Biochemistry, 1991 May 21, 30(20), 4835 - 43 Kinetic mechanism of DNA polymerase I (Klenow fragment): identification of a second conformational change and evaluation of the internal equilibrium constant; Dahlberg ME et al.; In a previously determined minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment (KF) of Escherichia coli DNA polymerase I, a nonchemical step that interconverted the KF'.DNAn+1.PPi and KF.DNAn+1PPi complexes was not observed in correct incorporation {Kuchta, R . D., Mizrahi, V., Benkovic, P.A., Johnson, K.A., & Benkovic, S.J . (1987) Biochemistry 26, 8410-8417} but was detected in misincorporation {Kuchta, R . D., Benkovic, P.A., & Benkovic, S.J . (1988) Biochemistry 27, 6716-6725} . In a pulse-chase experiment in this study, a burst amplitude of 100% of the enzyme concentration is observed; under pulse-quench conditions, the burst amplitude is 80%, indicative of the accumulation of the KF'.DNA.dNTP species owing to a slow step subsequent to chemical bond formation . This latter step was unequivocally identified by single-turnover pyrophosphorolysis and pyrophosphate-exchange experiments as one interconverting KF'.DNAn+1.PPi and KF.DNAn+1.PPi . The rate constants for this step in both directions were established through the rate constants for processive synthesis and pyrophosphorolysis . Pyrophosphorolysis of a 3'-phosphorothioate DNA duplex confirmed that the large elemental effect observed previously {Mizrahi, V., Henrie, R . N., Marlier, J.F., Johnson, K.A., & Benkovic, S.J . (1985) Biochemistry 24, 4010-4018} in this direction but not in polymerization is due to a marked decrease in the affinity of KF for the phosphorothioate-substituted duplex and not to the chemical step . The combination of the experimentally measured equilibrium constant for the bound KF.DNA species with the collective kinetic measurements further extends previous insights into the dynamics of the polymerization process catalyzed by KF. J Mol Biol, 1991 May 20, 219(2), 359 - 76 The role of internal packing interactions in determining the structure and stability of a protein; Lim WA et al.; Cassette mutagenesis has been used to investigate how internal packing interactions help to specify a protein's three-dimensional structure and stability . Three interacting residues in the hydrophobic core of the N-terminal domain of lambda repressor were randomized combinatorially . The randomization was restricted to the five amino acids Val, Leu, Ile, Met and Phe, thereby generating a sterically diverse set of core sequences composed solely of hydrophobic residues . We have isolated 78 of the 125 possible sequences generated by this randomization . Approximately 70% of the isolated sequences show some level of biological activity, and thus still carry sufficient information to encode the basic structure of lambda repressor . An assay based on the temperature dependence of activity in vivo has been used to estimate the relative activities and thermal stabilities of the set of mutants . In addition, nine mutants have been purified and their stabilities and DNA binding activities characterized in vitro . Of the 56 active sequences, only two, in addition to the wild-type, maintain the wild-type level of stability and activity . All three of these proteins satisfy stringent requirements for specifically shaped residues at each position . All of the remaining active sequences have reduced stabilities and/or reduced DNA binding affinities . These and previous results suggest that there are two levels of structural information encoded in core residues . At the first level, the basic structural information appears to reside largely in the hydrophobic character of these residues . The majority of sequences that simply maintain hydrophobicity at core positions are able to adopt the overall lambda repressor fold and maintain moderate stability . At the second, more detailed level, specific steric features of these residues and their packing interactions clearly act as important determinants of the protein's precise structure and stability . These results imply that many of the basic structural features of a protein could be predicted from relatively simple, degenerate sequence patterns. J Mol Biol, 1991 May 20, 219(2), 231 - 41 Effects of the nucleotide 3' to an amber codon on ribosomal selection rates of suppressor tRNA and release factor-1; Pedersen WT et al.; Rates of ribosomal selection of both release factor 1 (RF1) and a suppressor tRNA (Su7C33) were studied at an amber codon at which the 3' neighbor was permuted . Rates of RF1 selection vary 2.6-fold among contexts . The 3' neighbor-dependent variation of RF1 action correlates very strongly with the non-random frequencies of 3' neighbors at UAG terminators (r = 0.97), which argues that the rate of RF1 selection is an important determinant 3' neighbor choice at termination codons . The data are consistent with a model for RF1 selection in which RF1 makes a specific contact(s) to the 3' neighbor and that this interaction is most favorable to uridylic acid . Measured rates of Su7C33 selection vary fivefold among 3' contexts . We also develop a method to calculate rates of selection for other suppressors, based on the assumption that rates of RF1 selection at each 3' context can be generalized to other sites that have the same 3' neighbor . Rates for various suppressors appear to vary from two- to fivefold depending on the 3' neighbor . Generally, the rate of selection of suppressors at different contexts correlates with the stacking strength of the 3' neighbor as measured in vitro . The two- to fivefold range of 3' neighbor effects on rate of aminoacyl-tRNA selection is greater than that previously observed within sets of codons read by the same tRNA . It is suggested that the choice of codons to achieve favorable contexts may be more important than the choice of a common codon at some message sites. J Mol Biol, 1991 May 20, 219(2), 161 - 3 Crystallization of human thymidylate synthase; Schiffer CA et al.; Human thymidylate synthase has been crystallized in the absence of ligands and diffracts beyond 3.0 A . The protein was cloned and expressed in Escherichia coli and then crystallized from ammonium sulfate in the presence of beta-mercaptoethanol at a variety of pH values . The crystals are trigonal in the space-group P3(1)21; the unit cell dimensions are a = b = 96.7 A, c = 84.1 A. FEBS Lett, 1991 May 20, 283(1), 61 - 4 Human interleukin-5 expressed in Escherichia coli: assignment of the disulfide bridges of the purified unglycosylated protein; Proudfoot AE et al.; Human interleukin-5 is a homodimer; each subunit contains two cysteine residues that form two inter-subunit disulfide bonds . The topology of the disulfides in recombinant human interleukin-5 produced in Escherichia coli was studied by proteolytic digestion and peptide mapping . Disulfide linked peptides containing cysteine 42 linked to cysteine 84 were isolated . This indicated that cysteines 42 and 84 of one subunit were linked in an antiparallel manner to cysteines 84 and 42 of the other subunit. FEBS Lett, 1991 May 20, 283(1), 44 - 6 The conformations of trimethoprim/E . coli dihydrofolate reductase complexes . A 15N and 31P NMR study; Huang FY et al.; We have employed 15N and 31P NMR techniques to characterize the conformations of trimethoprim (TMP)/E . coli dihydrofolate reductase (DHFR) complexes in the presence and absence of NADPH and NADP+ . A single conformation was observed for TMP/DHFR, NADP+/DHFR, NADPH/DHFR, and TMP/NADPH/DHFR complexes . In the ternary complex of TMP/NADP+/DHFR both the 15N and 31P spectra revealed the presence of two conformations . However, the conformations of TMP and NADP+ in the ternary complex may not be correlated, resulting in the possible existence of four conformations for the protein ternary complex. FEBS Lett, 1991 May 20, 283(1), 23 - 6 Determination of DNA binding specificities of mutated zinc finger domains; Thiesen HJ et al.; By substituting non conserved amino acids present in the postulated alpha-helical region of zinc finger domains, we demonstrated that Cys2/His2 type zinc finger domains could be targeted to new DNA binding sites . The putative alpha-helical region of the second SP1 zinc finger (RSDELQRH) was replaced by amino acids (KSSALISH) occurring in analogous zinc finger positions of human zinc finger protein Kox 29 . The DNA binding specificity of the FPLC purified chimaeric protein (SP1-Kox 29) was determined by use of the target detection assay (TDA) . Chimaeric protein SP1-Kox 29 was subjected to randomized oligonucleotides (GGG NNNN GGC) that were designed on the basis that each SP1 zinc finger interacts with 3-4 nucleotides concerning its cognate target site GGG GCGG GGC . By this analysis the DNA binding specificity of SP1-Kox 29 was shown to have switched from the cognate SP1 binding site to GGG GGTG GGC . Structure-function analysis of this type should facilitate the determination of DNA binding specificities for any individual zinc finger of interest. FEBS Lett, 1991 May 20, 283(1), 135 - 9 Identification and characterization of a C-terminally extended form of recombinant murine IL-6; Danley DE et al.; Murine interleukin-6 (mIL-6) was expressed in Escherichia coli in the insoluble fraction of cell lysates . Approximately equal amounts of two polypeptide species, reactive with anti-IL-6 antibodies, were produced . The two forms of mIL-6 were isolated and found to have identical N-terminal sequences initiated by Met-Phe-Pro-Thr-Ser-Gln- . Peptide mapping after endoproteinase glu-C digestion led to isolation and characterization of the C-terminal peptides from each of the two forms and allowed the source of the heterogeneity to be identified as a C-terminal addition of three amino acids, Gln-Lys-Leu, to authentic mIL-6 . Inspection of the nucleotide sequence of the plasmid containing the mIL-6 gene and expression of the plasmid in other strains suggested that the addition of three amino acids was caused by a readthrough of the termination codon arising from an unexpected suppressor mutation in the original host strain . Although the C-terminus of IL-6 is critical for the activity of this cytokine, the IL-6 variant with extended C-terminus was fully active in two separate bioassays . This suggests that the additional amino acids do not disrupt the structure or function of this important region of the molecule. FEBS Lett, 1991 May 20, 283(1), 1 - 3 Aminoacyl-tRNA synthetases and DNA replication . Molecular mimicry between RNAII and tRNA(Lys); Mirande M; Recent data pertaining to different research areas, aminoacyl-tRNA synthetases and replication of ColE1 plasmids, have provided mutually attractive prospects . The gene encoding Escherichia coli lysyl-tRNA synthetase was first isolated as a host suppressor mutation that restores replication of a mutant Co1E1 replicon . Comparison of RNAII and tRNA(Lys) suggests that lysyl-tRNA synthetase is involved in the formation of the displacement loop required for ColE1 plasmids replication and provides major identity elements of tRNA(Lys). J Mol Biol, 1991 May 20, 219(2), 217 - 30 Synthetic DNA bending sequences increase the rate of in vitro transcription initiation at the Escherichia coli lac promoter; Gartenberg MR et al.; Appropriately phased DNA bending sequences replacing the CAP binding site upstream from the lac promoter increase by roughly tenfold the rate of specific transcription initiation from a superhelical promoter template in vitro; promoter occlusion results from polymerase binding to the upstream (dA)n.(dT)n tracts, but this phenomenon is not responsible for the observed phase-dependent transcriptional activity . The rates of open complex formation at both P1 and P2 promoters respond in a similar phase-dependent way to the synthetic curved DNA sequences. J Mol Biol, 1991 May 20, 219(2), 201 - 15 Catabolite activator protein-induced DNA bending in transcription initiation; Zinkel SS et al.; We describe experiments that enable us to track the presence and direction of the DNA bend induced by Escherichia coli catabolite activator protein (CAP) through the intermediate stages of transcription initiation at the lac promoter . Transcriptional complexes examined were formed on superhelical templates to enhance specific complex formation, and detected by electrophoretic analysis after restriction digestion . We found that the bend is maintained and even increased upon formation of closed and open complexes . Our results exclude the hypothesis that the energy of the CAP-induced bend is used to promote open complex formation . We now suggest a new model, in which DNA wraps around the CAP-polymerase complex to form a writhing structure equivalent to that at the end of an interwound superhelical domain . Formation of this structure may facilitate open complex formation . We further propose that the stored bend energy may be used to help counteract strong protein-protein or protein-DNA interactions, thus assisting the process of RNA polymerase escape from the promoter. J Mol Biol, 1991 May 20, 219(2), 189 - 99 Two mutations of phage mu transposase that affect strand transfer or interactions with B protein lie in distinct polypeptide domains; Leung PC et al.; Two mutations within the transposase (the A protein) gene of phage Mu with distinct effects on DNA transposition have been studied . The first mutation maps to the central domain (domain II) of A, a protein consisting of three major structural domains . The variant protein is normal in synapsis and cleavage of Mu ends but is temperature-sensitive in the strand transfer reaction, joining the Mu ends to target DNA . The second mutation is a deletion at the C terminus (within domain III); on the basis of genetic studies, the mutant protein is predicted to have lost the ability to interact with the Mu B protein . The B protein, in conjunction with A, promotes efficient intermolecular transposition, while inhibiting intramolecular transposition . We show that the purified mutant protein is proficient in intramolecular, but not intermolecular transposition in vitro . The interactions between A and B proteins have been followed by a proteolysis assay . The chymotrypsin sensitivity of the interdomainal Phe221-Ser222 peptide bond within the bidomainally organized B protein is exquisitely modulated by ATP, DNA and A protein . The sensitive or "open" state of this bond in native B protein becomes partially "open" upon binding of ATP by B, attains a "closed" or resistant configuration upon binding of DNA in presence of ATP, and is rendered "open" again upon addition of the A protein . In this test for the interaction of A protein with B protein-DNA complex, the domain II mutant behaves like wild-type A protein . However, the domain III mutant fails to restore chymotrypsin susceptibility of the Phe221-Ser222 bond. Cell, 1991 May 17, 65(4), 579 - 86 Sex-specific splicing and polyadenylation of dsx pre-mRNA requires a sequence that binds specifically to tra-2 protein in vitro; Hedley ML et al.; Somatic sex determination in Drosophila involves a hierarchy of regulated alternative pre-mRNA processing . Female-specific splicing and/or polyadenylation of doublesex (dsx) pre-mRNA, the final gene in this pathway, requires transformer (tra) and transformer-2 (tra-2) proteins . The mechanisms by which these proteins regulate RNA processing has not been characterized . In this paper we show that tra-2 produced in Escherichia coli binds specifically to a site within the female-specific exon of dsx pre-mRNA . This site, which contains six copies of a 13 nucleotide repeat, is required not only for female-specific splicing, but also for female-specific polyadenylation . These observations suggest that tra-2 is a positive regulator of dsx pre-mRNA processing. Gene, 1991 May 15, 101(1), 127 - 31 Cloning of coliphage-T4 gene pseT and high-level synthesis of polynucleotide kinase in Escherichia coli; Campos M et al.; The gene, pseT, of coliphage T4 which encodes polynucleotide kinase (PNK) was cloned directly into an expression plasmid using the polymerase chain reaction . When placed under the control of the trp promoter, the pse T gene can be maintained stably in Escherichia coli and yields high levels of the enzyme upon induction . The system described facilitates purification and provides very high yields of PNK. Biochem Biophys Res Commun, 1991 May 15, 176(3), 1142 - 8 A single Trp121 to Ala121 mutation in human cyclophilin alters cyclosporin A affinity and peptidyl-prolyl isomerase activity; Bossard MJ et al.; Fluorescence and NMR spectral data have suggested an interaction between the single tryptophan in cyclophilin (CyP) and its high affinity ligand cyclosporin A (CsA) . To study this interaction, a site mutation of Trp121 to Ala was introduced into human cyclophilin (CyP) and the encoded protein was expressed in E . coli . The Ala121 mutant was shown to catalyze the peptidyl-prolyl cis-trans isomerase (rotomase) reaction with several peptide substrates, albeit at less than ten percent the rate of the purified recombinant human CyP . Values for the apparent inhibition constant (Ki,app) of cyclosporin A with the human CyP and the Ala121 mutant were determined to be 1.6 +/- 0.4 nM and 640 +/- 90 nM, respectively by tight-binding inhibition analysis . The greater loss of affinity for CsA binding (400-fold) than for rotomase catalysis (20 fold) suggests that the catalytic and CsA binding properties associated with CyP can be decoupled as has been observed with an homologous protein found in E . coli (Liu, J . & Walsh, C.T . (1990) Proc . Natl . Acad . Sci . USA 87, 4028-4032). Biochem Biophys Res Commun, 1991 May 15, 176(3), 1086 - 92 Increase of O6-methylguanine-DNA-methyltransferase and N3-methyladenine glycosylase RNA transcripts in rat hepatoma cells treated with DNA-damaging agents; Laval F; A variety of DNA-damaging agents increase the O6-methylguanine-DNA-methyltransferase (transferase) and the N3-methyladenine (3-meAde)-DNA-glycosylase activities in a rat hepatoma cell line (H4 cells) . Using two cDNA expressing either the rat 3-meAde-DNA-glycosylase or the transferase, the level of mRNA transcripts was measured by hybridization in H4 cells treated with three different inducing agents, gamma-rays, cis-dichlorodiammine platinum II or N-methyl-9-hydroxy ellipticinium . The two mRNA increased 24 hours after the cell treatments but this enhanced transcription was a transient phenomenon, as it was no longer observed after 96 hours . No significant DNA amplification was detectable in the treated cells. Biochem J, 1991 May 15, 276 ( Pt 1), 63 - 71 Purification and characterization of recombinant tissue kallikrein from Escherichia coli and yeast; Wang J et al.; A full-length rat tissue kallikrein cDNA was constructed by oligonucleotide engineering through an extension of RSK 1105, a partial cDNA clone containing 534 bp of the 3' end of tissue kallikrein, followed by site-directed mutagenesis to remove the vector sequence from within the chimaeric coding sequence . The cDNA has been cloned both into the plasmid pET3b under the control of the T7 promoter/polymerase system, and into the shuttle vector PYE directed by the alpha-factor promoter . Expression in Escherichia coli was detected by direct radioimmunoassay, and recombinant kallikrein of 36 kDa was identified by Western-blot analysis using both polyclonal and monoclonal antibodies to rat tissue kallikrein, and by autoradiography of 14C-labelled L-amino acid-labelled-protein synthesis in the presence of rifampicin . Expression in yeast was also detected by direct radioimmunoassay, and recombinant kallikrein was identified by Western-blot analysis with a molecular mass of 39 kDa . The recombinant kallikrein from yeast, however, remained mostly inactive . Kallikrein was purified to apparent homogeneity from E . coli by DEAE-Sepharose CL-6B and aprotinin-affinity column chromatography and confirmed by the N-terminal ten-amino-acid sequence, which matched the deduced sequence from the cDNA . Both E . coli and yeast recombinant kallikreins have Tos-Arg-OMe-esterolytic and kininogenase activities similar to those of purified tissue kallikrein . Comparisons were made between recombinant kallikreins and rat tissue kallikrein with respect to size, charge, substrate specificity, susceptibility to inhibitors and immunological properties . Our results open the way for the study of kallikrein structure-function relationships through protein engineering. Proc Natl Acad Sci U S A, 1991 May 15, 88(10), 4443 - 7 Cloning, expression, and characterization of a class-mu glutathione transferase from human muscle, the product of the GST4 locus; Vorachek WR et al.; A class-mu glutathione transferase cDNA clone, GTHMUS, was isolated from human myoblasts and its sequence was determined . The sequence predicts a protein of molecular weight 25,599 whose 24 amino-terminal residues are identical to those of the class-mu isoenzyme expressed from the GST4 locus . The GTHMUS cDNA shares 93.7% nucleotide sequence identity with a human liver cDNA clone, GTH411, that is encoded at the GST1 locus . Comparison of the liver and muscle cDNA sequences shows two regions of remarkable sequence conservation: a 140-nucleotide region in the 5' coding portion of the molecule that has a single silent nucleotide substitution, and a 550-nucleotide region, including the entire 3' noncoding region, that has only three nucleotide substitutions or deletions . This sequence conservation suggests that gene conversion has occurred between the human GST1 and GST4 glutathione transferase gene loci . The human muscle and liver glutathione transferase clones GTHMUS and GTH411 have been expressed in Escherichia coli . The kinetic mechanism of the muscle enzyme was examined in product inhibition studies . The inhibition patterns are best modeled by a steady-state ordered bi-bi reaction mechanism . Glutathione is the first substrate bound and chloride ion is the first product released . Chloride ion inhibits the muscle enzyme. Proc Natl Acad Sci U S A, 1991 May 15, 88(10), 4260 - 4 Isolation and properties of a nitrile hydratase from the soil fungus Myrothecium verrucaria that is highly specific for the fertilizer cyanamide and cloning of its gene; Maier-Greiner UH et al.; A protein was purified from crude extracts of the soil fungus Myrothecium verrucaria by gel filtration and hydrophobic chromatography to homogeneity; this protein catalyzed the stoichiometric hydration of the fertilizer cyanamide to urea with high substrate specificity . This cyanamide hydratase (urea hydro-lyase; EC 4.2.1.69) contained zinc and consisted of six identical subunits with Mr = 27,700 . It was partially sequenced . The protein was detectable only when the fungus was grown on cyanamide as the sole nitrogen source . Genomic DNA from the fungus was cloned, and the gene encoding the enzyme was mapped with an oligonucleotide probe derived from the amino acid sequence within a 25,800-base-pair DNA region . The subunit of the enzyme is encoded by a 795-base-pair DNA sequence containing a 63-base-pair intron . A cDNA clone containing the intronless gene with an open reading frame encoding a sequence of 244 amino acids expressed the enzyme in active form in Escherichia coli with excellent yield. Proc Natl Acad Sci U S A, 1991 May 15, 88(10), 4114 - 8 Primary structure of a cerulenin-binding beta-ketoacyl-{acyl carrier protein} synthase from barley chloroplasts; Siggaard-Andersen M et al.; The radioactively labeled beta-ketoacyl thioester synthase inhibitor {3H} cerulenin was used to tag three dimeric barely chloroplast proteins (alpha alpha, alpha beta, and beta beta) from the stromal fraction . Oligonucleotides corresponding to amino acid sequences obtained from the purified proteins were used to generate with the polymerase chain reaction a probe for cDNAs encoding the beta subunit . cDNA sequencing revealed an open reading frame for 462 residues comprising the mature protein and a 35-amino acid transit peptide . The deduced amino acid sequence of the mature protein is homologous to the beta-ketoacyl-{acyl carrier protein} (ACP) synthase I {3-oxoacyl-ACP synthase; acyl-ACP:malonyl-ACP C-acyltransferase (decarboxylating), EC 2.3.1.41} of Escherichia coli . Under analogous experimental conditions {3H}cerulenin tagged a single dimeric protein from spinach chloroplasts. J Immunol, 1991 May 15, 146(10), 3599 - 603 Priming for in vitro and in vivo anti-human T lymphotropic virus type 1 cellular immunity by virus-related protein reconstituted into liposome; Noguchi Y et al.; In vitro and in vivo anti-human T lymphotropic virus type 1 (HTLV-1) cellular immunity was examined by immunizing rats with a truncated hybrid protein (228 amino acids) of gag and env of HTLV-1 produced by Escherichia coli . Animals were immunized with the hybrid protein reconstituted into mannan-derivative-coated liposomes (gag-env-lipo) . In vitro sensitization with a HTLV-1-positive cell line, TARS-1, of spleen cells obtained from these animals generated killer cells specific for syngeneic HTLV-1-positive cells . No killer activity was generated when spleen cells were obtained from animals immunized with the hybrid protein alone, the liposome alone, or the hybrid protein reconstituted into conventional liposomes with no polysaccharide coating . Killer cells were CD8+ CTL restricted to MHC class I . Analysis of CD8+ and CD4+ subsets in spleens showed the existence of primed CD8+ T cells in animals immunized with gag-env-lipo . Rats immunized with gag-env-lipo displayed accelerated rejection of TARS-1 but not of two other HTLV-1-negative tumor lines . Injection of carrageenan into animals strongly inhibited generation of killer cells, which indicates the necessity of macrophages for priming of CD8+ T cells with gag-env-lipo . Injection of carrageenan also cancelled in vivo immunity against HTLV-1+ cells induced with gag-env-lipo . These results, taken together, indicate that exogenous protein reconstituted into appropriate liposomes can effectively prime MHC class I restricted CD8+ T cells in vivo with macrophage dependency. J Biol Chem, 1991 May 15, 266(14), 9166 - 72 Localization of the calmodulin- and the actin-binding sites of caldesmon; Wang CL et al.; Expression of the C-terminal third of chicken gizzard caldesmon in Escherichia coli, using the Nagai vector (Nagai, K., and Thogersen, H.V . (1987) Methods Enzmol . 153, 461-481), produces a cII-caldesmon fusion protein (27 kDa) with caldesmon sequence beginning at Lys579 . Degradation during purification yields five peptides with molecular masses of 24, 22, 19 (two peptides), and 15 kDa . The 24-kDa peptide begins at Phe581; the 22-kDa peptide begins at Leu597, the two 19-kDa peptides begin at Phe581 and Val629, respectively; the 15-kDa peptide also begins at Val629 . We estimate that the 15-kDa and one of the 19-kDa peptides end near Leu710 . Site-directed mutagenesis was used to produce truncated peptides with known C termini; one peptide (17 kDa) terminates at Asn675 . Digestion of the fragments with chymotrypsin generates a second 15-kDa fragment that begins at Ser666 (15K') . All of the peptides, with the exception of 15K', bind Ca(2+)-calmodulin-Sepharose and share a common 37-amino acid peptide between Val629 and Ser666, suggesting this contains the calmodulin binding site . Comparison with published sequences (Takagi, T., Yazawa, M., Ueno, T., Suzuki, S., and Yagi, K . (1989) J . Biochem . (Tokyo) 106, 778-783 and Bartegi, A., Fattoum, A., Derancourt, J., and Kassab, R . (1990) J . Biol . Chem . 265, 15231-15238) for other calmodulin-binding fragments further restricts the binding site to 7 residues, Trp-Glu-Lys-Gly-Asn-Val-Phe, between Trp659 and Ser666 . All of the fragments, except the two 15-kDa peptides, co-sediment with F-actin, indicating that there are two segments in the C-terminal third of caldesmon that can interact with F-actin: one between Leu597 and Val629, the other between Arg711 and Pro756 . Although separated in the primary sequence, these domains may interact with the calmodulin-binding region in the folded structure. J Biol Chem, 1991 May 15, 266(14), 8946 - 51 Purification and characterization of the membrane-associated components of the maltose transport system from Escherichia coli; Davidson AL et al.; Maltose is transported across the cytoplasmic membrane of Escherichia coli by a binding protein-dependent transport system . The three membrane-associated components of the transport system, the MalK, MalF, and MalG proteins, have been solubilized from the membrane and maltose transport activity has been reconstituted in proteoliposome vesicles (Davidson, A . L., and Nikaido, H . (1990) J . Biol . Chem . 265, 4254-4260) . A modification of the reconstitution technique is presented which permits reconstitution from the detergent dodecyl maltoside . Utilizing reconstitution of maltose transport as an assay, we have purified these proteins in the presence of n-dodecyl-beta-D-maltoside . The purified proteins catalyze both maltose transport activity and ATP hydrolysis . In all experiments, the MalF, MalG, and MalK proteins behaved as a multiprotein complex; all three proteins were immunoprecipitated using antibody prepared against MalF, and they copurified, eluting from a gel filtration column between markers of Mr 160,000 and 200,000 . Each complex contains two MalK, one MalF, and one MalG proteins, providing two putative sites for ATP hydrolysis . Chemical cross-linking detected specific interactions between MalF and MalG and between MalF and MalK. J Biol Chem, 1991 May 15, 266(14), 8747 - 50 Effects of overproduction of superoxide dismutase on the toxicity of paraquat toward Escherichia coli; Liochev SI et al.; Gross overproduction of the manganese-containing superoxide dismutase in Escherichia coli, by virtue of a multicopy plasmid bearing the sodA gene, decreases enumeration on paraquat-containing agar plates . This reflects growth inhibition, not lethality, since cells on these plates can be rescued by exclusion of dioxygen . Growth in liquid medium revealed that the control strain adapted to growth in the presence of paraquat more rapidly than did the overproducer . Glucose-6-phosphate dehydrogenase, taken as a representative of the superoxide-inducible soxR regulon, was induced during exposure to paraquat to a much greater extent in the control than in the superoxide dismutase-over-producing strain . These results support the view that overproduction of superoxide dismutase interferes with induction of the soxR regulon and thus prevents a balanced adaptation to the multiple aspects of the toxicity of aerobic paraquat. FEMS Microbiol Lett, 1991 May 15, 64(2-3), 319 - 23 Improved methods for the detection of beta-galactosidase activity in colonies of Escherichia coli using a new chromogenic substrate: VBzTM-gal (2-(2-(4-(beta-D-galactopyranosyloxy)-3-methoxyphenyl)-vinyl)-3- methylbenzothiazolium toluene-4-sulphonate); Bainbridge BW et al.; The use of a new substrate 2-(2-(4-(beta-D-galactopyranosyloxy)-3- methoxyphenyl)-vinyl)-3-methylbenzothiazolium toluene-4-sulphonate (VB-zTM-gal) is described for the detection of beta-galactosidase activity in colonies of wild type and mutant strains of Escherichia coli . On enzymic hydrolysis this substrate, which is soluble in water, released a chromophore which is red at pH 7 and bound to cellulose and nitrocellulose . The best procedure for the detection of activity was to grow colonies on standard nitrocellulose membranes (pore size 0.45 microns) laid onto an agar plate and to float the membranes over a solution of the substrate . Coloured colonies developed within 3 min, which were stable at 4 degrees C for several days, and this identified the expression of beta-galactosidase activity . This was found to be more specific than methods using triphenyltetrazolium or Eosin Methylene Blue media, and more economical than methods using X-gal (5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside) . VBzTM-gal should have applications in gene cloning technology and in the detection of coliform organisms in polluted water. FEMS Microbiol Lett, 1991 May 15, 64(2-3), 127 - 34 Isolation and characterization of a temperature-sensitive conditional mutant of Escherichia coli altered for the control of phosphate-regulated proteins; Uribellarea JL et al.; We have identified a conditional mutation which confers a pleiotropic phenotype to Escherichia coli cells: no growth at temperature higher than 36 degrees C, an altered control of the synthesis of several phosphate-regulated polypeptides (including alkaline phosphatase, sn-glycerol-3-phosphate binding protein, phosphate binding protein and outer membrane porin protein PhoE) after growth at 36 degrees C and a wild-type phenotype at 30 degrees C . This mutation was located at minute 89.5 on the E . coli chromosome in a gene we have called cpr for conditional phosphate-regulated. Arch Biochem Biophys, 1991 May 15, 287(1), 60 - 7 Kinetic studies of L-aspartase from Escherichia coli: pH-dependent activity changes; Karsten WE et al.; The pH dependence of the kinetic parameters of the L-aspartase-catalyzed reaction have been examined in both the amination and the deamination directions . The enzyme isolated from Escherichia coli exists in a pH-dependent equilibrium between a higher pH form that has an absolute requirement for a divalent metal ion and for substrate activation, and a low pH form that does not require activation by either substrate or metal ions . The interconversion between these enzyme forms is observed near neutral pH in the profiles examined for the reaction in either direction . This pH-dependent activation has not been observed for other bacterial aspartases . Loss of activity is observed at high pH with a pK value of 9 . The pH profiles of competitive inhibitors such as 3-nitropropionic acid and succinic acid have shown that the enzyme group responsible for this activity loss must be protonated for substrate binding at the active site . An enzymatic group has also been identified that must be protonated in the amination reaction, with a pK value near 6.5, and deprotonated in the deamination reaction . This group, tentatively assigned as a histidyl residue, fulfills the criteria for the acid-base catalyst at the active site of L-aspartase. Arch Biochem Biophys, 1991 May 15, 287(1), 112 - 20 Reduction (dethiolation) of protein mixed-disulfides; distribution and specificity of dethiolating enzymes and N,N'-bis(2-chlorethyl)-N-nitrosourea inhibition of an NADPH-dependent cardiac dethiolase; Miller RM et al.; The S-thiolated proteins phosphorylase b (Phb) and carbonic anhydrase III (CAIII) were prepared with {3H}glutathione in a reaction initiated with diamide . These substrates were used to measure the rate of reduction (dethiolation) of protein mixed-disulfides by enzymes with properties similar to those of thioredoxin and glutaredoxin . This enzyme activity is termed a dethiolase since the identities of the enzymes are still unknown . The dethiolation of either S-{3H}glutathiolated Phb or S-{3H}glutathiolated CAIII was employed in tissue assays and for study of two partially purified dethiolases from cardiac tissue . NADPH-dependent dethiolase activity was most abundant except in rat liver and muscle . Total dethiolase activity was approximately 10-fold higher in neutrophils, 3T3-L1 cells, and Escherichia coli than in other sources . Rat skeletal muscle had 3- to 4-fold higher dethiolase activity than rat heart or liver . These data indicate that protein dethiolase activity is ubiquitous and that normal expression of the two dethiolase activities varies considerably . A partially purified cardiac NADPH-dependent dethiolase acted on Phb approximately 1.5 times faster than CAIII, and a glutathione (GSH)-dependent dethiolase acted on Phb 3 times faster than CAIII . The Km for glutathione for the GSH-dependent dethiolase was 15 microM with Phb as substrate and 10 microM with CAIII . Thus, the GSH-dependent dethiolase is probably not affected by normal changes in the cardiac glutathione content (normally approximately 3 mM) . Partially purified cardiac NADPH-dependent dethiolase was inactivated by BCNU (N,N'-bis(2-chloroethyl)-N-nitrosourea) and the GSH-dependent dethiolase was unaffected under similar conditions . In a soluble extract from bovine heart, 200 microM BCNU inhibited NADPH-dependent dethiolase by more than 60% but did not affect GSH-dependent activity . These results demonstrate that BCNU is a selective inhibitor of the NADPH-dependent dethiolase. Anal Biochem, 1991 May 15, 195(1), 177 - 81 Selective precipitation of proteins from guanidine hydrochloride-containing solutions with ethanol; Pepinsky RB; The solubility of guanidine hydrochloride in ethanol and conversely the low solubility of proteins have been used as the basis for a procedure for recovering proteins from guanidine-containing solutions by selective precipitation . Yields of greater than 94% were observed with as little as 28 ng protein and of 98% for larger quantities of protein up to 20 mg/ml . The precipitations were independent of molecular weight for proteins in the range of 6-100 kDa and could be run in as little as 5 min at room temperature . Unlike the conventional desalting methods for removing guanidine, ethanol precipitation is rapid, efficient, and can be applied simultaneously to a large number of samples . The approach should have a wide range of applications. FEMS Microbiol Lett, 1991 May 15, 64(2-3), 225 - 30 Clonal relationships among Escherichia coli serogroup 06 isolates from human and animal infections; Cherifi A et al.; The clonal relationship of thirty E . coli strains of 0 antigen serotype 06 isolated from human, dog, pig or cow infections were investigated . Two main clones with serotypes 06 : H1 or 06 : H31, H- were identified . Isolates from humans, dogs, pigs and cows were found in both clones, indicating that animals are a possible source for human extraintestinal Escherichia coli strains . Two human ETEC (06 : H16) and two pig isolates (06 : H10) were not related to the 06 : H1 or 06 : H31, H- E . coli clones. Proc Natl Acad Sci U S A, 1991 May 15, 88(10), 4419 - 23 P regulatory products repress in vivo the P promoter activity in P-lacZ fusion genes; Lemaitre B et al.; The transposition of P elements in Drosophila melanogaster is regulated by products encoded by the P elements themselves . The molecular mechanisms of this regulation are complex and still unclear . We have assayed in vivo the effects of P regulatory products on the P promoter itself by using P-lacZ fusion genes . We have found that all the P-lacZ insertions are repressed in a P background . This repression occurs in all the tissues observed and at all the developmental stages . The amount of transcripts specific for P-lacZ is substantially reduced in a P background . These results suggest that P trans-acting products can exert a direct repression on the P promoter transcription. Proc Natl Acad Sci U S A, 1991 May 15, 88(10), 4084 - 8 Polymerase chain reaction-mediated gene synthesis: synthesis of a gene coding for isozyme c of horseradish peroxidase; Jayaraman K et al.; The synthesis of a gene coding for horseradish peroxidase (HRP, isozyme c; EC 1.11.1.7) is described using a polymerase chain reaction (PCR)-mediated gene synthesis approach developed in our laboratory . In this approach, all the oligonucleotides making up the gene are ligated in a single step by using the two outer oligonucleotides as PCR primers and the crude ligation mixture as the target . The PCR facilitates synthesis and purification of the gene simultaneously . The gene for HRP was synthesized by ligating all 40 oligonucleotides in a single step followed by PCR amplification . The gene was also synthesized from its fragments by using an overlap extension method similar to the procedure as described {Horton, R . M., Hunt, H . D., Ho, S . N., Pullen, J . K . & Pease, L . R . (1989) Gene 77, 61-68} . A method for combining different DNA fragments, in-frame, by using the PCR was also developed and used to synthesize the HRP gene from its gene fragments . This method is applicable to the synthesis of even larger genes and to combine any DNA fragments in-frame . After the synthesis, preliminary characterization of the HRP gene was also carried out by the PCR to confirm the arrangement of oligonucleotides in the gene . This was done by carrying out the PCR with several sets of primers along the gene and comparing the product sizes with the expected sizes . The gene and the fragments generated by PCR were cloned in Escherichia coli and the sequence was confirmed by manual and automated DNA sequencing. J Biol Chem, 1991 May 15, 266(14), 9055 - 6 Crystallization and preliminary X-ray diffraction studies on the mutT nucleoside triphosphate pyrophosphohydrolase of Escherichia coli; Bessman MJ et al.; The mutT nucleoside triphosphatase, which prevents AT----CG transversions during DNA replication, has been crystallized from ammonium sulfate utilizing a novel technique involving vapor diffusion in capillaries . X-ray diffraction analysis has revealed that the crystals are monoclinic, space group P2(1), with cell constants a = 34.14, b = 72.54, c = 56.38, and beta = 98.90 . The Vm value of 2.31 A3/Da is consistent with two molecules of enzyme per asymmetric unit . The crystals are reasonably stable in the x-ray beam, and a data set to 2.5 A resolution has been collected for native protein . There is evidence that the crystals diffract to at least 2.1 A. J Biol Chem, 1991 May 15, 266(14), 9050 - 4 Characterization of the mutT nucleoside triphosphatase of Escherichia coli; Bhatnagar SK et al.; The mutT protein, which prevents A:T----C:G transversions during DNA replication, has the following enzymatic properties . Although it prefers dGTP as a substrate, it hydrolyzes all of the canonical nucleoside triphosphates to some extent . It has no activity in the absence of divalent cations, is maximally activated by magnesium ions, and has a pH optimum of 9.0 . Nucleoside triphosphates are hydrolyzed according to the following equation . dGTP----dGMP + PPi Studies with nucleotide analogues suggest that the enzyme may prefer the syn rather than the anti conformation of the nucleoside triphosphates, which might explain the role it plays in preventing mutations. J Biol Chem, 1991 May 15, 266(14), 8659 - 62 Negatively charged phospholipids restore prePhoE translocation across phosphatidylglycerol-depleted Escherichia coli inner membranes; Kusters R et al.; Translocation of outer membrane precursor proteins across the Escherichia coli inner membrane is severely hampered in lipid biosynthetic mutants with strongly reduced phosphatidylglycerol (PG) levels (De Vrije, T., De Swart, R . L., Dowhan, W., Tommassen, J., and De Kruijff, B . (1988) Nature 334, 173-175; Lill, R., Dowhan, W., and Wickner, W . (1990) Cell 60, 271-280) . Two independent methods were used to demonstrate that anionic lipids by virtue of their negative head-group charge are involved in membrane translocation of the precursor of the pore protein PhoE . Using a lipid transfer protein-based method we show that introduction from lipid vesicles of PG and other acidic phospholipids but not of phosphatidylcholine restores efficient translocation across the membrane of PG-depleted inner membrane vesicles . Moreover, translocation was found to be proportional to the PG content in vesicles isolated from strain HDL11 in which the PG content was altered by varying the synthesis of the PG-phosphate synthase. Biochem Biophys Res Commun, 1991 May 15, 176(3), 1062 - 7 Molecular cloning of cDNA encoding the 16 KDa subunit of vacuolar H(+)-ATPase from mouse cerebellum; Hanada H et al.; cDNA for the 16 kDa subunit of vacuolar H(+)-ATPase was cloned from mouse cerebellum and sequenced . The deduced polypeptide (155 amino acid residues; molecular weight, 15,808) was highly hydrophobic and homologous to the subunits of bovine adrenal medulla, Torpedo marmorata electric lobe, Drosophila and yeast . Glu-139 (supposed to be essential for proton transport) was also conserved as the potential dicyclohexylcarbodiimide binding site . The subunit had four transmembrane segments: Segment II and IV were highly homologous and Glu-139 was located in Segment IV . The roles of the non-conserved regions are discussed. Biochem Biophys Res Commun, 1991 May 15, 176(3), 1516 - 24 Molecular cloning and expression of mouse leukotriene A4 hydrolase cDNA; Medina JF et al.; A cDNA clone for mouse leukotriene A4 hydrolase encoding the full sequence of the enzyme was isolated from a mouse spleen lambda ZAP-II library . The identification was ascertained by expression of enzyme activity in Escherichia coli . The encoded protein has 610 amino acids and exhibits an extensive identity (93%) with the human leukotriene A4 hydrolase . A region spanning between residues 233 and 340, where the zinc binding site is located, was found to be perfectly conserved between the two species . We found six sites of polymorphism in the cDNA sequence of mouse LTA4 hydrolase, one of which leads to a difference in the encoded amino acid . The polymorphism of cDNA was confirmed by reverse transcription-PCR sequencing of mouse spleen total RNA, prepared as a mixture from ten different strains. FEMS Microbiol Lett, 1991 May 15, 64(2-3), 265 - 70 Analysis of the first two genes of the CS1 fimbrial operon in human enterotoxigenic Escherichia coli of serotype 0139:H28; Jordi BJ et al.; An oligonucleotide, derived from the N-terminal amino acid sequence of the CS1 fimbrial subunit protein was used to identify the subunit gene on recombinant plasmid pDEP23 containing the structural genes of the CS1 fimbrial operon . The nucleotide sequence of the subunit gene (csoA), encoding a protein of 171 amino acids, was determined . Flanking it upstream, a gene (csoB) encoding a protein of 238 amino acids was found . The CsoB and CsoA proteins are homologous to the CfaA and CfaB proteins in the CFA/I fimbrial operon . For all the CS1 producing strains investigated the structural genes are located on plasmids . Like CFA/I fimbriae, CS1 fimbriae are only expressed in the presence of a positive regulator, CfaD for CFA/I and Rns for CS1, respectively . The promoter region upstream of the csoB gene was cloned in front of the promoterless alkaline phosphatase (phoA) gene of the promoter-probe vector pCB267 . PhoA activity was enhanced approximately two-fold by the introduction of compatible plasmids containing either rns or cfaD. Gene, 1991 May 15, 101(1), 67 - 74 Classification of type-II restriction endonucleases and cloning of non-identical cohesive-end fragments without self-polymerization using nonpalindromic oligodeoxyribonucleotide adapters; Bellemare G et al.; Enzymatic partial filling-in of recessed 3'-end sequences, left after digestion of DNA by the restriction endonucleases (ENases) Sau3A and SalI, with the Klenow fragment of E . coli DNA polymerase I allows the forced ligation of the resulting fragments; this technology is already used for subcloning and for genomic bank construction . To simplify and generalize its utilization, class-II ENases have been arranged into 16 different families according to the composition of the 5'-protruding sequences present after cleavage . Moreover, this system was extended to allow the joining of noncompatible ends by the use of nonpalindromic complementary oligodeoxyribonucleotides (NPCOs) containing two nucleotides protruding at each 5' end . The use of these synthetic adapters maintains all the advantages of the initial gap-filling cloning technique: only one insert can be cloned per vector molecule and no self-ligation or -polymerization can occur with any of the DNA molecules involved . Only 22 such oligodeoxyribonucleotides are needed to generate the 60 NPCO pairs necessary to ligate to each other any member of twelve ENase families when the regeneration of ENase recognition sites is not required. Proc Natl Acad Sci U S A, 1991 May 15, 88(10), 4438 - 42 Isolation and expression of a cDNA encoding Renilla reniformis luciferase; Lorenz WW et al.; Renilla reniformis is an anthozoan coelenterate capable of exhibiting bioluminescence . Bioluminescence in Renilla results from the oxidation of coelenterate luciferin (coelenterazine) by luciferase {Renilla-luciferin:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.5} . In vivo, the excited state luciferin-luciferase complex undergoes the process of nonradiative energy transfer to an accessory protein, green fluorescent protein, which results in green bioluminescence . In vitro, Renilla luciferase emits blue light in the absence of any green fluorescent protein . A Renilla cDNA library has been constructed in lambda gt11 and screened by plaque hybridization with two oligonucleotide probes . We report here the isolation and characterization of a luciferase cDNA and its gene product . The recombinant luciferase expressed in Escherichia coli is identical to native luciferase as determined by SDS/PAGE, immunoblot analysis, and bioluminescence emission characteristics. Biochem Biophys Res Commun, 1991 May 15, 176(3), 958 - 65 Synthesis and biological properties of carba-analogs of heat-stable enterotoxin (ST) produced by enterotoxigenic Escherichia coli; Hidaka Y et al.; Analogs of a heat-stable enterotoxin (ST) that have a CH2-S linkage instead of an S-S linkage in the molecule were synthesized by conventional methods . The synthetic peptides showed toxicity, assayed as induction of fluid secretion in suckling mice, although their toxicities were hundredth that of native ST . This finding implies that ST is not recognized by its receptor protein through an exchange reaction between its disulfide linkages and thiol-groups of its receptor protein(s), but through hydrophobic or electrostatic interactions. Biochem Biophys Res Commun, 1991 May 15, 176(3), 1469 - 72 Recombinant cytochrome c peroxidase folds properly without conformational "annealing"; Ferrer JC et al.; Recombinant cytochrome c peroxidase isolated from Escherichia coli has recently been reported to exhibit an abnormal electronic absorption spectrum that is converted to the normal spectrum after conformational "annealing" of the recombinant enzyme by passage over a cytochrome c affinity column . The current report provides evidence that the abnormal spectrum observed in some preparations of recombinant cytochrome c peroxidase arises from the presence of contaminant, damaged forms cytochrome c peroxidase with altered spectra . Removal of these contaminant forms produces a major cytochrome c peroxidase fraction with a normal spectrum . We conclude that elution of recombinant cytochrome c peroxidase over a cytochrome c affinity column does not produce normal enzyme through conformational "annealing" but that it produces purified enzyme through removal of contaminants. Biochem Biophys Res Commun, 1991 May 15, 176(3), 1170 - 7 Human cDNA expressing a functional DNA glycosylase excising 3-methyladenine and 7-methylguanine; O'Connor TR et al.; A cDNA expression library from a human cell line was introduced into an E . coli strain deficient in the repair of 3-meAde bases in DNA . E . coli strains deficient in the repair of 3-meAde are unusually sensitive to DNA methylating agents . A plasmid pANPG10 (Alkyl-N-Purine-DNA Glycosylase) was rescued from the library based on its ability to reduce the sensitivity of the mutant strain to methylmethane sulfonate . Crude extracts of the E . coli mutant strain hosting the plasmid pANPG10 release both 3-meAde and 7-meGua from DNA . The longest open reading frame in the sequence codes for a polypeptide of 230 amino acids of molecular weight 25.5 kD, with a pI of 9.1 . The derived amino acid sequence of the human 3-meAde-DNA glycosylase has 85% sequence identity with the 3-meAde-DNA glycosylase from rat hepatoma cells. Biochemistry, 1991 May 14, 30(19), 4803 - 9 Structural characterization of functionally important regions of the Escherichia coli heat-stable enterotoxin STIb; Carpick BW et al.; The biological properties of the Escherichia coli enterotoxin STIb (STA-3, STh) reside in a 13 amino acid C-terminal domain, abbreviated STIb(6-18) . This tridecapeptide contains six cysteine residues involved in three intramolecular disulfide bridges . The solution structure of STIb(6-18) has been modeled as a series of three consecutive reverse turns {Gariepy et al . (1987) Proc . Natl . Acad . Sci . U.S.A . 83, 8907-8911} . Synthetic tridecapeptide analogues of STIb(6-18) with single amino acid substitutions at non-cysteine sites, as well as a truncated decapeptide lacking one of the three disulfide bridges, were prepared in order to examine the relationship between primary sequence and biological activity . The relative affinity of each analogue for intestinal cell receptors only partially correlates with their dose-dependent ability to cause diarrhea in suckling mice, suggesting that subsaturation doses of the enterotoxin with respect to receptor occupancy on intestinal cells may be sufficient to cause diarrhea . Two substitutions in the central-turn region of the molecule, namely, Asn12----Ala and Ala14----D-Ala, resulted in a large decrease or loss of receptor binding activity as compared to native STIb(6-18), pointing out the functional importance of this region . Analogues containing replacements at other sites showed moderate to slight reductions in biological activity . In particular, residues in the C-terminal region appear to be less important for activity, although their presence remains essential, since a truncated analogue missing the last three amino acids is inactive.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1991 May 14, 30(19), 4768 - 73 Sulfate transport in Neurospora crassa: regulation, turnover, and cellular localization of the CYS-14 protein; Jarai G et al.; Uptake of inorganic sulfate in Neurospora crassa is governed by the sulfur regulatory circuit and is under the control of positively and negatively acting regulatory genes . Two genetically and biochemically distinct systems are responsible for the uptake of sulfate from the environment . One of these, sulfate permease II, encoded by the cys-14 gene, functions primarily in mycelia . A defined region of the CYS-14 protein was highly expressed in Escherichia coli and purified . Anti-CYS-14 antibody was produced and used to detect the CYS-14 protein in N . crassa extracts . The CYS-14 protein has an approximate molecular weight of 95K, in agreement with its calculated size based on its predicted amino acid sequence . The steady-state level of the CYS-14 protein is highly regulated in wild-type mycelia and constitutive in an scon-1 mutant, whereas no CYS-14 protein could be detected in a cys-3 mutant . Following the accumulation of the cys-14 mRNA, that reaches its maximum in about 6 h, the CYS-14 protein accumulates to a maximum level in about 8 h after derepression . During conditions of sulfur repression, the CYS-14 protein turns over with a half-life of approximately 2 h . The CYS-14 protein appears to be localized in the plasma membrane, suggesting that it functions as a sulfate ion transporter. Biochemistry, 1991 May 14, 30(19), 4710 - 4 Assembly of the F0 proton channel of the Escherichia coli F1F0 ATPase: low proton conductance of reconstituted Fo sectors synthesized and assembled in the absence of F1; Pati S et al.; We have previously proposed that during assembly of the Escherichia coli F1F0 ATPase, the proton permeability of the Fo sector of the E . coli F1F0 ATPase is increased significantly by interactions with F1 subunits {Pati, S., & Brusilow, W.S.A . (1989) J . Biol . Chem 264, 2640-2644} . To test this model for Fo assembly, we purified F0 sectors synthesized in the presence and absence of F1 subunits and measured the abilities of these different preparations to bind purified F1 ATPase and to conduct protons when reconstituted into liposomes . The results of these studies demonstrated significant differences in proton-conducting abilities of the different Fo preparations . Fo sectors synthesized in the presence of F1 subunits were more permeable to protons than those synthesized in the absence of F1 subunits. Nucleic Acids Res, 1991 May 11, 19(9), 2485 - 8 Site-specific mutagenesis in cells with normal DNA repair systems: transitions produced from DNA carrying a single O6-alkylguanine; Chambers RW; This paper describes a systematic study of transition frequencies produced in vivo when a homologous series of O6-alkylguanine residues located at a preselected position in gene G of phi X174 form I' DNA (double-stranded, circular, covalently-closed, relaxed) is transfected into spheroplasts from two strains of Escherichia coli having normal DNA repair systems . Mutant frequencies were measured as percent of total phage produced by single bursts . The results are: (A) Synthetic DNA without any alkyl group gave a transition frequency of 0.02% . (B) In E . coli AB1157, the frequencies fall into two groups depending on the alkyl group: methyl and ethyl, 8-11%; n-propyl and n-butyl approximately 0.9% . (C) The average transition frequencies were higher in AB1157 than in C600 . These data demonstrate that a single O6-alkylguanine residue can produce a specific transition at significant frequencies in cells with normal repair systems and that the mutant frequency depends upon the nature of the alkyl group and the cell type. Nucleic Acids Res, 1991 May 11, 19(9), 2489 - 94 The computer simulation of RNA folding involving pseudoknot formation; Gultyaev AP; The algorithm and the program for the prediction of RNA secondary structure with pseudoknot formation have been proposed . The algorithm simulates stepwise folding by generating random structures using Monte Carlo method, followed by the selection of helices to final structure on the basis of both their probabilities of occurrence in a random structure and free energy parameters . The program versions have been tested on ribosomal RNA structures and on RNAs with pseudoknots evidenced by experimental data . It is shown that the simulation of folding during RNA synthesis improves the results . The introduction of pseudoknot formation permits to predict the pseudoknotted structures and to improve the prediction of long-range interactions . The computer program is rather fast and allows to predict the structures for long RNAs without using large memory volumes in usual personal computer. Nucleic Acids Res, 1991 May 11, 19(9), 2457 - 62 Translational frameshifting in the Escherichia coli dnaX gene in vitro; Tsuchihashi Z; Production of the gamma subunit of Escherichia coli DNA polymerase III holoenzyme is dependent on a very efficient translational frameshif in the dnaX gene . I used an E . coli in vitro translation system to analyze the mechanism of this frameshifting event . In this system, gamma was produced almost to the same extent as the inframe translation product, tau, suggesting that efficient frameshifting was reproduced in vitro . Coupling with transcription was not necessary for frameshifting . Addition of purified tau or gamma had no effect on the frameshifting process suggesting the absence of direct feedback regulation . By use of mutant genes, a strong pausing site was identified at or very close to the frameshift site . This pausing was apparently caused by a potential stem-loop structure which was previously shown to enhance frameshifting . Thus, enhancement of frameshifting by this putative stem-loop seems to be mediated by the translation pausing at the frameshift site . Despite the apparent structural similarity of the dnaX frameshift site to that of the eukaryotic retroviral genes, dnaX mRNA synthesized in vitro failed to direct the production of gamma in eukaryotic translation systems . This suggests that frameshifting in the dnaX gene depends on components specific to the E . coli translation system. Biochem Biophys Res Commun, 1991 Apr 30, 176(2), 705 - 10 A mixed valence form of the iron cluster in the B2 protein of ribonucleotide reductase from Escherichia coli; Hendrich MP et al.; A mixed valent form of the iron cluster (Fe(II)Fe(III) in the B2 protein of ribonucleotide reductase has been isolated and characterized . The irons in this state of the protein are ferromagnetically coupled as indicated by the observation of a novel S = 9/2 EPR spectrum . This is the first ferromagnetically coupled Fe(II)Fe(III) cluster reported for a protein and the first observation of the mixed valence form of ribonucleotide reductase. Eur J Biochem, 1991 May 8, 197(3), 643 - 53 Two-dimensional 1H, 15N-NMR investigation of uniformly 15N-labeled ribonuclease T1 . Complete assignment of 15N resonances; Schmidt JM et al.; Uniformly 15N-enriched ribonuclease T1 (RNase T1) was obtained from Escherichia coli by recombinant techniques . Heteronuclear 1H, 15N-shift correlation spectra were recorded utilizing proton detection . Direct 1H, 15N connectivities were established applying the heteronuclear multiple-quantum coherence technique . Additional 1H, 1H-TOCSY or 1H, 1H-NOESY transfer steps allowed for sequential assignments . Nitrogen atoms without directly bonded protons were detected by means of the heteronuclear multiple-bond correlation experiment . Signals emerging from 15NH and 15NH2 groups were distinguished by heteronuclear triple-quantum filtering methods . 119 nitrogen resonances out of the expected 127 were assigned unambiguously; in addition, previously obtained proton assignments were extended . Preliminary 1H, 15N NMR investigation were performed on the RNase-T1-3'GMP inhibitor complex . Results were interpreted with respect to nucleotide binding. Eur J Biochem, 1991 May 8, 197(3), 605 - 14 Structural and biological properties of human recombinant myeloperoxidase produced by Chinese hamster ovary cell lines; Moguilevsky N et al.; The cDNA encoding human myeloperoxidase carries three ATG codons in frame; 144, 111 and 66 bp upstream from the proprotein DNA sequence . In order to determine the most efficient signal sequence, three cDNA modules starting at each of the ATG were cloned into an eucaryotic expression vector and stably expressed in Chinese hamster ovary cell lines . In all three cases, recombinant human myeloperoxidase (recMPO) was secreted into the culture medium of transfected cells, indicating that each of the signal peptides functions efficiently . One of the recombinant cell lines, which was amplified using methotrexate, overexpresses enzymatically active recMPO up to 6 micrograms.ml-1.day-1 . The recombinant product was purified by a combination of ion-exchange and metal-chelate chromatography, and characterized in terms of molecular mass, amino-terminal amino acid analysis, glycosylation, physicochemical properties and biological activity . The data show that recMPO is secreted essentially as a monomeric, heme-containing, single-chain precursor of 84 kDa which exhibits peroxidase activity . Amino-terminal analysis indicated that cleavage of the signal peptide occurs between amino acids 48 and 49 . In addition, recMPO appeared to be glycosylated up to the last stage of sialylation, to an extent similar to that of the natural enzyme . Specific activity measurements as well as stability data, in various pH, temperature, ionic strength and reducing conditions, indicated that the recombinant single-chain enzyme behaves essentially in the same way as the natural two-chain molecule . Finally, recMPO was shown to exert potent cytotoxicity towards Escherichia coli when provided with its physiological substrates, i.e . hydrogen peroxide and chloride ions. Biochemistry, 1991 May 7, 30(18), 4565 - 72 Optically detected magnetic resonance study of the interaction of an arsenic(III) derivative of cacodylic acid with EcoRI methyl transferase; Tsao DH et al.; The interaction of the enzyme Escherichia coli RI methyl transferase (methylase) with an arsenic(III) derivative of cacodylic acid has been investigated by optical detection of triplet-state magnetic resonance (ODMR) spectroscopy in zero applied magnetic field . The reactive derivative (CH3)2AsSR is formed by the reduction of cacodylate by a thiol . The As(III) derivative binds to the enzyme by mercaptide exchange with a cysteine (Cys) residue located close to a tryptophan (Trp) site . The arsenical binding selectively induces an external heavy-atom effect, perturbing the nearby Trp residue in the enzyme . Zero-field splittings (ZFS) and total decay rate constants of the individual triplet-state sublevels of the Trp residue in the presence and absence of perturbation by As(III) have been determined . The perturbed Trp shows a large reduction in the overall decay lifetime compared with unperturbed Trp residue, exhibiting a high selectively for the Tx sublevel . This selectivity suggests that the As atom lies in the xz plane of the principal magnetic axis system of Trp, but not directly along the z (out-of-plane) axis . The accessibility of this enzyme binding site to the arsenical is decreased upon forming a ternary complex of methylase with sinefungin and a DNA oligomer, d{GCGAA(BrU)(BrU)CGC}, containing two 5-bromouracil (BrU) bases in place of thymine within the hexadeoxynucleotide recognition sequence . This result indicates that the arsenical binding site in methylase which produces the Trp heavy-atom effect is protected from this ligand by ternary complex formation or the enzyme undergoes a conformation change, removing the Cys from the Trp site . This protection is also observed in fluorescence quenching experiments.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1991 May 7, 30(18), 4432 - 43 Dimerization of Escherichia coli UvrA and its binding to undamaged and ultraviolet light damaged DNA; Mazur SJ et al.; The initial stages in the repair of damaged DNA by the Escherichia coli uvr system involve the recognition of damage by UvrA . We have examined in detail the binding of UvrA to DNA randomly damaged by ultraviolet light, undamaged DNA, and single-stranded DNA using nitrocellulose filter binding and gel mobility shift assays to arrive at the following model: UvrA dimers bind specifically to damaged DNA both in the presence and in the absence of ATP . The dimerization of UvrA is promoted by UvrA concentrations greater than 1 nM, the presence of ATP, or physiological temperatures, and the dimerization step dominates the temperature dependence of UvrA binding to DNA damaged by ultraviolet light . The apparent association constant for specific binding is dependent on the concentration of UvrA due to coupled dimerization, aggregation, and nonspecific binding reactions . At 1 nM UvrA, either with or without ATP, Kuv approximately 10(9) M-1 . The binding of UvrA to undamaged DNA is 10(3)-10(4)-fold weaker than the damage-specific binding . Both the strength of damage-specific binding and the discrimination between damaged and undamaged sites are affected by the salt concentration . The kinetics of association and dissociation reactions indicate that the primary effects of ATP are on the extent of UvrA dimerization rather than on the properties of the UvrA-uvDNA complex . The complexity of the interaction of UvrA, ATP, and DNA is indicated by the opposing effects of ATP binding and hydrolysis on UvrA dimerization. FEBS Lett, 1991 May 6, 282(2), 405 - 8 Recombinant aequorin as a probe for cytosolic free Ca2+ in Escherichia coli; Knight MR et al.; We describe a novel and simple method for the measurement of bacterial cytosolic free calcium ({Ca2+}i) using recombinant aequorin reconstituted within live bacterial cells . Using this method we have measured the effects of external calcium, complement, phagocytosis and antibiotics on the {Ca2+}i of Escherichia coli . In principle this method should be applicable to any genetically transformable organism and should suffer fewer problems than fluorescent dyes for subcellular calcium measurement. Thromb Haemost, 1991 May 6, 65(5), 560 - 4 Thrombolysis with an Escherichia coli-produced recombinant plasminogen activator (BM 06.022) in the rabbit model of jugular vein thrombosis; Martin U et al.; The recombinant plasminogen activator BM 06.022 consists of the kringle 2 and the protease domains of human t-PA and is unglycosylated because of the expression in Escherichia coli . The thrombolytic and pharmacokinetic properties as well as the hemostasis effects of BM 06.022 were investigated in the rabbit model of jugular vein thrombosis . The thrombi were 125I-fibrin labeled . Intravenous bolus injection of 50, 100, 200, and 400 kU/kg BM 06.022 or 400, 800, and 1600 kU/kg alteplase over 15 s to six rabbits/dose produced a dose-dependent increase of thrombolysis determined 2 h post injection . The dose-response curve of BM 06.022 was located left compared with that of alteplase . The effective dose of 50% thrombolysis (ED50) obtained by half-logarithmic regression analysis was 163 kU/kg (= 0.28 mg/kg) for BM 06.022 and 871 kU/kg (= 1.09 mg/kg) for alteplase . At equipotent doses (50% thrombolysis), the residual concentration of fibrinogen was 74.2% and 76.5%, that of plasminogen 66.7% and 69.4%, and that of alpha 2-antiplasmin 47.3% and 46% for BM 06.022 and alteplase, respectively . Pharmacokinetic analysis for plasma activity at a dose of 400 kU/kg revealed a half-life of 18.9 +/- 1.5 min for BM 06.022, whereas alteplase was distributed with a half-life of 2.1 +/- 0.1 min, accounting for 86.7 +/- 1.9% of the total AUC, followed by a beta-phase with a half-life of 13.8 +/- 0.9 min . Plasma clearance of BM 06.022 was 4.7 +/- 0.7 ml min-1 kg-1 compared with 20 +/- 1.2 ml min-1 kg-1 for alteplase.(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1991 May 6, 282(2), 415 - 8 Stoichiometry of metal-tetracycline/H+ antiport mediated by transposon Tn10-encoded tetracycline resistance protein in Escherichia coli; Yamaguchi A et al.; The tetracycline resistance protein (TetA) endoded by transposon Tn10 mediates the efflux of divalent cation-tetracycline chelating complexes {Yamaguchi, A., Udagawa, T . and Sawai, T . (1990) J . Biol . Chem . 265, 4809-4813} . It was confirmed that protons were antiported with the complexes through an electrically-neutral process because the antiport consumed delta pH but not delta psi . The quantitative relationship between delta pH and delta pTC determined by a flow-dialysis method clearly indicated a 1:1 stoichiometry of the monocationic metal-tetracycline/H+ exchange. J Mol Biol, 1991 May 5, 219(1), 27 - 36 Identification of the different intermediates in the interaction of (A)BC excinuclease with its substrates by DNase I footprinting on two uniquely modified oligonucleotides; Bertrand-Burggraf E et al.; (A)BC excinuclease is the enzymatic activity resulting from the joint actions of UvrA, UvrB and UvrC proteins of Escherichia coli . The enzyme removes from DNA many types of adducts of dissimilar structures with different efficiencies . To understand the mechanism of substrate recognition and the basis of enzyme specificity, we investigated the interactions of the three subunits with two synthetic substrates, one containing a psoralen-thymine monoadduct and the other a thymine dimer . Using DNase I as a probe, we found that UvrA makes a 33 base-pair footprint around the psoralen-thymine adduct and that UvrA-UvrB make a 45 base-pair asymmetric footprint characterized by a hypersensitive site 11 nucleotides 5' to the adduct and protection mostly on the 3' side of the damage . Conditions that favor dissociation of UvrA from the UvrA-UvrB-DNA complex, such as addition of excess undamaged DNA to the reaction mixture, resulted in the formation of a 19 base-pair UvrB footprint . In contrast, a thymine dimer in a similar sequence context failed to elicit a UvrA, a UvrA-UvrB or UvrB footprint and gave rise to a relatively weak DNase I hypersensitive site typical of a UvrA-UvrB complex . Dissociation of UvrA from the UvrA-UvrB-DNA complex stimulated the rate of incision of both substrates upon addition of UvrC, leading us to conclude that UvrA is not a part of the incision complex and that it actually interferes with incision . The extent of incision of the two substrates upon addition of UvrC (70% for the psoralen adduct and 20% for the thymine dimer) was proportional to the extent of formation of the UvrA-UvrB-DNA (i.e . UvrB-DNA) complex, indicating that substrate discrimination occurs at the preincision step. J Biol Chem, 1991 May 5, 266(13), 8545 - 50 Structure-function studies of bacteriorhodopsin XV . Effects of deletions in loops B-C and E-F on bacteriorhodopsin chromophore and structure; Gilles-Gonzalez MA et al.; Bacteriorhodopsin mutants containing deletions in loop B-C, delta Thr67-Glu74 or delta Gly65-Gln75 or a deletion in the loop E-F, delta Glu161-Ala168, were prepared . Following their expression in Escherichia coli, the mutant proteins were purified to homogeneity and refolded with retinal in detergent-phospholipid mixtures . The mutants containing deletions in the loop B-C were normal at 4 degrees C but showed the following changes at 20 degrees C . 1) The lambda max shifted from 540 to below 510 nm; 2) the rates of bleaching by hydroxylamine in the dark increased; and 3) the rate and steady state of proton pumping decreased . Deletion of the eight amino acids in loop E-F did not affect wild-type behavior . However, all the mutant proteins were more prone to thermal and sodium dodecyl sulfate denaturation than the wild-type bacteriorhodopsin . These observations show that the structures of the B-C and E-F loops are not essential for correct folding of bacteriorhodopsin, but they contribute to the stability of the folded protein. J Biol Chem, 1991 May 5, 266(13), 8455 - 63 Identification of two neutrophil chemotactic peptides produced by porcine alveolar macrophages; Goodman RB et al.; We have purified to homogeneity two distinct 10-kDa proteins with potent chemotactic activity for neutrophils from porcine alveolar macrophages incubated for 24 h with Escherichia coli endotoxin (lipopolysaccharide (LPS), 10 micrograms/ml) . Neutrophil chemotactic activity in alveolar macrophage supernatants was concentrated by adsorption to SP-Sephadex, and purified by cation exchange and reversed phase high performance liquid chromatography . The first peptide, alveolar macrophage chemotactic factor (AMCF)-I, had chemotactic activity for both porcine and human neutrophils . The chemotactic activity for porcine neutrophils was detectable at 3 x 10(-10) M, peaked at 3 x 10(-8) M, and was comparable to that of zymosan-activated porcine serum . Segmental instillation of AMCF-I into porcine lungs caused marked neutrophil accumulation at 4 h in both bronchoalveolar lavage fluid and in lung tissue . The second peptide, AMCF-II, was active at 1.4 x 10(-9) M for porcine neutrophils, but it was less active for human polymorphonuclear neutrophils than was AMCF-I . Oligonucleotide probes to regions of the N-terminal sequences of AMCF-I and AMCF-II hybridized to mRNA recovered from LPS-stimulated alveolar macrophages . The N-terminal sequences and amino acid compositions indicate that AMCF-I and AMCF-II are distinct proteins, but that both have homologies with a family of peptide chemoattractants produced by human blood monocytes and platelets . Thus, alveolar macrophages stimulated with LPS produce two distinct 10-kDa cytokines with potent chemotactic activity for neutrophils . This indicates that there are two different peptide pathways by which alveolar macrophages can recruit neutrophils into the lung. J Biol Chem, 1991 May 5, 266(13), 8176 - 83 Cytochalasin B as a probe of protein structure and substrate recognition by the galactose/H+ transporter of Escherichia coli; Cairns MT et al.; Cytochalasin B is a potent inhibitor of mammalian passive glucose transporters . The recent demonstration of sequence similarities between these proteins and several bacterial proton-linked sugar transporters suggested that cytochalasin B might be a useful tool for investigation of the galactose/H+ symport protein (GalP) of Escherichia coli . Equilibrium binding studies using membranes from a GalP-constitutive (GalPc) strain of E . coli revealed a single set of high affinity binding sites for cytochalasin B with a Kd of 0.8-2.2 microM . Binding was inhibited by D-glucose, but not by L-glucose . UV irradiation of the membranes in the presence of {4-3H}cytochalasin B photolabeled principally a protein of apparent Mr 38,000, corresponding to the GalP protein . Labeling was inhibited by greater than 80% in the presence of 500 mM D-glucose or D-galactose, the major substrates of the GalP system . The extent of inhibition of photolabeling by different sugars and sugar analogues showed that the substrate specificity of GalP closely resembles that of the mammalian passive glucose transporters . Structural similarity to the latter was revealed by tryptic digestion of {4-3H}cytochalasin B-photolabeled GalP, which yielded a radiolabeled fragment of apparent Mr 17,000-19,000, similar to that previously reported for the human erythrocyte glucose transporter. J Biol Chem, 1991 May 5, 266(13), 7988 - 94 Comparison of the activities of variant forms of eIF-4D . The requirement for hypusine or deoxyhypusine; Park MH et al.; Eukaryotic protein synthesis initiation factor 4D (eIF-4D) (current nomenclature, eIF-5A) contains the unique amino acid hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine) . The first step in hypusine biosynthesis, i.e . the formation of the intermediate, deoxyhypusine (N epsilon-(4-aminobutyl)lysine), was carried out in vitro using spermidine, deoxyhypusine synthase, and ec-eIF-4D(Lys), an eIF-4D precursor prepared by over-expression of human eIF-4D cDNA in Escherichia coli . In a parallel reaction, using N-(3-aminopropyl)cadaverine in place of spermidine, a variant form of eIF-4D containing homodeoxyhypusine (N epsilon-(5-aminopentyl)lysine) was prepared . Evidence that N-(3-aminopropyl)cadaverine can also act as the amine substrate for deoxyhypusine synthase in intact cells was obtained by incubating putrescine- and spermidine-depleted Chinese hamster ovary cells with {3H}cadaverine . In these cells, in which {3H}cadaverine is readily converted to N-(3-aminopropyl) {3H}cadaverine, small amounts of {3H}homodeoxyhypusine and another 3H-labeled compound, presumed to be N epsilon-(5-amino-2-hydroxy{3H}pentyl)lysine, were found . eIF-4D stimulates methionyl-puromycin synthesis, an in vitro model assay for translation initiation . Whereas the unmodified precursor ec-eIF-4D(Lys) appeared inactive, the deoxyhypusine-containing form provided a significant degree of stimulation . The variant form containing homodeoxyhypusine, on the other hand, showed little or no activity . These findings emphasize the importance of hypusine or deoxyhypusine for the biological activity of eIF-4D and demonstrate the influence of both the length and chemical nature of its amino alkyl side chain. J Biol Chem, 1991 May 5, 266(13), 8236 - 40 Alterations in the energetics of the carbamoyl phosphate synthetase reaction by site-directed modification of the essential sulfhydryl group; Mullins LS et al.; The change in reaction energetics of the bicarbonate-dependent ATPase reaction of Escherichia coli carbamoyl phosphate synthetase has been investigated for two site-directed mutations of the essential cysteine in the small subunit . Cysteine 269 has been proposed to facilitate the hydrolysis of glutamine by the formation of a glutamyl-thioester intermediate . The two mutant enzymes, C269S and C269G, along with the isolated large subunit, exhibit a 2-2.6-fold increase in the bicarbonate-dependent ATPase reaction relative to that observed for the wild type enzyme . In the presence of glutamine the overall enhancement is 3.7 and 9.0 for the C269G and C269S mutant enzymes, respectively . Carboxyphosphate is an intermediate in the bicarbonate-dependent ATPase reaction . The cause of the rate enhancements was investigated by measuring the positional isotope exchange rate in {gamma-18O4} ATP relative to the net rate of ATP hydrolysis . This ratio (Vex/Vchem) is a measure of the partitioning of the enzyme-carboxyphosphate-ADP complex . The partitioning ratio for the mutants is identical within experimental error to that observed for the wild type enzyme . This observation is consistent with the conclusion that the ground state for the enzyme-carboxyphosphate-ADP complex in the mutants is destabilized relative to the same complex in the wild type enzyme . If the increase in the absolute rate of ATP hydrolysis was due to a stabilization of the transition state for carboxyphosphate hydrolysis then the positional isotope exchange rate relative to the chemical hydrolysis rate would have been expected to decrease in the mutants. J Mol Biol, 1991 May 5, 219(1), 11 - 25 RNA-protein interactions in a Nus A-containing Escherichia coli transcription complex paused at an RNA hairpin; Dissinger S et al.; We have isolated Escherichia coli transcription complexes, paused in the presence and absence of Nus A, which contain RNA substituted at every UMP residue with a photocrosslinking nucleotide analog . The pause site is immediately downstream from an RNA stem-loop structure, and although pausing occurs in the absence of Nus A, it is substantially enhanced in the presence of Nus A . We have analyzed the secondary structure of this RNA and show that the analog does not interfere with the formation of the normal stem-loop structures . Additionally, the analog substrate does not alter transcriptional pausing, in the presence or absence of Nus A, indicating that Nus A recognition of the transcription complex is not affected by the presence of the crosslinking groups in the RNA . Ribonuclease digestion of the RNA in paused complexes identifies two accessible regions, two nucleotides in the loop and one near the base of the upstream side of the stem-loop . Cleavage at one loop nucleotide is enhanced by Nus A, while the nucleotide near the base of the stem-loop is partially protected . Upon irradiation of the transcription complex, Nus A is not photoaffinity labeled by the RNA, even at a high molar ration to RNA polymerase (250:1) . Both the beta and beta' subunits are labeled, however, indicating that the putative stem-loop binding domain on the core polymerase involves both subunits . Because the nucleotide protected from ribonuclease by Nus A is very near two analogs, yet Nus A is not crosslinked to the RNA, it is unlikely that Nus A could be protecting this position through direct contact . Furthermore, analog is substituted at positions in both the loop and at several positions in the stem, and again, no crosslinking to Nus A is observed . We conclude that enhancement of pausing by Nus A probably does not require direct interaction with the bases in the RNA stem-loop. J Biol Chem, 1991 May 5, 266(13), 8619 - 25 The combination of DNA methylation and H1 histone binding inhibits the action of a restriction nuclease on plasmid DNA; Higurashi M et al.; To investigate the potentials of DNA methylation and H1 histone in regulating the action of DNA binding proteins, well ordered complexes were formed by slow salt gradient dialysis of mixtures of H1 histone with either methylated or nonmethylated DNA . The sites methylated in the plasmids were CCGG . Methylation of cytosine in this site protects the DNA against HpaII endonuclease but not against MspI . However, when the methylated DNA was complexed to H1, it was protected against MspI . The protection was only effective for a subset of the MspI restriction sites . The protection of DNA afforded by the combination of H1 binding and DNA methylation did not apply to EcoRI, PstI, or BamHI sites and so did not seem to be due to aggregation of the DNA by H1 histone . Gel retardation assays indicated that the affinity of H1 for methylated DNA was not detectably different from its affinity for nonmethylated DNA . Probably methylated DNA when bound to H1 is in a conformation that is resistant to MspI endonuclease . Such conformational changes induced by DNA methylation and H1 binding might affect the action of other DNA binding proteins, perhaps in chromatin as well as in H1.DNA complexes. Pol Tyg Lek, 1991 May 27-Jun 3, 46(22-23), 430 - 2 {Effect of hydrocortisone and dopamine on activity of beta-glucuronidase in lung parenchyma during experimental septic shock}; Ladny JR et al.; An effect of hydrocortisone and dopamine on beta-glucuronidase activity in the lung tissue in experimental septic shock was analysed . The study involved 30 mongrel dogs . Septic shock was induced with i.v . E . coli endotoxin . Total and free activity of beta-glucuronidase was determined with Talalay et al . technique in pulmonary parenchyma homogenate, mitochondrial-lysosomal fraction and supernatant . It was found, that hydrocortisone and dopamine given together increase the stability of the pulmonary lysosomes indicating a value of such management of the septic shock in man. Eur J Pharmacol, 1991 May 2, 197(1), 17 - 25 Influence of SK&F 96148 on thromboxane-mediated responses in the airways of the cat; Dyson MC et al.; The effects of SK&F 96148, a thromboxane receptor blocking agent, on bronchoconstrictor responses were investigated in paralyzed, anesthetized, mechanically ventilated cats . I.v . injections of the thromboxane A2 (TXA2) mimics, U-46619 and U-44069, produced dose-related increases in transpulmonary pressure and lung resistance (RL) and decreases in dynamic compliance (Cdyn) . After administration of SK&F 96148, 5 mg/kg i.v., bronchoconstrictor responses to U-46619 and U-44069 were reduced markedly, whereas airway responses to prostaglandin (PG) F2 alpha, serotonin, PGD2, or the PGD2 metabolite, 9 alpha, 11 beta-PGF2, were not altered . The duration of action of SK&F 96148 was greater than 2 h, and the TXA2 receptor blockade was overcome when 10-fold larger doses of the TXA2 mimics were administered . Bronchoconstrictor responses to arachidonic acid, platelet-activating factor (PAF), endothelin-1, and E . coli endotoxin were blocked by SK&F 96148 . The present data suggest that SK&F 96148 has selective thromboxane receptor blocking activity in the airways of the cat, and that bronchoconstrictor responses to endothelin-1, arachidonic acid, PAF, and E . coli endotoxin are mediated in part by the formation of TXA2. Biochim Biophys Acta, 1991 May 2, 1089(1), 8 - 12 In vivo assembly of the cytochrome d terminal oxidase complex of Escherichia coli from genes encoding the two subunits expressed on separate plasmids; Newton G et al.; The cytochrome d terminal oxidase complex is a heterodimer located in the cytoplasmic membrane of Escherichia coli . Subunit II of the cytochrome d terminal oxidase complex was expressed independently of subunit I of the complex . It was found that the polypeptide is produced and is associated with the cytoplasmic membrane in the absence of subunit I, and is not associated with any of the three cytochrome components of the complex . Oxidase activity and heme binding are restored when the subunit I is expressed in the same cells using a second compatible plasmid . It has been previously demonstrated that subunit I, expressed in the absence of subunit II, contains cytochrome b-558, one of the three heme prosthetic groups found in the oxidase . Association of the two other heme moieties, cytochromes b-595 and d, apparently requires the association of the two subunits, and must be a late step in the assembly of the membrane-bound protein . It was also shown that under heme-deficient conditions, the two polypeptide subunits are expressed and are associated with the cytoplasmic membrane. Mol Biol Evol, 1991 May, 8(3), 261 - 81 Identification of adaptive changes in an evolving population of Escherichia coli: the role of changes with regulatory and highly pleiotropic effects; Kurlandzka A et al.; A population of Escherichia coli initiated with a single clone developed extensive morphological and physiological polymorphism after being maintained for 773 generations in glucose-limited continuous culture . To understand the mechanisms of adaptation to this environment, total protein patterns of four adaptive clones and of the parent strains were examined by two-dimensional gel electrophoresis . Approximately 20% of the proteins (approximately 160 in absolute numbers) showed significantly different levels of expression in pairwise comparisons of parent and adapted clones . The extent of these changes points to the importance of mutations with regulatory and/or highly pleiotropic effects in the adaptive process . The four evolved clones all expressed fewer proteins than did the parent strain, supporting the hypothesis of energy conservation during evolutionary change . Forty-two proteins that could be assigned to known cellular functions were identified . The changes in some of them indicated that the evolved clones developed different adaptive mechanisms to glucose-limited environment . Changes were observed in the expression levels of proteins associated with translation, membrane composition, shock response, and active transport . A fraction of the changes could not be either explained or predicted from a consideration of the nature of the environment in which the clones evolved. J Pediatr Surg, 1991 May, 26(5), 572 - 4 The role of pentoxifylline in endotoxin-induced alterations of red cell deformability and whole blood viscosity in the neonate; Mollitt DL et al.; Endotoxin induces alterations in the neonatal red cell membrane that result in decreased deformability and an increase in whole blood viscosity . These rheologic alterations are detrimental to flow in the microcirculation . Pentoxifylline (PTX), a methyl xanthine derivation, increases red cell deformability presumably through its effect on intracellular adenosine 5-triphosphate . The purpose of this study was to evaluate the effect of PTX on endotoxin-induced alterations in the neonatal red blood cell . Anticoagulated whole blood specimens obtained from the cord of 12 neonates at birth were used to study the effects of Escherichia coli endotoxin (LPS) with and without PTX (50 micrograms/mL) on red cell deformability and whole blood viscosity . LPS resulted in a significant (P less than .001) decrease in deformability compared with controls . PTX reversed these endotoxin-induced alterations (P less than .01), normalizing deformability to control values (P = NS) . LPS resulted in a significant increase (P less than .005) in blood viscosity that was reversed by PTX (P = NS) . Pentoxifylline reverses the detrimental rheologic effect of endotoxin in the neonate . This activity may be helpful in sustaining normal microcirculation in neonatal sepsis. Genetics, 1991 May, 128(1), 29 - 35 Mutant tryptophan aporepressors with altered specificities of corepressor recognition; Arvidson DN et al.; The Escherichia coli trpR gene encodes tryptophan aporepressor, which binds the corepressor ligand, L-tryptophan, to form an active repressor complex . The side chain of residue valine 58 of Trp aporepressor sits at the bottom of the corepressor (L-tryptophan) binding pocket . Mutant trpR genes encoding changes of Val58 to the other 19 naturally occurring amino acids were made . Each of the mutant proteins requires a higher intracellular concentration of tryptophan for activation of DNA binding than wild-type aporepressor . Whereas wild-type aporepressor is activated better by 5-methyltryptophan (5-MT) than by tryptophan, Ile58 and other mutant aporepressors prefer tryptophan to 5-MT as corepressor, and Ala58 and Gly58 prefer 5-MT much more strongly than wild-type aporepressor in vivo . These mutant aporepressors are the first examples of DNA-binding proteins with altered specificities of cofactor recognition. Proc Natl Acad Sci U S A, 1991 May 1, 88(9), 3802 - 6 Escherichia coli kgtP encodes an alpha-ketoglutarate transporter; Seol W et al.; The witA gene located between pss and rrnG on the Escherichia coli chromosome encodes a 432-amino acid protein . It is homologous to a human hepatoma glucose transporter and to E . coli membrane proteins that transport citrate (CitA), arabinose (AraE), and xylose (XylE), and, like these carrier proteins, WitA also contains 12 highly hydrophobic putative membrane-spanning regions . Gene disruption mutants constructed in two E . coli strains grew slowly or not at all, depending on genetic background, in M9 minimal medium containing alpha-ketoglutarate . Growth on alpha-ketoglutarate and uptake of alpha-{14C}ketoglutarate were restored by transformation with plasmids containing witA . These complementation studies indicate that WitA is an alpha-ketoglutarate transporter and should be renamed kgtP(alpha-ketoglutarate permease). Mutat Res, 1991 May, 254(3), 289 - 98 DNA damage by 8-methoxypsoralen plus near ultraviolet light (PUVA) and its repair in Escherichia coli: genetic analysis; Holland J et al.; Mutants of Escherichia coli, hyper-resistant and sensitive to 8-methoxypsoralen plus near ultraviolet light (PUVA) have been isolated and studied . Results show that a mutation, located at 57.2 min on the linkage map of E . coli, is responsible for the hyper-resistant phenotype . It is also responsible for the synthesis of a 55-kdal protein in high concentrations . In a wild-type cell the synthesis of this enzyme is inducible by mitomycin C . There are indications that the mutation may have occurred in a regulatory gene, puvR, and as a result the operon, including a putative puvA gene (the structural gene for the synthesis of the 55-kdal protein), is expressed constitutively . A model for the control of the PUV operon is proposed. Mutat Res, 1991 May, 254(3), 217 - 24 Effect of exogenous DNA fragments on human cell extract-mediated DNA repair synthesis; Biggerstaff M et al.; Extracts from HeLa cells were used to study the susceptibility of repair synthesis in UV-irradiated plasmid DNA to inhibition by exogenously added nucleic acid . Purified DNA restriction fragments have little inhibitory effect on repair synthesis . However, activated calf thymus DNA fragments, genomic DNA fragments in cell extracts, and sonicated plasmid DNA all inhibited repair synthesis . Degraded DNA fragments arising from E . coli during bacterial plasmid purification were found to be particularly inhibitory . tRNA is not a potent inhibitor of in vitro repair synthesis . In order to observe efficient DNA repair synthesis mediated by human cell extracts, it is essential to prepare highly purified closed circular plasmid DNA, and we describe a reliable method for doing so. Mol Gen Genet, 1991 May, 227(1), 160 - 4 Spontaneous and 9-aminoacridine-induced frameshift mutagenesis: second-site frameshift mutation within the N-terminal region of the lacI gene of Escherichia coli; Gordon AJ et al.; A novel forward mutational system, based on the acquisition of an Iq-d dominant phenotype from an initial Iq- recessive state, was used to identify second-site frameshift mutation {+/- 1(+/- 3n) events} within the N-terminal region of the lacI gene of Escherichia coli . The DNA sequences are described of forty-six spontaneous and twenty 9-aminoacridine(9-AA)-induced second site mutations . Although -1 frameshift events dominate both spectra, the nature and site specificity of these events clearly distinguish two mutational distributions . The spontaneous distribution contains two -(A:T) frameshift hotspots; one within a monotonic A5 run (9 occurrences), the other at a 5'-CACAACAAC-3' sequence (12 occurrences) . In contrast 17 of the 20 mutations recovered after 9-AA treatment involve the loss of a G:C pair, 14 of which occur at a single site (5'-CGGGC-3') . The striking specificity of the observed mutational hotspots is of interest since this open genetic target contains similar sequences which were infrequently recovered. Mol Gen Genet, 1991 May, 227(1), 155 - 9 Site-specific recombination in Escherichia coli between the att sites of plasmid pSE211 from Saccharopolyspora erythraea; Katz L et al.; pSE211 from Saccharopolyspora erythraea integrates site-specifically into the chromosome through conservative recombination between attP and attB, the plasmid and chromosomal attachment sites . Integration depends on the presence of int, an open reading frame (ORF) that lies adjacent to attP and encodes the putative integrase . Immediately upstream of int lies xis (formerly called orf2) which encodes a basic protein that is thought to exhibit DNA binding . xis and int were cloned in various combinations in pUC18 and expressed constitutively in Escherichia coli from the lac promoter . attP and attB were cloned in Streptomyces or E . coli plasmids containing kanamycin resistance (KmR) or chloramphenicol resistance (CmR) markers . Stable KmR CmR cointegrates formed by attP x attB or attP x attP recombination (integration) were obtained in E . coli hosts that expressed int . Co-integrates were not found in hosts expressing int + xis . Excision (intraplasmid att site recombination) was examined by constructing plasmids carrying attL and attR or two attP sites separating CmR from KmR and by following segregation of the markers in various hosts . Both attL x attR and attP x attP excision depended on both xis and int in E . coli . pSE211 att site integration and excision were not affected by a deletion in himA, the gene encoding a subunit of integration host factor. Biochem J, 1991 May 1, 275 ( Pt 3), 601 - 8 Chemical cleavage of plasmid DNA by glutathione in the presence of Cu(II) ions . The Cu(II)-thiol system for DNA strand scission; Reed CJ et al.; In the presence of Cu(II) ions, supercoiled DNA is cleaved in neutral solution by low concentrations of thiols . Supercoiled plasmid DNA is cleaved first to open circular DNA, which in turn produces linear DNA and eventually fragments . Cleavage is strongly temperature-dependent and is maximal at 0.10-0.25 M-NaCl concentration . In the presence of excess of either component of the Cu(II)-thiol pair, the extent of cleavage depended on the concentration of the limiting partner, and was easily detectable down to micromolar concentrations of limiting GSH . Scavengers of oxygen-derived species (such as hydrogen peroxide, superoxide radical ion and hydroxyl radical) indicated that the hydroxyl radical may be involved in the cleavage mechanism . DNA cleavage leads to some production of 2-thiobarbituric acid-reactive species and some of the cleavage sites, at least, had 5'-hydroxy and/or 3'-hydroxy groups . There was extensive base damage before cleavage . Studies with S1 nuclease indicated no gross sequence preference for Cu(II)-GSH cleavage of pSP64 plasmid DNA . The Cu(II)-thiol system did not appear to target special structural features in the DNA such as Z-DNA inserts, cruciform structures or left-handed (but non-Z) DNA . Cleavage might arise from a reagent generated either by the Cu(II)-thiol combination in free solution or by attack involving Cu(II) ions pre-bound to DNA . The attack of GSH plus Cu(II) ions on DNA may be a potential toxic lesion under physiological conditions unless special protective measures operate efficiently in the cell. Mol Gen Genet, 1991 May, 226(3), 473 - 83 Structural and functional analysis of the origin of conjugal transfer of the broad-host-range IncW plasmid R388 and comparison with the related IncN plasmid R46; Llosa M et al.; We cloned and sequenced a 402 bp DNA segment containing the origin of conjugal transfer (oriT) of the IncW plasmid R388 . Progressive deletions from each end of the sequence were assayed for oriT activity . Stepwise reductions in mobilization frequencies, representing the loss of functional elements, correlated with deletion of structural motifs in the sequence . A sequence of 330 bp of oriT was sufficient for efficient mobilization . The first 86 bp of the sequence contains five tandemly repeated DNA sequences of 11 bp, followed by a 10 bp perfect inverted repeat . Deletion of the first 95 bp reduced the frequency of transfer by a hundred-fold . The sequence between bp 183 and 218 was necessary and sufficient for low frequency mobilization and, thus, it was assumed to contain the nick site . This basis core was cloned as a 60 bp segment (from bp 176-236) that could be mobilized at low frequency . It includes two inverted repeats and a perfect integration host factor (IHF) consensus binding site . A third functionally important segment in oriT was located between bp 260 and 330 . The DNA sequence of the oriT of R388 could be aligned with that of the broad-host-range IncN plasmid R46 . Moreover, the relative positions of the three inverted repeats are also conserved . Overall sequence similarity was 52%, but was significantly higher in particular regions, which coincided with the functionally important segments mapped by deletion analysis . Conservation of these segments provided independent support for their essential role in oriT function. J Surg Res, 1991 May, 50(5), 436 - 41 Platelet-activating factor primes endotoxin-stimulated macrophage procoagulant activity; Kucey DS et al.; Macrophage procoagulant activity (PCA) at the site of inflammation may be induced by several stimuli including bacteria and endotoxin (LPS) . The local factors controlling PCA induction are poorly defined . The lipid mediator platelet-activating factor (PAF) is ubiquitous to inflammatory sites . To determine the effect of PAF on LPS-induced PCA, thioglycolate-elicited murine peritoneal macrophages were exposed to PAF (10(-7) M) or control medium for 30 min and then stimulated with LPS (10 micrograms/ml) for 2, 4, or 6 hr . The ability of macrophages to shorten the clotting time of plasma (ie., PCA) was then measured and clotting times were converted to PCA units using a thromboplastin standard . Cytosolic calcium ({Ca2+}i) measurements were made using the calcium-sensitive fluorescent dye indo-1 . PAF alone did not induce a rise in PCA expression (medium alone, 47 +/- 11 mU/10(6) cells; PAF alone, 49 +/- 12 mU/10(6) cells at t = 4 hr), but PAF treatment prior to LPS exposure resulted in a significant increase in the LPS-stimulated expression of PCA (LPS alone, 190 +/- 29 mU/10(6) cells; PAF/LPS, 329 +/- 57 mU/10(6) cells at t = 4 hr, P less than 0.05) . This priming effect was reversed by the PAF antagonist WEB 2086 (WEB/PAF/LPS, 196 +/- 31 mU/2 x 10(6) cells) . Stimulation of cells with PAF alone resulted in a rapid rise in {Ca2+}i (resting, 213 +/- 19 nmole; peak, 577 +/- 35 nmole) . This effect was also inhibited by WEB 2086 . These data suggest that PAF plays an important role in the modulation of PCA production by macrophages.(ABSTRACT TRUNCATED AT 250 WORDS) Am J Physiol, 1991 May, 260(5 Pt 2), H1474 - 81 Cardiac dysfunction after acute endotoxin administration in conscious sheep; Sugi K et al.; We evaluated cardiac function in an unanesthetized ovine model of hyperdynamic endotoxemia . The animals were instrumented for crystallographic dimension analysis of the left ventricle (LV) and measurement of LV, aortic, atrial, central venous, and pulmonary arterial pressures, and cardiac output . Seven sheep received 1.5 micrograms/kg of Escherichia coli endotoxin {lipopolysaccharide (LPS) LPS-P group} and were compared to a sham group . The sham group demonstrated no significant change in any of the variables . In the LPS-P group, the cardiac index increased (5.7 +/- 0.4 to 7.9 +/- 0.6 l.min-1.m-2) between 8 and 12 h after LPS . Concomitantly, the maximum elastance of LV end-systolic pressure-volume relations significantly decreased (2.88 +/- 0.27 mmHg/ml) compared with baseline (3.89 +/- 0.50 mmHg/ml) . Other indexes of the LV contractility (maximum pressure development and ejection fraction) were also reduced . There was a simultaneous increase in the LV end-systolic and diastolic volumes . These findings confirm the hypothesis that there is a myocardial depression during LPS in the ovine model. Am J Physiol, 1991 May, 260(5 Pt 2), H1415 - 23 Effect of fluid resuscitation from endotoxin shock on lung transvascular fluid and protein exchange; Mullins RJ et al.; The hypothesis that volume expansion during septic shock produces a greater transvascular protein flux than volume expansion alone was tested in anesthetized sheep by giving a high dose of endotoxin (40 micrograms/kg) intravenously . After 0.5 h of systemic hypotension, Ringer lactate, equivalent to 8% body wt, was infused followed by an additional 4 h of lymph collection . The results were compared with those from control animals receiving only Ringer lactate . The changes in plasma total protein with time were similar between groups . The increases in lymph flow and lymph protein flux were greater in the endotoxin-challenged group compared with the control group receiving Ringer lactate during the first 2 h but were similar thereafter . The interstitial volume was greater in the endotoxin-challenged animals compared with controls . The extravascular masses or apparent tissue concentrations for albumin or immunoglobulin G did not change in either group receiving Ringer lactate . The pulmonary edema following resuscitation from septic shock with Ringer lactate could be accounted for by either the pulmonary hypertensive effects of endotoxin or an initial, transient increase in microvascular protein permeability but not a sustained increase in microvascular permeability. Mol Pharmacol, 1991 May, 39(5), 643 - 9 Production of antisera selective for m1 muscarinic receptors using fusion proteins: distribution of m1 receptors in rat brain; Wall SJ et al.; A fragment of the cDNA encoding the third intracellular loop of the rat m1 muscarinic receptor was cloned, and the DNA was expressed in Escherichia coli as a fusion protein . The fusion protein was purified and utilized as an antigen to raise a polyclonal antiserum in rabbits . Chinese hamster ovary cells stably transfected with the cDNA encoding each of the five known subtypes of muscarinic receptor were used as tissue sources to test the antiserum . The antiserum was found to quantitatively immunoprecipitate m1 muscarinic receptors, while not precipitating m2, m3, m4, or m5 receptors . This selective antiserum was utilized to quantify the density of m1 muscarinic receptors in seven selected areas of the rat brain . Thus, cortex was found to contain approximately 0.8 pmol/mg of membrane protein, which represents 34% of the total density of muscarinic receptors . Similarly, hippocampus (1 pmol/mg; 47%), striatum (0.8 pmol/mg; 29%), and olfactory tubercule (0.9 pmol/mg; 35%) are rich in m1 receptors . In contrast, thalamus/hypothalamus contained only 0.15 pmol/mg, representing approximately 16% of the total density of muscarinic receptors, whereas pons/medulla (0.03 pmol/mg; 5%) and cerebellum (less than 0.01 pmol/mg; 2%) had very low levels of expression of m1 receptors . The development of a selective antiserum has provided a means for the quantification of a specific subtype of muscarinic receptor in tissues, such as the brain, that express multiple subtypes . This methodology will be applicable not only to the other subtypes of muscarinic receptor but also to the subtypes of several other neurotransmitter receptors that lack selective drugs with which to study them. J Med Chem, 1991 May, 34(5), 1606 - 12 Escherichia coli mediated biosynthesis and in vitro anti-HIV activity of lipophilic 6-halo-2',3'-dideoxypurine nucleosides; Murakami K et al.; A series of 6-substituted 2',3'-dideoxypurine ribofuranosides (ddP) was enzymatically synthesized with live E . coli in an effort to enhance the lipophilicity of this class of anti-human immunodeficiency virus (HIV) compounds and thereby facilitate drug delivery into the central nervous system . All 6-halo-substituted ddPs were substantially more lipophilic, as defined by their octanol-water partition coefficient (P), than their nonhalogenated congeners 2',3'-dideoxyinosine (ddI) or 2',3'-dideoxyguanosine (ddG) . For this class of compounds, log P's ranged from +0.5 to -1.2 in the following order: 6-iodo, 2-amino-6-iodo greater than 6-bromo, 2-amino-6-bromo greater than 6-chloro, 2-amino-6-chloro greater than 6-fluoro, 2-amino-6-fluoro much greater than ddG greater than ddI . These compounds were evaluated in vitro for ability to suppress the infectivity, replication, and cytopathic effect of HIV . 2-Amino-6-fluoro-, 2-amino-6-chloro-, and 6-fluoro-ddP exhibited a potent activity against HIV comparable to that of ddI or ddG and completely blocked the infectivity of HIV without affecting the growth of target cells . The comparative order of in vitro anti-HIV activity was 2-amino-6-fluoro, 2-amino-6-chloro, 6-fluoro greater than 2-amino-6-bromo greater than 2-amino-6-iodo, 6-chloro greater than 6-bromo greater than 6-iodo . These compounds also exhibited potent in vitro activity against HIV-2 and 3'-azido-3'-deoxythymidine-resistant HIV-1 variants . All 2-amino-6-halo-ddPs and 6-halo-ddPs were substrates for adenosine deaminase (ADA) and were converted to ddG or ddI, respectively . In the presence of the potent ADA inhibitor 2'-deoxycoformycin, 6-halo-substituted ddPs failed to exert an in vitro antiretroviral effect . These dideoxypurine nucleoside analogues represent a new class of lipophilic prodrugs of ddG and ddI that possess the potential for more effective therapy of HIV-induced neurologic disorders. J Gen Virol, 1991 May, 72 ( Pt 5), 1013 - 20 Molecular cloning and nucleotide sequencing of the coat protein gene of citrus tristeza virus; Sekiya ME et al.; Citrus tristeza virus (CTV) contains approximately 20,000 bases of positive-sense ssRNA, encapsidated by a coat protein of approximately 25,000 Mr that has previously been reported to consist of at least two size variants, cp1 and cp2 . In the present study, a cDNA library of the T36 isolate of CTV was prepared in a protein expression vector and screened with a polyclonal antibody against the coat protein . Five immunopositive clones produced proteins in Escherichia coli that reacted with monoclonal as well as polyclonal antibodies to the CTV coat protein . Nucleotide sequence analysis of a region common to the five clones revealed the presence of a 669 nucleotide open reading frame flanked by numerous in-frame termination codons . The encoded protein has a predicted Mr of 24,909 and an amino acid composition consistent with that previously reported for the CTV coat protein . Comparison of the predicted amino acid sequence of the coat protein with the amino-terminal sequences of cp1 and cp2 indicated that these coat protein species arise from the same primary translation product, as a result of post-translational proteolysis at sites approximately 12 to 15 and 26 amino acids from the amino terminus respectively . These results are the first reported cloning and sequencing of a CTV gene and provide evidence that CTV may be translated using subgenomic RNA. Mutat Res, 1991 May, 248(1), 35 - 43 Genotoxic and cell-transforming properties of (trans,trans)-muconaldehyde; Henschler D et al.; (trans,trans)-Muconaldehyde, a putative metabolite of benzene, should be expected to have mutagenic properties by virtue of its twin alpha,beta-unsaturated carbonylic function . It displayed definitely mutagenic properties in S . typhimurium TA100 without metabolic activation and with a 5-fold concentration of tester organisms in the preincubation assay and induced SOS response in E . coli . It induced micronucleus formation and morphological transformation in a dose-dependent manner in Syrian hamster embryofibroblasts . No DNA single-strand breaks or interstrand cross-links could be detected using the alkaline elution technique; however, strand-break generation by subsequent gamma-irradiation was found to be increased. Carcinogenesis, 1991 May, 12(5), 879 - 84 The action of 1-nitroso-8-nitropyrene in Escherichia coli: DNA adduct formation and mutational consequences in the absence of nucleotide excision-repair; Lambert IB et al.; To study the mechanisms of mutagenesis by the carcinogen 1,8-dinitropyrene we have determined the DNA adducts formed and mutations induced by its partially activated metabolite 1-nitroso-8-nitropyrene (1,8-NONP) in an Escherichia coli strain deficient in nucleotide excision-repair . Using DNA sequence analysis we have characterized a collection of 159 lacI- mutations recovered following treatment with 1,8-NONP . The mutational spectrum was dominated by -1 frameshifts (110 events) in runs of contiguous G or C residues . Frameshift frequency was observed to increase with the length of the reiterated sequence . Two mutations involved the loss of GpC from alternating (GpC)n sequences . The ratio of -1:-2 events observed following 1,8-NONP treatment was markedly different from that induced by N-acetoxy-N-acetyl-2-aminofluorene in the same genetic target . Other mutational classes recovered included 'spontaneous' hotspot