' chi psi) and tau complex (tau delta delta' chi psi) clamp loaders require ATP hydrolysis to load beta sliding clamps onto DNA . The beta sliding clamp tethers the polymerase (Pol) III* replicase to DNA for processive synthesis . Pol III* contains both gamma and tau, but only one each of the delta, delta', chi, and psi subunits . Hence, there is ambiguity with respect to which clamp loader, the gamma or tau complex, exists in the Pol III* replicase structure . In this study, ATP-binding site mutants of gamma and tau have been prepared, and these mutants, when assembled into either the gamma or tau complex, are inactive in clamp loading . These mutants have been used as a tool to determine the identity of the clamp loader in Pol III* . The nine-subunit Pol III* has been assembled using either mutant gamma or tau in place of wild-type gamma or tau . The results show that mutation of gamma inactivates Pol III* activity, but mutation of tau does not, indicating that the gamma complex (and not the tau complex) is the clamp loader of Pol III* . The tau subunit carries the task of dimerizing the core polymerase, and it is this association of tau with core that appears to direct the single copy subunits away from tau and onto gamma. J Biol Chem, 1995 Jun 2, 270(22), 13366 - 77 Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle . III . Interface between two polymerases and the clamp loader; Onrust R et al.; The nine-subunit DNA polymerase (Pol) III* coupled to its beta sliding clamp is a rapid and highly processive replicating machine . The multiple subunits are needed for the complicated task of duplicating the Escherichia coli chromosome . In this report, Pol III* was constituted from individual pure proteins, and its structure was studied . Constitution of the Pol III* particle requires an ordered addition of the subunits, and the final structure contains 14 polypeptides in the ratio alpha 2 epsilon 2 theta 2 tau 2 gamma 2 delta 1 delta' 1 chi 1 psi 1 . The structure can be summarized as being composed of two core polymerases (alpha epsilon theta) held together by a dimer of tau and one gamma complex clamp loader (gamma 2 delta 1 delta' 1 chi 1 psi 1) for loading beta onto DNA . At the center of the structure, the related tau and gamma subunits form a heterotetramer upon which the two core polymerases and clamp loader proteins assemble . The single copy nature of the delta, delta', chi, and psi subunits confers a structural asymmetry with respect to the two polymerases, presumably for the different functions of replicating the leading and lagging strands. J Biol Chem, 1995 Jun 2, 270(22), 13358 - 65 Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle . II . Intermediate complex between the clamp loader and its clamp; Naktinis V et al.; The Escherichia coli replicase, DNA polymerase III holoenzyme, derives its processivity from the beta subunit sliding clamp that encircles DNA and tethers the replicase to the template . The beta dimer is assembled around DNA by the gamma complex clamp loader in an ATP-dependent reaction . In this report, the essential contact between the clamp loader and beta is identified as mediated through the delta subunit of the gamma complex . The delta subunit appears to contact the face of the beta dimer ring that contains the two C termini . Surprisingly, ATP is required for the gamma complex to bind beta, but not for delta to bind beta . This indicates that delta is buried in the gamma complex and suggests a role for ATP in exposing delta for interaction with beta . A protease protection assay has been developed to specifically probe the delta subunit within the gamma complex . The results of the assay are consistent with an ATP-induced conformational change in the gamma complex that alters the state of the delta subunit within it . The implication of these key features to the clamp loading mechanism of the gamma complex is discussed. J Biol Chem, 1995 Jun 2, 270(22), 13348 - 57 Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle . I . Organization of the clamp loader; Onrust R et al.; The gamma complex of DNA polymerase III holoenzyme, the replicase of Escherichia coli, couples ATP hydrolysis to the loading of beta sliding clamps onto primed DNA . The beta sliding clamp tethers the holoenzyme replicase to DNA for rapid and processive synthesis . In this report, the gamma complex has been constituted from its five different subunits . Size measurements and subunit stoichiometry studies show a composition of gamma 2 delta 1 delta' 1 1 chi 1 psi 1 . Strong intersubunit contacts have been identified by gel filtration, and weaker contacts were identified by surface plasmon resonance measurements . An analogous tau complex has also been constituted and characterized; it is nearly as active as the gamma complex in clamp loading activity, but as shown in the fourth report of this series, it is at a disadvantage in binding the delta, delta', chi, and psi subunits when core is present (Xiao, H., Naktinis, V., and O'Donnell, M . (1995) J . Biol . Chem . 270, 13378-13383) . The single copy subunits within the gamma complex provide the basis for the structural asymmetry inherent within DNA polymerase III holoenzyme. J Biol Chem, 1995 Jun 2, 270(22), 13303 - 7 Crystallographic evidence for dimerization of unliganded tumor necrosis factor receptor; Naismith JH et al.; Activation of the cell surface receptors for tumor necrosis factor (TNF) is effected by the aggregation of cytoplasmic domains that occurs when the extracellular domains of two or three receptors bind to trimeric TNF alpha or TNF beta . The structure of the type I TNF receptor extracellular domain (sTNF-R1), crystallized in the absence of TNF, has now been determined at 2.25-A resolution . The receptor itself is an elongated molecule comprising four disulfide-rich domains in a nearly linear array . Contrary to expectations, the unliganded domains are found to associate into dimers of two distinct types, in which monomers are related by local two-fold axes of symmetry . In one case, the receptors are antiparallel to each other and associate through an interface that overlaps the TNF binding site . If intact receptors were capable of such an association, their cytoplasmic domains would be separated by over 100 A . This interaction could inhibit signaling in the absence of TNF . Parallel dimers are also observed in which the dimer interface is well separated from the TNF binding site . Associations among TNF-bound parallel dimers could cause receptor clustering . Both dimers bury substantial areas of protein surface and are formed by polar and non-polar interactions. J Biol Chem, 1995 Jun 2, 270(22), 13160 - 3 Identification of arginine residues in the putative L-aspartate binding site of Escherichia coli adenylosuccinate synthetase; Wang W et al.; Three arginine residues in the putative aspartate binding site of Escherichia coli adenylosuccinate synthetase were changed to leucines by site-directed mutagenesis . The mutant enzymes R303L, R304L, and R305L were purified to homogeneity on the basis of sodium dodecyl sulfate polyacrylamide gel electrophoresis and characterized by CD spectrometry and initial rate kinetics . CD spectral analysis indicated no differences in secondary structure between the mutants and the wild-type enzyme in the absence of substrates . The Km values for GTP and IMP for the mutants and the wild-type enzyme were comparable . However, the mutant enzymes exhibited 50-200-fold increases in their values of Km for the substrate aspartate relative to the wild-type enzyme . Although the kcat values for the mutant enzymes decreased, the changes were not as dramatic as those observed for the Km of aspartate . The modeling of aspartate in the crystal structure of the complex of adenylosuccinate synthetase with IMP and MgGDP-1 is consistent with the results of mutagenesis, placing the alpha- and beta-carboxylates of aspartate near the side chains of Arg-131, -303, and -305. J Biol Chem, 1995 Jun 2, 270(22), 12990 - 4 A novel activity of OmpT . Proteolysis under extreme denaturing conditions; White CB et al.; A novel property of the bacterial outer membrane protein T, OmpT, has been discovered . It is active under extreme denaturing conditions . This finding emerged during characterization of a protease associated with the degradation of recombinant proteins expressed as inclusion bodies in Escherichia coli . These inclusion body proteins are stable to proteolytic degradation until they are solubilized by denaturation . The protease that degrades them under denaturing conditions was identified as OmpT on the basis of substrate specificity, inhibitor profile, and confirmation that its N-terminal sequence is identical with that of OmpT . A previously unknown property of this enzyme, OmpT's preference for denatured substrates, may provide a clue to its physiological function . To facilitate further characterization of this proteolytic activity, we have optimized a system to extract and assay OmpT under denaturing conditions using a soluble substrate, rabbit muscle creatine kinase. J Biol Chem, 1995 Jun 2, 270(22), 12980 - 3 Inorganic polyphosphates in the acquisition of competence in Escherichia coli; Castuma CE et al.; A complex of polyhydroxybutyrate (PHB), Ca2+, and inorganic polyphosphate (polyP) was proposed as the membrane component responsible for competence for DNA entry in Escherichia coli (Reusch, R . N., and Sadoff, H . L . (1988) Proc . Natl . Acad . Sci . U.S.A . 85, 4176-4180) . While chemical and immunological assays and 1H NMR have unequivocally established the identity and content of PHB in the complex, comparable methods were not available for polyP . With specific enzyme assays developed for polyP, we have identified, in chloroform extracts of competent cell membranes, a novel form of polyP of about 60 to 70 residues in a stoichiometric ratio of PHB to polyP of 2:1 . In E . coli mutants, incapable of synthesizing the predominant, thousand-long polyP chains, appearance of this short polyP and its inclusion in membranes can account for their capacity to develop competence and indicates an auxiliary pathway for polyP synthesis . A variety of fluorescent lipid probes demonstrate the appearance of extensive rigid domains in membranes of competent cells . We propose that the PHB.Ca2+.polyP complex perturbs the conformation of the lipid matrix, making it more permeable to charged molecules and thus allowing the entry of DNA. J Biol Chem, 1995 Jun 2, 270(22), 12961 - 4 The Escherichia coli malonyl-CoA:acyl carrier protein transacylase at 1.5-A resolution . Crystal structure of a fatty acid synthase component; Serre L et al.; Endogenous fatty acids are synthesized in all organisms in a pathway catalyzed by the fatty acid synthase complex . In bacteria, where the fatty acids are used primarily for incorporation into components of cell membranes, fatty acid synthase is made up of several independent cytoplasmic enzymes, each catalyzing one specific reaction . The initiation of the elongation step, which extends the length of the growing acyl chain by two carbons, requires the transfer of the malonyl moiety from malonyl-CoA onto the acyl carrier protein . We report here the crystal structure (refined at 1.5-A resolution to an R factor of 0.19) of the malonyl-CoA specific transferase from Escherichia coli . The protein has an alpha/beta type architecture, but its fold is unique . The active site inferred from the location of the catalytic Ser-92 contains a typical nucleophilic elbow as observed in alpha/beta hydrolases . Serine 92 is hydrogen bonded to His-201 in a fashion similar to various serine hydrolases . However, instead of a carboxyl acid typically found in catalytic triads, the main chain carbonyl of Gln-250 serves as a hydrogen bond acceptor in an interaction with His-201 . Two other residues, Arg-117 and Glu-11, are also located in the active site, although their function is not clear. J Biol Chem, 1995 Jun 2, 270(22), 13573 - 9 The differential processing of homodimers of reverse transcriptases from human immunodeficiency viruses type 1 and 2 is a consequence of the distinct specificities of the viral proteases; Fan N et al.; Active, recombinant p68 reverse transcriptase (RT) from human immunodeficiency virus type 2 (HIV-2), with an NH2-terminal extension containing a hexahistidine sequence was isolated from extracts of Escherichia coli by immobilized metal affinity chromatography . Treatment of the purified p68/p68 homodimer of HIV-2 RT with recombinant HIV-2 protease generates stable, active heterodimer (p68/p58) that is resistant to further hydrolysis . Analysis of this p68/p58 HIV-2 RT heterodimer revealed that while one subunit is intact p68, the p58 subunit is COOH-terminally truncated by cleavage, not at Phe440 as is seen in processing of the p66/p66 HIV-1 RT homodimer by HIV-1 protease, but at Met484 . The expected COOH-terminal p10 fragment resulting from hydrolysis of p68 at Met484 is not released intact, but undergoes further cleavage at Asn494, Met503, and Tyr532 . Processing of p68/p68 HIV-2 RT with the HIV-1 protease led to cleavage of the Phe440-Tyr441 bond, exactly as is seen with p66/p66 HIV-1 RT, to give the analogous p53 subunit . Studies of a peptide substrate modeled after residues 437-444 in HIV-2 RT showed that while the HIV-1 protease was able to cleave the Phe440 bond, this bond was resistant to cleavage by the HIV-2 enzyme . Our findings provide a rationale for the previous observation that the RT heterodimer isolated from HIV-2 lysates is larger than that from HIV-1 . We conclude that the p68/p58 HIV-2 RT heterodimer, containing the Met484 truncated p58 subunit, is a biologically relevant form of the enzyme in vivo. J Biol Chem, 1995 Jun 2, 270(22), 13518 - 23 Unexpected structural requirements for GTPase activity of the interferon-induced MxA protein; Schwemmle M et al.; MxA is an interferon-induced 76-kDa GTPase that inhibits the multiplication of several RNA viruses . Deleting seven amino acids from the COOH terminus reduced the GTPase activity of purified MxA to 1.4% . MxA mutants with COOH-terminal deletions of 63 or more amino acids lost all ability to hydrolyze GTP and failed to bind guanine nucleotides . By contrast, an MxA deletion mutant consisting of 301 amino acids from the NH2 terminus and 87 amino acids from the COOH terminus retained about 9% of wild-type GTPase activity, underscoring the pivotal role of COOH-terminal sequences . Limited proteolysis of wild-type MxA with proteinase K resulted in two resistant polypeptides of 60 and 10 kDa, respectively, which copurified as a stable complex . The p60-p10 complex exhibited high GTPase activity, suggesting that it included all MxA domains required for this biochemical activity . Sequencing revealed that the NH2 terminus of the 60-kDa polypeptide mapped to leucine 41 and the NH2 terminus of the 10-kDa polypeptide to glutamine 564 of the MxA sequence . Based on these results we propose a model that suggests that the GTP-binding consensus element located in the NH2-terminal half of MxA is held in an active conformation by strong physical interactions with amino acids from the COOH-terminal region. J Biol Chem, 1995 Jun 2, 270(22), 13512 - 7 Interferon-induced MxA protein . GTP binding and GTP hydrolysis properties; Richter MF et al.; MxA is a GTPase encoded by an interferon-activated human gene which inhibits the multiplication of several RNA viruses . Recombinant histidine-tagged MxA protein (His-MxA) was expressed in Escherichia coli and purified to near homogeneity . Gel filtration showed that it formed high molecular weight oligomers . Purified His-MxA exhibited specific GTP hydrolysis rates of up to 350 nmol of GTP/min/mg of protein, corresponding to a turnover number of 27 min-1 . The Km for this reaction was 260 microM . Guanine nucleotides did not copurify with His-MxA . Binding experiments in solution with fluorescent-labeled nucleotides confirmed that His-MxA binds guanine nucleotides rather weakly and further showed that the fluorescent GDP analog N-methylanthraniloyl (mant)-GDP had a much lower affinity for His-MxA (Kd 20 microM, koff 8.5 s-1) than the nonhydrolyzable GTP analog mant-5'-guanylyl-beta,gamma-imidotriphosphate (mant-GMP-PNP) (Kd 0.75 microM, koff 0.012 s-1) . Competitive binding studies with nonlabeled nucleotides revealed a similar binding preference of His-MxA for GTP over GDP: the Kd for GTP was 20 microM, whereas the Kd for GDP was 100 microM . Thus, a high percentage of MxA molecules may be complexed with GTP in vivo. Berl Munch Tierarztl Wochenschr, 1995 Jun, 108(6), 221 - 3 {Development of a sandwich ELISA for detection of specific antibodies against fimbrial antigens K88, K99 and 987p of Escherichia coli}; Jakel C; For the assessment of the efficacy of a E . coli-vaccine it was important to detect the specific antibodies against the protective antigens . Under the use of monoclonal antibodies against the fimbrial antigens K88, K99 and 987p a sandwich-elisa was developed . With these elisa-tests we detected significant titre conversion after repeated immunisation of rabbits . The results are highly reproducible . That is why the elisa-tests are qualified for testing of vaccine batches . The use of an anti-pig-conjugate makes it possible to detect antibodies in colostrum and serum from pigs. Mol Endocrinol, 1995 Jun, 9(6), 647 - 58 Molecular characterization by mass spectrometry of the human estrogen receptor ligand-binding domain expressed in Escherichia coli; Seielstad DA et al.; The ligand binding domain of the human estrogen receptor (hER-LBD), encompassing the sequence MDPS282AG...V595, has been expressed at high levels in Escherichia coli from a pET-23d vector, and a purified preparation has been characterized both by mass spectrometry and biochemical methods . Inclusion bodies from the bacterial expression were solubilized by sonication and the hER-LBD was purified to near homogeneity by affinity chromatography over an estradiol-Sepharose column in urea-containing buffer . This material ran as a single peak on reversed-phase HPLC, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed a band with apparent molecular mass of 31-32 kilodaltons (kDa), somewhat smaller than that expected from the construct (35.6 kDa) . Edman degradation revealed a single sequence of MDPSAGDMRA, consistent with an intact N terminus . Further characterization of this material using low resolution matrix-assisted laser desorption ionization mass spectrometry indicated an apparent single protein species of average mass 33,143 daltons (Da) . Detailed molecular analysis by electrospray ionization mass spectrometry, however, revealed that this purified hER-LBD preparation was actually composed of a number of both modified and unmodified LBD protein fragments between 32,900-33,400 Da . The bulk of this 33-kDa protein mixture consisted of three LBD protein fragments with C termini at Ala571 (70%), Ala569 (23%), and Ser575 (4%), with only a trace amount (3%) of the full length expressed LBD (MDPS282...V595; 35, 602 Da) . These four protein species also appear to be partially chemically modified by carbamylation . The functional integrity of this hER-LBD preparation was investigated by a ligand-exchange assay, which showed that the hER-LBD retained full estradiol-binding capacity; specific, covalent labeling was also observed using either the electrophilic affinity-labeling ligand tamoxifen aziridine (TAZ) or the photoaffinity-labeling ligand hexestrol diazirine . Thus, this expressed hER-LBD preparation, while appearing nominally pure by conventional biochemical techniques and having the expected ligand-binding capacity, was shown by sensitive high resolution electrospray ionization mass spectrometry techniques to be largely truncated 20-26 amino acids from the expected C terminus and to have a substantial level of covalent modification arising from the urea.(ABSTRACT TRUNCATED AT 400 WORDS) Acta Virol, 1995 Jun, 39(3), 149 - 59 Primase activities constantly present in avian myeloblastosis virus core isolates: detection and basic characteristics; Riman J et al.; RNA-synthesizing activities (RNA-SAs) by its nature identical with primase activities (Pr-As) were found to be constantly present in avain myeloblastosis virus (AMV) core isolates . Their endogenous templates are molecules of the virus core-bound host cell DNA (AMV DNA) (Riman and Beaudreau, 1970) that have been recently recognized as a collection of still active early replicative structures (Riman et al., 1993b) . Like the Pr-As, the RNA-SAs are not inhibited by alpha-amanitin nor by aphidicolin and they show a mutually competitive affinity for ATP and GTP . Their reaction products treated with DNase I are short RNAs similar in length to initiator RNAs (iRNAs), their precursors and degradation products . In AMV core proteins separated in isopycnic CsCl gradients, they are chiefly located in the density region of reverse transcriptase activities (RT-As) but with a distinct peak fraction . Like Pr-As, they are able to use poly(dT) as template and to form, in the presence of {alpha-32P}ATP, products that after DNase I treatment consist of poly(rA) molecules similar in length to iRNA monomers and multimers . Like the Pr-As, they are able to complement E . coli DNA polymerase (pol) I reactions . They occur in the analyzed AMV core proteins as six distinct sedimentation species (PrA-SS) . This, together with other relevant properties, indicates the presence of Pr-As associated with molecules of a primase-alpha DNA polymerase enzyme complex, its degradation products and 'free' primase monomers. Mol Microbiol, 1995 Jun, 16(6), 1259 - 68 Identification of GroEL as a constituent of an mRNA-protection complex in Escherichia coli; Georgellis D et al.; An RNA-binding activity has been identified in Escherichia coli that provides physical protection of RNA against ribonucleases in an ATP- and Mg(2+)-dependent manner . This binding activity is stimulated under growth conditions known to cause a decrease in the rate of mRNA decay . RNA protection is mediated by a protein complex that contains a modified form of the chaperonin GroEL as an indispensable constituent . These results suggest a new role for GroEL as an RNA chaperone. Mol Microbiol, 1995 Jun, 16(6), 1243 - 57 Subcellular localization and topology of the K88 usher FaeD in Escherichia coli; Valent QA et al.; The subcellular localization of the K88 usher FaeD was studied in Escherichia coli whole cells by using isopycnic sucrose density gradient centrifugation of isolated membranes, the detergents Triton X-100 and sodium lauryl sarcosinate and immunoblotting with a specific FaeD antiserum . Cells containing the complete K88 operon, as well as cells containing the subcloned faeD gene in various expression vectors, were used . Most of the FaeD was present in the outer membranes in a detergent-resistant form . Agglutination experiments with E . coli cells expressing FaeD confirmed an outer membrane localization and indicated the presence of FaeD at the cell surface . Automated Edman degradation indicated that the mature FaeD contained 777 amino acid residues and confirmed that FaeD is synthesized with a rather long signal sequence of 35 amino acid residues . Twelve different FaeD-PhoA fusion proteins were prepared and characterized by nucleotide sequencing and immunoblotting . Most of these fusion sites were located in the amino-terminal and carboxyl-terminal regions of FaeD . Six amino-terminal fusion proteins were soluble proteins in the periplasm, whereas the other fusion proteins were associated with the outer membrane . The protease accessibility of FaeD and of the six outer membrane-bound FaeD-PhoA fusion proteins was studied using whole cells, cells with permeabilized outer membranes, and isolated membranes . Collagenase H, kallikrein, trypsin and proteinase K were used . Based on the results of these experiments and computer predictions, a model for the membrane topology of FaeD was developed in which FaeD contains a large central domain containing 24 membrane-spanning segments and two relatively large periplasmic regions, at the amino-terminal and carboxyl-terminal end of the protein, respectively. Mol Microbiol, 1995 Jun, 16(6), 1051 - 7 Hypothesis: chromosome separation in Escherichia coli involves autocatalytic gene expression, transertion and membrane-domain formation; Norris V; To explain how daughter chromosomes are separated into discrete nucleoids and why chromosomes are partitioned with pole preferences, I propose that differential gene expression occurs during DNA replication in Escherichia coli . This differential gene expression means that the daughter chromosomes have different patterns of gene expression and that cell division is not a simple process of binary fission . Differential gene expression arises from autocatalytic gene expression and creates a separate proteolipid domain around each developing chromosome via the coupled transcription-translation-insertion of proteins into membranes (transertion) . As these domains are immiscible, daughter chromosomes are simultaneously replicated and separated into discrete nucleoids . I also propose that the partitioning relationship between chromosome age and cell age arises because the poles of cells have a proteolipid composition that favours transertion from one nucleoid rather than from the other . This hypothesis forms part of an ensemble of related hypotheses which attempt to explain cell division, differentiation and wall growth in bacteria in terms of the physical properties and interactions of the principal constituents of cells. Microb Pathog, 1995 Jun, 18(6), 387 - 99 Plasmid coding for drug resistance and invasion of epithelial cells in enteropathogenic Escherichia coli 0111:H; Scaletsky IC et al.; Enteropathogenic Escherichia coli (EPEC) can adhere to, invade and multiply in human epithelial cells . To define the elements required for bacterial invasion, we isolated from an 0111:H- EPEC a 6.6 kb plasmid that is capable of conferring to an avirulent, non-adherent E . coli K12 strain (DK1) the capacity to invade epithelial cells . With this system a dissociation was possible between bacterial invasion and adherence to epithelial cells . Bacteria containing this plasmid synthesise a protein of 32 kDa (pl 4.93) which seemed to be required for cell invasion . The results provide a new basis for strategies to prevent EPEC infections. Braz J Med Biol Res, 1995 Jun, 28(6), 651 - 4 Determination of the efficiency of K99-F41 fimbrial antigen vaccine in newborn calves; Yano T et al.; Semipurified K99 and F41 fimbrial antigens were used to prepare an oil-emulsified vaccine against bovine enterotoxigenic colibacillosis . Nine Nelore cows about 7 months pregnant were divided into 3 groups (A, B and C) of 3 animals each, which received different doses of vaccine (1,500 HU, 750 HU and 380 HU, respectively) 8 and 2 weeks before delivery, in the neck by the subcutaneous route . As a control (group D), 3 pregnant cows of the same breed were not vaccinated for later challange of their calves . Vaccine efficiency was measured by the serological tests double diffusion and ELISA . Challenge of calves from the vaccinated and from the three control unvaccinated cows was carried out with the virulent Escherichia coli B41 strain (0101, STa+, K99+, F41+) . Two of the 3 calves from the unvaccinated cows died within 48 h with acute diarrhea . E . coli B41 was recovered as pure culture from their stools . In contrast, none of the calves born from vaccinated cows presented diarrhea . These data suggest that the antibody transfer to calves through colostrum gave full protection against the challenge . This semipurified fimbrial vaccine against K99-F41-harboring strains is the first oil-emulsified immunogen prepared in Brazil, which was not only efficient, but also had no adverse effects on vaccinated pregnant cows. Curr Genet, 1995 Jun, 28(1), 80 - 6 Characterization of the "promoter region" of the enolase-encoding gene enol from the anaerobic fungus Neocallimastix frontalis: sequence and promoter analysis; Fischer M et al.; The sequence of the Neocallimastix frontalis enolase gene promoter was determined up to 1800 nucleotides 5' to the major transcriptional start point . The base composition of the enolase upstream sequence revealed a very A + T-rich profile (13.5% G + C) leading to many putative hairpin structures . The functional organization of the N . frontalis enolase promoter was investigated by heterologous transient-expression assays . DNA fragments obtained by the sequential removal of sequences upstream of the translation start codon were fused to the Escherichia coli lacZ gene and the resulting plasmids were used to transform the ascomycetes Aspergillus nidulans and Penicillium roqueforti and the oomycete Saprolegnia monoica . Transient expression of the lacZ reporter gene was observed in regenerating proteoplasts of S . monoica when using the 0.3 kb or 1 kb upstream of the enolase coding region . In contrast no beta-galactosidase activity was detected in ascomycete protoplasts . DNA hybridization analysis revealed the integration of vector DNA in the genomic DNA of S . monoica and the presence of free copies of the transformation plasmid which could be rescued in E . coli . Our results indicate that the transcriptional machinery of the anaerobic chytrid N . frontalis may differ significantly from that of ascomycetes but that enough conservation exists within the lower fungi to allow a transient-driven expression of a reporter gene in an oomycete fungus. Res Microbiol, 1995 Jun, 146(5), 397 - 404 Phylogenetic characterization of the ubiquitous electron transfer flavoprotein families ETF-alpha and ETF-beta; Tsai MH et al.; Electron transfer flavoproteins (ETF) are alpha beta-heterodimers found in eukaryotic mitochondria and bacteria . We have identified currently sequenced protein members of the ETF-alpha and ETF-beta families . Members of these two families include (a) the ETF subunits of mammals and bacteria, (b) homologous pairs of proteins (FixB/FixA) that are essential for nitrogen fixation in some bacteria, and (c) a pair of carnitine-inducible proteins encoded by two open reading frames in Escherichia coli (YaaQ and YaaR) . These three groups of proteins comprise three clusters on both the ETF-alpha and ETF-beta phylogenetic trees, separated from each other by comparable phylogenetic distances . This fact suggests that these two protein families evolved with similar overall rates of evolutionary divergence . Relative regions of sequence conservation are evaluated, and signature sequences for both families are derived. Res Microbiol, 1995 Jun, 146(5), 363 - 70 Mutations which result in constitutive expression of the Bordetella pertussis filamentous haemagglutinin gene; Goyard S et al.; The expression of the filamentous haemagglutinin (FhaB) of Bordetella pertussis is positively regulated by the bvg locus which encodes a transcriptional activator, BvgA, and a transmembrane sensor protein, BvgS . The gene encoding FhaB, fhaB alone, is not expressed in Escherichia coli, but the introduction of the bvg locus in trans can restore fhaB expression . Using fhaB::lacZY fusions, we have isolated, in E . coli, partially bvg-independent constitutive mutants . The corresponding mutations have been localized to the upstream region of the fhaB promoter at position -15, -16 and -42 from the transcription initiation site . In the absence of the bvg locus, the strength of the mutated promoters was 15 and 200 times higher than the wild-type promoter in the absence of the bvg locus . The expression of these mutated promoters was still enhanced by the presence of the bvg locus. J Allergy Clin Immunol, 1995 Jun, 95(6), 1255 - 60 Homology of a bovine allergen and the oligomycin sensitivity-conferring protein of the mitochondrial adenosine triphosphate synthase complex; Parkkinen S et al.; We have characterized bovine allergens by constructing and analyzing a complementary DNA library from bovine skin . Clones producing proteins that reacted with IgE antibodies from persons with allergy were purified and sequenced . One set of the allergen-coding clones showed an almost complete homology with the bovine oligomycin sensitivity-conferring protein of the mitochondrial adenosine triphosphate synthase complex . The IgE antibodies adsorbed with the recombinant allergen reacted with an 11 kd protein in the cow dander extract . Binding of the IgE from patients allergic to the recombinant allergen expressed in Escherichia coli confirmed the allergen-coding ability of the complementary DNA sequence . The prevalence of the IgE-positive sera among patients with cow allergy and control subjects suggests that the recombinant allergen represents one of the minor allergens in cow dander . This is the first time a mammalian allergen has been identified as a protein with a known function. Genes Dev, 1995 Jun 1, 9(11), 1354 - 65 Activation of the TFIID-TFIIA complex with HMG-2; Shykind BM et al.; The nonhistone chromosomal protein HMG-2 was identified as a factor necessary for activation in a defined transcription reaction in vitro containing RNA polymerase II and purified factors . Activation occurred on all promoters assayed except that of the immunoglobulin IgH gene . TFIIA was required for stimulated levels of transcription . The activation process depended on the presence of TAFs in the TFIID complex and generated a preinitiation complex from which TFIIB dissociated more slowly . However, titration of TFIIB over three orders of magnitude did not obviate the requirement of activator and HMG-2 to achieve stimulated levels of transcription . Analysis of the activated reaction identified the TFIID-TFIIA complex as the first stage of modification during activation . These results suggest that activation can occur solely in the presence of the basal factors, activator protein, and an "architectural" HMG factor, which probably stabilizes an activated conformation of the TFIID-TFIIA-promoter complex. Biotechnol Appl Biochem, 1995 Jun, 21 ( Pt 3), 295 - 311 Recombinant human acetylcholinesterase expressed in Escherichia coli: refolding, purification and characterization; Fischer M et al.; A large-scale preparation of a recombinant human acetylcholinesterase (rhAChE) mutant harbouring a CyS580-->Ser substitution, expressed in Escherichia coli, was refolded following solubilization of the inclusion bodies . Refolded active rhAChE was purified by DEAE-Sepharose and affinity chromatography to apparent homogeneity with a specific activity (4572 units/mg) similar to that of erythrocyte AChE . The stability of the purified enzyme at 22-37 degrees C was dependent on the presence of 0.5 mg/ml BSA, and the optimum pH for stability was 9.0 . rhAChE has a UV-absorbance spectrum typical of a tryptophan-rich protein, with a distinct shoulder at 290 nm and a high absorption coefficient at 280 nm (epsilon 1% = 23.1) . The tryptophan residues in active rhAChE are located in an apolar environment, characteristic of a globular molecule . The difference in amino acid composition between red-blood-cell-derived and recombinant hAChE is probably reflected in their different pI values, namely 5.5-5.8 and 4.6-5.2 respectively . The CD spectrum of rhAChE is typical for an alpha/beta protein, indicating 39% alpha-helix and 22% beta-sheet . This secondary structure is similar to that determined for the Torpedo (electric fish) AChE, by both CD and X-ray crystallography . On the other hand, a purified misfolded and inactive molecule displays a decrease in alpha-helical content to 24%, accompanied by an increase in beta-sheet up to 42%, indicative of extensive changes in the conformation of the protein . On the whole, the recombinant enzyme has been refolded into a native-like conformation possessing full activity, and is thus similar to the naturally occurring red-blood-cell-derived hAChE. Appl Environ Microbiol, 1995 Jun, 61(6), 2428 - 30 The Lly protein protects Legionella pneumophila from light but does not directly influence its intracellular survival in Hartmannella vermiformis; Steinert M et al.; The lly locus (legiolysin) mediates the browning of the culture medium of Legionella pneumophila in the late stationary growth phase, presumably as a result of synthesis of homogentisic acid . Mutagenesis of the lly gene of the L . pneumophila Philadelphia I derivative JR32 did not affect intracellular replication in the natural host Hartmannella vermiformis . The Lly-negative mutant, however, showed a markedly decreased resistance to ordinary light . The cloned lly gene conferred an increased resistance to light in recombinant L . pneumophila and Escherichia coli K-12, indicating a contribution of the Lly protein to ecological adaptation of Legionella species. Mutat Res, 1995 Jun, 343(2-3), 95 - 107 The genotoxicity profile of atorvastatin, a new drug in the treatment of hypercholesterolemia; Ciaravino V et al.; While HMG-CoA reductase inhibitors such as fluvastatin, lovastatin, pravastatin and simvastatin demonstrate lack of in vitro and in vivo mutagenicity and clastogenicity in bacterial and mammalian cells, long term rodent carcinogenicity studies resulted in an increased incidence in neoplasms at high doses . These effects may be attributable to an exaggeration of the desired biochemical effect of the drug and/or a tumor promoting effect . The genotoxicity of atorvastatin, a newly developed HMG-CoA reductase inhibitor, was evaluated in a variety of test systems . In bacterial mutagenicity tests, the E . coli tester strain WP2(uvrA) and S . typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 were exposed to concentrations of atorvastatin as high as 5000 micrograms/plate both in the absence (S9-) and presence (S9+) of metabolic activation . Atorvastatin was not mutagenic in either E . coli or S . typhimurium . Chinese hamster lung V79 cell cultures were exposed to atorvastatin at concentrations of 50-300 micrograms/ml (S9-) and 100-300 micrograms/ml (S9+) and structural chromosome aberrations were assessed . Mutation at the hgprt locus was assessed at concentrations of 100-300 micrograms/ml (S9-) and 150-275 micrograms/ml (S9+) . Atorvastatin was neither mutagenic nor clastogenic in the absence or presence of S9 . The lack of in vitro genotoxicity was corroborated in vivo in a mouse micronucleus study in which single oral doses of atorvastatin were administered to male and female CD-1 mice at 1, 2500, or 5000 mg/kg . No biologically significant increases in the frequency of micronucleated polychromatic erythrocytes in bone marrow at 24, 48, or 72 h postdosing were observed . Thus, atorvastatin, as with the other tested HMG-CoA reductase inhibitors, is not genotoxic. Clin Radiol, 1995 Jun, 50(6), 364 - 70 Delayed CT findings in acute renal infection; Dalla-Palma L et al.; The computed tomography (CT) findings in twelve patients with acute renal infection examined immediately and 3 h after i.v . contrast medium are reported . Three patients also had delayed scans at 6 h . Three main features were observed on the delayed scans: 1 a delayed nephrogram with streaky, wedge shaped or round high density areas . The areas of high density were at the same sites of the inhomogeneous areas of reduced density on the early scans; 2 focal contrast medium staining or a rim of increased density around abscesses; 3 focal areas of increased density at sites distant from the low density areas seen on the early scans . It is postulated that the delayed areas of increased density replace early areas of reduced density caused by ischemia due to vasospasm and/or compressing oedema of the vascular bed or by tubular obstruction . Delayed CT appears to be useful because it improves diagnostic confidence and gives a more exact evaluation of the extent of infection. Carcinogenesis, 1995 Jun, 16(6), 1281 - 5 Spectrum of mutations induced by methyl and ethyl methanesulfonate at the hprt locus of normal and tag expressing Chinese hamster fibroblasts; Klungland A et al.; This work describes the isolation and characterization of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) induced 6-thioguanine-resistant mutants in normal and Escherichia coli tag gene expressing Chinese hamster fibroblast, RJKO, cells . It was previously shown that increased removal of 3-alkylated adenine, effected by 3-methyladenine DNA glycosylase I (Tag), reduces the frequencies of hprt mutations induced by alkylating agents which produce mostly N-alkylation (MMS and EMS) to half the normal rate . In order to identify which type of mutation is suppressed by increased 3-alkyladenine repair we have determined the DNA base sequence changes of the hprt cDNA in 61 independent MMS- and EMS-induced mutant clones . For both cell types and irrespective of the agent used, the majority of mutations were GC to AT transitions originating in the non-transcribed strand . Only 6/55 base substitutions occurred at AT base pairs: five AT to GC transitions and one AT to CG transversion . Six mutations were found to be deletions . These results indicate that 3-alkylated adenines in DNA are not directly premutagenic . The fact that the mutation frequency is reduced by increased 3-alkyladenine removal might be explained by postulating the existence in mammalian cells of an SOS-like response turned on by cytotoxic lesions like 3-alkyladenine, or, alternatively, that increased removal of 3-alkyladenine increases the number of single-strand breaks in DNA, which stalls DNA replication and allows a prolonged time for DNA repair by the alkyltransferase. Arch Biochem Biophys, 1995 Jun 1, 319(2), 574 - 8 Glucokinase of Escherichia coli: induction in response to the stress of overexpressing foreign proteins; Arora KK et al.; A variety of stressful conditions, such as heat shock, ethanol, osmotic shock, glucose deprivation, and oxidative stress, are known to induce the synthesis of specific proteins . Here, we report the induction in Escherichia coli of a protein elicited in response to a hitherto unidentified stress condition, i.e., the overexpression of foreign proteins . The induced protein identified as glucokinase (EC 2.7.1.2) is produced at a level > or = 20-fold higher than the level in wild-type E . coli when foreign proteins are expressed under the control of the alkaline phosphatase (phoA) promoter . The bacterial glucokinase is shown to have a mass of approximately 47 kDa determined by a "renaturation activity stain assay" in situ following sodium dodecyl sulfate-poly-acrylamide gel electrophoresis and exhibits a high specificity for the phosphorylation of glucose . The apparent Km values for glucose and ATP for the enzyme are 0.15 and 0.50 mM, respectively, indicating that the E . coli enzyme is a low Km glucose hexokinase . The enzyme cross-reacts with rabbit antisera raised against hexokinase from higher eukaryotes, implicating some sequence similarity with mammalian hexokinases . Under normal conditions, E . coli glucokinase plays a minor role in glucose metabolism . However, under anabolic stress conditions, this glycolytic enzyme may be required to supplement levels of glucose 6-phosphate . Alternatively, glucokinase, which is predicted in analogy to other hexose-utilizing kinases to have structural folds characteristic of hsp 70, may itself, or in combination with other E . coli proteins, function in the stabilization of newly synthesized proteins. Arch Biochem Biophys, 1995 Jun 1, 319(2), 540 - 50 Expression of cytochrome P450 2D6 in Escherichia coli, purification, and spectral and catalytic characterization; Gillam EM et al.; Cytochrome P450 (P450) 2D6 is the classic human liver debrisoquine 4-hydroxylase, the first human P450 for which genetic polymorphism was clearly demonstrated . We prepared 11 different constructs of P450 2D6, with modification at the N-terminus, for expression in Escherichia coli with the vector pCW . These varied considerably in levels of expression of apo- and holoprotein, with the best yield being obtained in a system in which much of the N-terminal hydrophobic segment was removed . Production of holoprotein was highly dependent upon the addition of delta-aminolevulinic acid and FeCl3 to cultures, even though heme production should not be limiting in this system . The expressed protein was not tightly bound to the "heavier" membrane fraction but did not appear to behave as a soluble protein either . A purification strategy was developed involving fractional centrifugation, Triton X-114 phase separation, and flavodoxin affinity chromatography, which led to recovery of apparently electrophoretically homogeneous protein in good yield . Purified P450 2D6 had the expected N-terminal amino acid sequence and catalytic activities toward debrisoquine (4-hydroxylation) and bufuralol (1'-hydroxylation) . The availability of a ready source of the recombinant protein should facilitate physical as well as functional studies and antibody production for other uses. Arch Biochem Biophys, 1995 Jun 1, 319(2), 508 - 11 Copper, zinc superoxide dismutase in Escherichia coli: periplasmic localization; Benov L et al.; Cu,ZnSOD purified from Escherichia coli has been used to raise antibodies in rabbits . The resultant antiserum was found to recognize a single band on Western blots of SDS-polyacrylamide gel electropherograms, and that single band coincided with the position of the Cu,ZnSOD . Ultrathin sections of fixed E . coli were treated with the antibody followed by protein A bearing 10-nm gold particles . Electron microscopy revealed that Cu,ZnSOD was largely localized in the periplasm in polar bays. J Gen Virol, 1995 Jun, 76 ( Pt 6), 1527 - 32 Expression of beta-galactosidase in neurons of dorsal root ganglia which are latently infected with herpes simplex virus type 1; Ecob-Prince MS et al.; Explanation into culture of dorsal root ganglia (DRG) latently infected with herpes simplex virus type 1 (HSV-1) causes reactivation of the virus . Previous studies have suggested that either latency-associated transcripts (LATs) were removed as an early consequence of reactivation or, alternatively, there was a population of latently infected cells which did not contain LATs . We have now attempted to detect this population of neurons by inserting a reporter gene (Escherichia coli lacZ gene), under the control of promoters other than LAT, into the HSV-1 strain 17 mutant in 1814, which was used in the earlier studies . One of these promoters, the human cytomegalovirus enhancer, resulted in weak expression of beta-galactosidase in DRG neurons for at least 5 months . The pattern of staining was predominantly homogeneous in neurons at 3 or 5 days post-infection or at 3 days post-explanation, and was predominantly speckled in latently infected neurons (1 to 5 months post-infection) . About 30% of the beta-galactosidase-positive neurons did not contain LATs by in situ hybridization . However, the detergents used to enable penetration of the substrate for beta-galactosidase had also reduced the levels of the LATs; in neurons which originally had only small numbers of LATs this may have reduced levels to below those detectable by the methods used . There was, therefore, no unequivocal evidence for a population of latently HSV-1-infected cells which did not express LATs. J Gen Virol, 1995 Jun, 76 ( Pt 6), 1515 - 20 Cloning, sequencing and expression of the S1 gene of avian reovirus; Shapouri MR et al.; The S1 genome segment of avian reovirus strain S1133 was cloned and completely sequenced . The sequence comprised 1636 bp with three distinct open reading frames (ORFs), suggesting the gene was polycistronic in nature . The three ORFs from 5' to 3' were predicted to encode polypeptides of 9.8, 3.8 and 34.9 kDa, respectively . Of the three ORFs, only the third possessed the AUG initiation codon in an optimum context for translation . The third ORF-encoded protein, 326 amino acids in length, was expressed in Escherichia coli and used as antigen in immunoblots . The protein was concluded to be sigma 3 on the basis of its recognition by a chicken anti-reovirus antiserum and due to the fact that a mouse antiserum raised against it recognized specifically the viral sigma 3 polypeptide . Sequence comparison of the avian reovirus S1 gene with its mammalian counterpart did not show any significant similarity between the two . However, amino acid sequence analysis and the predicted existence of a heptapeptide repeat pattern, as well as the relatively high frequency of alpha-helix structures in the amino terminal portion of sigma 3 suggests that this protein is structurally, and probably functionally, related to mammalian reovirus sigma 1 protein. J Gen Virol, 1995 Jun, 76 ( Pt 6), 1503 - 7 Expression, subcellular location and modification of the 50 kDa protein encoded by ORF2 of the apple chlorotic leaf spot trichovirus genome; Sato K et al.; A putative movement protein of molecular mass 50 kDa encoded by the ORF2 of the apple chlorotic leaf spot virus (ACLSV) genome was expressed in Escherichia coli using an expression vector and was then used to produce an antiserum . Immunoblot analysis using an antiserum raised against this protein showed that the ORF2 protein of ACLSV was detected in both cell wall and cell membrane fractions prepared from infected Chenopodium quinoa tissues . The ORF2 protein from infected tissues had a molecular mass of 52 kDa, larger than that of the full-length ORF2 protein (50 kDa protein) expressed in E . coli . Incubation of the 52 kDa protein with alkaline phosphatase resulted in a decrease in its apparent molecular mass from 52 kDa to 50 kDa, strongly suggesting that the ORF2 protein of ACLSV is phosphorylated in infected plant tissues. J Gen Virol, 1995 Jun, 76 ( Pt 6), 1493 - 6 The tobacco necrosis virus p7a protein is a nucleic acid-binding protein; Offei SK et al.; The two centrally located open reading frames (ORFs) of necroviruses may, by analogy with the similarly located and related ORFs of carmoviruses, be expected to have a function in virus movement in plants . In the case of tobacco necrosis virus (TNV) strain D these proteins both have a molecular mass of approximately 7 kDa and are thus known as p7a and p7b . We overexpressed p7a in Escherichia coli, separated it from bacterial proteins and renatured it on gels, and showed that p7a was able to bind single-stranded RNA and single-stranded DNA, but was unable to bind double-stranded DNA . These protein-nucleic acid complexes were stable at moderately high salt concentrations . Protein p7b could not be expressed in a number of bacterial systems . We speculate that in TNV, unlike some other viruses which encode a single movement protein with separate functional domains for RNA binding and plasmodesmatal targeting, p7a and p7b may respectively provide these functions on separate proteins. J Gen Virol, 1995 Jun, 76 ( Pt 6), 1417 - 31 Quiescent viral genomes in human fibroblasts after infection with herpes simplex virus type 1 Vmw65 mutants; Jamieson DR et al.; The development and utilization of a tissue culture system for the analysis of quiescent, nonreplicating herpes simplex virus type 1 (HSV-1) genomes is described . It was demonstrated previously that the HSV-1 Vmw65 mutant in1814, which is impaired for immediate early (IE) transcription, was retained for many days in human fetal lung (HFL) fibroblasts in a quiescent 'latent' state . Molecular analysis of the viral genome was not possible, however, due to residual expression of IE proteins and consequent cytotoxicity at high m.o.i . In the study reported here, IE transcription was reduced further by pretreatment of cells with interferon-alpha (IFN-alpha) and by the use of mutant in1820, a derivative of in1814 in which the Vmw110 promoter was replaced by the Moloney murine leukaemia virus (Momulv) enhancer . The Momulv enhancer was not expressed under IE conditions; thus in1820 was more impaired for replication than in1814 and behaved as if deficient for both Vmw65 and Vmw110 . In cells pretreated with IFN-alpha and subsequently infected with in1820 cytotoxicity was overcome, enabling a tissue culture system to be developed in which all cells stably retained at least one quiescent viral genome . To assist the analysis of gene expression, in1820 was further modified by insertion of the Escherichia coli lacZ gene controlled by the human cytomegalovirus enhancer (mutant in1883) or the HSV-1 immediate early Vmw110 promoter (in1884) . Expression of beta-galactosidase was not detected after infection of IFN-alpha-pretreated cells with in1883 or in1884 but could be induced in almost all cells containing a viral genome, by superinfection of cultures . In1820-derived viruses were retained for at least 9 days and were not reactivated by subculture of cells . A regular arrangement of nucleosomes, as found in cellular chromatin, was not detected on the viral genome at the thymidine kinase locus . The non-linear genome was a template for reactivation with no requirement for prior conversion to a linear form . A small number of remaining linear genomes resulted from incomplete uncoating of input virus. FEMS Microbiol Lett, 1995 Jun 1, 129(1), 63 - 7 Carbon dioxide regulated secretion of the EaeB protein of enteropathogenic Escherichia coli; Haigh R et al.; An eaeA mutant (intimin deficient) of enteropathogenic Escherichia coli stimulated phosphorylation of several host cell proteins, showing that intimate adherence is not required to activate signal transduction pathways in enteropathogenic E . coli-infected cells . Growth of enteropathogenic E . coli in tissue culture medium in 5% CO2, in the presence or absence of cultured cells, resulted in the secretion of several bacterial proteins . Two of these, 36 kDa and 20 kDa in size, were expressed at significantly lower levels in air . N-terminal sequencing and analysis of secreted proteins of an eaeB mutant indicated that the 36 kDa secreted protein was EaeB, previously implicated in the stimulation of signalling pathways in enteropathogenic E . coli-infected cells. FEMS Microbiol Lett, 1995 Jun 1, 129(1), 21 - 6 Sequence of cDNA coding for a 65 kDa adhesive protein for the specific detection of Trichomonas vaginalis by PCR; Rappelli P et al.; A Trichomonas vaginalis cDNA library was constructed and recombinant plaques were screened using rabbit immunoglobulins specific for P65, a protozoan protein involved in pathogenicity that we identified in a previous study . A 1.38 kilobases cDNA fragment coding for the P65 protein was cloned in E . coli and then sequenced . On the basis of of the sequence obtained, six primers were synthesised and used to set up a Polymerase Chain Reaction . The presence of a specific amplicon in all 30 clinical isolates tested shows that P65 is a conserved and stable gene . The reaction is highly sensitive (as few as 5 to 10 parasites can be detected) and specific for Trichomonas vaginalis; the gene coding P65 adhesin can be therefore considered a very good molecular target for polymerase chain reaction-based diagnostic purposes. EMBO J, 1995 Jun 1, 14(11), 2651 - 60 Effects of Holliday junction position on Xer-mediated recombination in vitro; Arciszewska L et al.; Site-specific recombination mediated by XerC and XerD functions in the segregation of circular replicons in Escherichia coli . A key feature of most models of recombination for the family of recombinases to which XerC and XerD belong is that a Holliday junction forms at the position of the first pair of recombinase-mediated strand exchanges and then branch migrates 6-8 bp to the position of the second pair of strand exchanges . We have tested this hypothesis for Xer recombination by studying the effects of junction position on XerC-mediated strand exchange in vitro . Recombination of synthetic Holliday junction substrates in which junction mobility was constrained to a region extending over or removed away from the normal cleavage and exchange point was analysed . All substrates undergo strand cleavage at the normal position . We infer that the Holliday junction need not be at this position during strand cleavage and exchange . With substrates in which the Holliday junction is constrained to a region away from the XerC-mediated cleavage point, strand exchange generates products with the predicted mispaired bases. EMBO J, 1995 Jun 1, 14(11), 2613 - 9 Codon-dependent conformational change of elongation factor Tu preceding GTP hydrolysis on the ribosome; Rodnina MV et al.; The mechanisms by which elongation factor Tu (EF-Tu) promotes the binding of aminoacyl-tRNA to the A site of the ribosome and, in particular, how GTP hydrolysis by EF-Tu is triggered on the ribosome, are not understood . We report steady-state and time-resolved fluorescence measurements, performed in the Escherichia coli system, in which the interaction of the complex EF-Tu.GTP.Phe-tRNAPhe with the ribosomal A site is monitored by the fluorescence changes of either mant-dGTP {3'-O-(N-methylanthraniloyl)-2-deoxyguanosine triphosphate}, replacing GTP in the complex, or of wybutine in the anticodon loop of the tRNA . Additionally, GTP hydrolysis is measured by the quench-flow technique . We find that codon-anticodon interaction induces a rapid rearrangement within the G domain of EF-Tu around the bound nucleotide, which is followed by GTP hydrolysis at an approximately 1.5-fold lower rate . In the presence of kirromycin, the activated conformation of EF-Tu appears to be frozen . The steps following GTP hydrolysis--the switch of EF-Tu to the GDP-bound conformation, the release of aminoacyl-tRNA from EF-Tu to the A site, and the dissociation of EF-Tu-GDP from the ribosome--which are altogether suppressed by kirromycin, are not distinguished kinetically . The results suggest that codon recognition by the ternary complex on the ribosome initiates a series of structural rearrangements resulting in a conformational change of EF-Tu, possibly involving the effector region, which, in turn, triggers GTP hydrolysis. EMBO J, 1995 Jun 1, 14(11), 2551 - 60 Escherichia coli FtsH is a membrane-bound, ATP-dependent protease which degrades the heat-shock transcription factor sigma 32; Tomoyasu T et al.; Escherichia coli FtsH is an essential integral membrane protein that has an AAA-type ATPase domain at its C-terminal cytoplasmic part, which is homologous to at least three ATPase subunits of the eukaryotic 26S proteasome . We report here that FtsH is involved in degradation of the heat-shock transcription factor sigma 32, a key element in the regulation of the E . coli heat-shock response . In the temperature-sensitive ftsH1 mutant, the amount of sigma 32 at a non-permissive temperature was higher than in the wild-type under certain conditions due to a reduced rate of degradation . In an in vitro system with purified components, FtsH catalyzed ATP-dependent degradation of biologically active histidine-tagged sigma 32 . FtsH has a zinc-binding motif similar to the active site of zinc-metalloproteases . Protease activity of FtsH for histidine-tagged sigma 32 was stimulated by Zn2+ and strongly inhibited by the heavy metal chelating agent o-phenanthroline . We conclude that FtsH is a novel membrane-bound, ATP-dependent metalloprotease with activity for sigma 32 . These findings indicate a new mechanism of gene regulation in E . coli. EMBO J, 1995 Jun 1, 14(11), 2545 - 50 Silencing of Escherichia coli bgl promoter by flanking sequence elements; Schnetz K; Silencing of a transcriptional unit by flanking sequence elements has so far only been described for eukaryotic systems . Here, a similar system is described in bacteria . The Escherichia coli bgl operon (beta-glucoside utilization) is normally cryptic due to very low promoter activity . However, low activity is not attributable to the quality of the promoter itself but is caused by its chromosomal context . The bgl promoter is perfectly active when tested outside of its normal context of a stretch of a few hundred base pairs . In addition, other promoters become inactivated when placed into the bgl region . Both the deletion of an upstream sequence element and the replacement of sequences located downstream result in promoter de-repression, demonstrating that silencing of promoters within this stretch of DNA in vivo is an active process brought about by the combined action of upstream and downstream chromosomal elements. EMBO J, 1995 Jun 1, 14(11), 2447 - 60 Epitope mapping and direct visualization of the parallel, in-register arrangement of the double-stranded coiled-coil in the NuMA protein; Harborth J et al.; NuMA, a 238 kDa protein present in the nucleus during interphase, translocates to the spindle poles in mitosis . NuMA plays an essential role in mitosis, since microinjection of the NuMA SPN-3 monoclonal antibody causes mitotic arrest and micronuclei formation . We have mapped the approximate position of the epitopes of six monoclonal NuMA antibodies using recombinant NuMA fragments . The SPN-3 epitope has been located to residues 255-267 at the C-terminus of the first helical subdomain of the central rod domain and several residues crucial for antibody binding have been identified . To gain insight into the ultrastructure of NuMA, several defined fragments, as well as the full-length recombinant protein, were expressed in Escherichia coli and purified to homogeneity . They were then characterized by chemical cross-linking, circular dichroism spectra and electron microscopy . The results directly reveal the tripartate structure of NuMA . A long central rod domain is flanked by globular end domains . The rod is 207 nm long and is at least 90% alpha-helical . It reflects a double-stranded coiled-coil with the alpha-helices arranged parallel and in register . The NuMA protein thus forms the longest coiled-coil currently known . Our analyses reveal no indication that recombinant NuMA assembles into filaments or other higher order structures. Virology, 1995 Jun 1, 209(2), 688 - 91 Fidelity of homologous recombination in vaccinia virus DNA; Ball LA; The fidelity of homologous recombination in vaccinia virus (VV) DNA was examined by constructing a viral recombinant whose genome contained a copy of the Escherichia coli lac z gene in which the central third of the gene was repeated on either side of the VV thymidine kinase (tk) gene . In this virus, homologous DNA recombination and consequent excision of the tk gene were necessary to restore the open reading frame for beta-galactosidase and thereby to confer a lac z+ phenotype . Imprecise recombination was predicted to increase the frequency of lac z- virus . However, after several passages during which almost every viral genome underwent homologous recombination, the frequency of lac z- plaques was indistinguishable from that of a control virus that could express beta-galactosidase without prior recombination . We conclude that homologous recombination in VV DNA occurs with perfect fidelity at least 99% of the time. Virology, 1995 Jun 1, 209(2), 480 - 8 Gene organization of chicken anemia virus; Kato A et al.; The genomic DNA of chicken anemia virus (CAV) was cloned and sequenced from a Japanese isolate CAA82-2 . The nucleotide sequence of CAA82-2 isolate was 98% identical with that of the European Cuxhaven-1 strain (Noteborn et al., J . Virol . 65, 3131-3139, 1991) . Nine open reading frames (ORFs) consisting of more than 100 nucleotides were found, i.e., four ORFs (CA1-CA4) on the plus strand and five ORFs (CA1R-CA5R) on the minus strand . These ORFs with the exception of CA4 are conserved between the two CAV isolates . All of these ORFs were expressed in Escherichia coli as fusion proteins with beta-galactosidase . By Western blot analysis, the CA2 and CA3 fusion proteins were found to react with CAV-infected chicken sera . Rabbit hyperimmune sera against the CA1, CA2, and CA3 fusion proteins were produced and tested their reactivity to CAV-infected cells . Two viral proteins with the apparent size of 54 and 16 kDa reacted with the antibodies against CA1 and CA3 fusion proteins, respectively . The 16-kDa protein, CA3, was suggested to be a major immunogen on CAV infection. Virology, 1995 Jun 1, 209(2), 304 - 14 Generation of thymidine kinase-deficient mutants of infectious laryngotracheitis virus; Schnitzlein WM et al.; Current vaccines for the avian respiratory disease infectious laryngotrachetitis consist of naturally attenuated strains of the causative agent--the herpesvirus infectious laryngotracheitis virus (ILTV) . Due to the dissemination of these viruses from vaccinated chickens as well as their possible reversion to more pathogenic forms, the use of genetically engineered viral vaccines lacking virulence factors while retaining antigenicity is being considered . Since the thymidine kinase (TK) activity of herpesviruses has been associated with virulence, inactivation of the encoding gene in the ILTV genome should attenuate the virus . Moreover, by analogy to other TK- herpesviruses, the ability of such ILTV mutants to induce a protective response in chickens should not be compromised . Therefore, the deliberate genetic alteration of ILTV was attempted . In order to prevent reversion and also to enable identification of the modified virus, a "marker" transcriptional unit (Escherichia coli lacZ gene fused to a SV-40 3'-polyadenylation signal sequence and regulated by the pseudorabies virus gX gene promoter) was inserted via homologous recombination at one of two loci within the ILTV TK gene . Recombinant viruses were identified and plaque-purified on the basis of their ability to produce beta-galactosidase . Retention of the foreign DNA at the predicted sites in the genomes of the recombinant ILTV was verified by Southern hybridization . Since their replication was unaffected by the thymine analog 1-(2-fluoro-2-deoxy-beta-D-arabinofuranosyl)-5-methyluracil, the recombinants appeared to have a TK- phenotype . Despite this apparent deficiency, prior inoculation of either recombinant virus into chickens afforded the birds protection against a lethal challenge of virulent ILTV . Moreover, the degree of respiratory distress in the chickens vaccinated with the recombinants was relatively mild compared to the severe reaction in birds receiving the parental virus . Thus, ILTV can be genetically attenuated without an accompanying loss of immunogenicity. J Cell Biol, 1995 Jun, 129(5), 1421 - 32 Separate cis-acting DNA elements of the mouse pro-alpha 1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice; Rossert J et al.; The genes coding for the two type I collagen chains, which are active selectively in osteoblasts, odontoblasts, fibroblasts, and some mesenchymal cells, constitute good models for studying the mechanisms responsible for the cell-specific activity of genes which are expressed in a small number of discrete cell types . To test whether separate genetic elements could direct the activity of the mouse pro-alpha 1(I) collagen gene to different cell types in which it is expressed, transgenic mice were generated harboring various fragments of the proximal promoter of this gene cloned upstream of the Escherichia coli beta-galactosidase gene . During embryonic development, X-gal staining allows for the precise identification of the different cell types in which the beta-galactosidase gene is active . Transgenic mice harboring 900 bp of the pro-alpha 1(I) proximal promoter expressed the transgene at relatively low levels almost exclusively in skin . In mice containing 2.3 kb of this proximal promoter, the transgene was also expressed at high levels in osteoblasts and odontoblasts, but not in other type I collagen-producing cells . Transgenic mice harboring 3.2 kb of the proximal promoter showed an additional high level expression of the transgene in tendon and fascia fibroblasts . The pattern of expression of the lacZ transgene directed by the 0.9- and 2.3-kb pro-alpha 1(I) proximal promoters was confirmed by using the firefly luciferase gene as a reporter gene . The pattern of expression of this transgene, which can be detected even when it is active at very low levels, paralleled that of the beta-galactosidase gene . These data strongly suggest a modular arrangement of separate cell-specific cis-acting elements that can activate the mouse pro-alpha(I) collagen gene in different type I collagen-producing cells . At least three different types of cell-specific elements would be located in the first 3.2 kb of the promoter: (a) an element that confers low level expression in dermal fibroblasts; (b) a second that mediates high level expression in osteoblasts and odontoblasts; and (c) one responsible for high level expression in tendon and fascia fibroblasts . Our data also imply that other cis-acting cell-specific elements which direct activity of the gene to still other type I collagen-producing cells remain to be identified. Invest Ophthalmol Vis Sci, 1995 Jun, 36(7), 1361 - 70 Measurement of blood-retinal barrier breakdown in endotoxin-induced endophthalmitis; Metrikin DC et al.; PURPOSE: Endophthalmitis is a severe inflammatory disorder with profound visual consequences . Treatment of this disorder has been limited by the lack of quantitative information regarding retinal responses to severe inflammation . The purpose of this study was to measure the effect of endotoxin-induced endophthalmitis on blood-retinal barrier (BRB) function in vivo using contrast-enhanced magnetic resonance imaging (MRI) . METHODS: Endophthalmitis was produced by injecting Escherichia coli endotoxin into the midvitreous of pigmented rabbits . Contrast-enhanced MRI was performed at selected intervals thereafter . In all cases, a clinical grading system was used to assess the severity of inflammation before imaging . In a dose-response experiment, total vitreous protein was measured from vitreous specimens obtained 1 day after endotoxin injection and immediately after the imaging procedure . RESULTS: At 1 day after injection, endotoxin produced a selective breakdown of the inner BRB at all doses evaluated (0.01 microgram to 500 micrograms) . Permeability-surface area product normalized to the area of leaky retina (PS') increased from 1.35 +/- 0.78 x 10(-4) cm/minute (mean +/- SEM, n = 4 eyes) at a dose of 0.01 microgram to 8.15 +/- 2.22 x 10(-4) cm/minute n = 4) eyes) at a dose of 10 micrograms . Inner BRB integrity was restored by day 28 after injection . In general, changes in PS', blood-aqueous barrier leakage, mean clinical score, and vitreous protein concentration were found, but the correlation between any two of these parameters was poor . CONCLUSION: Leakage of contrast appears early in the course of endotoxin-induced endophthalmitis and is a self-limited process . In future studies, these quantifiable changes in BRB permeability should prove useful in the assessment of various therapeutic interventions. Biochem J, 1995 Jun 1, 308 ( Pt 2), 641 - 4 The haem b558 component of the cytochrome bd quinol oxidase complex from Escherichia coli has histidine-methionine axial ligation; Spinner F et al.; The cytochrome bd ubiquinol oxidase from Escherichia coli is induced when the bacteria are cultured under microaerophilic or low-aeration conditions . This membrane-bound respiratory oxidase catalyses the two-electron oxidation of ubiquinol and the four-electron reduction of dioxygen to water . The oxidase contains three haem prosthetic groups: haem b558, haem b595 and haem d . Haem d is the oxygen binding site, and it is likely that haem d and b595 form a bimetallic site in the enzyme . Haem b558 has been previously characterized spectroscopically as being low spin and has been shown to be located within subunit I (CydA) of this two-subunit enzyme . It is likely that haem b558 is associated with the quinol oxidation site, which has also been shown to be within subunit I . In a previous effort to locate the specific amino acids axially ligated to haem b558, all six histidines within subunit I were altered by site-directed mutagenesis . Only one, histidine-186, was identified as a likely ligand to haem b558 . Hence it was suggested that haem b558 could not have bis(histidine) ligation . In the current work, a combination of low-temperature near-infrared magnetic circular dichroism (NIR-MCD) and EPR spectroscopies have been employed to identify the nature of the haem b558 axial ligands . The NIR-MCD spectrum at cryogenic temperatures is dominated by the low-spin haem b558 component of the complex, and the low-energy band near 1800 nm is strong evidence for histidine-methionine ligation . It is concluded that haem b558 is ligated to histidine-186 plus one of the methionines located within subunit I of the oxidase. Biochem J, 1995 Jun 1, 308 ( Pt 2), 543 - 6 Endotoxin-induced inflammation does not cause hepatic zinc accumulation in mice lacking metallothionein gene expression; Philcox JC et al.; The action of endotoxin lipopolysaccharide (LPS) on hepatic Zn uptake was examined in mice lacking expression of metallothionein (MT)-1 and MT-II genes . Hepatic Zn concentrations, which in normal control mice increased by a mean 29% (MT elevated 20-fold) 16 h post-LPS exposure, did not increase in MT-null mice . Plasma Zn fell by 68% in controls and 32% in MT-null mice . The time course of LPS action in normal mice was characterized by a rapid reduction (-74% at 4 h, -81% at 8 h) and partial recovery (-39% at 24 h) in plasma Zn, with a progressive increase over 24 h in hepatic concentrations of MT (by 36-fold) and Zn (by 40%) . In contrast, the MT-null mice had a linear decrease in plasma Zn (-15% at 8 h, -41% at 24 h) and early loss of Zn from the liver . The Zn changes seen in MT-null mice were largely attributable to LPS-associated anorexia . Food deprivation (20 h) alone caused respective 14% and 30% decreases in hepatic and plasma Zn concentrations and a 27% reduction in total liver Zn reserves, whereas fasted normal mice conserved Zn with a 4-fold increase in hepatic MT . This study confirms that MT synthesis is essential for endotoxin-induced liver Zn accumulation. Mutat Res, 1995 Jun, 329(1), 49 - 56 Starvation-associated mutation in Escherichia coli strains defective in transcription repair coupling factor; Bridges BA; When E . coli WU3610 (tyrA14 ochre) bacteria are starved of tyrosine on the surface of glucose-salts agar plates, there is a progressive accumulation of slow growing prototrophic mutants that are neither revertants at the ochre codon nor any of the well characterised tRNA ochre suppressors . Isogenic derivatives defective in transcription repair coupling factor (mfd) showed normal starvation-associated mutation (SAM) . WU361045, the original mfd strain, showed very much reduced SAM . At 37 degrees C this was associated with progressive loss of viability on plates but the defect in SAM was not due to loss of viability since incubation at 27 degrees C or addition of catalase prevented the loss of viability but did not restore SAM . Furthermore, mutants could not be rescued from starved WU361045 populations by a short period of tyrosine supplementation arguing that WU361045 was defective not in the survival of starvation-associated mutants, but in their formation . The SAM defect in WU361045 was not complemented by the katF gene on a low copy number plasmid . It is concluded that WU361045 carries an unidentified mutation, not under katF control, that greatly reduces SAM . If SAM is attributable to a spontaneously occurring DNA lesion, the latter is unlikely to be formed by hydrogen peroxide or active species derived from it. J Infect Dis, 1995 Jun, 171(6), 1636 - 9 HEp-2 cell-adherent Escherichia coli in patients with human immunodeficiency virus-associated diarrhea; Mathewson JJ et al.; Diarrhea occurs commonly in African human immunodeficiency virus (HIV) infections . A case-control (HIV-positive vs . -negative) study of adults with diarrhea was done in Lusaka, Zambia, to determine the prevalence of intestinal infection by HEp-2 cell-adherent Escherichia coli . Adherent E . coli were more common in HIV-positive patients with acute diarrhea than among HIV-negative controls (60% vs . 33%) and were found significantly more often in HIV-positive patients with chronic diarrhea than among HIV-negative controls with chronic diarrhea (79% vs . 17%, P < .002) . Adherent strains were found significantly more often among HIV-positive patients (69%) than in 22 asymptomatic subjects (36%, P < .02) . The HEp-2 cell adherence of the E . coli strains did not show a common pattern . Adherent bacteria were also observed in colonic biopsies from 32% of Zambians with chronic diarrhea who underwent endoscopy . Adherent E . coli may be an important cause of HIV-associated diarrhea in Zambia. J Bacteriol, 1995 Jun, 177(12), 3619 - 22 Topoisomerase activity during the heat shock response in Escherichia coli K-12; Camacho-Carranza R et al.; During the upshift of temperature from 30 to 42, 45, 47, or 50 degrees C, an increase in the level of supercoiling of a reporter plasmid was observed . This increase was present in groE and dnaK mutants but was inhibited in cells treated with chloramphenicol and novobiocin . The intracellular {ATP}/{ADP} ratio increased rapidly after an upshift in temperature from 30 to 47 degrees C and then decreased to reach a level above that observed at 30 degrees C . These results suggest that gyrase and proteins synthesized during heat shock are responsible for the changes seen in plasmid supercoiling . Proteins GroE and DnaK are probably not involved in this phenomenon. J Bacteriol, 1995 Jun, 177(12), 3610 - 2 SOS induction in Escherichia coli by single-stranded DNA of mutant filamentous phage: monitoring by cleavage of LexA repressor; Higashitani N et al.; Infection of Escherichia coli in the presence of chloramphenicol with mutant filamentous phage that are defective in the initiation of minus-strand DNA synthesis induces the SOS response as monitored by cellular LexA levels . This observation demonstrates that single-stranded DNA serves as a primary signal for SOS induction in vivo. J Bacteriol, 1995 Jun, 177(12), 3593 - 5 The putative fabJ gene of Escherichia coli fatty acid synthesis is the fabF gene; Magnuson K et al.; Siggaard-Andersen and coworkers (M . Siggaard-Andersen, M . Wissenbach, J . Chuck, I . Svendsen, J . G . Olsen, and P . von Wettstein-Knowles, Proc . Natl . Acad . Sci . USA 91:11027-11031, 1994) recently reported the DNA sequence of a gene encoding a beta-ketoacyl-acyl carrier protein synthase from Escherichia coli . These workers assigned this gene the designation fabJ and reported that the gene encoded a new beta-ketoacyl-acyl carrier protein synthase . We report that the fabJ gene is the previously reported fabF gene that encodes the known beta-ketoacyl-acyl carrier protein synthase II. J Bacteriol, 1995 Jun, 177(12), 3589 - 92 Anucleate cell production by Escherichia coli delta hns mutant lacking a histone-like protein, H-NS; Kaidow A et al.; Normal-sized anucleate cells were observed in the cultures of a delta hns mutant strain . Even in nucleate cells, some populations showed irregular intracellular localization of the nucleoids . The delta hns mutant showed reduced ploidy, although initiation of chromosome replication was essentially synchronous as defined by flow cytometric analysis . These results indicate that the delta hns mutant is defective in the mechanisms of chromosome partitioning and chromosome replication. J Bacteriol, 1995 Jun, 177(12), 3518 - 26 Suppression of signal sequence defects and azide resistance in Escherichia coli commonly result from the same mutations in secA; Huie JL et al.; The SecA protein of Escherichia coli is required for protein translocation from the cytoplasm . The complexity of SecA function is reflected by missense mutations in the secA gene that confer several different phenotypes: (i) conditional-lethal alleles cause a generalized block in protein secretion, resulting in the cytoplasmic accumulation of the precursor forms of secreted proteins; (ii) azi alleles confer resistance to azide at concentrations up to 4 mM; and (iii) prlD alleles suppress a number of signal sequence mutations in several different genes . To gain further insights into the role of SecA in protein secretion, we have isolated and characterized a large number of prlD mutations, reasoning that these mutations alter a normal function of wild-type SecA . Our results reveal a striking coincidence of signal sequence suppression and azide resistance: the majority of prlD alleles also confer azide resistance, and all azi alleles tested are suppressors . We suggest that this correlation reflects the mechanism(s) of signal sequence suppression . There are two particularly interesting subclasses of prlD and azi alleles . First, four of the prlD and azi alleles exhibit special properties: (i) as suppressors they are potent enough to allow PrlD (SecA) inactivation by a toxic LacZ fusion protein marked with a signal sequence mutation (suppressor-directed inactivation), (ii) they confer azide resistance, and (iii) they cause modest defects in the secretion of wild-type proteins . Sequence analysis reveals that all four of these alleles alter Tyr-134 in SecA, changing it to Ser, Cys, or Asn . The second subclass consists of seven prlD alleles that confer azide supersensitivity, and sequence analysis reveals that six of these alleles are changes of Ala-507 to Val . Both of the affected amino acids are located within different putative ATP-binding regions of SecA and thus may affect ATPase activities of SecA . We suggest that the four azide-resistant mutations slow an ATPase activity of SecA, thus allowing successful translocation of increased amounts of mutant precursor proteins. J Bacteriol, 1995 Jun, 177(12), 3479 - 84 Regulation of the Caulobacter crescentus dnaKJ operon; Avedissian M et al.; The bacterial heat shock proteins DnaK and DnaJ are members of a class of molecular chaperones that are required for a wide variety of cellular functions at normal growth temperatures . In Caulobacter crescentus, the expression of the dnaKJ operon is regulated both temporally during the normal cell cycle and by heat shock . Analysis of deletions and base substitutions in the 5' region of the operon established the presence of two functional promoters: a heat shock-inducible promoter, P1, with characteristics of a sigma 32 promoter, and an adjacent sigma 70-like promoter, P2 . Transcription initiating at the sigma 70-like promoter is under strict temporal control, whereas transcription initiating at the heat shock promoter at 30 degrees C is not . Transcription of dnaKJ occurs during a short period in the cell cycle, concomitant with the onset of DNA replication . Deletions in the 5' region have also revealed that all cis-acting sites required for temporal control of transcription reside within 50 bases of the P2 start site . Transcripts initiating from either the P1 or the P2 promoter have an RNA leader sequence with a high probability of forming an extensive secondary structure . Deletion of this leader sequence resulted in an increased rate of expression in both transcriptional and translational fusions . Although the temporal control of expression at physiological temperatures is not affected by the presence or absence of the leader sequence, changes in mRNA secondary structure may contribute to the modulation of DnaK and DnaJ levels at normal temperatures and during heat shock. J Bacteriol, 1995 Jun, 177(12), 3455 - 64 Role for the histone-like protein H-NS in growth phase-dependent and osmotic regulation of sigma S and many sigma S-dependent genes in Escherichia coli; Barth M et al.; The sigma S subunit of RNA polymerase (encoded by the rpoS gene) is the master regulator in a complex regulatory network that controls stationary-phase induction and osmotic regulation of many genes in Escherichia coli . Here we demonstrate that the histone-like protein H-NS is also a component of this network, in which it functions as a global inhibitor of gene expression during the exponential phase of growth . On two-dimensional gels, at least 22 sigma S-controlled proteins show increased expression in an hns mutant . H-NS also inhibits the expression of sigma S itself by a mechanism that acts at the posttranscriptional level . Our results indicate that relief of repression by H-NS plays a role in stationary-phase induction as well as in hyperosmotic induction of rpoS translation . Whereas certain sigma S-dependent genes (e.g., osmY) are only indirectly regulated by H-NS via its role in the control of sigma S expression, others are also H-NS-regulated in a sigma S-independent manner . (For this latter class of genes, rpoS hns double mutants show higher levels of expression than mutants deficient in rpoS alone.) In addition, we demonstrate that the slow-growth phenotype of hns mutants is suppressed in hns rpoS double mutants and that many second-site suppressor mutants that spontaneously arise from hns strains carry lesions that affect the expression of sigma S. J Bacteriol, 1995 Jun, 177(12), 3438 - 42 In vivo induction kinetics of the arabinose promoters in Escherichia coli; Johnson CM et al.; In Escherichia coli, the AraC protein represses transcription from its own promoter, PC, and when associated with arabinose, activates transcription from three other promoters, PBAD, PE, and PFGH . Expression from all four of these promoters is also regulated by cyclic AMP-catabolite activator protein; however, the arrangement of the protein binding sites is not identical for each promoter . We are interested in determining how the AraC protein is able to activate PBAD, PE, and PFGH despite their differences . We have characterized the induction response of the wild-type arabinose operons from their native chromosomal locations by primer extension analysis . In this analysis, mRNA from the four arabinose operons plus an internal standard could all be assayed in the RNA obtained from a single sample of cells . We found that each of the operons shows a rapid, within 15 to 30 s, response to arabinose . We also found that the expression of araFGH is more sensitive to catabolite repression but not to arabinose concentration than are araE and araBAD . Finally, we have determined the relative levels of inducibility in wild-type cells of araBAD, araFGH, and araE to be 6.5, 5, and 1, respectively . These results provide a basis for subsequent studies to determine the mechanism(s) by which AraC protein activates transcription from the different arabinose promoters. J Bacteriol, 1995 Jun, 177(12), 3420 - 6 Energy buffering of DNA structure fails when Escherichia coli runs out of substrate; Jensen PR et al.; To study how changes in the {ATP}/{ADP} ratio affect the level of DNA supercoiling in Escherichia coli, the cellular content of H(+)-ATPase was modulated around the wild-type level . A relatively large drop in the {ATP}/{ADP} ratio from the normal ratio resulted in a small increase in the linking number of our reporter plasmid (corresponding to a small decrease in negative supercoiling) . However, when cells depleted their carbon and energy source, the ensuing drop in energy state was accompanied by a strong increase in linking number . This increase was not due to reduced transcription of the DNA in the absence of growth substrate, since rifampin had virtually no effect on the plasmid linking number . To examine whether DNA supercoiling depends more strongly on the cellular energy state at low {ATP}/{ADP} ratios than at high ratios, we used cells that were already at a low energy state after substrate depletion; after the addition of an uncoupler to these cells, the {ATP}/{ADP} ratio decreased further, which resulted in a strong increase in plasmid linking number . Our results suggest that the strong thermodynamic control of DNA supercoiling takes over at low {ATP}/{ADP} ratios, whereas at high ratios homeostatic control mechanisms attenuate thermodynamic control. J Bacteriol, 1995 Jun, 177(11), 3347 - 50 Isolation and characterization of an Escherichia coli seryl-tRNA synthetase mutant with a large increase in Km for serine; Willison JC et al.; A mutant of Escherichia coli resistant to serine hydroxamate which has a large increase in Km for serine of seryl-tRNA synthetase is described . The mutant serS gene was cloned and sequenced and was found to contain a single-base-pair mutation, resulting in the substitution of the residue alanine 262 by valine in motif 2 . The methyl side chain of alanine 262 is not exposed at the active site, and molecular modeling indicated that replacement of alanine 262 by valine does not significantly affect the configuration of amino acids at the active site . This finding suggests that the residue at this position may be involved in a conformational change (possibly induced by ATP binding) which is necessary for optimal binding of the cognate amino acid. J Bacteriol, 1995 Jun, 177(11), 3344 - 6 Superoxide dismutase protects against aerobic heat shock in Escherichia coli; Benov L et al.; Exposure of a superoxide dismutase-null (sodA sodB) strain of Escherichia coli to aerobic heat stress (45 to 48 degrees C) caused a profound loss of viability, whereas the same heat stress applied anaerobically had a negligible effect . A superoxide dismutase-competent parental strain was resistant to the lethal effect of the aerobic heating . It follows that aerobic heating imposes an oxidative burden of which O2- must be a major component . This effect is not seen at 53 degrees C, presumably because, at this higher temperature, direct thermolability of vital cell components overrides the effect of superoxide radicals. J Bacteriol, 1995 Jun, 177(11), 3320 - 2 Transcriptional regulation from the cell surface: conformational changes in the transmembrane protein FecR lead to altered transcription of the ferric citrate transport genes in Escherichia coli; Wriedt K et al.; Ferric citrate induces the ferric citrate transport system in Escherichia coli without being taken up into cells . The cytoplasmic transmembrane protein FecR, required for the response to ferric citrate, was found to be cleaved by a cellular protease . FecR protein produced by fecR mutants impaired or constitutive in fecA transcription was protease resistant, indicating that conformational changes affect proper functioning of FecR. J Bacteriol, 1995 Jun, 177(11), 3277 - 82 Demonstration in vivo that interaction of maltose-binding protein with SecB is determined by a kinetic partitioning; Khisty VJ et al.; An early step in the export of maltose-binding protein to the periplasm is interaction with the molecular chaperone SecB . We demonstrate that binding to SecB in vivo is determined by a kinetic partitioning between the folding of maltose-binding protein to its native state and its association with SecB . A complex of SecB and a species of maltose-binding protein that folds slowly is shown to be longer-lived than a complex of the wild-type maltose-binding protein and SecB . In addition, we show that incomplete nascent chains, which are unable to fold, remain complexed with SecB. J Bacteriol, 1995 Jun, 177(11), 3241 - 50 Global regulation of a sigma 54-dependent flagellar gene family in Caulobacter crescentus by the transcriptional activator FlbD; Wu J et al.; Biosynthesis of the Caulobacter crescentus polar flagellum requires the expression of a large number of flagellar (fla) genes that are organized in a regulatory hierarchy of four classes (I to IV) . The timing of fla gene expression in the cell cycle is determined by specialized forms of RNA polymerase and the appearance and/or activation of regulatory proteins . Here we report an investigation of the role of the C . crescentus transcriptional regulatory protein FlbD in the activation of sigma 54-dependent class III and class IV fla genes of the hierarchy by reconstituting transcription from these promoters in vitro . Our results demonstrate that transcription from promoters of the class III genes flbG, flgF, and flgI and the class IV gene fliK by Escherichia coli E sigma 54 is activated by FlbD or the mutant protein FlbDS140F (where S140F denotes an S-to-F mutation at position 140), which we show here has a higher potential for transcriptional activation . In vitro studies of the flbG promoter have shown previously that transcriptional activation by the FlbD protein requires ftr (ftr for flagellar transcription regulation) sequence elements . We have now identified multiple ftr sequences that are conserved in both sequence and spatial architecture in all known class III and class IV promoters . These newly identified ftr elements are positioned ca . 100 bp from the transcription start sites of each sigma 54-dependent fla gene promoter, and our studies indicate that they play an important role in controlling the levels of transcription from different class III and class IV promoters . We have also used mutational analysis to show that the ftr sequences are required for full activation by the FlbD protein both in vitro and in vivo . Thus, our results suggest that FlbD, which is encoded by the class II flbD gene, is a global regulator that activates the cell cycle-regulated transcription from all identified sigma 54-dependent promoters in the C . crescentus fla gene hierarchy. J Bacteriol, 1995 Jun, 177(11), 3095 - 103 Molecular and biochemical characterization of two meta-cleavage dioxygenases involved in biphenyl and m-xylene degradation by Beijerinckia sp . strain B1; Kim E et al.; Beijerinckia sp . strain B1 is able to grow on either biphenyl or m-xylene as the sole source of carbon and is capable of cooxidizing many polycyclic aromatic hydrocarbons . The catabolic pathways for biphenyl and m-xylene degradation are coinduced and share common downstream enzymatic reactions . The catabolic pathway for biphenyl degradation involves two meta-cleavage steps, one for 2,3-dihydroxybiphenyl and a second for catechol . The catabolic pathway for m-xylene involves one m-cleavage step for 3-methylcatechol . The genes for two meta-cleavage dioxygenases were cloned from Beijerinckia sp . strain B1 on a single fragment of genomic DNA . The two genes are located approximately 5.5 kb away from one another . Expression of each gene separately in Escherichia coli and analysis of the meta-cleavage dioxygenase produced showed that one enzyme was more specific for 2,3-dihydroxybiphenyl while the second was more specific for catechol . The genes for the two meta-cleavage enzymes were thus labeled bphC and xylE for 2,3-dihydroxybiphenyl 1,2-dioxygenase and catechol 2,3-dioxygenase, respectively . Nondenaturing polyacrylamide gel electrophoresis followed by enzyme activity staining showed that the two meta-cleavage dioxygenases could be easily separated from each other . Similar analyses of Beijerinckia sp . strain B1 grown on succinate, biphenyl, or m-xylene indicate that both meta-cleavage enzymes are induced when cells are grown on either biphenyl or m-xylene . The nucleotide sequence was determined for both bphC and xylE . The two genes are transcribed in opposite directions, demonstrating that at least two operons must be involved in biphenyl degradation by Beijerinckia sp . strain B1 . Analysis of the deduced amino acid sequence indicates that 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC) falls into the class of meta-cleavage dioxygenases acting on dihydroxylated polycyclic aromatic hydrocarbons and is somewhat distinct from the main group of meta-cleavage dioxygenases acting on 2,3-dihydroxybiphenyl . Catechol 2,3-dioxygenase (XyIE) falls into the class of meta-cleavage enzymes acting on dihydroxylated monocyclic aromatic hydrocarbons but shows little similarity to the canonical TOL plasmid-encoded catechol 2,3-dioxygenase. J Bacteriol, 1995 Jun, 177(11), 2957 - 64 Characterization of traX, the F plasmid locus required for acetylation of F-pilin subunits; Maneewannakul K et al.; Acetylation of F-pilin subunits has previously been shown to depend upon expression of the F plasmid transfer operon gene traX . To assess the requirement for pilin acetylation in conjugative transfer of F, we constructed traX::kan insertion mutations and crossed them onto the transmissible F derivative pOX38 . Under standard conditions, the function of traX seemed to be dispensable . Although pilin synthesized by mutant plasmids pOX38-traX482 and pOX38-traX483 was not acetylated, F-pilus production and F-pilus-specific phage infection appeared to be normal and transfer occurred at wild-type frequency . Analysis of labeled products showed that TraX+ plasmids expressed two approximately 24- (TraX1) and 22-kDa (TraX2) polypeptides that localized in the cytoplasmic membranes of cells . No product that was similar in size to the product predicted from the traX open reading frame (27.5 kDa) was detected . Therefore, we used site-directed mutagenesis, stop codon linker insertions, and phoA fusion analysis to investigate traX expression . Both TraX1 and TraX2 appeared to be encoded by the traX open reading frame . Insertion of a stop codon linker into the traX C-terminal coding region led to synthesis of two correspondingly truncated products, and fusions to phoA indicated that only the traX reading frame was translated . Expression was also very dependent on the traX M1 start codon; when this was altered, no protein products were observed . However, pilin acetylation activity was still detectable, indicating that some other in-frame start codon(s) can also be used . All sequences that are essential for activity are contained between traX codons 29 and 225 . Sequence analysis indicated that traX mRNA is capable of forming a variety of base-paired structures . We suggest that traX expression is translationally controlled and that F-pilin acetylation activity may be regulated by physiological conditions in cells. Infect Immun, 1995 Jun, 63(6), 2120 - 5 Functional expression of falcipain, a Plasmodium falciparum cysteine proteinase, supports its role as a malarial hemoglobinase; Salas F et al.; Erythrocytic malaria parasites degrade hemoglobin as a principal source of amino acids for parasite protein synthesis . We have previously shown that a Plasmodium falciparum trophozoite cysteine proteinase, now termed falcipain, is required for hemoglobin degradation, and we have hypothesized that this proteinase is responsible for initial cleavages of hemoglobin . To further evaluate the biological role of falcipain, we expressed the enzyme in bacterial and viral expression systems . After expression in the baculovirus system, falcipain was enzymatically active and had biochemical properties very similar to those of the native proteinase . Recombinant falcipain rapidly hydrolyzed both denatured and native hemoglobin . Hemoglobin hydrolysis was blocked by cysteine proteinase inhibitors but not by inhibitors of other classes of proteinases . Our results support our hypothesis that falcipain is a critical malarial hemoglobinase that is responsible for both initial cleavages of hemoglobin and the subsequent hydrolysis of globin into small peptides. Clin Chem, 1995 Jun, 41(6 Pt 1), 862 - 6 Development and use of ELISA to quantify type II phospholipase A2 in normal and uremic serum; Dorsam G et al.; Previously we reported that uremic plasma contained eight times more phospholipase A2 (PLA2) activity than control plasma (Costello et al., Clin Chem 1990;36:198-200) . That study, however, did not distinguish between various PLA2s that could contribute to the observed increase . Therefore, we developed a sandwich ELISA to specifically quantify serum type II PLA2 . By ELISA, uremic sera contained significantly more type II PLA2 than control sera (median = 1025 micrograms/L, range = 52-3320 micrograms/L vs median = 9.2 micrograms/L, range = 4.6-17.5 micrograms/L; P = 0.002) . When serum samples were incubated with 1-{14C}oleate-labeled autoclaved Escherichia coli, activity was increased 14.6-fold in uremic vs normal serum, with a median of 6.5 mumol/min per liter (range 1.1-16.3) vs a control median of 0.49 mumol/min per liter (range 0.32-0.60; P = 0.002) . Thus, ELISA detects about eightfold more immunoreactive type II PLA2 in uremic serum than does enzymatic analysis . Evidently, the increase in PLA2 activity previously observed in uremic plasma is primarily due to increased concentrations of type II PLA2. Am J Respir Crit Care Med, 1995 Jun, 151(6), 1989 - 97 Surfactant protein-A deficiency in a primate model of bronchopulmonary dysplasia; King RJ et al.; Pathophysiologic and biochemical (surfactant protein and phospholipid) features were studied in a baboon model of hyperoxia-induced bronchopulmonary dysplasia (BPD) and superimposed infection . A total of 20 baboons were delivered by hysterotomy at 76% of gestation (140 d of gestational age) and were randomized into four groups, consisting of two control and two injury groups . Animals constituting a group that was managed on a pro re nata (PRN) basis were ventilated with clinically appropriate oxygen for the 16-d experimental period and served as ventilatory controls . They underwent an initial period of 42 h during which they demonstrated evidence of hyaline membrane disease (HMD), but began recovery at 42 h and by Day 6 appeared to have maximally recovered . At the time of these animals' killing, concentrations of surfactant proteins, messenger ribonucleic acids (mRNAs), and phospholipids were similar to those of normal adult baboons . Gestational control animals were delivered and killed without ventilation at 156 d gestational age . Surfactant protein-A (SP-A) and phospholipid concentrations in these animals' lavage fluids were about 10% of those in the PRN animals . Animals with BPD were subjected to positive-pressure ventilation and an FIO2 of 1.0 for 11 d, followed by 5 d of an FIO2 sufficient to maintain PaO2 at 40 to 50 mm Hg . The animals with BPD and infection were treated in the same way as the BPD group, except that 10(8) Escherichia coli were instilled intratracheally on Day 11, concomitantly with the reduction in FIO2.(ABSTRACT TRUNCATED AT 250 WORDS) Am J Respir Cell Mol Biol, 1995 Jun, 12(6), 597 - 604 CIC-2: a developmentally dependent chloride channel expressed in the fetal lung and downregulated after birth; Murray CB et al.; Growth and differentiation of the fetal lung are dependent on chloride and fluid secretion, yet the specific molecular identities of fetal chloride channels have not been fully determined . In this study, we demonstrate mRNA expression of the volume-activated chloride channel, CIC-2, in fetal rat lung using reverse-transcriptase polymerase chain reaction (RT-PCR) and ribonuclease (RNase) protection assay . By RNase protection assay, CIC-2 mRNA expression is most abundant in fetal lung and diminishes after birth until it is almost undetectable in adult rat lung . To confirm this result at the protein level, a C-terminal fragment of CIC-2 cDNA derived from 19-day fetal rat lung was cloned into an expression plasmid . The truncated 33-kD CIC-2 protein was expressed in Escherichia coli and purified by column chromatography . Polyclonal antibodies to this antigen were raised in chickens, and the antisera detected a 94-kD protein in fetal rat lung homogenates by Western blotting . Protein expression of CIC-2 was most abundant in mid and late gestation and decreased significantly shortly after birth, as would be predicted by the RNase protection data . CIC-2 protein was localized along the apical surface of fetal airway epithelium by immunocytochemistry . The abundant fetal expression of CIC-2 RNA and protein supports the hypothesis that CIC-2 is important to fetal lung development, and its apical location suggests that it may be involved in fluid secretion during normal lung morphogenesis. Nature, 1995 Jun 1, 375(6530), 397 - 400 A glutathione S-transferase involved in vacuolar transfer encoded by the maize gene Bronze-2; Marrs KA et al.; Glutathione S-transferases (GSTs) are enzymes that detoxify heterocyclic compounds (xenobiotics) by covalently linking glutathione to the substrate, forming a glutathione S-conjugate . A glutathione pump in the vacuolar membrane of barley actively sequesters herbicide-glutathione S-conjugates; glutathionation allows recognition and entry of the conjugates into vacuoles . The protein encoded by the Bronze-2 gene in maize performs the last genetically defined step in anthocyanin biosynthesis, resulting in the deposition of red and purple pigments in the vacuoles of maize tissues . We show here that Bz2 encodes a GST with activity in maize, transformed Arabidopsis thaliana plants and Escherichia coli . We demonstrate that anthocyanins extracted from maize protoplasts expressing BZ2 are conjugated with glutathione, and that vanadate, a known inhibitor of the glutathione pump in plant vacuolar membranes, inhibits the accumulation of anthocyanins in the vacuole . These results provide a biochemical function for BZ2, and suggest a common mechanism for the ability of plants to sequester structurally similar but functionally diverse molecules in the vacuole. Nature, 1995 Jun 1, 375(6530), 377 - 82 Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-alpha; Bourguet W et al.; The crystal structure of the human retinoid-X receptor RXR-alpha ligand-binding domain reveals a previously undiscovered fold of an antiparallel alpha-helical sandwich, packed as dimeric units . Two helices and one loop form the homodimerization surface, and hydrophobic heptad repeats participate in stabilizing the fold . The existence of a ligand-binding pocket is proposed that would allow 9-cis retinoic acid to interact with different functional modules, including the AF-2 activating domain . Several lines of evidence indicate that the overall structure is a prototype fold of ligand-binding domains of nuclear receptors. Cell Immunol, 1995 Jun, 163(1), 1 - 9 Serotonin upregulates mitogen-stimulated B lymphocyte proliferation through 5-HT1A receptors; Iken K et al.; Serotonin is a well-known neurotransmitter and neuroimmunomodulator which has been reported to modulate T cell and NK cell proliferation . In this study we investigated whether serotonin could regulate mitogen-stimulated proliferation of the mature B lymphocyte . Mouse and rat spleen cells were cultured with serotonin in the presence or absence of a combination of Escherichia coli lipopolysaccharide and dextran sulfate, and proliferation was assessed by {3H}thymidine uptake or propidium iodide staining of DNA . Serotonin alone had no effect on spleen cell proliferation, while it increased mitogen-stimulated B cell proliferation in a dose- and time-dependent manner . These effects were reproduced by the selective 5-HT1A receptor agonist 8-OH-DPAT . Serotonin- or 8-OH-DPAT-induced increase in proliferation could be blocked by the 5-HT1A receptor antagonists (+)WAY 100135 and propranolol . Moreover, lipopolysaccharide-activated mouse spleen cells expressed specific binding sites for {3H}8-OH-DPAT . These results show that serotonin upregulates mitogen-stimulated B lymphocyte proliferation through 5-HT1A receptors, thus providing an important link between this neurotransmitter and the immune system. Am Heart J, 1995 Jun, 129(6), 1051 - 7 Adenoviral mediated gene transfer to atherosclerotic arteries after balloon angioplasty; Landau C et al.; The purpose of this study was to examine the pattern of catheter-mediated adenoviral gene transfer into atherosclerotic vessels subjected to balloon injury . Atherosclerotic lesions were created in the iliac arteries of New Zealand white rabbits fed with cholesterol . Balloon dilatation was performed at the angiographically defined region of maximal stenosis . Instillation of a recombinant adenoviral vector encoding beta Galactosidase was performed at the angioplasty site with either (1) a double-balloon catheter (n = 7 arterial segments), (2) a hydrogel-coated balloon (n = 3), (3) a perforated balloon (n = 3), or (4) a catheter with an inflatable circumferential helical ring (n = 4) . Successful gene transfer reflected by expression of nuclear-localizing beta-galactosidase activity was observed in all sections displaying angioplasty effect . Genetically modified cells were located in pockets within the deep portions of the neointima, the media, and the adventitia immediately adjacent to dissection planes . Gene transfer to an atherosclerotic vessel subjected to balloon angioplasty is feasible with recombinant adenovirus vectors and currently available delivery catheters . The regions of the vessel wall that express the foreign protein are those which contribute most importantly to the proliferative cellular response which characterizes postangioplasty restenosis. Mutat Res, 1995 Jun, 334(3), 283 - 92 A rapid method for detection of mutations in the lacI gene using PCR-single strand conformation polymorphism analysis: demonstration of its high sensitivity; Ushijima T et al.; The lacI gene has been used as a target gene in various mutation assays . We modified single strand conformation polymorphism (SSCP) analysis by introducing restriction digestion to detect mutations in the gene rapidly, and determined the sensitivity of the method . The entire coding sequence and partial promoter region of the lacI gene were amplified by the polymerase chain reaction with {alpha-32P}dCTP in a 1247 base pair fragment, digested into eight restriction fragments, and analyzed by SSCP . The sensitivity of the method was assessed using 160 phages with lacI mutations, which were selected by assay of expression of beta-galactosidase after their infection into E . coli . Of the 160 mutants, 146 (91.3%) showed shifted bands in the first condition of SSCP analysis (without glycerol, 20 degrees C) . The remaining 14 mutants were analyzed in a second condition (with 5% glycerol, 20 degrees C), and eight of them showed shifted bands (cumulatively 96.3% of the 160 mutants) . The remaining six mutants were analyzed in a third condition (with 5% glycerol, 10 degrees C), and all of them showed shifted bands (cumulatively 100%) . Sequencing of the restriction fragments with mobility shifts in the 160 mutants revealed 108 kinds of mutations, 100 (92.6%) being detected in the first condition, seven (cumulatively 99.1%) in the second condition, and one (cumulatively 100%) in the third condition . This method greatly reduced the time to identify lacI mutations, and allowed the detection of multiple mutations in one lacI mutant . The results also show that in general PCR-SSCP analysis is very sensitive when test fragments are shorter than about 250 base pairs and electrophoresis is performed under at least two conditions. J Immunol, 1995 Jun 1, 154(11), 6048 - 57 Expression and biologic characterization of the murine chemokine KC; Bozic CR et al.; KC, the product of an immediate early gene induced in mouse fibroblasts by platelet-derived growth factor, was expressed in Escherichia coli by using a maltose binding protein vector and biochemically characterized as a ligand for both murine and human polymorphonuclear neutrophils (PMN) . On murine PMN, KC is both a potent chemoattractant and up-regulator of Mac-1 cell surface expression . On human PMN, in contrast, KC exhibits dissociation of its chemoattractant and Mac-1 up-regulatory activities . Although KC strongly increases Mac-1 expression on human PMN, it does not induce chemotaxis in vitro . 125I-KC-Tyr binds to both mouse and human PMN with two classes of binding sites, including high affinity sites of 0.8 and 2 nM, with approximately 9,000 and 10,000 sites per cell, respectively . On mouse PMN, human macrophage inflammatory protein (MIP)-2 alpha and MIP-2 beta compete for 125I-KC-Tyr binding with high affinity, whereas the murine beta-chemokine TCA-3 does not compete . KC binds to human PMN by the IL-8 type B receptor and to murine PMN by a murine IL-8 type B receptor homologue . 125I-KC-Tyr also binds to human RBC with a single class of high affinity sites . KC mRNA is constitutively expressed in multiple murine tissues . With human IL-8 and KC cDNA as probes, a mouse neutrophil exudate library was screened: KC and MIP-2 were the dominant chemokine species found . Thus, KC appears to be intimately involved in murine inflammation and its constitutive expression may have a role in the basal trafficking of neutrophils. Endocrinology, 1995 Jun, 136(6), 2477 - 84 Molecular characterization of a testis-specific estrogen sulfotransferase and aberrant liver expression in obese and diabetogenic C57BL/KsJ-db/db mice; Song WC et al.; Sulfation represents a major pathway for the inactivation of steroid hormones such as estrogens and is catalyzed by a group of enzymes called sulfotransferases . Aberrant regulation of an estrogen sulfotransferase has been demonstrated previously in the livers of obese and diabetogenic C57BL/KsJ-db/db strain mice . In this paper, we report the molecular cloning and functional characterization of a full-length complementary DNA for estrogen sulfotransferase from mouse testis . The mouse estrogen sulfotransferase complementary DNA encodes 295 amino acids . It shares 88%, 77%, 75%, and 68% identity in amino acid sequence with the rat liver, human liver, guinea pig adrenal, and bovine placental estrogen sulfotransferase, respectively . The mouse enzyme was expressed as a glutathione-S-transferase fusion protein in Escherichia coli . The fusion protein was affinity purified, and milligram quantities of pure enzyme were obtained after cleavage of the fusion protein with thrombin . The expressed enzyme exhibits a high substrate specificity toward estrogens, including estradiol and estrone . Neither dehydroepiandrosterone, pregnenolone, testosterone, nor a simple phenolic compound, 4-nitrophenol appears to be a substrate . Northern hybridization indicates that messenger RNA (1.3 kilobases) for the estrogen sulfotransferase is expressed exclusively in the testes in control C57BL/KsJ mice . However, both the messenger RNA and protein are dramatically induced in the livers of obese and diabetogenic C57BL/KsJ-db/db mice . In contrast to the liver, the constitutive expression of the enzyme in the testis is not affected by the db/db genotype . These results recapitulate the species-specific nature in the tissue distribution of estrogen sulfotransferase and suggest complex regulatory mechanisms in its expression under normal and pathophysiological conditions. J Vet Pharmacol Ther, 1995 Jun, 18(3), 204 - 9 Kinetics, dose response, tachyphylaxis and cross-tachyphylaxis of vascular leakage induced by endotoxin, zymosan-activated plasma and platelet-activating factor in the horse; Mills PC et al.; Vascular leakage induced by intradermal injection of endotoxin, zymosan-activated plasma (ZAP) and platelet-activating factor (PAF) was measured in nine Thoroughbreds using 125-iodine human serum albumin (125I-HSA) as a marker in the blood . ZAP and PAF produced dose-dependent increases in vascular permeability with the maximum occurring within the first 15 min after injection . The vascular leakage induced by endotoxin was also dose-dependent, but the maximum occurred 2 h after intradermal injection . Intradermal sites previously injected with endotoxin were refractory to a second injection of endotoxin for up to 5 days . However, sites injected with endotoxin and re-injected with either ZAP or PAF remained responsive with increased vascular leakage compared to saline injected control sites re-injected with either ZAP or PAF . Diminished response to endotoxin challenge may contribute to the poor prognosis of endotoxaemia in the horse. Yakugaku Zasshi, 1995 Jun, 115(6), 401 - 19 {Gene expression of human eukaryotic initiation factor-4E for protein synthesis and study of its recognition mechanism of mRNA cap structure}; Morino S et al.; Being stimulated by the insights from model studies that (i) the intimate combination of hydrogen-bonding pairing and aromatic stacking interactions is important for the specific binding of guanine base by peptide and (ii) the pi-pi stacking force of Trp is significantly strengthened by the guanine N7-methylation (m7G), this research project was started, because (a) the mRNA cap structure is characterized by the existence of m7G and (b) an eukaryotic initiation factor-4E (eIF-4E), a protein which specifically recognizes the mRNA cap structure and opens the protein biosynthesis, contains 8 Trp residues irrespective of its relatively low molecular weight of about 25 kDa . In order to prepare the sufficient amount of sample for carrying out the analysis of the recognition mechanism of mRNA cap structure by eIF-4E at the atomic level, firstly, the expression of human eIF-4E gene in Escherichia coli was attempted . An artificial gene encoding for human eIF-4E was chemically synthesized and succeeded in the expression with two different forms, i.e., as a fusion protein with human growth hormone and a direct expression of soluble protein . The isolation of eIF-4E and its purification procedure using the m7GTP affinity chromatography were accomplished . It was shown by spectroscopic methods that the recombinant eIF-4E exhibits essentially the same tertiary structure as the native one and the binding ability with mRNA cap analog was identical with each other . In order to analyze the functional amino acid residues which are essential for specific recognition of mRNA cap structure, next a series of eIF-4E mutants were prepared by the site-directed mutagenesis, and His37, His200, Trp102 and Glu103 were suggested to be important for binding of mRNA cap structure, as judged from comparison of the binding abilities of respective mutants with a m7GTP affinity column . Since the crystals of recombinant eIF-4E-m7GTP complex suitable for X-ray crystallography are now in preparation, the detailed interaction mode between them will be opened in near future. Biochem Mol Biol Int, 1995 Jun, 36(2), 275 - 83 The production of nitrating species by the reaction between nitrite and hypochlorous acid; Kono Y; Nitrite inhibited the killing of Escherichia coli by hypochlorous acid . The protection curve was sigmoid . Complete protection occurred at nitrite concentrations greater than that of hypochlorous acid . Hypochlorous acid reacts rapidly with nitrite, as shown by phenolic nitration, using 4-hydroxyphenylacetic acid . The nitration was biphasic . Amines and amino acids inhibited the nitration, but metal chelators and hydroxyl radical scavengers except for dimethylsulfoxide did not . The reaction between hypochlorous acid and nitrite yields nitrating species such as nitrogen dioxide or nitronium ion . Nitrite could protect E . coli by removing toxic nitrating species by hypochlorous acid. Protein Expr Purif, 1995 Jun, 6(3), 379 - 87 dUTPase from the retrovirus equine infectious anemia virus: high-level expression in Escherichia coli and purification; Bergman AC et al.; Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, and plays important roles in nucleotide metabolism and DNA replication . The dUTPase gene of the retrovirus equine infectious anemia virus (EIAV) was cloned and overexpressed in Escherichia coli using the T7 RNA polymerase expression system . The recombinant vector (pET-3a/EDU), constructed by mutagenic PCR, was transformed into E . coli BL21 (DE3) pLysS cells, resulting in expression of EIAV dUTPase at about 40% of the extracted protein . This level of overproduction is very high compared to previous reports on heterologous expression of dUTPases in E . coli . A one-step purification procedure using phosphocellulose chromatography results in a homogeneous preparation of the enzyme in a yield of 45 mg liter-1 of bacterial culture . The purified EIAV dUTPase, run on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, shows an apparent molecular mass of 15.1 kDa in accordance with the gene structure . The isoelectric point (pI) was determined to 5.6 . Gel filtration under nondenaturating conditions gives a retention volume corresponding to a molecular mass of 40.6 kDa, suggesting a trimeric organization of the enzyme . The amino acid composition and amino-terminal sequence of the recombinant dUTPase are in agreement with predictions from the DNA sequence. Protein Expr Purif, 1995 Jun, 6(3), 363 - 70 Expression of the rat alpha 1 thyroid hormone receptor ligand binding domain in Escherichia coli and the use of a ligand-induced conformation change as a method for its purification to homogeneity; Apriletti JW et al.; The rat alpha 1 thyroid hormone receptor (rTR alpha 1) mediates hormone-dependent gene regulation by utilizing several distinct structural domains, including those containing DNA and ligand binding sites . Binding of the hormone to the ligand binding domain (TR-LBD) induces conformational changes in the receptor that are involved in affecting the receptor's transcriptional regulatory and other functions . A 33-kDa protein fragment (Met122-Val410) of rTR alpha 1, which includes the entire TR-LBD, was expressed in Escherichia coli, yielding typically 1.5 mg of soluble TR-LBD/liter of bacteria . The protein was purified to > 99% homogeneity with a final yield of 24% by hydrophobic interaction, DEAE anionic exchange, and heparin cationic exchange chromatographic steps . The Kd of the purified TR-LBD for 3,3',5-triiodo-L-thyronine (T3) was 0.06 nM, identical to that for full-length rTR alpha 1 . T3 analogs had affinities consistent with values obtained for full-length rTR alpha 1 . In all three chromatography steps, TR-LBD prebound to {125I}T3 eluted earlier than the unliganded TR-LBD, like the full-length receptor . These studies indicate that the binding affinity and specificity of the TR-LBD are similar to those of the intact rTR alpha 1 and that the ligand-induced conformational changes occur in the LBD itself . These studies also provide methodology for obtaining milligram quantities of protein useful for biochemical and biophysical studies of the thyroid hormone receptor and its ligand-induced changes. Protein Expr Purif, 1995 Jun, 6(3), 357 - 62 High yields of interleukin-8 produced by a synthetic gene expressed in Escherichia coli and purified with a single antibody affinity column; Miller EJ et al.; We developed a highly efficient expression system for the production of interleukin-8 (IL-8) in Escherichia coli . A synthetic gene used in the vector was designed to code for the 72-amino-acid form of IL-8 and incorporate additional new restriction sites . IL-8 was expressed in very large amounts in the periplasmic space and extracted by a gentle method which did not utilize denaturants . About 69% of the protein extracted from the periplasmic space was properly processed IL-8 . A single anti-IL-8 monoclonal antibody affinity chromatography column yielded homogeneous IL-8 as determined by HPLC molecular sieve chromatography and amino-terminal sequencing . Between 14 and 22 mg of IL-8 was purified per liter of bacterial culture, in which the wet weight of E . coli was 7.6 g/liter . The recombinant IL-8 was fully active compared to published data and a commercially available preparation of recombinant IL-8 . Our IL-8 and the commercial product had identical neutrophil binding isotherms, chemotactic activities, and enzyme release properties. Protein Expr Purif, 1995 Jun, 6(3), 312 - 8 Cloning, expression, and purification of rat brain protein L-isoaspartyl methyltransferase; David CL et al.; Protein L-isoaspartyl methyltransferase (PIMT) methylates isoaspartyl residues in peptides and proteins using S-adenosyl-L-methionine as the methyl donor . A cloned source of this enzyme should be useful in the identification of cellular substrates and for quantitation and localization of isoaspartyl sites in purified proteins, including recombinant proteins used as pharmaceuticals . Rat brain PIMT cDNA was amplified using the polymerase chain reaction . The reaction product was directionally cloned into the expression vector p delta blue (M . E . Brandt and L . E . Vickery, Arch . Biochem . 294, 735-740, 1992) . The vector contains the strong promoter lambda pL and allows for the direct expression of cloned genes . After transformation, Escherichia coli cells containing the plasmid constitutively produced recombinant rat brain PIMT (rrPIMT) at levels between 2 and 3% of total soluble protein . Recombinant enzyme was purified to homogeneity by ammonium sulfate precipitation of the crude extract followed by anion-exchange chromatography . The specific activity was 14,000 pmol methyl groups transferred/min/mg protein at 30 degrees C using bovine gamma-globulin as the methyl acceptor . A typical yield was 12 mg of purified rrPIMT per liter of bacterial culture . Subsequent dye ligand chromatography increased the specific activity of the preparation to 16,800 pmol methyl groups transferred/min/mg protein with an overall yield of 5 mg per liter of bacterial culture . Using isoaspartyl delta sleep-inducing peptide as the methyl acceptor, rrPIMT exhibited normal Michaelis-Menten kinetics that yielded the following constants: Km (S-adenosyl-L-methionine) = 1.1 microM, Km (peptide) = 16 microM, Vmax = 60,000 pmol/min/mg. Protein Expr Purif, 1995 Jun, 6(3), 298 - 304 Spinach ferredoxin i: overproduction in Escherichia coli and purification; Piubelli L et al.; Ferredoxin I is the most abundant form of photosynthetic-type ferredoxin present in spinach chloroplasts . A cDNA clone encoding the precursor of spinach ferredoxin I has been engineered to synthesize the mature form of the plant protein in Escherichia coli . Among several different plasmid constructions, the expression system based on phage T7 promoter (vector pET-11d) was found to be the most efficient for spinach ferredoxin overproduction . Upon induction, ferredoxin I accounted for about 2.5% of soluble E . coli protein . A rapid procedure for the purification of the recombinant protein, which yielded at least 1 mg of homogeneous ferredoxin I per gram of cells (fresh wt), was developed . The recombinant protein was found to be identical to ferredoxin I isolated from spinach, both by mass spectrometry analysis and by N-terminal protein sequencing, indicating in vivo removal of the N-terminal methionine . Ferredoxin I was synthesized as the holoprotein, correctly assembled with the {2Fe-2S} cluster as judged by its absorption spectrum, and was fully active in the assay with its physiological partner (ferredoxin-NADP+ reductase) . The expression system described here is amenable to the structure-function relationship study of spinach ferredoxin I through site-directed mutagenesis and NMR spectroscopy. Protein Expr Purif, 1995 Jun, 6(3), 284 - 90 High-level expression of prochymosin in Escherichia coli: effect of the secondary structure of the ribosome binding site; Wang G et al.; Regulation of the expression of prochymosin cDNA in Escherichia coli at the translational level was studied by mutating the regions between the Shine-Dalgarno (SD) sequence and the initiation codon and upstream of the SD signal . Results revealed that expression plasmids with a distance of 7-10 bp from SD to ATG have the potential to be expressed at higher levels . However, an approximately 20-fold variation in expression was observed with plasmids harboring different base composition but identical distance in the spacer . Analysis of the predicted secondary structure of ribosome binding sites (RBS) indicates that the control of expression by base composition is mediated by the secondary structure of the RBS . An unfolded state of the RBS is required for high expression . Therefore, a vector for enhanced translation can be designed and constructed via prediction of the secondary structure of the proposed RBS and mutagenesis . Based on this strategy, high-level expression of prochymosin, up to 39% of the total cellular proteins, was achieved . The 9-base sequence proposed by Olins and Rangwala as a translational enhancer did not exhibit an additive effect on prochymosin expression . This is probably because the affinity of the SD sequence used in this study to 16S rRNA is strong enough that no additional element is required to facilitate the formation of the translation initiation complex. Protein Expr Purif, 1995 Jun, 6(3), 272 - 7 One-step magnetic purification of recombinant DNA-binding proteins using magnetizable phosphocellulose; Risoen PA et al.; Magnetizable solid phase technology was used to develop a method for the rapid purification of recombinant proteins expressed in Escherichia coli . We describe the purification of two recombinant DNA-binding proteins: the minimal DNA-binding domain of the oncoprotein Myb and full-length yeast TFIIIA . Both were purified in one step directly from an E . coli lysate by means of magnetizable phosphocellulose particles (PhosphoMagnaCel) . All operations were performed in microcentrifuge tubes and could be completed within 15 min . High purity and excellent recovery of proteins active in sequence specific DNA-binding were obtained . The procedure allowed the simultaneous purification of eight mutant Myb-proteins within 30 min. Protein Expr Purif, 1995 Jun, 6(3), 256 - 64 Expression in Escherichia coli and affinity purification of a CKS-troponin I fusion protein; Hayden M et al.; The human cardiac troponin I gene was subcloned and expressed at high levels in Escherichia coli as a fusion protein to CMP-KDO synthetase (CKS) . Expression levels of the CKS-troponin I fusion were 8% of total cellular protein 4 h after induction with IPTG . The fusion was expressed primarily as an insoluble protein as shown by SDS-PAGE analysis . Expressed CKS-troponin I fusion from a crude lysate was antigenic against anti-CKS and anti-troponin I monoclonal antibodies in Western blots . The fusion was affinity-purified over a TnC affinity column using a urea-solubilized extract of a crude cell lysate . Serial dilutions of crude soluble extracts of the troponin I fusion were assayed in several microparticle enzyme immunoassays and found to exhibit similar immunogenic responses relative to cardiac troponin I isolated from human heart tissue. Protein Expr Purif, 1995 Jun, 6(3), 244 - 50 Glycosomal glyceraldehyde-3-phosphate dehydrogenase of Trypanosoma brucei and Trypanosoma cruzi: expression in Escherichia coli, purification, and characterization of the enzymes; Hannaert V et al.; The Trypanosoma brucei and Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenases have been overexpressed in Escherichia coli using a T7 expression system . These enzymes have been highly purified by ammonium sulfate precipitation, followed by phenyl-Sepharose and phospho-ultrogel chromatography . From 1 liter of bacterial culture, we obtained 4.4 mg of T . brucei enzyme, with a specific activity of 147 units/mg, and 26.6 mg of T . cruzi enzyme, with a specific activity of 122 units/mg . Both proteins have a similar subunit mass of 38 kDa . Some physicochemical and kinetic properties have been determined and compared with those reported for the authentic T . brucei enzyme . The two enzymes appear to be very similar, except for the dependence of their activity on ionic strength. Plant Cell, 1995 Jun, 7(6), 747 - 57 Molecular characterization of BET1, a gene expressed in the endosperm transfer cells of maize; Hueros G et al.; A cDNA clone, BET1 (for basal endosperm transfer layer), was isolated from a cDNA bank prepared from 10-days after pollination (DAP) maize endosperm mRNA . BET1 mRNA was shown to encode a 7-kD cell wall polypeptide . Both the mRNA and protein were restricted in their distribution to the basal endosperm transfer layer and were not expressed elsewhere in the plant . BET1 expression commenced at 9 DAP, reached a maximum between 12 and 16 DAP, and declined after 16 DAP . The initial accumulation of the BET1 polypeptide reached a plateau by 16 DAP and declined thereafter, becoming undetectable by 20 DAP . The antibody raised against the BET1 protein reacted with a number of polypeptides of higher molecular mass than the BET1 monomer . Most of these were present in cytosolic fractions and were found in nonbasal cell endosperm extracts, but three species appeared to be basal cell specific . This result and the reactivity of exhaustively extracted cell wall material with the BET1 antibody suggest that a fraction of the protein is deposited in a covalently bound form in the extracellular matrix . We propose that the BET1 protein plays a role in the structural specialization of the transfer cells . In addition, BET1 provides a new molecular marker for the development of this endosperm domain. J Biomol NMR, 1995 Jun, 5(4), 367 - 75 Rapid corepressor exchange from the trp-repressor/operator complex: an NMR study of {ul-13C/15N}-L-tryptophan; Lee W et al.; {ul-13C/15N}-L-tryptophan was prepared biosynthetically and its dynamic properties and intermolecular interaction with a complex of Escherichia coli trp-repressor and a 20 base-pair operator DNA were studied by heteronuclear isotope-edited NMR experiments . The resonances of the free and bound corepressor (L-Trp) were unambiguously identified from gradient-enhanced 15N-1H HSQC, 13C-1H HSQC, 13C- and 15N-edited 2D NOESY spectra . The exchange off-rate of the corepressor between the bound and free states was determined to be 3.4 +/- 0.52 s-1 at 45 degrees C, almost three orders of magnitude faster than the dissociation of the protein-DNA complex . Examination of the experimental NOE buildup curves indicates that it may be desirable to use longer mixing times than would normally be used for a large molecule, in order to detect weak intermolecular NOEs in the presence of exchange . Intermolecular NOEs from bound corepressor to trp-repressor and DNA were analyzed with respect to the mechanism of ligand exchange . This analysis suggests that, in order for the ligand to diffuse out of the complex, there must be significant movement or 'breathing' of the protein and/or DNA. J Biomol NMR, 1995 Jun, 5(4), 339 - 44 High-level 2H/13C/15N labeling of proteins for NMR studies; Venters RA et al.; The protein human carbonic anhydrase II (HCA II) has been isotopically labeled with 2H, 13C and 15N for high-resolution NMR assignment studies and pulse sequence development . To increase the sensitivity of several key 1H/13C/15N triple-resonance correlation experiments, 2H has been incorporated into HCA II in order to decrease the rates of 13C and 1HN T2 relaxation . NMR quantities of protein with essentially complete aliphatic 2H incorporation have been obtained by growth of E . coli in defined media containing D2O, {1,2-13C2, 99%} sodium acetate, and {15N, 99%} ammonium chloride . Complete aliphatic deuterium enrichment is optimal for 13C and 15N backbone NMR assignment studies, since the 13C and 1HN T2 relaxation times and, therefore, sensitivity are maximized . In addition, complete aliphatic deuteration increases both resolution and sensitivity by eliminating the differential 2H isotopic shift observed for partially deuterated CHnDm moieties. Zhongguo Zhong Yao Za Zhi, 1995 Jun, 20(6), 369 - 70, inside back cover {Protective effect of semen Ziziphi spinosae on superoxide dismutase reduction in mice with endotoxin fever}; Peng Z et al.; An animal model with decreasing SOD was established by fever from intravenous injection of endotoxin . The SOD level was measured by RIA in the animal serum and liver tissues . The results indicated that the SOD level of the model group was obviously lower than that of the normal group (P < 0.05), but the level of the group treated with Semen Ziziphi Spinosae was higher than that of the model group . The study shows that Semen Ziziphi Spinosae can protect mice with endotoxin fever from SOD decrease. Poult Sci, 1995 Jun, 74(6), 998 - 1010 Effects of early immune stress and changes in dietary metabolizable energy on the development of newly hatched turkeys . 2 . Selected characteristics of immune function; Piquer FJ et al.; Two 21-d experiments were conducted to document the effects of an early immunologic stress and changes in dietary ME(n) on selected characteristics of immune function of newly hatched turkeys . Eight treatments were included in each experiment . Treatments were the result of complete factorial arrangements of two types of injection and four isonitrogenous diets . Turkeys in both experiments were injected i.p . with .5, .5, and .2 mL of saline (SAL) or .5, .5, and .2 mL of a solution of Escherichia coli lipopolysaccharide (LPS) (100 micrograms LPS/mL SAL) at 1, 3, and 5 d of age, respectively . In Experiment 1, two diets were formulated to contain 2,800 kcal ME(n)/kg . One was a corn-soybean meal based diet (CSBM) and the other contained 8% Solkafloc (SKF) . A third diet (3,100 kcal ME(n)/kg) was formulated by substituting 8% sucrose (SUC) for the 8% SKF . The fourth diet (HE) included in Experiment 1 was formulated to contain 3,700 kcal ME(n)/kg . The CSBM and SUC diets and two additional diets were tested in Experiment 2 . The latter were the CSBM diet containing 74.5 mg ibuprofen/kg (IBU) and a corn-soybean meal diet formulated to contain 3,100 kcal ME(n)/kg (CS31) . Concentrations of plasma IgG and jejunal IgG and IgA were not affected by injection or diet . Age-related changes in Ig concentrations were consistently observed in Experiments 1 and 2 . Injection with LPS reduced the number or responses of blood leukocytes to mitogens at 8 d of age (P < .01), as compared with samples from turkeys injected with SAL . Leukocytes in whole blood samples from turkeys fed the HE diet responded less to LPS stimulation than those fed the SUC diet (P < .01) . Injection with LPS did not markedly affect the characteristics of immune function studied, and feeding a diet with 3,100 kcal ME(n)/kg and 28.5% crude protein did not measurably affect the characteristics of immune function of young turkeys. Poult Sci, 1995 Jun, 74(6), 983 - 97 Effects of early immune stress and changes in dietary metabolizable energy on the development of newly hatched turkeys . 1 . Growth and nutrient utilization; Piquer FJ et al.; Two experiments were conducted to document the effects of an early immunologic stress and changes in dietary ME(n) on growth and nutrient utilization of newly hatched turkeys . Treatments in both experiments consisted of a complete factorial arrangement of two types of injection and four isonitrogenous diets . Turkeys were injected i.p . with saline (SAL) or a solution of lipopolysaccharide (LPS) (100 micrograms LPS/mL SAL) at 1, 3, and 5 d of age . In Experiment 1, two diets were formulated to contain 2,800 kcal ME(n)/kg . One was a corn-soybean meal-based diet (CSBM) and the other contained 8% Solkafloc (SKF) . A third diet (3,100 kcal ME(n)/kg) was formulated by substituting 8% sucrose (SUC) for the 8% SKF . The fourth diet included in Experiment 1 was formulated to contain 3,700 kcal ME(n)/kg . The CSBM and SUC diets were also included in Experiment 2 . Two additional diets tested in Experiment 2 were the CSBM diet containing 74.5 mg ibuprofen/kg (IBU) and a corn-soybean meal-based diet with a ME(n) value of 3,100 kcal/kg (CS31) . Injection with LPS reduced (P < .05) BW of turkeys throughout Experiment 1 and until 9 d of age in Experiment 2, as compared with injection with SAL, irrespective of dietary treatment . The reduction in BW was mainly due to a decrease in feed intake (FI) (P < .05) . Turkeys fed diets with 3,100 kcal ME(n)/kg were heavier (P < .05) than those fed diets with 2,800 kcal ME(n)/kg, irrespective of injection . Inclusion of ibuprofen to the CSBM diet from 1 to 14 d improved (P < .05) BW and feed efficiency (P < .01) of turkeys at 14 d of age, compared with turkeys fed the CSBM diet . Determined ME(n) was not affected by LPS injection . Adverse effects of LPS injection on growth of turkey poults were mainly the consequence of a reduced FI and not of altered nutrient utilization . These effects were not fully alleviated by feeding a diet with 3,100 kcal ME(n)/kg. Neuroscience, 1995 Jun, 66(3), 737 - 50 Delivery of a foreign gene to sympathetic preganglionic neurons using recombinant herpes simplex virus; Levatte MA et al.; Two recombinant herpes simplex type 1 viruses expressing beta-galactosidase (encoded by the Escherichia coli lacZ gene) inserted into the unique long 41 (encoding virus host shutoff) or unique short 5 (encoding glycoprotein J) open reading frames were generated . Purified recombinants or wild-type herpes simplex type 1 were injected into the left adrenal gland of hamsters . Three days later, virus-infected neurons were detected in spinal cord sections from all infected hamsters . Neurons were visualized with beta-galactosidase histochemistry in spinal cord sections from hamsters infected with either of the recombinants but not with the wild-type virus . Wild-type virus could only be detected with immunocytochemistry . Insertional mutagenesis into the unique long 41 or unique short 5 regions of the herpes simplex genome by lacZ did not disrupt the neurotropic properties of the virus . Both recombinant viruses labelled the central nervous system sympathoadrenal preganglionic neurons as well as brainstem neurons . Because the virus host shutoff recombinant more readily crossed synapses to reach the brainstem compared to the glycoprotein J recombinant, the presence of glycoprotein J may facilitate cell to cell transmission in vivo . Both recombinants may be useful for the study of synaptic organization of neural circuits . Our recombinant viruses were less lytic yet neurovirulent after mutation of either glycoprotein J or virus host shutoff of herpes simplex virus type 1 wild-type . These recombinant viruses express the bacterial beta-galactosidase which is readily detectable using simple histochemistry . Inoculation of the adrenal gland or kidney with these viruses led to clear labelling of spinal cord cells . These viruses may be useful markers of specific neural circuits. Neurol Res, 1995 Jun, 17(3), 209 - 16 Adenovirus-mediated p53 gene delivery inhibits 9L glioma growth in rats; Badie B et al.; Adenoviral vectors have recently been shown to effectively deliver genes into a variety of tissues . Since these vectors have some advantages over the more extensively investigated retroviruses, we studied the effect of two replication-defective adenovectors bearing human wild type tumor suppressor gene p53 (Adp53) and Escherichia coli beta-galactosidase gene (AdLacZ) on 9L glioma cells . Successful in vitro gene transfer was shown by DNA polymerase chain reaction (PCR), and expression was confirmed by reverse transcriptase RNA PCR and Western blot analyses . Transduction of 9L cells with the Adp53 inhibited cell growth and induced phenotypic changes consistent with cell death at low titers, while AdLacZ caused cytopathic changes only at high titers . Stereotactic injection of AdLacZ (10(7) plaque forming units) into tumor bed stained 25 to 30% of tumor cells at the site of vector delivery . Injection of Adp53 (10(7) plaque forming units), but not AdLacZ (controls), into established 4-day old 9L glioma brain tumors decreased tumor volume by 40% after 14 days . As a step toward gene therapy of brain tumors using replication-defective adenoviruses, these data support the use of tumor suppressor gene transfer for in vivo treatment of whole animal brain tumor models. Immunology, 1995 Jun, 85(2), 311 - 7 IL-10 augments CD23 expression on U937 cells and down-regulates IL-4-driven CD23 expression on cultured human blood monocytes: effects of IL-10 and other cytokines on cell phenotype and phagocytosis; Spittler A et al.; The effects of human recombinant interleukin-10 (IL-14) on the expression of several markers on U937 and human peripheral blood monocytes was studied by immunofluorescence and fluorescence-activated cell sorter (FACS) analysis . IL-10 augmented Fc IgE receptor (Fc epsilon RII/CD23) further enhanced by cotreatment with IL-4 or interferon-gamma (IFN-gamma) . In contrast, the basal level of Fc epsilon RII expression on blood monocytes appeared to fall in response to IL-10, and this effect became more evident on IL-4-treated cells . Furthermore, the constitutive and IFN-gamma-triggered Fc gamma RI/CD64 expression was augmented on both monocytes and U937 cells . Thus the expression of Fc gamma RII/CD32, Fc gamma/RIII/CD16, Fc alpha R/CD89, the receptor for complement components (CR1/CD35, CD3/CD11b, CR4/CD11c) and the receptor for transferrin/CD71 was not significantly influenced on IL-10-treated cells . IL-10 modestly triggered CD14 antigen expression on monocytes but not U937 . The expression of intercellular adhesion molecule-1 (ICAM-1)/CD54 on monocytes was significantly inhibited by IL-10 . As expected, a marked reduction of the constitutive as well as of the IFN-gamma or IL-4-driven expression on HLA-DR, HLA-DP and HLA-DQ was observed on IL-10-cultured monocytes . On the other hand, the expression of major histocompatibility complex (MHC) class I molecules was slightly and dose-dependently induced on IL-10-treated monocytes . The ability of blood monocytes to phagocytose IgG-sensitized ox erythrocytes, and to bind and ingest opsonized Escherichia coli or latex particles, was amplified by IL-10 . Our data demonstrate that IL-10 modulates the expression of a wide variety of structures on human mononuclear phagocytes, and augments their phagocytic capacity. Plant Cell Physiol, 1995 Jun, 36(4), 669 - 80 Cytosolic aconitase participates in the glyoxylate cycle in etiolated pumpkin cotyledons; Hayashi M et al.; Two different aconitases are known to be expressed after the germination of oil-seed plants . One is a mitochondrial aconitase that is involved in the tricarboxylic acid cycle . The other participates in the glyoxylate cycle, playing a role in gluconeogenesis from stored oil . We isolated and characterized a cDNA for an aconitase from etiolated pumpkin cotyledons . The cDNA was 3,145 bp long and capable of encoding a protein of 98 kDa . N-terminal and C-terminal amino acid sequences deduced from the cDNA did not contain mitochondrial or glyoxysomal targeting signals . A search of protein databases suggested that the cDNA encoded a cytosolic aconitase . Immunoblotting analysis with a specific antibody against the aconitase expressed in Escherichia coli revealed that developmental changes in the amount of the aconitase were correlated with changes in levels of other enzymes of the glyoxylate cycle during growth of seedlings . Further analysis by subcellular fractionation and immunofluorescence microscopy revealed that aconitase was present only the cytosol and mitochondria . No glyoxysomal aconitase was found in etiolated cotyledons even though all the other enzymes of the glyoxylate cycle are known to be localized in glyoxysomes . Taken together, the data suggest that the cytosolic aconitase participates in the glyoxylate cycle with four glyoxysomal enzymes. Brain Res Mol Brain Res, 1995 Jun, 30(2), 336 - 46 The human D1A dopamine receptor gene promoter directs expression of a reporter gene to the central nervous system in transgenic mice; Severynse DM et al.; Dopamine receptors are involved in many aspects of dopaminergic neurotransmission including regulation of motor control, cognition, affect and neuroendocrine function . The D1A receptor is the most widely distributed dopamine receptor in the brain and is expressed at high levels in the striatum and nucleus accumbens, but is also found throughout cortical, limbic, hypothalamic and thalamic brain regions . We have cloned a 6.4 kb fragment 5' of the human D1A dopamine receptor gene and shown that this region activates transcription of the chloramphenicol acetyltransferase (CAT) gene in a cell-specific manner . To study the expression of these sequences in vivo we analyzed the expression of the E . coli lac Z gene under the regulation of the 6.4 kb fragment in transgenic mice . Expression of the transgene was primarily detected in the brain, with only low levels detected in peripheral tissues . The 5' flanking sequences were able to direct the tissue-specific expression of lac Z in three different lines of transgenic mice, to a number of brain regions including the caudate-putamen, thalamus, amygdala, cerebral cortex, hippocampus and hypothalamus . Greatest expression of the lac Z gene was detected in areas of the thalamus and amygdaloid complex . In the striatum, beta-galactosidase activity was restricted to neurons within the matrix and was not detected within striosomes . Results of this study demonstrate that the 6.4 kb region upstream of the human D1A receptor gene is sufficient to confer tissue-specific expression in the CNS of transgenic mice . Furthermore, expression of the transgene to neurons within the matrix of the striatum, but not the striosomes suggests that expression of the D1A receptor may be regulated differently within these areas. J Trop Pediatr, 1995 Jun, 41(3), 153 - 7 Antibody titre and avidity in saliva and serum are not impaired in mildly to moderately undernourished children; Nagao AT et al.; Avidities and titres of serum IgA, IgM, IgG, and salivary IgA antibodies for poliovirus type 3, beta-lactoglobulin and E . coli O antigens were analysed in well-nourished (n=58) and mildly to moderately undernourished (n = 18) 1-7-year-old children, living in slums in Sao Paulo, Brazil, with irregular water supply and no sewage system . The serum titres of antibodies in the undernourished children were not statistically different from the well-nourished ones, except for the IgA anti-beta-lactoglobulin titres which were lower in the youngest undernourished group . In respect to avidity of the serum antibodies, no statistical difference was observed, except that the youngest undernourished children presented avidities of serum IgA anti-E . coli O antigens higher than those of the well-nourished children of the same age range . With respect to salivary titres and avidities no difference was observed between well-nourished and undernourished children . The fact that the undernourished children were not deficient in antibody amount or quality, as measured in this study, suggests that in this respect their immune system was not impaired. Genetika, 1995 Jun, 31(6), 784 - 7 {Mutagenic effect of oxidizing agents on the thermally-induced prophage lambda cI857: effect of a system of oxidative stress}; Kalinin VL et al.; The lethal and mutagenic effects of hydrogen peroxide H2O (2-20 mM), cumene hydroperoxide (0.2-2.0 mM), and potassium permanganate KMnO4 (0.25-1.0 mM) on the heat-inducible lambda cI857 prophage were studied under conditions of heat-induction immediately after the mutagenic treatment of lysogenic cells of Escherichia coli oxyR+ or oxyR delta 3 . Within the range of the doses used, these agents decreased prophage survival by 3-5 orders of magnitude and increased mutation frequency by up to 0.2% under the action of hydrogen peroxide and cumene hydroperoxide, and up to 1.5% in the case of KMnO4 (in oxyR+ cells) . In the absence of the inducible OxyR system of oxidative stress, both lethal and mutagenic effects of H2O were enhanced . The oxyR delta 3 mutation increased lethal and mutagenic effects of cumene hydroperoxide and KMnO4 only at the highest concentrations used . Apparently, the OxyR system does not repair lesions induced by oxidative agents, but only prevents their formation. Genetika, 1995 Jun, 31(6), 759 - 66 {Analysis of expression of the RSV-lacZ-gene in transgenic embryos of the loach Misgurnus fossilis L . during different variations of injection}; Andreeva LE et al.; Several series of microinjection of the RSV-lacZ gene (the Escherichia coli beta-galactosidase gene under the control of the long terminal repeat of the Raus sarcoma virus) into fertilized mud loach eggs were carried out . The expression of the transgene in 3-5 day-old fry was shown to depend neither upon the stage of fry development at which the RSV-lacZ gene was introduced (early blastodisc, late blastodisc, 2-blastomere embryo) nor upon the region of transgene injection (blastodisc cytoplasm or egg yolk) . Additionally, when injected into yolk, a smaller number of transgene expression points were observed and their distribution was confined to the surface of the yolk sac . In some experimental series, transgene expression was observed in 100% of embryos . The presence of exogenous genetic material in one of the experimental series was proved for 100% of 5- to 6-week old juveniles, when injected into cytoplasm, and for 67% of juveniles, when injected into yolk . This work provides evidence of the possibility of 100% transfer and expression of exogenous DNA when transgenic loachs are generated by injection into embryos and fry at different stages of their development. Br J Rheumatol, 1995 Jun, 34(6), 525 - 8 OM-89 modulation of chronic inflammation: relevance to clinical use; Willis D et al.; The modulatory effects of a glycoprotein-rich endotoxin-free extract of Escherichia coli (OM-89) have been studied using the cotton pellet model of chronic inflammation in the male Wistar rat . OM-89 had a suppressive effect on the size of granuloma surrounding implanted cotton pellets at both 4 and 40 mg/kg given three times weekly . The lower dosage of 4 mg was effective throughout and there was little to be gained by increasing the dose as further reduction of granuloma size was not obtained . Whether given prior to, at the same time as, or after an inflammatory stimulus, OM-89 had suppressive effects . However, if given before, animals at first went through a phase of 'sensitization' before suppressive effects were seen on further exposure to OM-89 antigens, a phenomenon which might have bearing on clinical findings in rheumatoid arthritis . In animals presensitized to a cotton pellet, OM-89 was statistically as effective as indomethacin in suppressing a second granuloma . OM-89 combined with indomethacin showed additive effects and was highly effective . The results indicate that OM-89 could be efficacious in the treatment of chronic inflammatory conditions and there is the possibility that in appropriate circumstances OM-89 might replace some drugs currently used and in others reduce their dosage. Plant Mol Biol, 1995 Jun, 28(3), 549 - 67 The Arabidopsis MADS-box gene AGL3 is widely expressed and encodes a sequence-specific DNA-binding protein; Huang H et al.; The Arabidopsis AGL3 gene was previously identified on the basis of sequence similarity to the floral homeotic gene AGAMOUS (AG), which encodes a protein with a conserved MADS domain that is also found in human and yeast transcription factors (SRF and MCM1, respectively) . Analysis of newly isolated full-length cDNA clones as well as genomic clones indicates that AGL3 is indeed a MADS-box gene with a general intron-exon structure similar to other plant MADS-box genes . However, unlike the others, which are expressed specifically in flowers, AGL3 is expressed in all above-ground vegetative organs, as well as in flowers, but not in roots . Furthermore, since AGL3 is MADS-domain protein, it is likely that it is also a DNA-binding protein regulating transcription . To characterize AGL3 as a DNA-binding protein in vitro, we expressed the AGL3 protein in Escherichia coli, and characterized its DNA-binding properties . We show that AGL3 binds to sequences which resemble the target sequences of SRF and MCM1, and have determined the consensus sequence to which AGL3 binds using random oligonucleotides . These results suggest that AGL3 is a widely distributed DNA-binding protein, which may be involved the transcriptional regulation of genes in many cells. Plant Mol Biol, 1995 Jun, 28(3), 391 - 403 Characterisation of two tomato fruit-expressed cDNAs encoding xyloglucan endo-transglycosylase; Arrowsmith DA et al.; Xyloglucan endo-transglycosylase (XET) catalyses the cleavage and concomitant transfer of one xyloglucan molecule to another . It is thought to be an important component of cell wall metabolism, particularly in expanding tissue and ripening fruits . The recently reported cloning of a cDNA encoding a seed-expressed XET from nasturtium {9} has enabled two XET-encoding cDNAs to be isolated from a tomato fruit (breaker stage) cDNA library . Their deduced amino acid sequences exhibit ca . 40% identity to nasturtium XET . One of the tomato cDNA clones (tXET-B2) was over-expressed in Escherichia coli; following purification and refolding, the recombinant protein was shown to have XET activity, with no detectable hydrolytic activity . Southern hybridisation analysis suggests that these clones are members of a small multi-gene family encoding tomato XET . Ribonuclease protection assays show that transcripts protected by one of the clones (tXET-B1) are most abundant in pink fruit pericarp and were also detected in stems. Trends Biochem Sci, 1995 Jun, 20(6), 248 - 9 Methods and reagents . Electro-transformation of Escherichia coli with plasmid DNA; Hengen PN; Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet . This month's column discusses some reasons for switching to electroporation to transform bacteria with plasmid DNA . For details on how to partake in the newsgroup, see the accompanying box. Br J Surg, 1995 Jun, 82(6), 844 - 8 Effect of carbon dioxide pneumoperitoneum on bacteraemia and endotoxaemia in an animal model of peritonitis; Gurtner GC et al.; Laparoscopy is increasingly used in conditions complicated by peritonitis . A theoretical concern is that carbon dioxide pneumoperitoneum may increase bacteraemia . This study examines the effect of carbon dioxide pneumoperitoneum on bacteraemia, endotoxaemia and physiological correlates of sepsis in an animal model of peritonitis . New Zealand white rabbits were assigned to three groups of six animals . Group 1 received an intraperitoneal inoculation of 10(9) colony-forming units of Escherichia coli followed by a 10-cm midline laparotomy . Group 2 received an identical bacterial inoculum followed by a 12-mmHg carbon dioxide pneumoperitoneum for 1 h . Group 3 received no bacteria but had a 12-mmHg carbon dioxide pneumoperitoneum for 1 h . Groups 1 and 2 had significantly higher levels of bacteraemia (P < 0.01) and endotoxaemia (P < 0.01) accompanied by significantly lower mean arterial pressures (P < 0.05) and higher heart rates (P < 0.05) compared with group 3 . After 6 h groups 1 and 2 were significantly hypocarbic (P < 0.01), leucopenic (P < 0.01) and thrombocytopenic (P < 0.01) . There was no difference between group 1 and group 2 . A carbon dioxide pneumoperitoneum of 12 mmHg does not increase bacteraemia or endotoxaemia, nor does it adversely affect physiological or laboratory correlates of sepsis compared with laparotomy in this animal model of peritonitis. Nippon Rinsho, 1995 Jun, 53(6), 1537 - 47 {Recent studies on mammalian DNA repair mechanisms}; Tsuzuki T et al.; Errors in the replication of DNA are a major source of spontaneous mutation and a number of cellular functions correct these errors to maintain a low frequency of spontaneous mutation . First, we focused on two enzymes, 8-oxo-dGTPase and O6-methylguanine-DNA methyltransferase, which may be involved in the processes of spontaneous and induced mutagenesis, respectively . These enzymes are present in various organisms, from bacteria to mammalian cells, and appears to be responsible for preventing the occurrence of such mutations at the pre-replicational step . In addition to these two, the other mechanisms, such as nucleotide excision repair and mismatch repair, that also keep mutation rate low we reviewed . Genetic defects in one of the genes involved in DNA repair mechanisms have been linked to the high incidence of cancer, such as Xeroderma pigmentosum and hereditary nonpolyposis colorectal cancer. Kansenshogaku Zasshi, 1995 Jun, 69(6), 678 - 83 {Four cases of hemolytic uremic syndrome (HUS) associated with serotype O165 verotoxin producing Escherichia coli (VTEC) identified by LPS-solid phase enzyme-linked immunosorbent assay (ELISA)}; Uchida H et al.; Enzyme-linked immunosorbent assay using LPS derived from newly recognized serotype O165 verotoxin producing Escherichia coli (VTEC) could identify 4 cases of hemolytic uremic syndrome (HUS) associated with O165 VTEC . All 4 cases showed a typical clinical course seen in VTEC-associated HUS . We screened 33 cases of HUS whose pathogen was not identified by culture of serodiagnosis . The O165 serotype was not thought to be important not only as a VTEC but also as an enteropathogenic E . coli . However, the prevalence, 4 cases, was as high as of O111 serotype, which is the second major serotype of VTEC in Japan . We have to be careful for this serotype when we look for the pathogen of the patients with hemorrhagic colitis or with HUS. Biosci Biotechnol Biochem, 1995 Jun, 59(6), 1133 - 4 A method for the re-isolation of an autonomously replicating plasmid from Aspergillus transformants; Ozeki K et al.; An autonomously replicating plasmid is expected to increase the frequency of Aspergillus transformation . To construct this type of plasmid, we developed a rapid method of re-isolating autonomously replicating plasmids from Aspergillus transformants . Transformants grown in MM medium under selective pressure for 1-2 days were converted to protoplasts with a cell wall lytic enzyme (e.g . Yatalase) . The protoplasts were lysed with phenol/chloroform followed by precipitation with ethanol . The total DNA was treated with RNaseA, re-precipitated with PEG, and then used to transform E . coli . These re-isolated plasmids were mainly the plasmid monomer.
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