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