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| Cell, 1989 Dec 22, 59(6), 1027 - 34 Excision and insertion of the conjugative transposon Tn916 involves a novel recombination mechanism; Caparon MG et al.; The covalently closed circular form of the conjugative transposon Tn916, which acts as an intermediate in transposition, is produced by a novel type of recombination . Excision of the element pairs noncomplementary base pairs, which flank the transposon in a heteroduplex, at the joint of a circular form . By a reversal of the excision process, the base pairs from the heteroduplex are inserted into the next target . We present a detailed molecular model for the movement of conjugative transposons that involves the initial formation of staggered nicks in the "coupling regions" that flank the inserted element . The different products of excision and insertion of Tn916 can be explained by this model. Eur J Biochem, 1989 Dec 22, 186(3), 563 - 9 Immobilization and affinity purification of recombinant proteins using histidine peptide fusions; Ljungquist C et al.; A gene fusion approach to simplify protein immobilization and purification is described . A gene encoding the protein of interest is fused to a gene fragment encoding the affinity peptide Ala-His-Gly-His-Arg-Pro . The expressed fusion proteins can be purified using immobilized metal affinity chromatography . A vector, designed to ensure obligate head-to-tail polymerization of oligonucleotide linkers was constructed by in vitro mutagenesis . A linker encoding the affinity peptide, was synthesized and polymerized to two, four and eight copies . These linkers were fused to the 3' end of a structural gene encoding a two-domain protein A molecule, ZZ, and to the 5' end of a gene encoding beta-galactosidase . Fusion proteins, of both types, with zero or two copies of the linker showed little or no binding to immobilized Zn2+, while a relatively strong interaction could be observed for the fusions based on four or eight copies of the linker . Using a pH gradient, the ZZ fusions were found to be eluted from the resin at different pHs depending on the number of the affinity peptide . These results demonstrate that genetic engineering can be used to facilitate purification and immobilization of proteins to immobilized Zn2+ and that the multiplicity of the affinity peptide is an important factor determining the binding characteristics. Eur J Biochem, 1989 Dec 22, 186(3), 523 - 33 Purification of active human plasminogen activator inhibitor 1 from Escherichia coli . Comparison with natural and recombinant forms purified from eucaryotic cells; Lawrence D et al.; Plasminogen activator inhibitor 1 (PAI-1) inhibits both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) and, therefore, is an important regulator of plasminogen activation . We have developed eucaryotic and procaryotic expression systems for PAI-1 and characterized the recombinant glycosylated and non-glycosylated products, together with a non-recombinant natural control, produced in the histosarcoma cell line HT 1080 . For eucaryotic expression, the PAI-1 cDNA was stably transfected into chinese hamster ovary cells (CHO cells), while procaryotic expression in Escherichia coli was examined after inserting the DNA sequence encoding the mature PAI-1 protein into an inducible expression vector . Recombinant PAI-1 from CHO cells was purified approximately 50-fold in two steps and was indistinguishable from natural PAI-1 . Between 3% and 4% of total cellular protein in the procaryotic expression system consisted of PAI-1, from which it was purified approximately 30-fold, with yields of between 15% and 20% . This PAI-1 formed 1:1 complexes with uPA and also with the single- and two-chain forms of tPA . Kinetic analysis demonstrated that the procaryote-produced PAI-1 had an inhibitory activity towards all three forms of PA that resembled that of natural PAI-1 with association rate constants of approximately 10(7) M-1 s-1 . In contrast to PAI-1 from eucaryotic cells, the PAI-1 from E . coli had an inherent activity equal to that of guanidine/HCl-activated natural PAI-1 . The activity could not be increased by treatment with denaturants suggesting that the latent form of PAI-1 was absent . However, at 37 degrees C the procaryote-produced PAI-1 lost activity at the same rate as natural PAI-1, with approximately 50% of the activity remaining after 3 h . This activity could be partially restored by treatment with 4 M guanidine/HCl . E . coli-derived PAI-1, added to human plasma and fractionated by Sephacryl S-200 chromatography, eluted in two peaks that were similar to those obtained with guanidine-activated PAI-1 from eucaryotic cells, suggesting that it bound to the PAI-1-binding protein (vitronectin). Biochim Biophys Acta, 1989 Dec 22, 1009(3), 251 - 6 Construction of an EBV-derived shuttle vector for studying the influence of transcription on mutagenesis; Palombo F et al.; We have constructed an EBV-derived shuttle vector, pF1-EBV, which replicates in human cells as an extrachromosomal element . The structural sequences of the gene encoding the bacterial xanthine-guanine-phosphoribosyltransferase (gpt) were fused to the promoter and presumptive control region of the mouse metallothionein I (MT-I) gene . Human 293 cells transformed with the recombinant plasmid synthesized gpt mRNA and the expression of the gene was inducible by zinc . The gpt gene offers a convenient system of selection for mutant plasmids by transformation into the appropriate gpt- E . coli strain . A clonal cell line created by establishment of the pF1-EBV shuttle vector showed a spontaneous gpt- frequency of 2.10(-5) . An increase in mutation frequency above background was induced by mutagenizing this cell line with the alkylating agent N-methyl-N-nitrosourea (MNU) . The recombinant molecule that we have constructed should provide a tool for studying the role of gene expression in DNA repair and mutagenesis. Science, 1989 Dec 22, 246(4937), 1578 - 84 Specific interactions in RNA enzyme-substrate complexes; Guerrier-Takada C et al.; Analysis of crosslinked complexes of M1 RNA, the catalytic RNA subunit of ribonuclease P from Escherichia coli, and transfer RNA precursor substrates has led to the identification of regions in the enzyme and in the substrate that are in close physical proximity to each other . The nucleotide in M1 RNA, residue C92, which participates in a crosslink with the substrate was deleted and the resulting mutant M1 RNA was shown to cleave substrates lacking the 3' terminal CCAUCA sequence at sites several nucleotides away from the normal site of cleavage . The presence or absence of the 3' terminal CCAUCA sequence in transfer RNA precursor substrates markedly affects the way in which these substrates interact with the catalytic RNA in the enzyme-substrate complex . The contacts between wild-type M1 RNA and its substrate are in a region that resembles part of the transfer RNA "E" (exit) site in 23S ribosomal RNA . These data demonstrate that in RNA's with very different cellular functions, there are domains with similar structural and functional properties and that there is a nucleotide in M1 RNA that affects the site of cleavage by the enzyme. Gene, 1989 Dec 21, 85(1), 193 - 7 Phage Trojan horses: a conditional expression system for lethal genes; Heitman J et al.; The EcoRI restriction enzyme (ENase) cleaves DNA molecules within the sequence GAATTC . Cells expressing this lethal activity normally make a second enzyme, the M.EcoRI methyltransferase (MTase), which protects their chromosomal DNA by modifying the EcoRI recognition sites . To isolate mutants of the EcoRI ENase, its gene was cloned into a filamentous phage vector (M13mp18) under control of the lac promoter . Normally, filamentous phages (M13, f1 and their derivatives) form turbid plaques by impairing the growth of their host cell without killing it . In contrast, phages expressing the EcoRI ENase kill the host cell, but survive long enough to produce plaques which are very clear . Expression of the M.EcoRI MTase rescues the host and restores turbid plaque formation . EcoRI ENase mutants were isolated by screening for mutants that make turbid, instead of clear, plaques on an M- host . This conditional expression system may be useful for cloning and mutating genes for other toxic proteins. Gene, 1989 Dec 21, 85(1), 1 - 13 Comparison of the nucleotide and amino acid sequences of the RsrI and EcoRI restriction endonucleases; Stephenson FH et al.; The RsrI endonuclease, a type-II restriction endonuclease (ENase) found in Rhodobacter sphaeroides, is an isoschizomer of the EcoRI ENase . A clone containing an 11-kb BamHI fragment was isolated from an R . sphaeroides genomic DNA library by hybridization with synthetic oligodeoxyribonucleotide probes based on the N-terminal amino acid (aa) sequence of RsrI . Extracts of E . coli containing a subclone of the 11-kb fragment display RsrI activity . Nucleotide sequence analysis reveals an 831-bp open reading frame encoding a polypeptide of 277 aa . A 50% identity exists within a 266-aa overlap between the deduced aa sequences of RsrI and EcoRI . Regions of 75-100% aa sequence identity correspond to key structural and functional regions of EcoRI . The type-II ENases have many common properties, and a common origin might have been expected . Nevertheless, this is the first demonstration of aa sequence similarity between ENases produced by different organisms. Gene, 1989 Dec 21, 85(1), 91 - 7 Semisynthetic promoters activated by cyclic AMP receptor protein of Escherichia coli; Aiba H et al.; Semisynthetic promoters activated by Escherichia coli cyclic AMP receptor protein (CRP) were created by combining a synthetic CRP-binding site (crb) and nucleotide sequences derived from cryptic promoter regions . A 22-bp oligodeoxyribonucleotide corresponding to an idealized crb was randomly placed into DNA regions that precede a promoterless lacZ gene on a plasmid . Several plasmid clones were obtained which allowed the expression of lacZ in crp+ cya+ cells carrying a chromosomal deletion of lac genes . The beta-galactosidase and the quantitative S1-nuclease assays of crp+ and delta crp cells harboring these plasmids indicated that the transcription from newly created promoters is dependent on CRP . Sequence analysis revealed that these promoters are divided into two types based on the location of the crb relative to the transcription start point (tsp) . The distance from the center of the crb to the tsp is 70 bp in the first type and 38 bp in the second type . The sequences of all these promoters exhibit poor homology with the consensus promoter sequence. Gene, 1989 Dec 21, 85(1), 35 - 43 Sumo15A: a lambda phasmid that permits easy selection for and against cloned inserts; Kurachi S et al.; We report the construction of a phasmid vector, Sumo15A, designed for recombination-based screening of recombinant DNA libraries {Seed, Nucleic Acids Res . 11 (1983) 2427-2445} . This vector permits rapid selection in Escherichia coli for homology-mediated integration and excision between homologous DNA inserts cloned in a supF-carrying plasmid and in Sumo15A . The region available for recombination spans the homologous sequence shared by the plasmid and the phasmid . SupF is the selection tool that we used . Efficient selection for supF expression by Sumo15A requires recombination mediated by the lambda phage red gene, which promotes homologous recombination between phage and plasmid DNAs . Counterselection against supF expression by Sumo15A occurs because the presence of a pSC101-derived plasmid replicon in this phasmid permits the growth of Sumo15A as a plasmid in a specialized host, E . coli strain DK37 . In strain DK37, Sumo15A cannot replicate as a phage, and the presence of a plasmid-carrying supF is lethal to cells plated on galactose plates . This scheme was developed to select for sequences that are transcribed from chromosomes of interest. Gene, 1989 Dec 21, 85(1), 243 - 6 Development of a shuttle vector and a conjugative transfer system for Actinobacillus pleuropneumoniae; Lalonde G et al.; An original genetic system for Actinobacillus pleuropneumoniae has been developed . A shuttle cloning vector, pYG53, was constructed from the wild-type plasmid pYG10 . It permits, in conjunction with electroporation, the introduction of cloned genes into this species . A conjugal transfer system between Escherichia coli and A . pleuropneumoniae involving pYG54, a mobilizable derivative of pYG53, is also described . Conjugation efficiencies of 8.3 x 10(-3) exconjugants per donor can be obtained. Gene, 1989 Dec 21, 85(1), 109 - 14 High-level expression of recombinant genes in Escherichia coli is dependent on the availability of the dnaY gene product; Brinkmann U et al.; We have observed that proteins, such as human tissue-type plasminogen activator, pro-urokinase or gp41 of human immunodeficiency virus, which have a high content of rare codons in their respective genes, are not readily expressed in Escherichia coli . Furthermore induction of these heterologous genes leads to growth inhibition and plasmid instability . Supplementation with tRNA(AGA/AGG(Arg)) by cotransfection with the dnaY gene, which supplies this minor tRNA, resulted in high-level production with greatly improved cell viability and plasmid stability. Gene, 1989 Dec 21, 85(1), 215 - 20 Cloning, expression and nucleotide sequence of the gene fragment encoding an antigenic portion of the nucleoside triphosphate hydrolase of Toxoplasma gondii; Johnson AM et al.; Toxoplasma gondii expresses high levels of an active nucleoside triphosphate hydrolase (NTPase; EC 3.6.1.3) with several unique properties . It has been detected as a circulating antigen in mice, making it an ideal candidate for diagnostic tests for toxoplasmosis . A cDNA library constructed from T . gondii poly(A)+RNA was made in lambda gt11 . One hundred thousand members of this library were immunoscreened with a rabbit polyclonal antibody to the purified NTPase . Six positive clones were subcloned into the plasmid, pGEX-IN, and the inserts were restriction-mapped . All clones had identical partial restriction enzyme maps . One insert was subcloned into M13mp18 and sequencing by the deoxy/dideoxy method showed an NTPase-encoding gene (ntp) fragment of 571 bp . The insert was also purified, radiolabelled, and used to hybridize to Northern blots of tachyzoite RNA and quantitative Southern blots of tachyzoite DNA . Northern blotting revealed that the NTPase mRNA was in great abundance and had a length of about 2800 nucleotides . Southern blotting showed a gene copy number of between one and five, and the possibility that ntp is tandemly repeated over a large length of DNA . The NTPase was expressed as a glutathione S-transferase (EC 2.5.1.18) fusion protein of about 50 kDa, which reacted with polyclonal rabbit antibody on Western blotting. J Mol Biol, 1989 Dec 20, 210(4), 881 - 2 Preliminary crystallographic analysis of 5-carboxymethyl-2-hydroxymuconate isomerase from Escherichia coli; Wigley DB et al.; Escherichia coli 5-carboxymethyl-2-hydroxymuconate (CHM) isomerase was purified from an overexpressing cell line . The enzyme has been crystallized from ammonium sulphate in two different crystal forms . One of these has been analysed and found to be orthorhombic I222 or I2(1)2(1)2(1) with cell dimensions a = 88 A, b = 89 A, c = 121 A . The asymmetric unit contains two dimers (Vm = 2.11 A3/dalton) . The crystals diffract to beyond 3.0 A resolution and are stable to irradiation with X-rays . Data have been collected to 3.0 A resolution and a search for potential heavy-metal derivatives is in progress. J Mol Biol, 1989 Dec 20, 210(4), 859 - 67 Molecular genetic, biochemical and nuclear magnetic resonance studies on the role of the tryptophan residues of glutamine-binding protein from Escherichia coli; Shen QC et al.; The results of molecular genetic, biochemical and nuclear magnetic resonance studies on glutamine-binding protein of Escherichia coli suggest that the only two tryptophan residues, at positions 32 and 220, in the protein molecule are likely to be involved in (or sensitive to) interactions with the membrane-bound protein components of the glutamine transport system . It has been found that both tryptophan residues have limited motional freedom, are located away from the surface of the protein molecule and are not close to the ligand-binding site . Their presence, however, is required for the optimal transport of L-glutamine across the cytoplasmic membrane, though not essential for the ligand-binding process . The relevance of these results to the structure and function of the glutamine-binding protein in the glutamine transport system is discussed. J Mol Biol, 1989 Dec 20, 210(4), 849 - 57 Proton nuclear magnetic resonance studies on glutamine-binding protein from Escherichia coli . Formation of intermolecular and intramolecular hydrogen bonds upon ligand binding; Shen QC et al.; Proton nuclear magnetic resonance studies have revealed several structural and dynamic properties of the glutamine-binding protein of Escherichia coli . When this protein binds L-glutamine, six low-field, exchangeable proton resonances appear in the region from +5.5 to +10 parts per million downfield from water (or +10.2 to +14.7 parts per million downfield from the methyl proton resonance of 2,2-dimethyl-2-silapentane-5-sulfonate) . This suggests that the binding of L-glutamine induces specific conformational changes in the protein molecule, involving the formation of intermolecular and intramolecular hydrogen bonds between the glutamine-binding protein and L-glutamine, and within the protein molecule . The oxygen atom of the gamma-carbonyl group of L-glutamine is likely to be involved in the formation of an intermolecular hydrogen bond between the ligand and the binding protein . We have shown that at least one phenylalanine and one methyl-containing residue are spatially close to this intermolecular hydrogen-bonded proton . The intermolecular and intramolecular hydrogen-bonded protons of the ligand-protein complex undergo solvent exchange . The local conformations around these intermolecular and intramolecular hydrogen bonds are quite stable when subjected to pH and temperature variations . From these results, the utility of proton nuclear magnetic resonance spectroscopy for investigating such binding proteins has been shown, and a picture of the ligand-binding process can be drawn. EMBO J, 1989 Dec 20, 8(13), 4315 - 23 tRNA hopping: enhancement by an expanded anticodon; O'Connor M et al.; At a low level wild-type tRNA(1Val) inserts a single amino acid (valine) for the five nucleotide sequence GUGUA which has overlapping valine codons . Mutants of tRNA(1Val) with an insertion of A or U between positions 34 and 35 of their anticodons have enhanced reading of the quintuplet sequences . We propose that this decoding occurs by a hopping mechanism rather than by quintuplet pairing . Such hopping involves disengagement of the paired codon and anticodon with the mRNA slipping two (or more) bases along the ribosomal--peptidyl tRNA complex and subsequently re-pairing at a second codon--the landing site . The mutant with the anticodon sequence 3'CAAU5' 'hops' over the stop codon in the mRNA sequence GUG UAA GUU with the insertion of a single amino acid (valine) . In contrast, in reading the same sequence, the mutant with the anticodon 3'CAUU5' hops onto the stop with the insertion of two valine residues . It is likely that in some instances of hopping alternate anticodon bases are used for the initial pairing and at the landing site. EMBO J, 1989 Dec 20, 8(13), 4335 - 44 The DNA unwinding element: a novel, cis-acting component that facilitates opening of the Escherichia coli replication origin; Kowalski D et al.; We have discovered that DNA supercoiling, in the absence of replication proteins, induces localized unwinding in the Escherichia coli replication origin (oriC) at the same sequence opened by the dnaA initiator protein . The DNA helix at the tandemly repeated, 13mer sequence is thermodynamically unstable, as evidenced by hypersensitivity to single-strand-specific nuclease in a negatively supercoiled plasmid, and demonstrated by stable DNA unwinding seen after two-dimensional gel electrophoresis of topoisomers . A replication-defective oriC mutant lacking the leftmost 13mer shows no nuclease hypersensitivity in two remaining 13mers and no detectable DNA unwinding on two-dimensional gels . The replication defect in the oriC mutant can be corrected by inserting a dissimilar DNA sequence with reduced helical stability in place of the leftmost 13mer . Thus, the helical instability of the leftmost 13mer, not the specific 13mer sequence, is essential for origin function . The rightmost 13mer exhibits helical instability but differs from the leftmost 13mer in its strict sequence conservation among related bacterial origins . The repeated 13mer region appears to serve two overlapping functions: protein recognition and helical instability . We propose that the cis-acting sequence whose helical instability is required for origin function be called the DNA unwinding element (DUE). EMBO J, 1989 Dec 20, 8(13), 4345 - 50 P22 repressor mutants deficient in co-operative binding and DNA loop formation; Valenzuela D et al.; We show here, both in vivo and in vitro, that P22 repressor binds co-operatively to operator sites separated by an integral number of turns of the DNA helix . We measure this co-operativity in vivo using an assay in which repression of a promoter requires co-operative binding of P22 repressors to two separated (non-adjacent) operator sites . We report the isolation of mutant repressors that have high affinity for single operator sites, but are defective in co-operative binding . Six different mutants, all bearing single amino acid changes in the carboxyl domain, have been isolated . We purified the two mutants most deficient in co-operative binding, and found that they bind non-co-operatively in vitro to adjacent as well as to non-adjacent pairs of operator sites. J Mol Biol, 1989 Dec 20, 210(4), 771 - 84 Structure of vaccinia virus late promoters; Davison AJ et al.; Functional elements of vaccinia virus late promoters were characterized by mutagenesis . Synthetic oligonucleotides were inserted into a plasmid vector containing the lacZ gene of Escherichia coli flanked by sequences from the thymidine kinase (TK) gene of vaccinia virus . The lacZ gene, under control of the synthetic promoter, was introduced into the vaccinia virus genome at the TK locus by homologous recombination, and each of the 122 recombinants thus obtained was assayed for beta-galactosidase expression . The relative amounts and 5' ends of lacZ mRNAs specified by a subset of the recombinants were determined by primer extension . The analysis indicated that late promoters may be considered in terms of three regions; an upstream sequence of about 20 base-pairs, rich in T and A residues, separated by a spacer region of about six base-pairs from a highly conserved (-1)TAAAT(+4) element within which transcription initiates . All single nucleotide substitutions within the three A residues of the TAAAT, as well as the addition of a fourth A residue, caused drastic reductions in promoter strength . All substitutions of the T residues at -1 and +4 were also detrimental to promoter activity, to an extent that depended on the strength of the promoter as determined by the upstream sequence . mRNA synthesis appeared to initiate within the three A residues regardless of promoter strength . The 5'-poly(A) leader, which is a unique feature of poxvirus late mRNAs, was diminished in length when either of the T residues at -1 and +4 was mutated, was absent or limited to a few nucleotides when any of the three A residues was substituted, but was unaffected by changes outside the TAAAT sequence . The data are consistent with a model for the generation of the normal 5'-poly(A) leader by an RNA polymerase slippage mechanism requiring three consecutive A residues . Single nucleotide substitutions within the six base-pairs upstream and three base-pairs downstream from the TAAAT sequence had modest effects on promoter strength . The most and least favourable changes led to a fourfold increase and an eightfold decrease in activity, respectively . Sequences further upstream were essential for late promoter function; tracts of T or A residues enhanced expression up to 20-fold, the former conferring much greater activity . Highest expression was obtained with a tract of 18 or 20 T residues.(ABSTRACT TRUNCATED AT 400 WORDS) J Mol Biol, 1989 Dec 20, 210(4), 749 - 69 Structure of vaccinia virus early promoters; Davison AJ et al.; Functional elements of a vaccinia virus early promoter were characterized by making a complete set of single nucleotide substitutions, as well as more complex mutations, and assaying their effects on gene expression . Synthetic oligonucleotides, based primarily on the sequence of the 7.5-kD early promoter, were inserted into a plasmid vector containing the lacZ gene of Escherichia coli flanked by sequences from the thymidine kinase (TK) gene of vaccinia virus . The lacZ gene, under control of the synthetic promoter, was introduced into the vaccinia virus genome at the TK locus by homologous recombination, and each of the 331 different recombinant viruses thus obtained was assayed for beta-galactosidase expression . The relative amounts and precise 5' ends of lacZ mRNAs specified by a subset of the recombinants were determined by primer extension . Many promoters were tested for their ability to direct specific transcription in vitro . A generally good correlation was noted between measurements of promoter strength estimated by beta-galactosidase expression, primer extension of in vivo mRNA and transcription in vitro . A relatively simple picture emerged from the analysis . The early promoter consists of a 16 base-pair critical region, in which most single nucleotide substitutions have a major effect on expression, separated by 11 base-pairs of a less critical T-rich sequence from a seven base-pair region within which initiation with a purine usually occurs . For the critical region of the 7.5-kD promoter, AAAAgTaGAAAataTA, any substitution of an upper-case nucleotide reduced expression, usually drastically, whereas certain substitutions of lower-case nucleotides maintained or significantly enhanced expression . On the basis of this analysis, the wide range of activities of natural promoters could be attributed to the presence of one or more non-optimal nucleotides in the critical region . Moreover, single nucleotide substitutions in such promoters had the predicted enhancing effects . Most mutations in the critical region of the 7.5-kD promoter behaved independently, but some nucleotide substitutions compensated for potentially detrimental nucleotides at other positions . Promoters substantially stronger than any natural ones examined were constructed by combining several up-mutations within the critical region of the 7.5-kD promoter and by repeating the critical region sequence . Like the TATA box of eukaryotic RNA polymerase II promoters, the critical region specifies the site of transcriptional initiation. EMBO J, 1989 Dec 20, 8(13), 4289 - 96 Synthetic curved DNA sequences can act as transcriptional activators in Escherichia coli; Bracco L et al.; Can a transcriptional activator known to bend DNA be functionally replaced by a sequence-directed bend in Escherichia coli? To investigate this question, a partially truncated promoter was used, deleted of its -35 region and of its CRP binding site, leaving only two Pribnow boxes as functional elements . Synthetic and naturally occurring curved DNA sequences introduced upstream from these elements could restore transcription at either one of the two natural starts . Some of these hybrid promoters turned out to be more efficient than the CRP activated wild-type gal promoter in vivo . Control experiments performed with very similar sequences devoid of any curvature produced weak promoters only . Minimal changes in the location of the centre of curvature or perturbation in the amount of curvature strongly affected the level of expression . No significant stimulation of transcription could be detected in vitro . Furthermore, both gal P1 and P2 starts could be activated in vivo but also in vitro via a properly positioned CRP binding site . This partial analogy suggests that bending induced by the cAMP-CRP complex upon binding to its site may be biologically relevant to the mechanism of transcriptional activation. EMBO J, 1989 Dec 20, 8(13), 4297 - 305 The unusual stability of the IS10 anti-sense RNA is critical for its function and is determined by the structure of its stem-domain; Case CC et al.; IS10 transposition is regulated by an approximately 70 nt anti-sense RNA, RNA-OUT . RNA-OUT folds into a duplex 'stem-domain' topped by a loosely paired 'loop-domain' . The loop-domain is critical for RNA-RNA pairing per se; pairing initiates by interaction of the RNA-OUT loop with the 5' end of the target mRNA . We show here that RNA-OUT is unusually stable in vivo (half-life 60 min) and that this stability is conferred by specific features of the RNA-OUT stem-domain . One critical feature is stable base-pairing: mutations that disrupt stem pairing destabilize RNA-OUT in vivo and abolish anti-sense control; combinations of mutations that restore pairing also restore both stability and control . We propose that the stem renders RNA-OUT resistant to 3' exoribonucleases . Other features of the stem-domain prevent this essential duplex from being an effective substrate for double-strand nucleases: two single base mutations disrupt antisense control by making RNA-OUT susceptible to RNase III . Mutations in the loop region have little effect on RNA-OUT stability . Implications for IS10 biology and the design of efficient anti-sense RNAs are discussed. FEBS Lett, 1989 Dec 18, 259(1), 185 - 8 Loss of calcium sensitivity of plasma gelsolin is associated with the presence of calcium ions during preparation; Pope B et al.; Gelsolin is a calcium-dependent actin severing and capping protein . Calcium 'opens' the molecule to make actin binding sites accessible, but removal of calcium from the medium does not necessarily fully reverse this process . The calcium sensitivity of actin monomer binding and actin filament severing is here shown to vary considerably with the source of gelsolin and conditions of preparation . Plasma gelsolin undergoes irreversible loss of calcium sensitivity when prepared in the presence of calcium ions . This is not due solely to effects of bound calcium, because purified human plasma gelsolin expressed in E . coli and stored in calcium shows no comparable loss of calcium sensitivity when prepared or stored in calcium . These results suggest the presence of factors in plasma which, in the presence of calcium, promote an irreversible structural change in gelsolin resulting in permanent loss of calcium sensitivity. FEBS Lett, 1989 Dec 18, 259(1), 121 - 4 Recombinant core particles of hepatitis B virus exposing foreign antigenic determinants on their surface; Borisova GP et al.; Insertion of foreign oligopeptide sequences (40-50 amino acids in length) into the Pro144 position of hepatitis B core antigen (HBcAg) leads to the formation of chimeric capsids in Escherichia coli cells . These capsids are morphologically and immunologically similar to native HBcAg, but expose the inserted oligopeptides on their outer surface and exhibit antigenic and immunogenic characteristics of the latter . As a source of model antigenic determinants, the appropriate DNA copies excised from cloned viral genes such as the pre-S region of hepatitis B virus, the transmembrane protein gp41 of human immunodeficiency virus 1 and the envelope protein gp51 of bovine leukemia virus have been used . The localization of the inserted antigenic determinants on the surface of chimeric capsids does not depend on the presence or absence of the arginine-rich, 39 amino acid-long C terminus of HBcAg. Vet Rec, 1989 Dec 16, 125(25), 620 - 4 A comparison of two oral rehydration solutions in experimental models of dehydration and diarrhoea in calves; Dupe RJ et al.; Two oral rehydration solutions (ORS 1 and ORS 2) were evaluated in isolated intestinal loops of anaesthetised calves, in an experimental model of dehydration in the calf, in calves with experimentally induced diarrhoea and in 164 calves with clinical diarrhoea . The studies in isolated intestinal loops indicated that water absorption was significantly greater from ORS 2 than from ORS 1 . After the intraperitoneal administration of hypertonic mannitol combined with intravenous diuretics, the plasma volume of calves was reduced by about 30 per cent, and was more rapidly expanded after treatment with ORS 2 than ORS 1 . The plasma volume remained significantly reduced (P less than 0.01) three hours after dosing with ORS 1 whereas after treatment with ORS 2 it was not significantly different from the initial value . Acidosis was corrected to a significantly (P less than 0.01) greater extent after treatment with ORS 2, and peripheral perfusion also returned to normal more rapidly in calves given ORS 2 . In newly purchased calves in which diarrhoea was induced experimentally with an E coli challenge, base deficit and diarrhoea were corrected more rapidly in the calves receiving ORS 2 . When the solutions were tested in the treatment of 164 clinical cases of diarrhoea and dehydration there was no statistically significant difference in mortality between the formulations, although the overall mortality was 4.8 per cent in the calves treated with ORS 2, compared with 8.6 per cent in the calves treated with ORS 1 . It was concluded that ORS 2 performed better than ORS 1 especially in the expansion of plasma volume and the correction of acidosis. Biochem Biophys Res Commun, 1989 Dec 15, 165(2), 685 - 8 Discontinuous DNA replication in a lig-7 strain of Escherichia coli is not the result of mismatch repair, nucleotide-excision repair, or the base-excision repair of DNA uracil; Wang TC et al.; After pulse-labeling with 3H-thymidine for 30 s at 42 degrees C, the newly-synthesized DNA from uvrB5 lig-7, uvrB5 lig-7 ung-1 (or ung152), uvrB5 lig-7 mutL218 (or mutS215), and uvrB5 lig-7 ung-1 mutL218 (or mutS215) cells sedimented very slowly in alkaline sucrose gradients . The bulk of these DNA molecules were smaller than 2,000 nucleotides long (i.e., about the size of Okazaki fragments), and none of the 3H-radioactivity was found to sediment as high-molecular-weight DNA . These results indicate that the apparent discontinuous DNA replication observed in lig-7 strains is not the result of mismatch repair, nucleotide-excision repair, or the base-excision repair of DNA uracil. J Biol Chem, 1989 Dec 15, 264(35), 21422 - 30 AMP deaminase from yeast . Role in AMP degradation, large scale purification, and properties of the native and proteolyzed enzyme; Merkler DJ et al.; Eukaryotes have been proposed to depend on AMP deaminase as a primary step in the regulation of intracellular adenine nucleotide pools . This report describes 1) the role of AMP deaminase in adenylate metabolism in yeast cell extracts, 2) a method for large scale purification of the enzyme, 3) the kinetic properties of native and proteolyzed enzymes, 4) the kinetic reaction mechanism, and 5) regulatory interactions with ATP, GTP, MgATP, ADP, and PO4 . Allosteric regulation of yeast AMP deaminase is of physiological significance, since expression of the gene is constitutive (Meyer, S . L., Kvalnes-Krick, K . L., and Schramm, V . L . (1989) Biochemistry 28, 8734-8743) . The metabolism of ATP in cell-free extracts of yeast demonstrates that AMP deaminase is the sole pathway of AMP catabolism in these extracts . Purification of the enzyme from bakers' yeast yields a proteolytically cleaved enzyme, Mr 86,000, which is missing 192 amino acids from the N-terminal region . Extracts of Escherichia coli containing a plasmid with the gene for yeast AMP deaminase contained only the unproteolyzed enzyme, Mr 100,000 . The unproteolyzed enzyme is highly unstable during purification . Substrate saturation plots for proteolyzed AMP deaminase are sigmoidal . In the presence of ATP, the allosteric activator, the enzyme exhibits normal saturation kinetics . ATP activates the proteolyzed AMP deaminase by increasing the affinity for AMP from 1.3 to 0.2 mM without affecting VM . Activation by ATP is more efficient than MgATP, with half-maximum activation constants of 6 and 80 microM, respectively . The kinetic properties of the proteolyzed and unproteolyzed AMP deaminase are similar . Thus, the N-terminal region is not required for catalysis or allosteric activation . AMP deaminase is competitively inhibited by GTP and PO4 with respect to AMP . The inhibition constants for these inhibitors decrease in the presence of ATP . ATP, therefore, tightens the binding of GTP, PO4, and AMP . The products of the reaction, NH3 and IMP, are competitive inhibitors against substrate, consistent with a rapid equilibrium random kinetic mechanism . Kinetic dissociation constants are reported for the binary and ternary substrate and product complexes and the allosteric modulators. J Biol Chem, 1989 Dec 15, 264(35), 21369 - 75 The adaptive response to alkylation damage . Constitutive expression through a mutation in the coding region of the ada gene; Hughes SJ et al.; We have shown by genetic mapping, molecular cloning, and DNA sequencing that four Escherichia coli mutants, which express the adaptive response to alkylation damage constitutively, are mutated in the ada gene . All four mutant ada genes have two GC to AT transition mutations in the coding region and encode altered Ada proteins with two amino acid substitutions in the N-terminal domain . E . coli carrying the mutated ada genes on recombinant plasmids overexpressed both the mutated ada gene and the chromosomal alkA gene . This observation indicates that the mutant Ada proteins act as strong positive regulators of the ada and alkA genes in the absence of DNA alkylation . One mutant protein, Ada-11, was shown to be a strong activator of ada gene expression in a cell-free system . An altered pattern of tryptic digestion of the Ada-11 protein compared with the wild-type Ada protein suggested that it has a different conformation . One amino acid substitution, namely methionine residue 126 replaced by isoleucine, occurred in all four mutant Ada proteins, and this mutation alone was sufficient to convert the Ada protein into a strong activator of ada and alkA gene expression in vivo. J Biol Chem, 1989 Dec 15, 264(35), 21277 - 85 Site-directed chemical modification for probing DNA-protein interactions . Osmium tetroxide modification of the -10 site of the lacUV5 promoter enhances open complex formation; Chan PT et al.; A new experimental approach, site-directed chemical modification, was used to explore relationships between RNA polymerase-promoter interactions and function . For this study, the lacUV5 promoter with an exposed -10 thymine on the non-template strand was constructed . Osmium tetroxide was selected as the thymine modifying reagent . Modification occurred predominantly at the exposed -10 T with 5-fold less reactivity at the -12 T residue . The isolated modified strand was used to reconstitute a lacUV5 promoter with -10 (-12) adducts . OsO4 modification at both the -10 and -12 positions of the lacUV5 promoter significantly enhances Escherichia coli RNA polymerase-promoter open complex formation relative to the unmodified promoter . DNase I cleavage sites at -7, -8, and -10 of the unmodified promoter were rendered insusceptible to scission in the modified promoter . However, no difference can be detected in the RNA polymerase footprints for unmodified versus modified open complexes . The latter are fully capable of productive transcription with comparable amounts of identical run-off transcripts to unmodified open complexes . A 16 degrees C reduction in Tm was found for a 14-base pair oligonucleotide duplex containing a single OsO4-bispyridine adduct . The latter result suggests that open complex formation appears to be enhanced due to promoter unpairing at the -10 (-12) adduct sites. J Biol Chem, 1989 Dec 15, 264(35), 21116 - 21 Recombinant bovine alpha-lactalbumin obtained by limited proteolysis of a fusion protein expressed at high levels in Escherichia coli; Wang M et al.; A cDNA clone containing the entire coding region for bovine pre-alpha-lactalbumin (LA) together with 27 base pairs of 5'-noncoding and 268 base pairs of 3'-noncoding sequences was isolated from a bovine mammary cDNA plasmid library in the Okayama-Berg vector system using a synthetic oligonucleotide probe and sequenced . The coding segment for mature LA was subcloned into the T7 expression system of Studier and co-workers (Studier, F.W., and Moffatt, B.A . (1986) J . Mol . Biol . 189, 113-130; Rosenberg, A.H., Lade, B.N., Chui, D.S., Lin, S.W., Dunn, J.J., and Studier, F.W . (1987) Gene (Amst.) 56, 125-135) and expressed as a 21-kDa fusion protein that consisted of the mature bovine LA sequence connected to the NH2-terminal 50 residues of human cathepsin D by a linker sequence containing protease cleavage sites . This fusion protein was expressed in an insoluble form and accumulated to about 50% of the total bacterial protein within 3 h after induction of T7 RNA polymerase synthesis . The protein was solubilized, purified by gel filtration, and converted to an active form by treatment with mixtures of reduced and oxidized glutathione in the presence of Ca2+ . The maximum specific activity of the fusion protein was about 25% of that of native LA, suggesting that the attachment of an NH2-terminal extension sterically hinders but does not prevent the interaction with galactosyltransferase . The extension also does not block the binding of the regulatory Ca2+ ion that is required for folding from the reduced denatured state . Trypsin cleaved the folded fusion protein specifically at a Lys-Glu bond at the junction with the mature LA sequence to give a product indistinguishable in structure and activity from native LA. J Biol Chem, 1989 Dec 15, 264(35), 21073 - 9 Site of pyruvate formation and processing of mammalian S-adenosylmethionine decarboxylase proenzyme; Stanley BA et al.; Mammalian S-adenosylmethionine decarboxylase was expressed at a high level in an Escherichia coli mutant deficient in this enzyme . The proenzyme form of this enzyme was cleaved and processed to the mature decarboxylase which contains two pairs of nonidentical subunits, the larger of which contains a pyruvate prosthetic group . In order to determine the site of formation of the pyruvate, two approaches were used . First, the mammalian S-adenosylmethionine decarboxylase produced in E . coli was purified to homogeneity and the pyruvate converted to alanine by a reductive amination . The large subunit was then isolated by reversed phase high pressure liquid chromatography and the amino-terminal sequence determined and compared with the sequence of the proenzyme derived from its cDNA . These results indicated that the bond between glutamic acid 67 and serine 68 was the site of cleavage . Second, each of the serine residues in portion of the proenzyme likely to contain the cleavage site were altered by site-directed mutagenesis and the RNA produced from plasmids containing these mutations was translated in a reticulocyte lysate . The translation products were tested for processing and for S-adenosylmethionine decarboxylase activity . Altering the serine residues at positions 50, 66, and 69 to alanines had little effect but changing serine at position 68 to alanine completely prevented both processing and activity . These results indicate that the serine residue at position 68 of the proenzyme which is in the underlined position in the sequence -Leu-Ser-Glu-Ser-Ser-Met- is the residue which is converted to the pyruvate prosthetic group in human S-adenosylmethionine decarboxylase. J Biol Chem, 1989 Dec 15, 264(35), 21031 - 7 Purification of a DNA replication terminus (ter) site-binding protein in Escherichia coli and identification of the structural gene; Hidaka M et al.; In Escherichia coli cells, there is a protein that specifically binds to DNA replication terminus (ter) sites on the host and plasmid genome and then blocks progress of the DNA replication fork . We reported that extract of the cells carrying the plasmid with the tau gene, which was identified to be an essential gene for the termination reaction at the ter site, contained about an 8-fold increase in ter-binding activity of the plasmid-free cells . With improvement of the promoter region of the tau gene on the plasmid by site-directed mutagenesis, the host cells produced the ter-binding protein (Ter protein) over 2,000-fold . Using these over-producing cells as the enzyme source, the Ter protein was purified to apparent homogeneity . Molecular mass 36,000, amino-terminal amino acid sequence (45 residues) and composition of the protein were in good agreement with those deduced from DNA sequence of the tau gene . Footprinting using the purified Ter protein revealed a specific binding to the ter sequences. J Biol Chem, 1989 Dec 15, 264(35), 20940 - 6 Expression of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli; Slice LW et al.; A cDNA clone for the catalytic subunit of murine cAMP-dependent protein kinase was placed into two expression vectors, pLWS-3 and pLSW-4 . For pLWS-3, the entire coding region of the catalytic subunit was inserted into the NdeI site of pT7-7 under the control of the T7 promoter . pLWS-4 contains a polycistronic transcript under control of the lac UV5 promoter encoding for the type I regulatory subunit followed by the catalytic subunit . Significant expression was achieved with pLWS-4 in Escherichia coli 222 and JM101; however, the catalytic subunit was produced in an insoluble form . In the case of the catalytic subunit produced in E . coli BL21(DE3) by pLWS-3, the catalytic subunit accounted for approximately 30% of the total bacterial protein . Up to 5 mg of this catalytic subunit per liter of culture was in the soluble extract . Solubility was improved substantially when induction was carried out at 30 degrees C instead of 37 degrees C . This recombinant catalytic subunit was purified by phosphocellulose chromatography, followed by ammonium sulfate precipitation and gel filtration . A Mr of 38,000 was estimated based on size exclusion chromatography and on polyacrylamide gel electrophoresis . The recombinant protein had a free alpha-amino-terminal Gly in contrast to the mammalian enzyme which is myristylated at the amino-terminal glycine . The lack of acylation did not significantly alter the activity of the enzyme . The specific activity of 19 mumol/min/mg is comparable to the mammalian enzyme . The Km values for Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) (43 microM) and MgATP (18.5 microM also were comparable . The absence of the acyl group also did not prevent holoenzyme formation . Holoenzyme activation by cAMP was indistinguishable for holoenzyme made with mammalian catalytic subunit and recombinant catalytic subunit . The recombinant enzyme was more sensitive than the mammalian enzyme to heat denaturation at 49 degrees C . The t1/2 for the recombinant catalytic subunit was 0.7 min in contrast to 3.9 min for the mammalian enzyme . This difference in stability may be attributable to the lack of the acyl group . The recombinant enzyme was particularly sensitive to heat denaturation in the presence of low concentrations (0.01%) of Triton X-100. Cancer Res, 1989 Dec 15, 49(24 Pt 1), 6997 - 7001 A novel, sensitive assay for O6-methyl- and O6-ethylguanine in DNA, based on repair by the enzyme O6-alkylguanine-DNA-alkyltransferase in competition with an oligonucleotide containing O6-methylguanine; Souliotis VL et al.; A novel assay for O6-alkylguanine-type adducts in DNA is reported . It is based on the use of the suicide repair enzyme O6-alkylguanine-DNA-alkyltransferase (AGT) to repair such adducts in DNA in competition with an oligonucleotide containing a single residue of O6-methylguanine, end labeled to high specific activity . The stoichiometric mode of action of AGT results in decreased amounts of oligonucleotide being repaired in the presence of increasing levels of adducts in the competing DNA . The extent of oligonucleotide repair is determined by immunoprecipitation of the unrepaired form with rabbit antiserum directed against O6-alkyldeoxyguanosine and radiocounting . The amount of O6-alkylguanine in competing DNA is calculated by reference to a standard curve constructed using DNA of known alkylation . In view of its relatively wide spectrum of alkyl group specificity, use of AGT from rat liver permits the determination of both O6-methyl- and O6-ethylguanine (detection limits, 0.8 fmol and 3 fmol, respectively) . On the other hand, the restricted specificity of Escherichia coli AGT to repair of O6-methylguanine makes the assay based on it specific for this type of lesion (detection limit, 0.5 fmol) . The maximum amount of DNA which can be included in the assay is 15 micrograms and 10 micrograms for the rat liver and E . coli AGT-based assays, respectively, leading to a limit of sensitivity of 8 x 10(-8) mol O6-methylguanine/mol guanine (50 fmol/mg DNA) (both enzymes) and 3 x 10(-7) mol O6-ethylguanine/mol guanine (200 fmol/mg DNA) (rat liver AGT-based assay) and making this one of the most sensitive assays for these important precarcinogenic adducts . The new assay has been validated by assaying DNA from rat liver methylated in vivo with dimethylnitrosamine to a known extent and has been found to give results in close agreement with those of radioimmunoassay . Six h after i.p . administration of dimethylnitrosamine (0.01-1 mg/kg) to rats, O6-methylguanine was detectable by the competitive-repair assay in liver or lymphocyte DNA at levels of 0.14-14.4 mumol/mol guanine. J Biol Chem, 1989 Dec 15, 264(35), 21167 - 76 Defective homologous pairing and proficient processive unwinding by the recA430 mutant protein and intermediates of homologous pairing by recA protein; Ikawa S et al.; The recA protein promotes the formation and processing of joint molecules of homologous double- and single-stranded DNAs in vitro . Under a set of specified conditions, we found that the substitution of a single amino acid in the recA protein (recA430 mutation) depresses its activity for the homologous pairing to about 1/100 of that by the wild type protein when compared by the rate for the first 2-3 min of the reaction, but that the mutation only slightly, if at all, affects its ability to bind progressively to double-stranded DNA to unwind the double helix ("processive unwinding") . This is in striking contrast to an anti-recA protein monoclonal IgG, ARM193, which severely inhibits the processive unwinding but not the homologous pairing, providing further support for our conclusion that the homologous pairing and processive unwinding are functionally independent of each other . Antibody ARM193 caused the breakdown of spontaneously formed filaments of the recA protein, but the recA430 mutation did not affect the self-polymerization of the protein . The recA430 protein was apparently proficient in the functional binding to a single-stranded DNA and in the hydrolysis of ATP . However, we found that under the above conditions the mutant protein was defective as to homology-independent conjunction of DNA molecules to form a "ternary complex" (of macromolecules) . These results suggest that (i) only one DNA-binding site is sufficient for the recA protein to promote the processive unwinding (the ability of the protein to form spontaneous filaments is closely related to this process) and that (ii) two DNA-binding sites on each of the recA polypeptides or those composed of a dimer (or oligomer) of the polypeptide are required for the recA protein to promote both the conjunction of parental DNA molecules and the homologous pairing (the ability to form the spontaneous filaments is not essential to this process) . (iii) The simultaneous inactivation of the activity to promote the homologous pairing and that to form the ternary complex by the single substitution of the amino acid provides a physical support for the conclusion that the ternary complex is an indispensable intermediate in the homologous pairing. J Biol Chem, 1989 Dec 15, 264(35), 21146 - 52 Characterization of the relA1 mutation and a comparison of relA1 with new relA null alleles in Escherichia coli; Metzger S et al.; The most widely studied "relaxed" mutant of the relA locus, the relA1 allele, is shown here to consist of an IS2 insertion between the 85th and 86th codons of the otherwise wild-type relA structural gene, which normally encodes a 743-amino acid (84 kDa) protein . The RelA protein is a ribosome-dependent ATP:GTP (GDP) pyrophosphoryltransferase that is activated during the stringent response to amino acid starvation and thereby occasions the accumulation of guanosine 3',5'-bispyrophosphate (ppGpp) . We propose that the IS2 insertion functionally splits the RelA protein into two (alpha and beta) peptide fragments which can complement each other in trans to yield residual ppGpp synthetic activity; neither fragment shows this activity when expressed alone . Cell strains with a single copy relA null allele show physiological behavior that is much the same as relA1 mutant strains . Both relA1 and relA null strains accumulate ppGpp during glucose starvation and do not accumulate ppGpp during the stringent response . The presence of ppGpp in verifiable relA null strains is interpreted as unequivocal evidence for an alternate route of ppGpp synthesis that exists in addition to the relA-dependent reaction. J Biol Chem, 1989 Dec 15, 264(35), 20827 - 30 SecB protein stabilizes a translocation-competent state of purified prePhoE protein; Kusters R et al.; Efficient translocation of pure precursor of PhoE protein (prePhoE) could be accomplished in an in vitro system consisting of only inverted Escherichia coli inner membrane vesicles, ATP, and SecA and SecB protein . In this in vitro system SecB and not trigger factor could stabilize a translocation-competent state of prePhoE . In contrast, translocation competency of proOmpA could be induced by both trigger factor and SecB protein, suggesting specificity in interactions between cytosolic factors and precursors in outer membrane protein translocation. J Biol Chem, 1989 Dec 15, 264(35), 21230 - 8 The organization of the purL gene encoding 5'-phosphoribosylformylglycinamide amidotransferase of Escherichia coli; Sampei G et al.; Escherichia coli 5'-phosphoribosylformylglycinamide (FGAR) amidotransferase (EC 6.3.5.3) encoded by the purL gene catalyzes the conversion of FGAR to formylglycinamidine in the presence of glutamine and ATP for the de novo purine nucleotide biosynthesis . On the basis of the nucleotide sequence of purL, the enzyme was dissected along the polypeptide chain into at least three discrete regions, designated as domains I, II, and III, by genetic complementation tests . Domain III (255 amino acids), which resides in the C-terminal region, is similar in amido acid sequence to several glutamine amidotransferases and exerts the transfer of the amide nitrogen of glutamine . Domain I (791 amino acids) resides in the N-terminal region and contains a potential ATP binding motif . Domain II (249 amino acids) locates between domains I and III and is composed of an alternating structure of at least eight predicted beta-strand and alpha-helix elements, as has been observed in the family of triosephosphate isomerases . The functions of domains I and II have been discussed in relation to the transfer of the carbonyl oxygen of FGAR into the gamma-phosphorus moiety of ATP . These results support a model that the E . coli purL gene is a fused gene of at least three different gene families . The highly repetitive sequences of the E . coli genome appeared to play an important role in the process of the gene fusion. J Biol Chem, 1989 Dec 15, 264(35), 21122 - 30 Functional domains of the Escherichia coli dnaK heat shock protein as revealed by mutational analysis; Cegielska A et al.; The employment of a set of truncated dnaK peptides produced by deletion and insertion mutations in the Escherichia coli dnaK gene allowed us to define regions of the dnaK protein which are involved in particular enzymatic functions . The results obtained suggest that the dnaK polypeptide is organized into at least two distinct functional domains . The highly conserved amino-terminal portion is required for the ATPase activity . The carboxyl-terminal portion, characterized by relatively low similarity among species, is responsible for the autophosphorylating activity . The mutant dnaK protein C{74}, which lacks amino acid sequences at the extreme carboxyl-terminal portion of the protein, retains both the ATPase and the autophosphorylating activities . The results obtained with the full-length (70-kDa) dnaK756 protein suggest that the thermolabile defect of the dnaK756 mutation affects directly or indirectly the ATPase active site of the enzyme . The autophosphorylating activity of the dnaK+, dnaK756, and C{74} polypeptides was activated at least 10-fold by the addition of CaCl2. J Biol Chem, 1989 Dec 15, 264(35), 21066 - 72 Nucleotide binding and cofactor activities of purified bovine brain and bacterially expressed ADP-ribosylation factor; Weiss O et al.; The ADP-ribosylation factor (ARF) is a member of the small molecular weight GTP-binding protein family and serves as the cofactor in the cholera toxin-catalyzed activation of the stimulatory regulatory subunit (Gs) of adenylate cyclase . Bovine Arf1 has been expressed at high levels and purified from bacteria . The recombinant Arf1 was compared with purified bovine brain Arf and shown to be nearly identical with respect to immunoblotting, guanine nucleotide binding, GTP hydrolysis, and cholera toxin cofactor activities . The only known chemical difference between the recombinant and brain proteins is the lack of myristic acid at the amino terminus of the expressed protein . The preparation of nucleotide-free Arf1 has allowed a more accurate determination of the binding constants for guanine nucleotides and revealed a significantly higher affinity for GDP than was previously determined . The effect of magnesium ions on nucleotide affinities was also determined and found to be quite different for the different guanine nucleotides . We have shown that GDP binds to the protein in the absence of magnesium, while GTP or guanosine 5'-O-(thiotriphosphate) can only bind to Arf1 in the presence of nanomolar (or higher) levels of the free metal . This characterization of the nucleotide binding and the ability to produce large amounts of a single species of ARF with full retention of a range of activities should greatly facilitate subsequent studies on the structure and function of ARF. Thromb Res, 1989 Dec 15, 56(6), 697 - 708 Methylprednisolone affects inhibitors of the complement and the contact systems; functional and immunochemical studies on alpha 2-macroglobulin and C1 inhibitor; Roeise O et al.; The effects of methylprednisolone sodium succinate (MP) on main inhibitors of the classical pathway of complement and on the contact system were studied in citrated pool plasma . Endotoxin (2.10(9) ng/l, lipopolysaccharide B, E . coli 026: B6 Difco Laboratories, Detroit, Michigan, USA) and/or MP in doses of 0.1, 1, 5 and 10 mg/ml were incubated with plasma at 37 degrees C . Plasma samples were obtained at timed intervals up to 24 hours for determination of C1 inhibitor (C1inh) and alpha 2-macroglobulin (alpha 2M) values using both functional and immunochemical assays . Plasma containing endotoxin without MP revealed decreases of C1inh and alpha 2M values after 12 hours . Addition of MP in high doses (10 mg/ml) gave an additive effect on the endotoxin-induced decreases of C1inh and alpha 2M values, evident 1 and 12 hours after the beginning of incubation, respectively . When MP alone was added to plasma (5 and 10 mg/ml) also significant decreases in C1inh and alpha 2M values were seen . MP in low doses (0.1 and 1 mg/ml) did not either influence the endotoxin-induced changes in the protease inhibitor functions, or induce significant changes in C1inh and alpha 2M values when incubated in plasma without endotoxin . This study demonstrates that MP in high doses induces marked decreases in plasma C1inh and alpha 2M inhibitory functions and that MP has an additive effect on the endotoxin-induced decreases of these inhibitors. Biochem J, 1989 Dec 15, 264(3), 695 - 701 Isolation and sequence analysis of a cDNA encoding rat liver alpha-L-fucosidase; Fisher KJ et al.; cDNA clones for alpha-L-fucosidase were isolated from a rat liver lambda gt11 expression library by using both monospecific polyclonal antibodies against the affinity-purified enzyme and biotinylated rat liver fucosidase cDNA sequences as probes . The largest clone, lambda FC9, contained a 1522 bp full-length cDNA insert (FC9) that encoded the 434-amino acid-residue subunit (Mr 50439) of rat liver alpha-L-fucosidase . A putative signal peptide 28 amino acid residues in length preceded the sequence for the mature protein . In addition, FC9 specified for 11 nucleotide residues of 5' untranslated sequence, 78 nucleotide residues of 3' untranslated sequence and a poly(A) tail . The deduced amino acid sequence from FC9 in conjunction with the experimentally determined N-terminus of the mature enzyme suggested that rat liver fucosidase did not contain a pro-segment . However, there was the possibility of limited N-terminal processing (one to five amino acid residues) having occurred after removal of the predicted signal peptide . Amino acid sequences deduced from FC9 were co-linear with amino acid sequences measured at the N-terminus of purified fucosidase and on two of its CNBr-cleavage peptides . An unusual aspect of rat liver alpha-L-fucosidase protein structure obtained from the FC9 data was its high content of tryptophan (6%) . The coding sequence from FC9 showed 82% sequence identity with that from a previously reported incomplete human fucosidase sequence {O'Brien, Willems, Fukushima, de Wet, Darby, DiCioccio, Fowler & Shows, (1987) Enzyme 38, 45-53}. Gene, 1989 Dec 14, 84(2), 487 - 91 Efficient synthesis of mouse thymidylate synthase in Escherichia coli; Zhang HC et al.; The coding region of the mouse thymidylate synthase (TS)-encoding cDNA (ts) was inserted downstream from the phage T7 promoter and translation initiation signals of the expression vector, pET-3a, and transformed into Escherichia coli BL21(DE3){pLysS} . When the wild-type (wt) cDNA sequence was used, mouse TS was synthesized in the bacterial cells in response to induction, but the level of expression was low . When the second codon (Leu) was changed from CUG, found in the normal mRNA, to CUU, the level of expression increased 17-fold and TS represented 5-10% of total cell protein . The recombinant enzyme was purified to homogeneity by affinity chromatography . The recombinant TS had the same Mr as the enzyme from cultured mouse fibroblasts . Kinetic studies with the recombinant enzyme showed that the apparent Km values for deoxyuridylate and 5,10-methylenetetrahydrofolate were 10.5 and 22 microM, respectively, which were similar to the values for TS from mouse cell extracts . The mouse ts expression vector will be useful for the large-scale production of the wt enzyme and for the creation and analysis of mutant enzymes by protein engineering techniques. Gene, 1989 Dec 14, 84(2), 481 - 5 Asparaginyl-tRNA synthetase from Escherichia coli has significant sequence homologies with yeast aspartyl-tRNA synthetase; Anselme J et al.; The Escherichia coli asnS gene codes for asparaginyl-tRNA synthetase (NRSEC) . We have sequenced the asnS region, including 382 bp of the 5'-untranslated region, 1398 bp of the coding region and 280 bp of the 3'-untranslated region . The DNA-derived NRSEC amino acid (aa) sequence was confirmed by direct aa sequencing of the N-terminal parts of the native protein and of a 28-kDa internal fragment generated by trypsin digestion . The asnS gene product has been purified to homogeneity using three chromatographic steps . Sequence comparison of the deduced NRSEC sequence with all aminoacyl-tRNA synthetase sequences showed significant homologies with the yeast aspartyl-tRNA synthetase and weaker relationships with other aminoacyl-tRNA synthetases for aa with an XAX codon. Gene, 1989 Dec 14, 84(2), 463 - 6 Gene expression in a cell-free system on the preparative scale; Baranov VI et al.; A cell-free system for preparative gene expression is described . It is composed of DNA-free Escherichia coli extract and added plasmid DNA; coupled transcription-translation proceeds with a continuous flow of the feeding solution containing nucleoside triphosphates and amino acids . The system works at a high constant rate for tens of hours . The yield of synthesised proteins after 20-50 h is hundreds of micrograms from 1 ml of the reaction mixture . Electrophoretic analysis of translation products confirms synthesis of proteins of the expected molecular mass. Gene, 1989 Dec 14, 84(2), 311 - 8 Genetic transformation of the filamentous yeast, Trichosporon cutaneum, using dominant selection markers; Glumoff V et al.; An efficient transformation system for the filamentous yeast, Trichosporon cutaneum, has been developed . Transformation was obtained with plasmids carrying either the Escherichia coli hygromycin B phosphotransferase-encoding gene (hph) or the Streptoalloteichus hindustanus phleomycin-resistance gene (ble), as dominant selection markers . Expression of both resistance-conferring genes was controlled by the gpd promoter and the trpC terminator, from Aspergillus nidulans . The transformation frequency was up to 500 colonies/micrograms of transforming DNA, using the ble gene, and up to 100 colonies/micrograms of transforming DNA, using the hph gene . Co-transformation frequencies using unselected DNA varied between 50 and 65% . The transforming DNA was found to consist of multiple tandem plasmid copies of high Mr . This polymeric structure, in nonselective media, was mitotically unstable, possibly indicating that it existed in an episomal state. Gene, 1989 Dec 14, 84(2), 227 - 36 Recognition of nucleotide sequences at the Escherichia coli galactose operon P1 promoter by RNA polymerase; Chan B et al.; Specific nucleotide (nt) sequences in the -35 region are not essential for galP1 promoter activity, whereas nt sequences in the spacer region are needed for transcription initiation: a G:C base pair at nt -14 and sequences upstream from this position are necessary . In the absence of these sequences, transcription initiation is dependent on the insertion of oligodeoxyribonucleotides carrying -35 region consensus hexamer sequences . Additionally, for maximal promoter activity, specific sequences just upstream from nt -49 are required . Because galP1 carries no sequence resembling the -35 region consensus hexamer, we propose that recognition by RNA polymerase proceeds via an unusual mechanism involving contacts upstream from the -10 hexamer, distortion of the spacer region and a contact upstream from nt -49. Gene, 1989 Dec 14, 84(2), 237 - 45 The nature of an intragenic suppressor of the Escherichia coli dnaA508 temperature-sensitive mutation; Eberle H et al.; Escherichia coli strain E508 (dnaA508) is temperature-sensitive for dnaA function . A mutant with an intragenic suppressor of the dnaA508 mutation, called PR1, has been isolated . The suppressor mutation(s) allow initiation of DNA synthesis at 42 degrees C and, like dnaA cold-sensitive mutants, PR1 grows poorly at 32 degrees C . Two-dimensional gel analysis indicates that DnaA protein is overproduced in PR1 . Transcriptional analysis indicates two to three times the number of dnaA and dnaN transcripts in PR1, as compared to a wild-type dnaA+ strain . The dnaA gene from PR1 has been cloned and found to complement the original dnaA508 mutation, as well as dnaA46, but not dnaA5 . Sequencing of the dnaAPR1 gene reveals three separate base changes, two of which result in nonconservative amino acid substitutions and the third is a change in the start codon from GTG to ATG. Gene, 1989 Dec 14, 84(2), 257 - 66 A promoter-probe vector-host system for the cyanobacterium, Synechocystis PCC6803; Ferino F et al.; A vector-host system for testing promoters in the cyanobacterium Synechocystis PCC6803 has been constructed . It relies on a small Escherichia coli promoter-probe plasmid, pFF11, which has four unique restriction sites in a polylinker upstream from the cat reporter gene . This plasmid is able to obtain a cyanobacterial origin of replication by homologous recombination with the resident plasmid of the recipient host, generating a new E . coli-Synechocystis PCC6803 shuttle vector . This plasmid does not confer any detectable chloramphenicol acetyl transferase activity to this cyanobacterium in the absence of a promoter insert . Several heterologous promoters were tested in Synechocystis PCC6803 using this system . Results obtained with the lambda pR promoter and the repressor-encoding cI857 gene demonstrate that these elements can be used for high-level and tightly regulated gene expression in Synechocystis PCC6803. Gene, 1989 Dec 14, 84(2), 407 - 17 Construction and properties of an Epstein-Barr-virus-derived cDNA expression vector for human cells; Belt PB et al.; A cDNA expression vector containing the element oriP and the sequence encoding the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) as well as the hygromycin B-resistance dominant marker gene has been constructed . Its characteristics have been compared to a similar vector lacking the EBV sequences . (a) The EBV+ vector is maintained as an episome with a copy number of approx . 50 per cell, whereas the number of the integrated EBV- copies is in general smaller than 10, when simian virus 40-transformed xeroderma pigmentosum fibroblasts (XP20S-SV) constitute the recipient cell line . (b) The presence of the EBV sequences in the vector resulted in a five- to ten-fold higher transfection efficiency with the Ca.phosphate precipitation technique . (c) cDNA inserts in the EBV+ vector are shown to be efficiently and properly expressed in the recipient cell . (d) If transfection is performed with a mixture of EBV+ vectors with different inserts, transfectants are shown to harbour different plasmids within one cell . (e) The ratio between these plasmids in one cell can be shifted in favour of a vector with a particular insert, when selection for this insert is performed . (f) Reconstruction experiments indicated that isolation of a low-abundance sequence from a mixture of vectors is at least 100-fold more efficient with the EBV+ system, than with the EBV- system . (g) Rescue of the episomal vector from transfected cells can be readily achieved. Gene, 1989 Dec 14, 84(2), 359 - 69 Structure and expression analyses of the S-adenosylmethionine synthetase gene family in Arabidopsis thaliana; Peleman J et al.; The plant, Arabidopsis thaliana, contains two S-adenosylmethionine synthetase-encoding genes (sam) . Here, we analyze the structure and expression of the sam-2 gene and compare it with the previously described sam-1 gene . Northern-blot analysis using gene-specific probes shows that both sam-1 and sam-2 are highly expressed in stem, root, and callus tissue . This similar expression pattern might be mediated by the presence of three highly conserved sequences in the 5' region of both sam genes . Using a chimeric beta-glucuronidase (GUS)-encoding gene, we show that in transgenic tobacco plants, 748 bp of 5' sam-1 sequences generate high GUS activity in the same type of tissues as previously observed in transgenic A . thaliana plants . A deletion analysis of these 5' sam-1 sequences indicates that 224 bp of 5' sam-1 sequences can still induce higher expression of the gene in stem and root relative to leaf . However, the level of expression is reduced when compared to the expression level obtained with the full-length promoter. Nature, 1989 Dec 14, 342(6251), 816 - 9 Specific binding of HIV-1 recombinant Rev protein to the Rev-responsive element in vitro; Daly TJ et al.; The human immunodeficiency virus type 1 (HIV-1) genome encodes the regulatory protein Rev, of relative molecular mass 13,000, which is synthesized from fully processed viral transcripts before synthesis of HIV-1 structural proteins . Rev has been postulated to exert control within the nucleus at the level of messenger RNA processing . The availability of Rev in the nucleus serves to increase the proportion of unspliced and singly spliced mRNA species relative to fully spliced mRNA molecules, resulting in an increased synthesis of viral structural proteins . A highly conserved cis-acting sequence termed the Rev-responsive element (RRE) has been identified in the envelope gene (env) of the viral transcript that seems to control mRNA processing in a Rev-dependent manner . Genetic studies have identified rev gene mutants with dominant phenotypes, supporting the hypothesis that Rev interacts directly with the RRE . Here we demonstrate that Rev protein, purified from Escherichia coli, binds in a sequence-specific manner to the RRE element in vitro. Biochemistry, 1989 Dec 12, 28(25), 9586 - 93 Resonance energy transfer measurements between substrate binding sites within the large (Klenow) fragment of Escherichia coli DNA polymerase I; Allen DJ et al.; Resonance energy transfer was used to determine separation distances between fluorescent derivatives of substrates for Klenow fragment and a unique sulfhydryl, cysteine 907, on the enzyme . Fluorescent derivatives of duplex DNA, deoxynucleotide triphosphates (dNTP), and deoxynucleotide monophosphates (dNMP), modified with aminonaphthalenesulfonates (ANS), served as energy-transfer donors to the fluorophore used to modify cysteine 907, 4-{N-{(iodoacetoxy)ethyl}-N-methylamino}-7-nitrobenz-2-oxa-1,3-diazole (IANBD) . The labeling of cysteine 907 with NBD caused no decrease in the enzyme's polymerase activity, suggesting that the probe did not significantly alter the conformation of the enzyme . The efficiency of singlet-singlet resonance energy transfer was determined from the quantum yield of the donor in the presence and absence of acceptor . By Forster's theory, the measured distances between cysteine 907 and binding sites for duplex DNA, dNTP, and dNMP were 25-39, 19-28, and 17-26 A, respectively . As the fluorophores, attached to the substrates via a tether arm, are separated from the substrates by approximately 12 A, the distances measured between binding sites are subject to this uncertainty . To measure the separation between binding sites for duplex DNA and dNMP, and to reduce the uncertainty introduced by the tether arm, two experiments were carried out . In the first, duplex DNA was labeled with the acceptor fluorophore NBD and used with the donor ANS-modified dNMP to yield a measured distance separating these two sites of 19-28 A.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1989 Dec 12, 28(25), 9602 - 7 Alternative proton donors/acceptors in the catalytic mechanism of the glutathione reductase of Escherichia coli: the role of histidine-439 and tyrosine-99; Deonarain MP et al.; The cloned Escherichia coli gor gene encoding the flavoprotein glutathione reductase was placed under the control of the tac promoter in the plasmid pKK223-3, allowing expression of glutathione reductase at levels approximately 40,000 times those of untransformed cells . This greatly facilitated purification of the enzyme . By directed mutagenesis of the gor gene, His-439 was changed to glutamine (H439Q) and alanine (H439A) . The tyrosine residue at position 99 was changed to phenylalanine (Y99F), and in another experiment, the H439Q and Y99F mutations were united to form the double mutant Y99FH439Q . His-439 is thought to act in the catalytic mechanism as a proton donor/acceptor in the glutathione-binding pocket . The H439Q and H439A mutants retain approximately 1% and approximately 0.3%, respectively, of the catalytic activity of the wild-type enzyme . This reinforces our previous finding {Berry et al . (1989) Biochemistry 28, 1264-1269} that direct protonation and deprotonation of the histidine residue are not essential for the reaction to occur . The retention of catalytic activity by the H439A mutant demonstrates further that a side chain capable of hydrogen bonding to a water molecule, which might then act as proton donor, also is not essential at this position . Tyr-99 is a further possible proton donor in the glutathione-binding pocket, but the Y99F mutant was essentially fully active, and the Y99FH439Q double mutant also retained approximately 1% of the wild-type specific activity.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1989 Dec 12, 28(25), 9594 - 602 Inactivation of the pyruvate dehydrogenase complex of Escherichia coli by fluoropyruvate; Flournoy DS et al.; The pyruvate dehydrogenase complex (PDH complex) of Escherichia coli and its pyruvate dehydrogenase component (E1) are rapidly inactivated by low concentrations of fluoropyruvate in a thiamin pyrophosphate (TPP) dependent process . The inactivation rates for the PDH complex and for its E1 component are similar . Pyruvate protects the PDH complex and the E1 component against inactivation by fluoropyruvate . Dihydrolipoamide protects the E1 component from inactivation . TPP is not covalently bound to the PDH complex or to the E1 component by the inactivating reaction . When {14C}fluoropyruvate is used to inactivate the PDH complex, 14C remains bound to the complex after gel filtration . This bound radioactivity is cleaved from the protein by NH2OH, -OH, and NaBH4 but not by dilute acid . When released by -OH, greater than 90% of the 14C cochromatographs with acetate on DEAE-Sephadex . When released by NaBH4, and 14C is recovered as {14C}ethanol . Colorimetric analysis for sulfhydryl groups on the native E1 component and the inactivated E1 component, using 5,5'-dithiobis(2-nitrobenzoate), reveals that complete inactivation results from covalent modification of 1.37 +/- 0.03 sulfhydryl residues . Fluoropyruvate is known to generate acetyl-TPP at the active site of E1 . The available evidence indicates that acetylation of a sulfhydryl group by acetyl-TPP at the active site of the E1 component inactivates the enzyme. Nucleic Acids Res, 1989 Dec 11, 17(23), 9979 - 91 Nucleotide sequence of katF of Escherichia coli suggests KatF protein is a novel sigma transcription factor; Mulvey MR et al.; The katF gene of Escherichia coli has been sequenced revealing a 1086 base pair open reading frame from which the sequence of a 362 amino acid protein has been deduced . The direction of transcription of katF was confirmed by expression of the gene cloned in both directions behind a T7 promoter . The KatF protein expressed in vitro migrates with an apparent size of 42 kDa . Comparison of the katF sequence to the sequence of rpoD, which encodes the sigma subunit of RNA polymerase, revealed a 181 bp region with 65% homology and a 38 bp segment that was 87% homologous . A 62 amino acid region of the predicted KatF protein sequence was found to be 85% homologous to the corresponding sequence of sigma 70, including a segment implicated in core polymerase binding . Homology was also observed with the heat shock regulatory protein encoded by htpR. Nucleic Acids Res, 1989 Dec 11, 17(23), 9889 - 908 Site-directed cross-linking of mRNA analogues to the Escherichia coli ribosome; identification of 30S ribosomal components that can be cross-linked to the mRNA at various points 5' with respect to the decoding site; Stade K et al.; Three different mRNA analogues (28 to 34 nucleotides long) were prepared by T7 transcription from synthetic DNA templates . Each message contained the sequence ACC-GCG (coding for threonine and alanine, respectively), together with a single thio-U residue located at a variable position on the 5'-side of these coding triplets . A photo-reactive group was introduced by substitution of the thio-U with 4-azidophenacyl bromide . The messages were bound to E . coli 70S ribosomes in the presence of the appropriate tRNA-Thr or tRNA-Ala, and the azidophenyl group was photoactivated . Cross-linking was found to occur exclusively within the 30S subunit, with the 32P-label in the cross-linked mRNA being divided roughly equally between 30S ribosomal proteins and 16S RNA . Immunological analysis of the cross-linked proteins showed that, in the presence of either tRNA species, protein S7 was the primary target, whereas in the absence of tRNA only small amounts of protein S21 were cross-linked . The cross-link site to 16S RNA lay in all cases very close to its extreme 3'-terminus . These data indicate that the outgoing message leaves the cleft of the 30S subunit in a "northerly" direction. Nucleic Acids Res, 1989 Dec 11, 17(23), 9735 - 47 Initiation complex formation on Euglena chloroplast 30S subunits in the presence of natural mRNAs; Wang CC et al.; An in vitro system has been developed that allows the formation of translation initiation complexes with Euglena chloroplast 30S ribosomal subunits and natural mRNAs . For these experiments two regions of the Euglena chloroplast genome have been cloned behind the T7 transcriptional promoter and the corresponding RNAs synthesized in vitro . These mRNAs are capable of forming initiation complexes with chloroplast 30S subunits in the presence of fMet-tRNA and E . coli initiation factors . Deletion of the normal translation start site results in a message that is no longer recognized by the chloroplast subunits suggesting that the correct AUG initiation codon on the mRNA is being selected by the small ribosomal subunit . Initiation complex formation with the chloroplast 30S subunits is specific for chloroplast mRNAs and mRNA from the phage MS2 is not active in this system. Nucleic Acids Res, 1989 Dec 11, 17(23), 9719 - 33 Interaction of novel bis(platinum) complexes with DNA; Roberts JD et al.; Bis(platinum) complexes {{cis-PtCl2(NH3)}2H2N(CH2)nNH2} are a novel series of potential anticancer agents in which two cis-diamine(platinum) groups are linked by an alkyldiamine of variable length . These complexes are potentially tetrafunctional, a unique feature in comparison with known anticancer agents . Studies of DNA interactions of bis(platinum) complexes in comparison with cisplatin demonstrate significant differences . Investigations of interstrand crosslink formation in which crosslinking of a short DNA fragment is detected by gel electrophoresis under denaturing conditions demonstrate that interstrand crosslinks are 250 fold more frequent among bis(platinum) adducts than among cisplatin-derived adducts under the conditions examined . These investigations indicate that bis(platinum) adducts contain a high frequency of structurally novel interstrand crosslinks formed through binding of the two platinum centers to opposite DNA strands . Unlike cisplatin, bis(platinum) complex binding does not unwind supercoiled DNA . Studies with the E . coli UvrABC nuclease complex demonstrate that both linear and supercoiled DNA containing bis(platinum) adducts are subject to incision by the repair enzyme complex . Initial studies using UvrABC nuclease as a probe to define the base and sequence specificity for bis(platinum) complex binding suggest that the specificity of the bis(platinum)s is similar, but not identical, to that of cisplatin. Nucleic Acids Res, 1989 Dec 11, 17(23), 9621 - 36 An enlarged largest subunit of Plasmodium falciparum RNA polymerase II defines conserved and variable RNA polymerase domains; Li WB et al.; We have isolated the gene encoding the largest subunit of RNA polymerase II from Plasmodium falciparum . The RPII gene is expressed in the asexual erythrocytic stages of the parasite as a 9 kb mRNA, and is present as a single copy gene located on chromosome 3 . The P . falciparum RPII subunit is the largest (2452 amino acids) eukaryotic RPII subunit, and it contains enlarged variable regions that clearly separate and define five conserved regions of the eukaryotic RPII largest subunits . A distinctive carboxyl-terminal domain contains a short highly conserved heptapeptide repeat domain which is bounded on its 5' side by a highly diverged heptapeptide repeat domain, and is bounded on its 3' side by a long carboxyl-terminal extension. Nucleic Acids Res, 1989 Dec 11, 17(23), 9749 - 59 The presence of the region on pBR322 that encodes resistance to tetracycline is responsible for high levels of plasmid DNA knotting in Escherichia coli DNA topoisomerase I deletion mutant; Shishido K et al.; Plasmid pBR322 DNA isolated from Escherichia coli DNA topoisomerase I deletion mutant DM800 is estimated to contain about 10% of the knotted forms (Shishido et al., 1987) . These knotted DNA species were shown to have the same primary structure as usual, unknotted pBR322 DNA . Analysis of the knotting level of deletion, insertion and sequence-rearranged derivatives of pBR322 in DM800 showed that the presence of the region on pBR322 encoding resistance to tetracycline (tet) is required for high levels of plasmid knotting . When the entire tet region is present in a native orientation, the level of knotting is highest . Inactivating the tet promoter is manifested by a middle level of knotting . For deletion derivatives lacking various portions of the tet region, the level of knotting ranges from lowest to high depending on the site and length of the tet gene remaining . Inverting the orientation of tet region on the pBR322 genome results in a middle level of knotting . Deleting the ampicillin-resistance (bla)gene outside of its second promoter does not affect the level of knotting, if the entire tet gene remains . A possible mechanism of regulation of plasmid knotting is discussed. Eur J Biochem, 1989 Dec 8, 186(1-2), 227 - 32 Expression and processing of cyanobacterial Mn-stabilizing protein in Escherichia coli; Kuwabara T et al.; The woxA gene of cyanobacterium Anacystis nidulans R2, which encodes the precursor of the Mn-stabilizing protein involved in photosynthetic water oxidation, was found to be expressed in Escherichia coli . The 30-kDa expression product was indistinguishable from the authentic mature protein on SDS/PAGE . Upon fractionation of E . coli cells, the expression product was co-precipitated with the membrane fraction, which is consistent with the water-insoluble nature of the authentic mature protein . Analysis of the amino-terminal amino acid sequence of the product revealed that it is identical to the sequence from the 28th residue of the precursor, indicating that the precursor is processed in E . coli . The expression product was digested by trypsinization of E . coli spheroplasts, but not by that of intact cells . This observation suggests that the product is secreted from the cytoplasmic membrane, but not from the outer membrane . The occurrence of both processing and secretion suggests that a signal peptidase of E . coli can recognize the structure for translocation across the thylakoid membrane . Comparison of the signal sequence and the presequence of sweet potato sporamin A suggests that the processing enzymes of the thylakoid membrane and endoplasmic reticulum possess a common substrate specificity. Eur J Biochem, 1989 Dec 8, 186(1-2), 137 - 43 Expression of recombinant diaminopimelate epimerase in Escherichia coli . Isolation and inhibition with an irreversible inhibitor; Higgins W et al.; Recombinant diaminopimelate epimerase is overproduced to give 1% of soluble protein when grown under the appropriate conditions in Escherichia coli . This compares with 0.02% of the constitutive level of wild-type enzyme . A new purification procedure now yields milligram quantities of homogeneous enzyme of high specific activity (192 U/mg) . This has enabled sufficient amounts of enzyme both to compare with wild-type enzyme and to enable active site modification studies to be performed . Incubation of the enzyme with 2-(4-amino-4-carboxybutyl)-2-aziridine-carboxylic acid (AZIDAP), results in time-dependent irreversible inhibition . Tryptic digestion of the inactivated enzyme and peptide-mapping show that AZIDAP is specifically and covalently bound to the enzyme at a unique peptide . Determination of the amino acid sequence of this peptide and comparison with the sequence deduced from the DNA sequence of the dapF gene shows that Cys73 is labelled . Finally based on limited sequence similarities around this cysteine and active-site cysteines of proline racemase and 1-hydroxyproline 2-epimerase, together with mechanistic considerations, we propose that all three non-pyridoxal-phosphate-containing racemases/epimerases derive from a common evolutionary origin. Eur J Biochem, 1989 Dec 8, 186(1-2), 87 - 93 The synthesis and functional evaluation of RNA and DNA polymers having the sequence of Escherichia coli tRNA(fMet); Perreault JP et al.; Stepwise, solid-phase chemical synthesis has provided long RNA and DNA polymers related to the sequence of Escherichia coli tRNA(fMet) . The 34-ribonucleotide oligomer corresponding to the sequence of the 5'-half tRNA molecule has been synthesized and then characterized by gel purification, terminal nucleotide determinations and sequence analysis . This 34-nucleotide oligomer serves as an acceptor in the RNA-ligase-catalyzed reaction with a phosphorylated 43-ribonucleotide oligomer corresponding to the sequence of the 3'-half molecule of tRNA(fMet) . The DNA molecule having the sequence of tRNA(fMet) is a 76-deoxyribonucleotide oligomer with a 3'-terminal riboadenosine residue and all U residues replaced by T . These polymers have been compared with an oligodeoxyribonucleotide lacking all 2'-hydroxyl groups except for the 3'-terminal 2'-OH, an oligoribonucleotide lacking modified nucleosides and E . coli tRNA(fMet) . The all-RNA 77-nucleotide oligomer can be aminoacylated by E . coli methionyl-tRNA synthetase preparation from E . coli with methionine and threonylated in the A37 position using a yeast extract . In agreement with work by Khan and Roe using tDNA(Phe) and tDNA(Lys), the rA77-DNA(fMet) can be aminoacylated, and preliminary evidence suggests that it can be threonylated to a small extent . Kinetic data support the notion that aminoacylation of tRNA(fMet) does not depend on the presence of 2'-hydroxyl groups with the exception of that in the 3'-terminal nucleotide. Eur J Biochem, 1989 Dec 8, 186(1-2), 303 - 8 The voltage-dependent activity of Escherichia coli porins in different planar bilayer reconstitutions; Lakey JH et al.; Because of conflicting results from differing techniques, the degree of voltage sensitivity of Escherichia coli porins in planar bilayers is still a matter of debate . In order to provide the first comparative study, OmpF porin was purified in three ways; firstly as native outer membrane vesicles, secondly as salt-extracted porin trimers in sodium dodecyl sulphate and thirdly as solubilised trimers extracted with octyl-polyoxyethylene (Octyl-POE) . These methods represent the major approaches to porin isolation and purification . All three were reconstituted into Schindler-type bilayers . Detergent-solubilised OmpF was also reconstituted into Montal-Mueller- and Mueller-Rudin-type bilayers . In all cases voltage-dependent closing of OmpF was observed . Octyl-POE-extracted PhoE porin was similarly investigated in all three types of planar bilayer . Two membrane-formation techniques appeared genuinely to alter the voltage sensitivity of the porins they contained . Firstly, porins in membranes formed by the Montal-Mueller technique sometimes showed an increase in voltage sensitivity during the first 30 min after bilayer formation . Secondly, membranes formed by the Mueller-Rudin technique on thick polyethylene septa showed both poor solvent drainage and a significantly reduced porin voltage sensitivity. Gene, 1989 Dec 7, 84(1), 17 - 22 Distinct functional contributions of three potential secondary structures in the phage G4 origin of complementary DNA strand synthesis; Hiasa H et al.; Three potential secondary structures, stem-loops I, II, and III, are contained in the phage G4 origin of complementary DNA strand synthesis, G4oric, and are believed to be involved in its recognition by dnaG-encoded primase and the synthesis of primer RNA . In a previous publication {Sakai et al., Gene 71 (1988) 323-330}, we suggested that base pairing between the loops of stem-loops I, and II, and/or II and III, might play a role in G4oric function . To test this hypothesis, site-directed mutagenesis was used to construct mutants which carried base substitutions in loops I, II and III that destroyed possible interloop base pairing . These mutations, however, did not seriously affect G4oric activity . This indicates that base pairing between the loops is not essential for G4oric functional activity, and also that base substitutions which do not affect the secondary structure of stem-loops I, II and III, do not affect G4oric activity . To complete an analysis of the effects of altering the structure of the G4oric stem-loops, insertions were made into stem-loop III . In contrast to stem-loops I and II, all insertions into stem-loop III destroyed in vivo G4oric activity. Gene, 1989 Dec 7, 84(1), 165 - 72 Gene engineering by selectable intraplasmid recombination: construction of novel dihydrofolate reductase minigenes; Crouse GF et al.; An intraplasmid recombination system in Escherichia coli has been designed to make possible the engineering of various genes using methods that greatly reduce dependence on appropriately placed restriction enzyme sites . This system has been used to manipulate intervening sequences in dihydrofolate reductase minigenes and to vary the number of 48-bp repeats in the promoter region . In this method, the two fragments to be recombined are cloned into a plasmid separated by a fragment of DNA containing an expressible galactokinase-encoding gene (galK) . Selection for loss of the galK gene, but for retention of the plasmid in E . coli, results in a plasmid in which the two fragments have undergone homologous recombination . Several new plasmids are reported here which contain an expressible galK gene flanked by multiple restriction sites . These plasmids should be useful in recombination and as convenient sources of a gene for which both positive and negative selections are available in E . coli. Gene, 1989 Dec 7, 84(1), 159 - 64 Construction of linker-scanning mutations using a kanamycin-resistance cassette with multiple symmetric restriction sites; Smith ML et al.; We demonstrate how a kanamycin-resistance (KmR) cassette flanked by polylinkers with multiple restriction sites can be used to introduce nucleotide (nt) sequence replacements into a region of interest . This method differs in two significant ways from traditional methods of linker mutagenesis . First, the presence of the KmR gene allows for selection of the polylinker, greatly facilitating formation of linker-containing molecules . Second, the polylinker with multiple restriction sites allows a given linker insertion to be combined with a second linker insertion in a variety of different ways and makes possible a range of novel nt to remain in the resulting linker replacement . The result of this flexibility is that fewer different molecules are needed to cover a region, and that relatively large replacements (greater than 40 nt) are possible . We have used this method to introduce a series of sequence replacements that span the mouse dihydrofolate reductase promoter region. Gene, 1989 Dec 7, 84(1), 143 - 51 A reliable method for random mutagenesis: the generation of mutant libraries using spiked oligodeoxyribonucleotide primers; Hermes JD et al.; A new procedure for the production of a defined library of random mutants is described . Long spiked oligodeoxyribonucleotides (oligos), in which a predetermined level of the three 'wrong' phosphoramidites are used at each position, are made as primers for a standard oligo-directed mutagenesis protocol . Spiked oligo synthesis on a DNA synthesizer is achieved using an in-line mixing procedure that only requires five phosphoramidite reservoirs and which avoids contamination of any of the pure phosphoramidite reagents . Immutable positions (i.e., positions in the oligo for which pure reagents are used) can be specified, and a silent 'marker' base can be included that allows an early estimate of the mutagenesis efficiency . The randomness of the library in respect to the number, type, and position of the altered bases, is easily verified by DNA sequencing . This procedure has been used to generate a random mutant library of the gene encoding a sluggish triosephosphate isomerase . Among the transformants from this library, a number of second-site suppressor mutations have been found that increase the specific catalytic activity of the starting isomerase . This approach provides a more complete library than a method using chemical mutagenic reagents. Nature, 1989 Dec 7, 342(6250), 714 - 6 Sequence-specific RNA binding by the HIV-1 Rev protein; Zapp ML et al.; The human immunodeficiency virus type 1 (HIV-1) Rev protein acts post-transcriptionally to increase the amounts of the viral gag-pol and env messenger RNAs in the cytoplasm of infected cells . The mechanism of Rev action is uncertain . Possibilities include an accelerating effect on the rate of export of its mRNA targets from the nucleus and/or modulation of the splicing of pre-mRNAs . Both the gag-pol and env mRNAs contain a sequence that is required for responsiveness to Rev--the Rev responsive element, RRE . Here we show that Rev is a sequence-specific binding protein, whose binding site is the RRE . This information should help to clarify the mechanism by which Rev acts. Gene, 1989 Dec 7, 84(1), 73 - 81 Synthesis in Escherichia coli of the major glycoprotein of human rotavirus: analysis of the antigenic regions; Johnson MA et al.; Various regions of the gene encoding the major neutralization antigen, VP7, of human rotavirus have been expressed in Escherichia coli, as N-terminal fusions to beta-galactosidase under the control of the lac promoter . We have determined that the fusion products of two clones containing regions AB (aa 69-158) and ABC (aa 69-319) were antigenic, reacting with antibodies raised against whole virus . When guinea pigs were immunized with fusion protein purified by monoclonal antibody affinity columns, no neutralizing or virus-binding antibodies were detected, but antibodies binding to denatured VP7 were present. J Mol Biol, 1989 Dec 5, 210(3), 659 - 63 Ribosomal affinity and translational initiation in Escherichia coli . In vitro investigations using translational initiation regions of differing efficiencies from the atp operon; Lang V et al.; The atp operon of Escherichia coli comprises nine genes that are differentially expressed . The control of the atp genes' expression rates has been shown to be exercised primarily at the level of translational initiation, but how is this achieved in molecular terms? In order to study the interactions of 30 S ribosomal subunits with specifically the translational initiation regions (TIRs) of atpB, atpE and atpG, restriction fragments bearing these TIRs were excised from the atp operon and cloned into an SP6 promoter transcription vector . mRNA transcripts were made in vitro and used in primer extension inhibition studies and equilibrium mRNA-30 S ribosomal subunit binding measurements . The binding of 30 S ribosomal subunits blocked primer extension 14 to 15 bases downstream from the respective translational start codons . The affinities of binding of 30 S ribosomal subunits showed the relationship atpE greater than atpB greater than atpG . This was also the order of the efficiency of translation promoted by the respective TIRs, both in vivo and on the in vitro synthesized mRNA fragments . Thus, the affinity of 30 S ribosomal subunits is at least to some extent correlated with the rate of translational initiation. J Mol Biol, 1989 Dec 5, 210(3), 551 - 9 Signal transduction in the phosphate regulon of Escherichia coli involves phosphotransfer between PhoR and PhoB proteins; Makino K et al.; PhoB protein is the transcriptional activator for genes in the phosphate regulon of Escherichia coli, such as phoA and pstS, that are induced by phosphate deprivation . PhoR protein activates PhoB when phosphate is limiting and inactivates it when phosphate is in excess . We constructed a plasmid with a mutant phoR gene (phoR1084), which encoded a PhoR protein (PhoR1084) lacking the amino-terminal hydrophobic region of the intact protein . The cells carrying the plasmid overproduced PhoR1084, which was recovered in the soluble fraction of the cell lysate . We purified the Phor1084 protein and showed that it was autophosphorylated in the presence of ATP, and the phosphate group on the protein was rapidly transferred to PhoB . The phosphorylation of PhoB protein occurred concurrently with the acquisition of the ability to activate transcription from the pstS promoter . On the basis of these findings, we propose that phosphorylated PhoB protein activates transcription from the promoters of the phosphate regulon, and that the role of PhoR is to catalyze the formation and breakdown of phosphorylated PhoB in response to phosphate concentrations in the medium. J Mol Biol, 1989 Dec 5, 210(3), 521 - 30 Intermediates in the formation of the open complex by RNA polymerase holoenzyme containing the sigma factor sigma 32 at the groE promoter; Cowing DW et al.; The interaction of E sigma 32 with the groE promoter at temperatures between 0 degrees C and 37 degrees C was studied using DNase I footprinting and dimethyl sulfate methylation . Three distinct complexes were observed . At 0 degrees C E sigma 32 fully protected sequences between -60 and -5 from DNase I digestion on the top (non-template) strand of the promoter . At 16 degrees C the majority of the E sigma 32 promoter complexes had a DNase I footprint almost identical with that seen at 37 degrees C, protecting the DNA from about -60 to +20; however, little DNA strand separation had occurred, and the changes in sensitivity of guanine residues to dimethyl sulfate methylation caused by E sigma 32 differed from those seen at 37 degrees C . DNA strand separation, and changes in the pattern of protections from and enhancements of methylation by dimethyl sulfate to those characteristic of the open complex, occurred at temperatures between 16 degrees C and 27 degrees C . It is plausible to assume that these temperature-dependent isomerizations are analogous to the time-dependent sequence of intermediates on the pathway to open complex formation at 37 degrees C . Therefore we propose that the formation of an open complex by E sigma 32 at the groE promoter involves three classes of steps: E sigma 32 initially binds to the promoter in a closed complex (RPC1) in which the enzyme interacts with a smaller region of the DNA than in the open complex . E sigma 32 then isomerizes to form a second closed complex (RPC2) in which the enzyme interacts with the same region of the DNA as in the open complex . Finally, a process of local DNA denaturation (strand opening) leads to formation of the open complex (RPO). J Mol Biol, 1989 Dec 5, 210(3), 513 - 20 Interaction of Escherichia coli RNA polymerase holoenzyme containing sigma 32 with heat shock promoters . DNase I footprinting and methylation protection; Cowing DW et al.; The DNase I protection pattern of E sigma 32 was assayed on three heat shock promoters, the E sigma 32 promoter for the groESL operon, P2 of the dnaKJ operon, and rpoD PHS, the E sigma 32 promoter upstream from rpoD . E sigma 32 protected each of these promoters from DNase I digestion from around -60 to around +20 . Protection from dimethyl sulfate methylation was assayed at the groE promoter . E sigma 32 binding altered the sensitivity to methylation of bases in the vicinity of both the -10 and -35 regions . The DNase I footprints for the E sigma 32 promoters were very similar to the DNase I footprint of E sigma 70 on the lacUV5 promoter . After analyzing the DNase I footprints by taking into account the contacts predicted to be made by DNase I, it appeared that E sigma 32, like E sigma 70, contacts the DNA primarily on one face of the helix in the -35 region and on both faces in the -10 region. J Mol Biol, 1989 Dec 5, 210(3), 473 - 84 Visualization of RecA protein and its complexes with DNA by quick-freeze/deep-etch electron microscopy; Heuser J et al.; Freeze-etch electron microscopy of pure RecA protein aggregates, as well as of RecA protein complexes on single-stranded and double-stranded DNA formed with various nucleotides, has permitted a clearer discrimination between the two different helical polymers that this protein forms . Both are continuous, single-start, right-handed helices; however, the form observed when ATP or non-hydrolyzable ATP analogs are present has a pitch of 9.5 nm and a diameter of 10 nm, while the other form, observed in the absence of ATP or its analogs, or in the presence of ADP, has a pitch of 6 nm and a diameter of 12 nm . The former "long pitch" helix is found only when RecA protein is bound to DNA . The latter "short pitch" helix is also observed in pure RecA protein polymers (also termed rods) and in the needle-like paracrystals of RecA protein that form in the presence of magnesium or spermidine ions, representing bundles of rods closely packed in register . Addition of ATP or non-hydrolyzable ATP analogs in the absence of DNA dissociates the pure RecA protein crystals, as well as individual helical rods, into short curvilinear chains of attached monomers . These chains typically form closed, circular rings of 7(+/- 1) protein monomers, similar in construction to a single turn of the RecA protein helix, but significantly broader in diameter . The role of ATP in interconverting the various polymeric forms of RecA protein is discussed within the context that ATP functions as a reversible allosteric effector of RecA protein, much as it mediates reversible conformational changes in other vectoral motor proteins such as myosin, dynein, kinesin and the 70,000 Mr "heat shock" ATPases . We discuss how cyclic conversions back and forth between the short- and long-pitch conformations of RecA protein could mediate in reversible single-stranded and double-stranded DNA interactions during the search for homology. J Mol Biol, 1989 Dec 5, 210(3), 439 - 52 Autodigestion and RecA-dependent cleavage of Ind- mutant LexA proteins; Lin LL et al.; The LexA repressor of Escherichia coli undergoes a specific cleavage reaction in vivo, an event that leads to derepression of the SOS regulon and requires an activated form of RecA protein . In vitro, cleavage requires RecA at neutral pH; at alkaline pH, a spontaneous cleavage reaction termed autodigestion takes place . Both autodigestion and RecA-mediated cleavage cut the same bond, and are observed for the same set of substrates, suggesting that RecA acts indirectly to stimulate LexA self-cleavage at neutral pH, perhaps binding to LexA and acting as an allosteric effector . We previously isolated a set of lexA(Ind-) mutants that are deficient in in vivo RecA-mediated cleavage but retain significant repressor function . Here, we describe the in vitro cleavage of purified mutant proteins . All of those tested were deficient in both cleavage reactions . Although most of them were equally deficient in both reactions, some were more deficient in one reaction than the other . Several mutant proteins appeared to have defects in binding to RecA . Autodigestion of all but one of the poorly cleavable mutant proteins reached a maximum rate at pH around 10, as does wild-type LexA . The exception was KR156, which changed Lys156, a residue previously implicated in the mechanism of cleavage, to Arg, another basic residue: for this protein, the rate of autodigestion increased with pH at values above 11 . RecA-mediated cleavage of KR156 was 1% the wild-type rate at pH 7, but increased with increasing pH to a plateau at pH 9.5, where the rate was 40% the wild-type rate . In contrast, an essentially constant rate was observed for wild-type LexA over the pH range 6 to 11 . We suggest, first, that deprotonation of Arg156 and, by inference, Lys156 in the wild-type protein, is required for both autodigestion and RecA-mediated cleavage: and second, that RecA acts to reduce the pKa of Lys156, allowing efficient cleavage of wild-type repressor under physiological conditions. Eur J Pharmacol, 1989 Dec 5, 172(6), 443 - 51 HIV-1 protease: mutagenesis of asparagine 88 indicates a domain required for dimer formation; Guenet C et al.; Considerable interest exists in the HIV-1 protease for biochemical studies as a potential therapeutic target of acquired immunodeficiency syndrome . We have produced the retroviral enzyme in E . coli from a synthetic gene encoding the protease that was constructed by assembling six overlapping and complementary oligonucleotides into the vector pKK223-3 . When expressed in E . coli, the recombinant protease was able to correctly process the HIV-1 core protein p24 from a beta-galactosidase-gag fusion protein and to use a heptapeptide as a substrate for proteolytic cleavage . A single base pair mutation was identified in a recombinant that resulted in the substitution of lysine for asparagine at position 88 and a significant loss of enzyme activity . Through site-directed mutagenesis, the Asn88 was changed to five other residues representative of all classes of amino acids . The correlation between enzyme activity and amino acid substitution suggests that the protease domain surrounding position 88 affects the protein's potential for forming an active homodimeric protein and hence, indicates a biochemical interaction that could be inhibited by novel antiviral compounds. J Biol Chem, 1989 Dec 5, 264(34), 20817 - 21 The calcium-binding site in the galactose chemoreceptor protein . Crystallographic and metal-binding studies; Vyas MN et al.; We have determined the relative affinities in solution for various metals which bind to the lone calcium-binding site of the D-galactose-binding protein which resembles the EF-hand loop . In order of affinity the metals are: Ca2+ approximately Tb3+ approximately Pb2+ greater than Cd2+ greater than Sr2+ greater than Mg2+ much greater than Ba2+ . The binding affinity for calcium (Kd = 2 microM) and the slow off-rate determined for terbium (1 x 10(-3) s-1) and that the metal-binding site is unperturbed by sugar binding argue for a structural role . Furthermore, we have crystallographically refined the structure of the binding protein with the calcium substituted by cadmium, compared it with the calcium-bound structure, and found them to be identical . The results of these structural and solution studies support the hypothesis that for a given metal-binding loop, cation hydration energy, size, and charge are major factors contributing to binding affinity. J Biol Chem, 1989 Dec 5, 264(34), 20786 - 95 Probing the role of two hydrophobic active site residues in the human dihydrofolate reductase by site-directed mutagenesis; Schweitzer BI et al.; In the x-ray structure of the human dihydrofolate reductase, phenylalanine 31 and phenylalanine 34 have been shown to be involved in hydrophobic interactions with bound substrates and inhibitors . Using oligonucleotide-directed mutagenesis and a bacterial expression system producing the wild-type and mutant human dihydrofolate reductases at levels of 10% of the bacterial protein, we have constructed, expressed, and purified a serine 31 (S31) mutant and a serine 34 (S34) mutant . Fluorescence titration experiments indicated that S31 bound the substrate H2folate 10-fold tighter and the coenzyme NADPH 2-fold tighter than the wild-type human dihydrofolate reductase . The serine 31 mutation had little effect on the steady-state kinetic properties of the enzyme but produced a 100-fold increase in the dissociation constant (Kd) for the inhibitor methotrexate . The serine 34 mutant had much greater alterations in its properties than S31; specifically, S34 had a 3-fold reduction in the Km for NADPH, a 24-fold increase in the Km for H2folate, a 3-fold reduction in the overall reaction rate kcat, and an 80,000-fold increase in the Kd for methotrexate . In addition, the pH dependence of the steady-state kinetic parameters of S34 were different from that of the wild-type enzyme . These results suggest that phenylalanine 31 and phenylalanine 34 make very different contributions to ligand binding and catalysis in the human dihydrofolate reductase. J Biol Chem, 1989 Dec 5, 264(34), 20770 - 7 Recognition and cleavage signals for mRNA processing lie within local domains of the phage f1 RNA precursors; Blumer KJ et al.; In Escherichia coli infected with the filamentous phage f1, a number of the abundant phage mRNAs, species C-G, are products of post-transcriptional processing . The approach of cloning the phage sequences likely to include the processing signals in a plasmid under transcriptional control of the lambda PL promoter (Blumer, K . J., and Steege, D . A . (1984) Nucleic Acids Res . 12, 1847-1861) was extended to additional sites to show that processing at all five major sites is mediated by nuclease activity encoded by the bacterial genome . Primer extension methods were used to map more accurately in the f1 DNA sequence the 5' end points of the processed RNAs . The DNA segments that encode the mRNA processing signals were delimited from parallel series of 5'-3' and 3'-5' deletions made into the regions in which the RNA 5' ends map . For each deletion variant, in vivo f1 mRNA processing activity was assessed by primer extension and S1 nuclease mapping methods . The data indicate that the processing signals are comprised of relatively local regions near the point of RNA cleavage . Whereas cleavage occurs at the 5' border of the sequences that comprise the D, E, and F processing sites and thereby places most of the recognition information in the mature or product portion of the precursor, it occurs more centrally within the region comprising the C site . The C and D sites function independently as substrates for cleavage, with the necessary information contained in regions of 70 and 90 nucleotides, respectively . Cleavage at the E site appears to require a region of 130 nucleotides which completely contains the F site . From the effects on processing activity of deleting sequences in this region, the overlapping E and F processing sites appear to consist functionally of two subdomains . Each has a cleavage site at its immediate 5' end which can be substituted by foreign sequences, but both utilize a common recognition domain downstream from the point of strand scission. J Biol Chem, 1989 Dec 5, 264(34), 20697 - 704 UvrABC incision of N-methylmitomycin A-DNA monoadducts and cross-links; Pu WT et al.; The Escherichia coli UvrABC endonuclease is a multisubunit enzyme that initiates the repair of a wide variety of DNA lesions in vivo by making dual incisions on a damaged strand at the eighth or ninth phosphodiester bond 5' and the fourth or fifth phosphodiester bond 3' to the modified base . It has been hypothesized that UvrABC is able to recognize a broad spectrum of lesions because it does not recognize the lesion per se but rather gross helical distortions that the lesion induces in the DNA . Several lesions have recently been studied which are thermal stabilizing and are not believed to distort the DNA grossly, including the CC-1065-N-3-adenine and anthramycin-N-2-guanine adducts . We have studied the activity of UvrABC in vitro on another thermal stabilizing and nondistortive adduct, N-methylmitomycin A (NMA), a bifunctional DNA-alkylating agent that reacts with guanine on the side facing the minor groove, yielding either monoadducts or interstrand cross-links . NMA adducts increase the thermal stability of DNA, and theoretical calculations indicate that NMA adducts do not grossly distort the DNA helix . Our results show that UvrABC makes incisions at the eighth phosphodiester bond 5' and the fifth phosphodiester bond 3' to an NMA monoadduct, consistent with the incision pattern observed for the majority of other lesions that are also recognized by UvrABC . DNA containing a site-specific NMA cross-link was also recognized and incised by UvrABC . The rate of incision of NMA cross-linked DNA was about 200-fold higher in supercoiled molecules than in relaxed molecules, whereas the rate of incision of DNA containing NMA monoadducts was stimulated approximately 2-fold by supercoiling . The signal for UvrABC recognition and incision of damaged DNA is discussed in relation to the ability of UvrABC to incise NMA adducts as well as other nondistortive lesions. J Biol Chem, 1989 Dec 5, 264(34), 20591 - 5 Cloning the polB gene of Escherichia coli and identification of its product; Chen H et al.; Using an in vivo mini-Mu cloning system, we have cloned the polB gene of Escherichia coli into the multicopy plasmid, pUC18 . A chromosomal insert of 4.9 kilobases gave 30-40-fold overproduction of DNA polymerase II, and the cells containing the plasmid showed normal growth . The restriction pattern of the polB gene does not match that of either the polA gene or polC gene . Plasmid-directed protein synthesis demonstrates peptides of 99 and 82 kDa which are not expressed by derivative plasmids without DNA polymerase II activity . It appears from in situ gel assays and high performance liquid chromatography that 82- and 55-kDa proteins are derived from the 99-kDa protein by degradation, but all retain activity . DNA polymerase I or DNA polymerase III antibody does not inhibit the synthesis reaction of partially purified DNA polymerase II, but DNA polymerase II antibody does . By the criteria of restriction pattern of the polB gene, molecular weight of the protein, and antibody inhibition of reaction, DNA polymerase II can be demonstrated to be a distinct DNA polymerase. J Biol Chem, 1989 Dec 5, 264(34), 20547 - 51 The in vitro conversion of chorismate to isochorismate catalyzed by the Escherichia coli entC gene product . Evidence that EntA does not contribute to isochorismate synthase activity; Tummuru MK et al.; The entC and entA genes, coding for the enzymes isochorismate synthase and 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase, respectively, were subcloned behind the T7 promoter in the expression plasmid pGEM3Z . Their protein products were overproduced and partially purified for in vitro analysis of the conversion of chorismate to isochorismate . Whereas previous genetic experiments suggested that the EntA enzyme has a role in this conversion, this study clearly indicates that EntC alone catalyzes the reaction . Addition of EntA had no effect on isochorismate synthase activity . As a result, the mutation (previously designated entC401) in strain AN191 was characterized by nucleotide sequence analysis . The lesion is a single base substitution in the entA gene, resulting in a glutamic acid-for-glycine substitution at the penultimate amino acid (residue 247) of the EntA enzyme . The mutant protein was partially purified and shown to be devoid of 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase activity, whereas the entC gene product from strain AN191 exhibited normal isochorismate synthase function . These results conflict with the earlier characterization of the entC401 mutation in a different genetic background . The data presented herein establish that the EntA protein does not contribute to isochorismate synthase activity and that the mutant strain that led to this suggestion harbors a defective allele of entA rather than entC. J Biol Chem, 1989 Dec 5, 264(34), 20487 - 95 Activity and deletion analysis of recombinant human cathepsin L expressed in Escherichia coli; Smith SM et al.; A cDNA clone encoding the human cysteine protease cathepsin L was expressed at high levels in Escherichia coli in a T7 expression system . The insoluble recombinant enzyme was solubilized in urea and refolded at alkaline pH . 38-kDa procathepsin L was purified by gel filtration at pH 8.0, and a 29-kDa form of the enzyme was purified by gel filtration after autoprocessing of the proenzyme at pH 6.5 . The kinetic properties of the 29-kDa species of recombinant cathepsin L were similar to those published for the human liver enzyme (Mason, R . W., Green, G . D . J., and Barrett, A.J . (1985) Biochem . J . 226, 233-241), using benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide as substrate . However, the stability of the recombinant enzyme, and its pH optimum for this substrate was shifted to a higher pH . Structure-function studies of cathepsin L were performed by constructing mutations in either the propeptide portion or the carboxyl-terminal light chain portion of the protein . These constructions were expressed in the E . coli system, and enzymatic activities were assayed following solubilization, renaturation, and gel filtration chromatography of the mutated proteins . Deletions of increasing size in the propeptide resulted in large proportional losses of activity, indicating that the propeptide is essential for proper enzyme folding and/or processing in this renaturation system . Deletion of part of the light chain containing a disulfide-forming cysteine residue or a single amino acid substitution of alanine for this cysteine residue resulted in almost complete loss of activity . These data suggest that the disulfide bond joining the heavy and light chains of cathepsin L is essential for enzymatic activity. J Biol Chem, 1989 Dec 5, 264(34), 20363 - 71 The solution structure of the Escherichia coli initiator tRNA and its interactions with initiation factor 2 and the ribosomal 30 S subunit; Wakao H et al.; The conformation of the Escherichia coli initiator tRNA has been investigated using enzymatic and chemical probes . This study was conducted on the naked tRNA and on the tRNA involved in the various steps leading to the formation of the 30 S.IF-2.GTP.fMet-tRNA.AUG complex . A three-dimensional model of the initiator tRNA is presented, which displays several differences with yeast tRNAPhe: (i) the anticodon arm is more rigid; (ii) the presence of an additional nucleotide in the D loop results in specific features in both T and D loops; (iii) C1 and A72 might form a noncanonical base pair . Aminoacylation and formylation induce subtle conformational adjustments near the 3' end, the T arm and the D loop . Initiation factor (IF) 2 interacts with a rather limited portion of the tRNA, covering the T loop and the minor groove of the T stem, and induces an increased flexibility in the anticodon arm . The specific structural features observed in the T loop are probably recognized by IF-2 . In the 30 S.IF-2.GTP.fMet-tRNA.AUG complex, additional protections are observed in the acceptor stem and in the anticodon arm, resulting from a strong steric hindrance and from the codon-anticodon interaction within the subunit decoding site. J Biol Chem, 1989 Dec 5, 264(34), 20297 - 302 Deletion of ant in Escherichia coli reveals its function in adaptation to high salinity and an alternative Na+/H+ antiporter system(s); Padan E et al.; We have deleted the chromosomal ant gene from Escherichia coli by substitution with the kan gene, which encodes kanamycin resistance . The delta ant strains obtained cannot adapt to high sodium concentrations (700 mM, pH 6.8), which do not affect the wild type . The Na+ sensitivity of delta ant is pH dependent, increasing at alkaline pH . Thus at pH 8.5, 100 mM NaCl retard growth of delta ant with no effect on the wild type . The delta ant strains also cannot challenge the toxic effects of Li+ ions, a substrate of the Na+/H+ antiporter system . However, growth of these strains is normal on carbon sources which require Na+ ions for transport and growth . Moreover, antiporter activity, as measured in everted membrane vesicles, is not significantly impaired . A detailed analysis of the remaining antiporter activity in a delta ant strain reveals kinetic properties which differ from those displayed by the ant protein: (a) Km for transport of Li+ ions is about 15 times higher and (b) the activity is practically independent of intracellular pH . Our results demonstrate the presence of an alternative Na+/H+ antiporter(s) in E . coli, additional to ant system. J Biol Chem, 1989 Dec 5, 264(34), 20482 - 6 Phosphorylation inactivates Escherichia coli isocitrate dehydrogenase by preventing isocitrate binding; Dean AM et al.; Equilibrium binding studies demonstrate that purified Escherichia coli isocitrate dehydrogenase binds isocitrate, alpha-ketoglutarate, NADP, and NADPH at 1:1 ratios of substrate to enzyme monomer . The phosphorylated enzyme, which is completely inactive, is unable to bind isocitrate but retains the ability to bind NADP and NADPH . Replacement of serine 113, which is the site of phosphorylation, by aspartate results in an inactive enzyme that is unable to bind isocitrate . Replacement of the same serine with other amino acids (lysine, threonine, cysteine, tyrosine, and alanine) produces active enzymes that bind both substrates . Hence, the negative charge of an aspartate or a phosphorylated serine at site 113 inactivates the enzyme by preventing the binding of isocitrate. J Mol Biol, 1989 Dec 5, 210(3), 561 - 72 Insertion sequence IS10 anti-sense pairing initiates by an interaction between the 5' end of the target RNA and a loop in the anti-sense RNA; Kittle JD et al.; Transposition of insertion sequence IS10 is regulated by an anti-sense RNA which inhibits transposase expression when IS10 is present in multiple copies per cell . The anti-sense RNA (RNA-OUT) consists of a stem domain topped by a flexibly paired loop; the 5' end of the target molecule, RNA-IN, is complementary to the top of the loop, and complementarity extends for 35 base-pairs down one side of RNA-OUT . We present here genetic evidence that anti-sense pairing, both in vitro and in vivo, initiates by interaction of the 5' end of RNA-IN and the loop domain of RNA-OUT; other features of the reaction are discussed . In the context of this model, we discuss features of this anti-sense system which are important for its biological effectiveness, and suggest that IS10 provides a convenient model for design of efficient artificial anti-sense RNA molecules. FEBS Lett, 1989 Dec 4, 258(2), 335 - 8 Expression in Escherichia coli of catalytically active phenylalanine ammonia-lyase from parsley; Schulz W et al.; In parsley (Petroselinum crispum), phenylalanine ammonia-lyase (PAL) is encoded by 4 structurally similar genes . The nucleotide sequence of a near full-length cDNA and the deduced amino acid sequence of PAL-4 are presented and compared with the corresponding sequences of PAL-1, a previously described representative of the gene family . Transformation of Escherichia coli cells with PAL-1 or PAL-4 cDNA yielded catalytically active PAL, suggesting that the catalytic center of the enzyme is formed spontaneously rather than by a plant-specific mechanism. FEBS Lett, 1989 Dec 4, 258(2), 277 - 80 The intracellular localization of glycolate oxidoreductase in Escherichia coli; Sallal AK et al.; Distribution of glycolate oxidoreductase in cell-free extracts of E . coli grown in mineral medium containing sodium glycolate has been studied . The enzyme was found to be largely associated with the cytoplasmic membranes . Homologous antiserum to the membranes inhibited the enzyme activity completely after the solubilization of the intact membranes with Triton X-100 . Results on the effect of pronase on glycolate oxidoreductase activity confirmed that part of the enzyme is exposed to the surface of the membrane. FEBS Lett, 1989 Dec 4, 258(2), 274 - 6 Irradiation of the template with high-intensity (pulse-laser) ultraviolet light results in DNA-polymerase termination events at deoxyguanosine residues; Panyutin IG et al.; During primer elongation by Escherichia coli DNA-polymerase I large fragments on the template were irradiated with UV laser pulses at an intensity greater than or equal to 10(10) W/m2 . In addition to the termination events at photoproducts typical of low-intensity UV irradiation, termination is observed before deoxyguanosine residues . The effect of the UV light intensity on the ratio of termination efficiencies before dPy and dG suggests that the termination of polymerization before deoxyguanosine residues results from the formation of photoproducts yielded by two-quantum reactions . The results obtained herein, together with data published previously, imply that photomodification of dG residues is the major two-quantum reaction under the action of high-intensity UV radiation on DNA. FEBS Lett, 1989 Dec 4, 258(2), 251 - 4 New crystal forms of the small subunit of ribonucleotide reductase from Escherichia coli; Nordlund P et al.; The small subunit of ribonucleotide reductase from Escherichia coli has been crystallized in two new crystal forms . The form most suitable for X-ray analysis belongs to the orthorhombic space group P2(1)2(1)2(1) . It has the cell dimensions 74.3 A, 85.5 A, 115.7 A and diffracts to about 2.1 A resolution . The asymmetric unit most probably contains one dimer . Absorption spectra of single crystals confirm that the crystals contain a binuclear iron center . Crystals of the iron-depleted apoenzyme have also been obtained. Infect Immun, 1989 Dec, 57(12), 3808 - 15 Cloning and characterization of Treponema hyodysenteriae antigens and protection in a CF-1 mouse model by immunization with a cloned endoflagellar antigen; Boyden DA et al.; We cloned genes that code for Treponema hyodysenteriae antigens into Escherichia coli with the purpose of identifying protective antigens for vaccine development . Three different genomic libraries were screened with various antisera reactive with T . hyodysenteriae antigens . The cloned antigens and corresponding native T . hyodysenteriae antigens were analyzed for molecular size, serum reactivity, solubility in sarcosine, and segregation during phase partitioning with the nonionic detergent Triton X-114 . The results from these analyses suggested that the gene products were components of either the cytoplasmic membrane, periplasm, or endoflagella of T . hyodysenteriae . The cloned antigens were tested as vaccine candidates in a CF-1 mouse model of T . hyodysenteriae infection and immunity . Intraperitoneal injection of crude E . coli extracts containing cloned antigens did not protect mice from challenge . However, serum from mice injected with a crude extract of an E . coli clone which expressed an endoflagellar antigen killed T . hyodysenteriae in vitro . Partially purified preparations of this cloned endoflagellar antigen protected mice against oral challenge with both the homologous serotype (B204) and a heterologous serotype (B234) of T . hyodysenteriae . These results suggest that the endoflagellar proteins could be used as an effective subunit vaccine against T . hyodysenteriae. Infect Immun, 1989 Dec, 57(12), 3743 - 50 In vivo formation of hybrid toxins comprising Shiga toxin and the Shiga-like toxins and role of the B subunit in localization and cytotoxic activity; Weinstein DL et al.; Shiga toxin, Shiga-like toxin I (SLT-I) and Shiga-like toxin II (SLT-II) are cell-associated cytotoxins that kill both Vero cells and HeLa cells, whereas Shiga-like toxin II variant (SLT-IIv) is an extracellular cytotoxin that is more cytotoxic for Vero cells than for HeLa cells . The basis for these differences in cytotoxin localization and host cell specificity were examined in this study . The A and B subunit genes of Shiga toxin and the SLTs were recombined by two methods so that hybrid toxins would be formed in vivo . Complementation of heterologous subunits was accomplished by cloning the individual A and B subunit genes of SLT-I, SLT-II, and SLT-IIv on plasmid vectors of different incompatibility groups so that they could be maintained in double transformants of Escherichia coli . In addition, six operon fusions were constructed so that the A and B subunit genes of Shiga toxin, SLT-II, and SLT-IIv could be expressed as a single operon . The activities of the hybrid cytotoxins were assessed in three ways: (i) level of cytotoxicity, (ii) ratio of HeLa to Vero cell cytotoxicity, and (iii) ratio of extracellular to cell-associated cytotoxicity . Neither the A subunit of Shiga toxin nor SLT-I associated with a heterologous B subunit to form an active cytotoxin . However, in all other cases the hybrid molecules formed by subunit complementation or operon fusion were cytotoxic . Furthermore, the cytotoxic specificity and localization of the hybrid cytotoxins always corresponded to the activities of the native toxin possessing the same B subunit. Clin Immunol Immunopathol, 1989 Dec, 53(3), 488 - 98 Endotoxin, tumor necrosis factor-alpha and interleukin 1 induce interleukin 6 production in vivo; Shalaby MR et al.; The ability of Escherichia coli-derived lipopolysaccharide (LPS), recombinant (r) interleukin 1-beta (rIL-1 beta), and r murine tumor necrosis factor-alpha (rMuTNF-alpha) to induce interleukin 6 (IL-6) production in vivo was investigated . Peak serum IL-6 concentration was attained after 2 hr of LPS injection into mice . The coinjection of antiserum against rMuTNF-alpha with LPS resulted in a reduction of the induced serum IL-6 level, indicating the involvement of endogenous TNF-alpha in LPS induction of IL-6 . Recombinant IL-1 beta and rMuTNF-alpha injected directly caused the production of substantial amounts of IL-6 within 30 min . The injection of a combination of rIL-1 beta and rTNF-alpha induced a significantly greater level of IL-6 than either agent alone . The greater level of serum IL-6 was associated with hypothermia and an increased lethality among mice injected with both cytokines . These data demonstrate the abilities of IL-1 beta and TNF-alpha to induce IL-6 production in vivo and indicate that LPS induction of IL-6 may be mediated, at least partially, through TNF-alpha action . The data describe a new in vivo biologic activity shared between IL-1 beta and TNF-alpha and suggest that IL-6 may be an important effector in the manifestation of TNF-alpha and IL-1 beta actions in vivo. J Commun Dis, 1989 Dec, 21(4), 313 - 7 Enterotoxigenic Escherichia coli from an outbreak with cholerigenic syndromes of gastroenteritis; Kulshrestha SB et al.; In an outbreak of food-poisoning, 76 out of 200 students who had dined in an Institute mess experienced acute cholerigenic syndromes of gastroenteritis . Processing of the seven stool samples of affected students and remnants of 4 out of 5 type of food for isolation of bacterial pathogen(s) revealed only the presence of Escherichia coli which were serotyped as 020, 026, 045, 053 and one untypable (UT) . Enterotoxigenicity testing of these isolates revealed serotypes 020, 026 of E . coli to be heat-labile enterotoxin producer when subjected to Biken test, Latex agglutination test and co-agglutination test . Based on the laboratory findings there are good reasons to believe that serotype 020 was responsible for this episode. Tsitologiia, 1989 Dec, 31(12), 1478 - 84 {The partial recA-lexA dependence of the adaptive response of Escherichia coli to exposure to methylmethane sulfonate}; Zhestianikov VD et al.; A study was made of the adaptive response to methylmethane sulfonate (MMS) in E . coli . (18 strains of B, WP2, and H/r30 groups, including three strains of bacteria with pKM101 plasmid) . The adaptation of wild type cells and uvrA- and uvrB- mutants to non-lethal concentrations of MMS (10-30 mkg/ml during 90-120 min) leads to a significant increase in their resistance to lethal MMS concentrations (10-30 mM for 10-120 min): the dose modifying factor (DMF) being 1.5-1.8 . In single recA or lexA mutants (or double recA uvr- and lexA uvr- mutants) the efficiency of adaptive response to MMS was significantly lower: the DMF being 1.1-1.2 . In Bs-1 gamma R strain with intragenic suppressor of lexA gene the adaptive response efficiency was the same as in B/r (recA+lexA+) strain . There is no adaptive response to MMS in polA- strains . The adaptive response to MMS in E . coli is different from that to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methylnitrosourea (MNM), because in these two cases it is absolutely lexA-recA dependent . It is supposed that a partial recA-lexA dependence of the adaptive response to MMS in E . coli may be due to a specific MMS-induced lethal damage that induces an adaptive repair non-related to the system of recA-lexA-independent adaptive responses to MNNG and MNM . The presence of a plasmid of drug resistance pKM101 exerts no influence on the value, efficiency and recA-lexA-dependence of the adaptive response of E . coli to MMS. J Gen Microbiol, 1989 Dec, 135 ( Pt 12), 3413 - 20 Comparison of the uptake systems for the entry of various BtuB group colicins into Escherichia coli; Benedetti H et al.; Colicins A, E1, E2 and E3 belong to the BtuB group of colicins . The NH2-terminal region of colicin A is required for translocation, and defects in this region cannot be overcome by osmotic shock of sensitive cells . In addition to BtuB, colicin A requires OmpF for efficient uptake by sensitive cells . The roles of BtuB and OmpF in translocation and binding to the receptor of the colicins A, E1, E2 and E3 were compared . The results suggest that for colicin A OmpF is used both as a receptor and for translocation across the outer membrane . In contrast, for colicin E1, OmpF is used neither as a receptor nor for translocation . For colicins E2 and E3, the situation is intermediate: only BtuB is used as a receptor but both BtuB and OmpF are involved in the translocation step. Biochem Int, 1989 Dec, 19(6), 1297 - 307 High-molecular-mass proteases (possible proteasomes) in Escherichia coli K12; Vaithilingam I et al.; Two high-molecular-mass proteases have been detected in E.coli K12 and isolated from the periplasmic fraction released by osmotic shock . The two proteases, designated Protease peri7 and Protease peri8, have similar molecular masses (greater than 2000 kDa) and degrade alpha- and beta-casein, but not insulin B chain . Protease peri7 is a metalloprotease activated 3-6 fold by ATP, dATP and GTP but inhibited by AMP . Nucleotide hydrolysis occurs during protein breakdown . Protease peri8, in contrast, is a serine protease unaffected by nucleotides or metal chelators . The two proteases appear by electron microscopy to be ring-shaped particles of approximately 125 A degrees in diameter . These proteases appear to be very similar to the multi-protease complexes (Proteasomes) detected in a variety of eukaryotic cells. Pharmazie, 1989 Dec, 44(12), 840 - 2 {The effects and properties of sodium nucleinate as a pyrogen working standard . 6 . Pyrogen inactivation by incubation with blood serum or blood plasma}; Dressel H; The 24-h storage of horse serum at 4 degrees C to which bacterial lipopolysaccharide (P) or sodium nucleinate (NN) was added, did not change the pyrogenic effect of these mixtures . Even after 2-h incubation at 37 degrees C changes could not be clearly detected . 24-h incubation at 37 degrees C leads to a clear, but only in case of NN, statistically significant reduction of pyrogenic effect . 24-h incubation of human heparin plasma at 37 degrees C, to which P or NN was added, in both cases resulted in statistically significant reductions of pyrogenic effect . Thus, when incubated with horse serum or human heparin plasma, the pyrogenic effect of NN is reduced in principle as well as that of P treated in the same way. Biokhimiia, 1989 Dec, 54(12), 1994 - 9 {The effect of composition and ionic strength of external solution on the aspartate-ammonia lyase and fumarate hydratase activity in Escherichia coli cells}; Verevkin AN et al.; It was found that the nonspecific effect of ionic strength of the external solution on the enzymatic activity of E . coli cells consists in rapid changes in the permeability of cell membranes interacting with the substrate . This effect depends on the initial substrate concentration, i.e., ionic strength of the external solution, and is maintained for some time as the substrate concentration decreases . Chloramphenicol, a protein synthesis inhibitor, and sodium azide, a respiration inhibitor (300 micrograms/ml and 200 microM, respectively) do not change the enzymatic activity of E . coli cells during the synthesis of L-aspartic and L-malic acids from fumaric acid . The kinetic equations of L-aspartate and L-malate synthesis are described by equations of zero and intermediate (between zero and first) order, respectively. Ultramicroscopy, 1989 Dec, 31(4), 333 - 44 Automatic selection of macromolecules from electron micrographs by component labelling and symbolic processing; Harauz G et al.; A new solution to the problem of extracting images of individual biological macromolecules from electron micrographs is described . There are three distinct steps in the process . The initial stage of low-level image processing consists of noise suppression and edge detection . An intermediate stage of component labelling and feature computation bridges the gap between the iconic (low-level) processing and the final phase of symbolic (high-level) processing . Simple symbolic objects (bounding boxes) are derived from the edges, and are easily represented and manipulated in the decision-making process . The efficacy of the algorithm is demonstrated using electron micrographs of ribosomes and ribosomal subunits . The hierarchical nature of the analysis embodies a reduction in the amount of data and a change in its nature . Initially, thousands of pixels of continuous gray levels must be dealt with . After component labelling, there are fewer than a hundred bounding boxes whose manipulation can easily be defined and articulated by an expert . The software package that has been written can thus serve as a basis for applying artificial intelligence methodologies to analysis of electron micrographs. Arch Histol Cytol, 1989 Dec, 52(5), 485 - 91 A histological and experimental study on the fate of an increased number of lymph follicles produced in the mouse popliteal lymph node by exogenous antigen stimulation; Hoshi H et al.; Eight-week-old female C57B1/6 mice were injected with endotoxin LPS and/or other antigens into the left hind footpad, and then the number of lymph follicles in the draining popliteal lymph nodes was examined . In untreated mice each popliteal node contained 10-12 lymph follicles at both 8 and 15 weeks of age . Animals given 50 micrograms of LPS at 8 weeks of age showed an increase in the number of lymph follicles 3 weeks later, but this number returned to normal levels by 15 weeks after the LPS injection . After a 2-micrograms-LPS injection at 23 weeks of age, the number of lymph follicles in the draining lymph node was unchanged, but that in animals given the 2-micrograms-injection 15 weeks after the 50-micrograms-LPS injection was significantly increased . In animals receiving 2 Lf of diphtheria toxoid, instead of the 2-micrograms-LPS, at 15 weeks after a 50-micrograms-LPS injection, the number of lymph follicles per draining node was within the normal range . In one group of mice, the initial injection of 50-micrograms-LPS at 8 weeks of age was followed by injections every third week of several kinds of antigens which had been shown to be ineffective in inducing follicle formation . Here, the number of lymph follicles in the draining popliteal node was kept to significantly increased levels at 25 weeks of age . The present results suggest that, while most lymph follicles normally developing in the lymph node are maintained for a long time under normal conditions, many lymph follicles induced by antigenic challenge have a limited life span and undergo atrophy unless they are periodically activated by additional antigenic stimuli, and that atrophied follicles finally become unable to respond to antigenic stimulation . It is also suggested that antigenic materials which trigger the formation of lymph follicles in the primary challenge can evoke follicle formation more efficiently in the secondary challenge. Can J Microbiol, 1989 Dec, 35(12), 1076 - 80 Kinetic properties of 7 alpha-hydroxysteroid dehydrogenase from Escherichia coli 080; Prabha V et al.; Results on the kinetics of 7 alpha-hydroxysteroid dehydrogenase 7 alpha-HSDH showed that this enzyme could oxidize all bile acids having an -OH group at the C-7 position . Lineweaver-Burk plots showed Michaelis constant (Km) values of 0.83 and 0.12 mM for cholic acid and chenodeoxycholic acid, respectively . The effect of enzyme concentration on the reaction velocity showed a constant increase in the enzyme activity with increase in enzyme-protein concentration . 7 alpha-HSDH was activated by Na+, K+, Ca2+, and Mn2+ ions and by reducing agents having a thiol group (dithiothreitol, 2-mercaptoethanol) . Co2+, Hg2+, Fe3+, Mg2+, Zn2+, Ba2+, and Cu2+ ions, chelating agents (potassium oxalate, heparin, EDTA) oxidizing agents (sodium perchlorate, sodium periodate, sodium persulphate), and detergents (Tween 20, Tween 40, Tween 80, Triton X-100, sodium lauryl sulphate) were inhibitory to 7 alpha-HSDH activity. Vet Immunol Immunopathol, 1989 Dec, 23(3-4), 333 - 44 Humoral response in neonatal calves following immunization with Escherichia coli (strain J5): the effects of adjuvant, age and colostral passive interference; Tyler JW et al.; Serologic responses in 61 calves 3 to 34 days of age following immunization with bacterins containing a heat-killed rough mutant, Escherichia coli 0111:B4 (strain J5) were determined by an enzyme-linked immunosorbent assay specific for the IgG isotype . Administration of either heat-killed bacteria or oil-based adjuvants alone failed to enhance serologic recognition of common core antigens when comparing to nonvaccinate controls . Increased titers were uniquely and specifically limited to calves receiving the antigen in an oil emulsion . In a second experiment, age and initial, passively acquired titer recognizing the vaccinal antigen were not found to have any effect on the magnitude of the humoral response of 57 calves following immunization. Indian J Med Res, 1989 Dec, 90, 478 - 83 Modulation of immunosuppression in obstructive jaundice by Tinospora cordifolia; Rege NN et al.; A clinical study was undertaken to determine the immune status of patients with obstructive jaundice . Screening of 16 patients for phagocytic and microbicidal activity of polymorphonuclear cells (PMN) revealed a significant depression (21.2 +/- 3.7% phagocytosis and 20.85 +/- 4.5% intracellular killing) of these functions, as compared to normal values (30.37 +/- 5.1% and 26.41 +/- 4.3% respectively) . An animal model of cholestasis was also established, using rats, in which a significant depression of activity of PMN and peritoneal macrophages was observed . These cellular abnormalities were found to precede and predispose to infection . The rats also showed an increased susceptibility to Escherichia coli infection (mortality rate 77.78%) . A defect was detected in their serum responsible for depressing the function of phagocytic cells . An attempt was made to improve this immunosuppression by treating the rats with water extract of T . cordifolia 100 mg/kg for 7 days, following development of cholestasis . The extract improved the cellular immune functions . Mortality rate following Esch . coli infection was significantly reduced to 16.67 per cent . This study showed that cholestasis results in immunosuppression and therefore indicates the need for an immunomodulator in management of obstructive jaundice . The plant T . cordifolia seems to meet this need by consolidating host defence mechanism. Br Poult Sci, 1989 Dec, 30(4), 919 - 25 Factors affecting chicken thrombocyte morphology and the relationship with heterophil:lymphocyte ratios; Gross WB; 1 . Thrombocytes were observed in a haemocytometer chamber and cells were classified into 5 morphological groups which were related to the extent of environmental stress . Thrombocyte morphology scores (TMS) were calculated for each blood sample . 2 . Following exposure to social stress, chilling, or the injection of killed Escherichia coli, both TMS and heterophil:lymphocyte (H:L) ratios were increased . From maximum values 1 d after chilling H:L and TMS values returned to normal within 2 and 11 d respectively . 3 . The addition of corticosterone (200 mg/kg) to the food resulted in increased H:L ratios whereas TMS were not affected . 4 . The injection of an adrenal blocker, 1,1-dichloro-2,2 bis p-choro-phenyl ethane, resulted in decreased H:L values whereas TMS were not affected . 5 . Resistance to E . coli challenge infection was not affected by TMS. Photochem Photobiol, 1989 Dec, 50(6), 745 - 51 Photobiological properties of a novel, naturally occurring furoisocoumarin, coriandrin; Ashwood-Smith MJ et al.; The photobiological properties of a novel, naturally occurring furoisocoumarin isolated from coriander and named coriandrin are described . Photosensitized lethal and mutagenic effects in bacteria indicate that it is more active than psoralen . It is a weak frameshift mutagen in the dark . Mammalian cells in tissue culture are photosensitized more actively with coriandrin than with psoralen even though preliminary evidence from interrupted radiation experiments and DNA analysis suggest that coriandrin does not form DNA interstrand crosslinks . Sister chromatid exchanges were induced with a unit dose of 1.1 x 10(-2) with coriandrin; the value for psoralen is 3 x 10(-3) . Coriandrin appears to be metabolized more rapidly than furocoumarins by liver mixed function oxidases . Skin photosensitizing activity is very weak compared with psoralen, a surprising observation considering its potency in biological test systems. Photochem Photobiol, 1989 Dec, 50(6), 733 - 8 Sanguinarine, a phototoxic H2O2-producing alkaloid; Tuveson RW et al.; Sanguinarine chloride, a quaternary salt of a benzophenanthrene alkaloid, was phototoxic to catalase-deficient strains of Escherichia coli but not to Trichoplusia ni (cabbage looper moth larvae), an insect with high levels of catalase activity . Chemical analyses confirm that sanguinarine is an efficient producer of H2O2 . This differential toxicity suggests that the mode of phototoxic action involves production of H2O2 which could be detoxified in many organisms by catalase. Protein Seq Data Anal, 1989 Dec, 2(6), 463 - 6 High sequence conservation between isocitrate lyase from Escherichia coli and Ricinus communis; Beeching JR; The deduced amino acid sequences of isocitrate lyase (EC 4.1.3.1) from Escherichia coli and Ricinus communis (castor bean) were compared and regions of high homology between the two enzymes were identified . The castor-bean enzyme had a 14 amino acid amino-terminal, and a 25 amino acid carboxy-terminal extension and a 102 amino acid central insertion compared to the E . coli enzyme . Enzymatic data were used to attempt to identify specific amino acids in the active site . Comparisons with putative peroxisomal/gloxysomal targeting sequences were made and a region including part of the central insertion of the castor bean enzyme was tentatively identified. Zhonghua Liu Xing Bing Xue Za Zhi, 1989 Dec, 10(6), 333 - 6 {An outbreak of diarrhoea in newborns caused by EIEC}; She SL; An outbreak of 18 acute infant diarrhoea cases caused by enteroinvasive E . coli O152:K? and O112ac: K66 were reported in a nursery in the obstetrics department of a hospital in Huangshi city in 1987 . Among them 2 cases died . Epidemic state, etiology biochemical and serological tests, clinical feature and resistance to drugs were discussed . The importance of the management of nurseries and stool examination for the pathogens in intestinal tract in puerpera were emphasized. Poult Sci, 1989 Dec, 68(12), 1631 - 6 Response of White Leghorn chicks fed ascorbic acid and challenged with Escherichia coli or with corticosterone; van Niekerk T et al.; This study was undertaken to assess the effects of dietary ascorbic acid on the growth and immunoresponsiveness of chickens when subjected to particular types of stress . White Leghorn chicks were fed diets containing no supplemental ascorbic acid, and ascorbic-acid diet (330 ppm) for 2 days or for 19 days before challenge . Then, half of the females were inoculated with Escherichia coli; half of the males were challenged with dietary corticosterone (30 ppm) for 12 days; and the remaining chicks were maintained as controls . These chicks, reared under "good" husbandry procedures, did not realize advantages in growth or feed efficiency due to the short- or long-term consumption of diets containing ascorbic acid . Incubation with E . coli resulted in considerably higher heterophil-to-lymphocyte ratios 24 h after inoculation, and E . coli-induced mortality was higher for pullets on short-term ascorbic acid than for those on long-term or no ascorbic acid . Dietary corticosterone caused differences in body weight and the relative weights of certain organs, regardless of dietary levels of ascorbic acid . The antibody response to red-blood-cell antigens from sheep was enhanced in unchallenged cockerels (no dietary corticosterone) fed ascorbic acid on a long-term basis, but dietary corticosterone overshadowed the advantageous effects of dietary ascorbic acid . These data showed that the effects of supplemental ascorbic acid on growth and on immunoresponsiveness were related to the quality of the husbandry, length of supplemental feeding, age of the chicks, endogenous-exogenous balance for ascorbic acid, and the relationship with corticosterone. Mol Microbiol, 1989 Dec, 3(12), 1745 - 52 Stability of ColE1-like and pBR322-like plasmids in Escherichia coli; Ayala-Sanmartin J et al.; The average copy number, the level of ampicillin resistance conferred by one plasmid, and the degree of plasmid multimerization were determined for several ColE1-like and pBR322-like plasmids . From the results obtained, the variance of the units of partition corresponding to each plasmid studied was calculated . Experimentally determined plasmid stability was compared with that calculated using the variance of the units of partition and the ratio between the generation times of plasmid-free and of plasmid-carrying cells, assuming that the units of partition are distributed randomly between daughter cells . Stability of the pBR322-like plasmids present mainly as monomers in the bacterial host was consistent with random partitioning, whereas pBR322-like plasmids, present mainly as dimers, and the ColE1-like plasmid showed greater stability than that predicted with random partitioning at cell division. Genes Dev, 1989 Dec, 3(12A), 2003 - 10 The activity of sigma 32 is reduced under conditions of excess heat shock protein production in Escherichia coli; Straus DB et al.; The expression of heat shock genes in Escherichia coli is controlled by the action of an alternate sigma-factor of RNA polymerase, sigma 32, which directs core RNA polymerase to recognize the promoters for heat shock genes . After a shift from 30 degrees C to 42 degrees C, both the level of sigma 32 and transcription initiation at heat shock promoters transiently increase, indicating that heat shock gene expression is regulated by changes in the concentration of sigma 32 . Here, we report that heat shock gene expression is regulated by changes in the activity of sigma 32 under some conditions . Our results show that the transient repression of heat shock protein synthesis, which follows a shift down from 42 degrees C to 30 degrees, occurs as a result of decreased transcription initiation at heat shock promoters, but this repression is accompanied by only a small decrease in the level of sigma 32 . In addition, the induction of heat shock proteins following overproduction of sigma 32 from a multicopy plasmid is only transient, despite the fact that the level of sigma 32 remains elevated . Constitutive overproduction of sigma 32 also fails to cause a proportionate increase in heat shock gene transcription . These three examples suggest that the activity of sigma 32 is reduced under conditions of excess heat shock gene expression. Genes Dev, 1989 Dec, 3(12A), 1899 - 912 Selection of the initiator tRNA by Escherichia coli initiation factors; Hartz D et al.; We have developed a new technique, called 'toeprinting,' which has allowed a study of the tRNA-binding properties of Escherichia coli translation initiation complexes . In response to natural mRNAs, the initiator tRNA and a variety of elongator tRNAs bind to the same tRNA-binding site on the 30S ribosomal subunit as long as a cognate codon is present near the Shine and Dalgarno sequence . The selection of the initiator tRNA in 30S initiation complexes is accomplished by initiation factors IF2 and IF3 . 70S ribosomes accept both initiator tRNA and elongator tRNAs on natural mRNAs, much like 30S ribosomal subunits; IF3 and IF2 do not, however, select the initiator tRNA on 70S initiation complexes unless the initiation factor IF1 is present. Genet Res, 1989 Dec, 54(3), 167 - 71 RelA mutation and pBR322 plasmid amplification in amino acid-starved cells of Escherichia coli; Riethdorf S et al.; Plasmid pBR322 is amplified following amino-acid limitation in Escherichia coli relA hosts . In relA+ hosts there was no significant amplification or a much smaller one . Plasmid amplification is due to the relA mutation; when the relA+ allele is transferred into the relA mutant CP79 this strain no longer amplifies plasmid DNA during amino acid starvation . It is concluded that ppGpp is a negative effector of plasmid replication . Amplification is temperature dependent, being maximal at 32 degrees C and negligible at 37 degrees C. Appl Environ Microbiol, 1989 Dec, 55(12), 3091 - 4 Comparison of membrane filtration and Autoanalysis Colilert presence-absence techniques for analysis of total coliforms and Escherichia coli in drinking water samples; Lewis CM et al.; Over a 4-month period, 950 samples of treated drinking water were analyzed for total coliforms (TC) and Escherichia coli by both membrane filtration (MF) and Autoanalysis Colilert presence-absence (AC) techniques . The two tests agreed 97% of the time on the basis of presumptive TC results and 98.5% of the time on the basis of verified TC results . Samples which produced disagreement between the two tests were most often TC positive by MF and TC negative by AC . E . coli was recovered four times: twice by MF only, and twice by AC only but without the diagnostic fluorescence reaction . In two samples, E . coli could not be isolated from fluorescence-positive AC tests . On the basis of these results, the AC test was implemented as the routine analytical procedure for TC but not for E . coli. Antimicrob Agents Chemother, 1989 Dec, 33(12), 2042 - 8 Gene amplification contributes to sulfonamide resistance in Escherichia coli; Nichols BP et al.; A sulfathiazole-resistant strain of Escherichia coli was isolated and shown to contain a fourfold tandemly amplified segment of DNA 18 kilobase pairs in length in addition to a mutationally altered dihydropteroate synthase, the target enzyme for sulfonamide inhibition . The amplified DNA contained a gene designated sur that contributed to sulfathiazole resistance when present in greater amounts than those in the wild type . Sulfathiazole resistance was markedly decreased upon loss of the amplified DNA after nonselective growth . Plasmids that contained sur also conferred only weak sulfathiazole resistance on wild-type strains . Comparison of the restriction maps of the amplified DNA, wild-type DNA, and sur-containing plasmids showed that a DNA rearrangement occurred before or concomitant with the DNA amplification event . The DNA rearrangement resulted from an IS5 insertion, which, in conjunction with an IS5 element residing near sur in the wild-type strain, resulted in an -IS5-sur-IS5- configuration . Homologous recombination could account for duplication and subsequent amplification of the sur region . High-copy-number plasmids containing the sur locus did not express a sulfathiazole-resistant dihydropteroate synthase, nor did they overexpress wild-type dihydropteroate synthase . These data suggest that the high level of sulfathiazole resistance in this strain results from a synergistic effect of two different mutations. J Clin Chem Clin Biochem, 1989 Dec, 27(12), 941 - 6 Chromogenic endotoxin assay in plasma . Selection of plasma pretreatment and production of standard curves; Fukui H et al.; The aim of this study was to define the optimal conditions for the plasma pretreatment and to improve the production of standard curves for plasma endotoxin determination by a chromogenic substrate assay . Endotoxin standard from E . coli O 111:B 4 (0-50 ng/l) was added to pyrogen-free water or to plasma samples from 12 healthy subjects and 24 alcoholics, before pretreatment by heating (75 degrees C, 5 minutes) or with perchloric acid (0.32 mol/l) . When endotoxin standard curves were determined using a microprocessor-controlled reader, the slopes of the curves obtained with plasma differed from those with pyrogen-free water . The slope of the standard curve prepared with plasma samples from different patients exhibited marked interindividual variations . Compared with the heating method, the perchloric acid method gave more variable results and a lower recovery of added endotoxin, especially in plasma from alcoholics . The results permit the following conclusion: 1 . For plasma endotoxin determination, a standard curve should be prepared for each individual plasma sample . 2 . The endotoxin standard should be added before pretreatment of the plasma . 3 . Pretreatment of the plasma by heating at 75 degrees C for 5 minutes provides more reliable results than pretreatment with perchloric acid. Br J Pharmacol, 1989 Dec, 98(4), 1383 - 91 Evidence for the involvement of a plasma kallikrein-kinin system in the immediate hypotension produced by endotoxin in anaesthetized rats; Katori M et al.; 1 . In vitro incubation of normal rat plasma with endotoxin from E . coli (3-10 mg ml-1) in the incubation mixture) caused a dose-dependent increase in levels of free kinin and plasma kallikrein in the presence of o-phenanthroline, together with a mirror-image, dose-dependent decrease in the residual levels of the precursors, plasma prekallikrein and high-molecular-weight kininogen . Low-molecular-weight kininogen levels were not modified . 2 . Intravenous injection of endotoxin (3-30 mg kg-1) into the femoral vein of anaesthetized rats resulted in dose-dependent hypotension . In blood collected up to 15 min after injection, the levels of prekallikrein and high-molecular-weight kininogen in plasma were decreased while levels of the active forms, plasma kallikrein and free kinin, showed a transient increase in the blood 1 min after administration of endotoxin . 3 . A degradation product of bradykinin, des-Phe8-Arg9-bradykinin, as measured by a newly developed enzyme immunoassay, was detectable up to 5 min after administration of endotoxin . 4 . Intravenous infusion of soybean trypsin inhibitor inhibited both the formation of bradykinin and des-Phe8-Arg9-bradykinin and the initial hypotension . 5 . It can be concluded from our results that plasma prekallikrein is activated in the blood immediately after administration of endotoxin to rats and that bradykinin is a major cause of the immediate hypotension. AIDS Res Hum Retroviruses, 1989 Dec, 5(6), 663 - 70 Lymphotoxin cDNA clones from a HTLV-I-carrying T cell line HUT-102; Kato S et al.; The conditioned medium of a HTLV-I-carrying T cell line HUT-102 showed cytotoxic activity against a mouse fibroblast cell line L-M . We prepared the cDNA library from HUT-102 poly(A)+ RNA and screened it using oligonucleotide probes that correspond to the amino acid sequences conserved in tumor necrosis factor (TNF) and lymphotoxin (LT) . As a result we obtained two kinds of cDNA clones encoding LT in which amino acid residue 26t of mature LT is different; one is Asn and another is Thr . The sequence of the genomic clones obtained using a polymerase chain reaction method showed that the HUT-102 genome also contains two types of LT genes . Recombinant LTs expressed in Escherichia coli exhibited the same level of cytolytic activity against L-M cells . These results indicate that the cytotoxin constitutively produced by HUT-102 cells include two kinds of LT. AIDS Res Hum Retroviruses, 1989 Dec, 5(6), 621 - 8 Low antigenicity of HIV-1 rev: rev-specific antibody response of limited value as correlate of rev gene expression and disease progression; Reiss P et al.; An enzyme immunoassay based on an E . coli-produced HIV-1 rev gene product was used to detect rev-specific antibodies in longitudinally collected serum samples from 196 initially symptom-free men who were seropositive for antibodies to HIV-1 structural proteins and 72 men who seroconverted for such antibodies . In 61% of men no rev-specific antibodies were detected at all, 30% had persistently detectable rev-specific antibodies, and in 9% rev-specific antibodies were only transiently or intermittently detected . When a persistent rev-specific antibody response occurred in subjects who seroconverted to structural proteins, it was always, with one exception, found within 12 months of seroconversion . The rev-specific antibodies were also studied in a transectional sample of sera from the men who remained symptom-free and from those who developed AIDS-related conditions or AIDS, as well as in sera from 31 other men with AIDS-related conditions and in sera from 6 of these men at the time they developed AIDS . The rev-specific antibodies were found in 34% of symptom-free men, in 28% of patients with AIDS-related conditions, and in 16% of patients with AIDS . The low incidence of rev-specific antibodies early after infection may be due to low antigenicity of rev . The lower prevalence of rev-specific antibodies in sera from patients with AIDS, compared with patients with AIDS-related conditions and symptom-free HIV-1-infected individuals, may be explained by a progressive HIV-1-induced immunodeficiency.(ABSTRACT TRUNCATED AT 250 WORDS) AIDS Res Hum Retroviruses, 1989 Dec, 5(6), 577 - 91 Cleavage of HIV-1 gag polyprotein synthesized in vitro: sequential cleavage by the viral protease; Erickson-Viitanen S et al.; The virally encoded protease of human immunodeficiency virus is responsible for the processing of the gag and gag-pol polyprotein precursors to their mature polypeptides . Since correct processing of the viral polypeptides is essential for the production of infectious virus, HIV protease represents a potential target for therapeutic agents that may prove beneficial in the treatment of AIDS . In this study, full-length gag polyprotein has been synthesized in vitro to serve as a substrate for bacterially expressed HIV-1 protease . Expression of the protease in E . coli from the lac promoter was enhanced approximately five-fold by deletion of a potential hairpin loop upstream from the codon determining the amino terminus of mature protease . Extracts of induced cultures of E . coli harboring a protease-containing plasmid served as the source of protease activity . The gag polyprotein synthesized in vitro was cleaved by such lysates, producing fragments corresponding in size to p17 plus p24 and mature p24 . Immunoprecipitations with monoclonal antibodies to p17 and p24 polypeptides suggest that initial cleavage of gag polyprotein occurs near the p24-p15 junction . The proteolysis was inhibited by pepstatin with an IC50 of 0.15 mM for cleavage at the p24-p15 junction and 0.02 mM for cleavage at the p17-p24 junction. Mol Gen Genet, 1989 Dec, 220(1), 69 - 72 Down regulation of the mercury resistance operon by the most promoter-distal gene merD; Nucifora G et al.; The effect of the merD gene on the expression of the mer operon was determined from the rates of accumulation of merA-lacZ fusion protein in the presence and absence of an active merD gene in trans . In the presence of the merD gene, beta-galactosidase activity was 2- to 4-fold lower . The merD gene was cloned in a T7 promoter expression vector and the MerD protein product was visualized by autoradiography. Mol Gen Genet, 1989 Dec, 220(1), 60 - 4 A mutation of Streptomyces lividans which prevents intraplasmid recombination has no effect on chromosomal recombination; Kieser HM et al.; A mutation (rec-46) of Streptomyces lividans, previously shown to prevent (or greatly diminish) homologous and illegitimate intraplasmid recombination, was shown to have no effect on generalised chromosomal recombination occurring in matings or in protoplast fusions, nor to affect homologous recombination between a recombinant plasmid and the host chromosome . By comparison with Escherichia coli mutants defective in various aspects of recombination, the rec-46 mutation is similar to those in recF, recJ, recO and topA. J Trop Med Hyg, 1989 Dec, 92(6), 379 - 82 Development of Vero cell miniculture assay for detection of heat-labile enterotoxin of Escherichia coli and its significance; Choudhry MA et al.; In the present study the Vero cell miniculture assay was developed for the detection of heat-labile enterotoxin of E . coli . The test was found to be reliable and efficient and was comparable with other tissue culture assays . The new cell lines BHK21, MDBK, LM and CK included in the present study did not respond positively to the E . coli toxins. Eur J Immunol, 1989 Dec, 19(12), 2367 - 73 Identification by sequence analysis of chemotactic factors for monocytes produced by normal and transformed cells stimulated with virus, double-stranded RNA or cytokine; Van Damme J et al.; A monocyte chemotactic activity was found to be released by various types of cultured human cells after appropriate stimulation: normal diploid fibroblasts, peripheral blood mononuclear cells or monocytes isolated therefrom, and a number of tumor cell lines, including osteosarcoma (MG-63) and hepatoma (Malavu) but not melanoma (Bowes) cells . Cultures of diploid human fibroblasts and these tumor cells stimulated with interleukin (IL) 1 or double-stranded RNA {poly(rI).poly(rC)}, or infected with viruses (measles or rubella viruses) were found to produce chemotactic activity for both monocytes and granulocytes . Media collected from fibroblasts treated with E . coli or IL 6 did not contain such activity . Granulocyte and monocyte chemotactic activities were serologically distinct, and could be separated by successive chromatographical procedures . While the granulocyte chemotactic activity of both fibroblasts and MG-63 cells had previously been identified as granulocyte chemotactic protein/IL 8, the monocyte chemotactic activity from MG-63 cells was identified by amino acid sequence analysis as a different protein recently described to be released by human glioma and myelomonocytic cell lines . In view of the similarity in their chromatographical behavior, monocyte chemotactic activities from fibroblasts, MG-63 cells and fresh monocytes can probably be assigned to identical molecules . Cultures of unfractionated peripheral blood cells, however, were found to release an additional monocyte chemotactic protein, identifiable by amino acid sequence analysis as platelet factor 4. Br J Exp Pathol, 1989 Dec, 70(6), 697 - 704 Lactoferrin can protect mice against a lethal dose of Escherichia coli in experimental infection in vivo; Zagulski T et al.; Experiments were undertaken to demonstrate and partially explain the protective effect of bovine lactoferrin (LB) when administered intravenously to mice 24 h before a challenge with a lethal dose of Escherichia coli . About 70% of mice pretreated with LB survived challenge . The survival rates in control mice treated with E . coli alone and pretreated with bovine serum albumin (BSA), were 4 and 8%, respectively . Human lactoferrin (LH) had almost the same protective effect as LB . Sufficient amounts of ferric ions were given to mice, in single and multiple doses, for full serum transferrin saturation 30 min before or after E . coli administration . The multiple dose of ferric ions did not change considerably the survival rate of mice pretreated with LB . In contrast, a single dose of ferric ions gradually decreased the survival rate of the mice after the first week of experiment . From day 14 this decrease was statistically significant in all groups of mice treated with a single dose of ferric ions when compared with mice pretreated only with LB, and the difference ranged from 25 to 35% on day 30 . The possible mechanism(s) of protective effect of LB and role of iron ions are discussed. Biochem J, 1989 Dec 1, 264(2), 381 - 8 Mutational analysis of the channel and loop sequences of human ferritin H-chain; Levi S et al.; Human ferritin H-chain mutants were obtained by engineering the recombinant protein expressed by Escherichia coli . The mutagenesis were directed to the C-terminal sequence forming the hydrophobic channel, to the hydrophilic channel and to the loop sequence . The mutants were analysed for extent of expression, for stability, for capacity to incorporate iron and for kinetics of iron uptake and iron oxidation . Of the 22 mutants analysed only two with deletions of single residues in the loop sequence and one with deletion of the last 28 amino acid residues did not assemble into ferritin-like proteins . The other mutants assembled correctly and showed similar chemical/physical properties to the wild-type; they included duplication of an 18-amino acid-residue stretch, deletion of the last 22 and the last seven residues and various mutations of single amino acid residues . Two mutants with extensive alteration in the C-terminal sequence had a diminished thermostability associated with incapability to incorporate iron though they still catalysed iron oxidation . The mutants with alterations of the sequence around the hydrophilic channel showed diminished iron uptake and oxidation kinetics, together with a slightly larger apparent molecular size . The results indicate (i) that two of the sequences are important for ferritin assembly/stability, (ii) that the presence of the hydrophobic channel is essential for formation of the iron core and (iii) that the sites of iron interaction and the path of iron penetration into ferritin remain unidentified. Proc Natl Acad Sci U S A, 1989 Dec, 86(24), 9991 - 5 Construction of human chromosome 21-specific yeast artificial chromosomes; McCormick MK et al.; Chromosome 21-specific yeast artificial chromosomes (YACs) have been constructed by a method that performs all steps in agarose, allowing size selection by pulsed-field gel electrophoresis and the use of nanogram to microgram quantities of DNA . The DNA sources used were hybrid cell line WAV-17, containing chromosome 21 as the only human chromosome and flow-sorted chromosome 21 . The transformation efficiency of ligation products was similar to that obtained in aqueous transformations and yielded YACs with sizes ranging from 100 kilobases (kb) to greater than 1 megabase when polyamines were included in the transformation procedure . Twenty-five YACs containing human DNA have been obtained from a mouse-human hybrid, ranging in size from 200 to greater than 1000 kb, with an average size of 410 kb . Ten of these YACs were localized to subregions of chromosome 21 by hybridization of RNA probes (corresponding to the YAC ends recovered in Escherichia coli) to a panel of somatic cell hybrid DNA . Twenty-one human YACs, ranging in size from 100 to 500 kb, with an average size of 150 kb, were obtained from approximately equal to 50 ng of flow-sorted chromosome 21 DNA . Three were localized to subregions of chromosome 21 . YACs will aid the construction of a physical map of human chromosome 21 and the study of disorders associated with chromosome 21 such as Alzheimer disease and Down syndrome. Proc Natl Acad Sci U S A, 1989 Dec, 86(24), 9836 - 40 Conformational characterization of a single-site mutant of murine epidermal growth factor (EGF) by 1H NMR provides evidence that leucine-47 is involved in the interactions with the EGF receptor; Moy FJ et al.; Epidermal growth factor (EGF) is a small protein containing 53 amino acids and three disulfide bonds . There is significant current interest in structure-function relationships in EGF and EGF-like proteins, including the homologous type-alpha transforming growth factors . The Leu-47 residue of murine EGF (mEGF) is one of several that are strongly conserved among the EGF-like growth factors, suggesting that it may contribute to the active site of mEGF . In several different binding assays, the activity of the mutant analog in which Leu-47 is replaced by Ser {( Ser47}mEGF) ranges from 8 to 18 times weaker than that of wild-type mEGF . The NMR data summarized in this paper demonstrate that the significant differences in the binding activities of wild-type and {Ser47}mEGF cannot be attributed to structural changes remote from the three-dimensional site of mutation . The only minor conformational changes that are indicated by these data involve side chains of residues proximal to Leu-47 in the three-dimensional structure . Therefore, Leu-47 and/or residues spatially adjacent to Leu-47 constitute part of the active site of mEGF. Proc Natl Acad Sci U S A, 1989 Dec, 86(24), 9827 - 31 Internal dynamics of lactose permease; Dornmair K et al.; The transport protein lactose permease was reconstituted in vesicles of dimyristoylphosphatidylcholine, and the internal dynamics were studied by measuring the fluorescence anisotropy decay of the tryptophan residues and of a covalently bound pyrene label . For the tryptophans three relaxation processes and for the pyrene two relaxation processes with relaxation times in the nanosecond range were observed . The slowest process, of approximately 50 ns, is assigned to orientational fluctuations of membrane-spanning helices . When the temperature is decreased below the lipid-phase transition, this relaxation process is slowed down and restricted in amplitude . Because the transport rate is known to also decrease below the phase transition, this observation suggests a coupling between internal dynamics and transport . This coupling is analyzed on the basis of the Kramers relation for chemical reactions. Proc Natl Acad Sci U S A, 1989 Dec, 86(24), 9752 - 6 Inhibition of human immunodeficiency virus 1 protease in vitro: rational design of substrate analogue inhibitors; Dreyer GB et al.; Inhibitors of the protease from human immunodeficiency virus 1 (HIV-1) were designed, synthesized, and kinetically characterized . Analogues of a heptapeptide substrate of HIV-1 protease with sequence similar to the p17-p24 cleavage site in the natural substrate, Pr55gag, were synthesized in which the scissile dipeptide bond was replaced with bonds from six categories of stable mimics of an aspartic proteolysis transition state or intermediate . These mimics included an analogue of statine, hydroxyethylene isosteres, two categories of phosphinic acids, a reduced amide isostere, and an alpha,alpha-difluoroketone . The resulting peptide analogues were linear competitive inhibitors of purified recombinant HIV-1 protease with inhibition constants ranging from 18 nM to 40 microM depending on the type of inhibitor . A truncated inhibitor, an analogue of a hexapeptide, retained full inhibitory potency . The most potent inhibitors, containing the hydroxyethylene isostere, effectively blocked the proteolytic processing of a recombinant form of Pr55gag by HIV-1 protease in a cell-free assay. Proc Natl Acad Sci U S A, 1989 Dec, 86(24), 9657 - 61 Cloning and expression of a protective antigen from the cattle tick Boophilus microplus; Rand KN et al.; Glycoproteins located on the luminal surface of the plasma membrane of tick gut epithelial cells, when used to vaccinate cattle, are capable of stimulating an immune response that protects cattle against subsequent tick infestation . One such tick gut glycoprotein, designated Bm86, has been purified to homogeneity and the amino acid sequences of peptide fragments generated by endoproteinase Lys-C digestion have been determined . We report here the isolation and characterization of a cDNA that encodes Bm86 . The nucleotide sequence of the cDNA contains a 1982-base-pair open reading frame and predicts that Bm86 contains 650 amino acids including a 19-amino acid signal sequence and a 23-amino acid hydrophobic region adjacent to the carboxyl terminus . The main feature of the deduced protein sequence is the repeated pattern of 6 cysteine residues, suggesting the presence of several epidermal growth factor-like domains . A fusion protein consisting of 599 amino acids of Bm86 and 651 amino acids of beta-galactosidase was expressed in Escherichia coli as inclusion bodies . Ticks engorging on cattle vaccinated with these inclusion bodies were significantly damaged as a result of the immune response against the cloned antigen. Experientia, 1989 Dec 1, 45(11-12), 1097 - 9 Purification and amino acid sequencing of naturally occurring N-formyl-methionyl oligopeptides from Escherichia coli; Broom MF et al.; A novel purification procedure for N-formyl methionyl oligopeptides from bacterial culture supernatants has been developed . C-terminal carboxypeptidase microsequencing of purified peptides from E . coli supernatants enabled identification of 6 naturally occurring formyl-oligopeptides. Circ Shock, 1989 Dec, 29(4), 329 - 34 Endotoxin-induced mortality in rats is reduced by nitrones; Hamburger SA et al.; The goal of these investigations was to determine if nitrone spin-trapping agents can alter mortality associated with endotoxemia in the rat . Reactive free radicals attack nitrone spin-trapping agents forming relatively reactive, persistent free radical spin adducts . We administered 85 mM (10 ml/kg) of alpha-phenyl N-tert-butyl nitrone (PBN), alpha-4-pyridyl-N-oxide N-tert-butyl nitrone (4-POBN), 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), or vehicle (saline i.p.) 30 min before endotoxin (25 mg/kg i.p.) or vehicle to Sprague-Dawley (SD) or Holtzman virus-free (HVF) rats (n = 10-17/group) . All vehicle-treated rats receiving endotoxin were dead by 1 day . At 7 days, 83% of PBN-treated SD, 42% of PBN- or POBN-treated HVF, and 25% of DMPO-treated HVF rats were alive . The difference in survival of PBN-treated animals between strains may reflect the higher susceptibility of HVF rats to endotoxin . The observed reduction in mortality may be related to the well-established capacity of spin-trapping agents to capture reactive free radicals that may be generated in target tissues in response to endotoxin, and that would otherwise react with cell components and produce tissue injury. Radiat Res, 1989 Dec, 120(3), 532 - 6 Deinococcus radiodurans DNA increases the radiation resistance of Escherichia coli; Dalrymple GV et al.; A genomic DNA library of Deinococcus radiodurans DNA has been prepared using the plasmid vector pBR322 . The recombinant plasmid was used to transform a more radiation-sensitive organism, Escherichia coli RR1 . Following selection of transformed organisms by their ability to grow on ampicillin, radiation-resistant organisms were selected by irradiation with 137Cs gamma radiation . Increased radiation resistance correlates with the presence of a 3-kb fragment of DNA in these cells which is derived from D . radiodurans. Proc Natl Acad Sci U S A, 1989 Dec, 86(23), 9213 - 7 Physiological role during export for the retardation of folding by the leader peptide of maltose-binding protein; Liu G et al.; It has been shown that folding of precursor maltose-binding protein of Escherichia coli in vitro is retarded by the leader peptide . We now present evidence that this modulation of folding plays a role during the export of maltose-binding protein in vivo . Maltose-binding protein synthesized in vivo without a leader sequence did not engage the cellular export apparatus . However, the requirement for the leader in at least one step, that of binding the export factor SecB, could be overcome by an amino acid substitution in the mature portion of maltose-binding protein . This substitution retarded the folding of the polypeptide even in the absence of a leader . Investigations using purified proteins in vitro demonstrated that SecB would stably bind to species of maltose-binding protein devoid of a leader when the folding of the binding proteins was sufficiently slow . Thus, we conclude that one of the roles of the leader is to retard folding and expose the binding site for SecB. J Clin Microbiol, 1989 Dec, 27(12), 2751 - 7 Single primer pair for amplifying segments of distinct Shiga-like-toxin genes by polymerase chain reaction; Karch H et al.; By using a single synthetic oligonucleotide primer pair in the polymerase chain reaction, we amplified specific Shiga-like-toxin (SLT) gene segments from DNAs of 20 clinical Escherichia coli isolates, irrespective of whether they produce SLT-I, SLT-II, or heretofore uncategorized SLTs . These segments were not detectable in any of 20 nontoxigenic E . coli strains . The primers deduced from a conserved region among SLT genes are so-called degenerate-sequence primers; i.e., they contain intentionally introduced sequence ambiguities to overcome minor sequence variations within different SLT genes . In direct gel hybridization with genomic DNA, both primers recognized SLT-I and SLT-II DNA sequences . Amplified sequences of target DNA obtained by polymerase chain reaction were visualized after gel electrophoresis by ethidium bromide staining, and definitive identification of the amplification product as an SLT gene segment was achieved by hybridization to SLT-I- and SLT-II-specific 20-base oligonucleotide probes complementary to a portion of the amplified sequences but not to the primers . The detecting oligonucleotide probes shared only 30% base homology and were shown to recognize specifically SLT-I or SLT-II sequences within genomic DNA . Moreover, they were used to distinguish whether the amplified sequence originated from SLT-I or SLT-II genes . The PCR system with the primers described here is a powerful technique to amplify SLT sequences in E . coli strains that produce serologically distinct SLTs and will facilitate identification of these pathogens, particularly among a multitude of nonpathogenic E . coli strains. J Cell Biol, 1989 Dec, 109(6 Pt 1), 2939 - 50 Mutations in the SAC1 gene suppress defects in yeast Golgi and yeast actin function; Cleves AE et al.; The budding mode of Saccharomyces cerevisiae cell growth demands that a high degree of secretory polarity be established and directed toward the emerging bud . We report here our demonstration that mutations in SAC1, a gene identified by virtue of its allele-specific genetic interactions with yeast actin defects, were also capable of suppressing sec14 lethalities associated with yeast Golgi defects . Moreover, these sac1 suppressor properties also extended to sec6 and sec9 secretory vesicle defects . The genetic data are consistent with the notion that SAC1p modulates both secretory pathway and actin cytoskeleton function . On this basis, we suggest that SAC1p may represent one aspect of the mechanism whereby secretory and cytoskeletal activities are coordinated, so that proper spatial regulation of secretion might be achieved. J Cell Biol, 1989 Dec, 109(6 Pt 1), 2895 - 903 Partial deduced sequence of the 110-kD-calmodulin complex of the avian intestinal microvillus shows that this mechanoenzyme is a member of the myosin I family; Garcia A et al.; The actin bundle within each microvillus of the intestinal brush border is laterally tethered to the membrane by bridges composed of the protein complex, 110-kD-calmodulin . Previous studies have shown that avian 110-kD-calmodulin shares many properties with myosins including mechanochemical activity . In the present study, a cDNA molecule encoding 1,000 amino acids of the 110-kD protein has been sequenced, providing direct evidence that this protein is a vertebrate homologue of the tail-less, single-headed myosin I first described in amoeboid cells . The primary structure of the 110-kD protein (or brush border myosin I heavy chain) consists of two domains, an amino-terminal "head" domain and a 35-kD carboxy-terminal "tail" domain . The head domain is homologous to the S1 domain of other known myosins, with highest homology observed between that of Acanthamoeba myosin IB and the S1 domain of the protein encoded by bovine myosin I heavy chain gene (MIHC; Hoshimaru, M., and S . Nakanishi . 1987 . J . Biol . Chem . 262:14625-14632) . The carboxy-terminal domain shows no significant homology with any other known myosins except that of the bovine MIHC . This demonstrates that the bovine MIHC gene most probably encodes the heavy chain of bovine brush border myosin I (BBMI) . A bacterially expressed fusion protein encoded by the brush border 110-kD cDNA binds calmodulin . Proteolytic removal of the carboxy-terminal domain of the fusion protein results in loss of calmodulin binding activity, a result consistent with previous studies on the domain structure of the 110-kD protein . No hydrophobic sequence is present in the molecule indicating that chicken BBMI heavy chain is probably not an integral membrane protein . Northern blot analysis of various chicken tissue indicates that BBMI heavy chain is preferentially expressed in the intestine. J Cell Biol, 1989 Dec, 109(6 Pt 1), 2653 - 64 SEC62 encodes a putative membrane protein required for protein translocation into the yeast endoplasmic reticulum; Deshaies RJ et al.; Yeast sec62 mutant cells are defective in the translocation of several secretory precursor proteins into the lumen of the endoplasmic reticulum (Rothblatt et al., 1989) . The deficiency, which is most restrictive for alpha-factor precursor (pp alpha F) and preprocarboxypeptidase Y, has been reproduced in vitro . Membranes isolated from mutant cells display low and labile translocation activity with pp alpha F translated in a wild-type cytosol fraction . The defect is unique to the membrane fraction because cytosol from mutant cells supports translocation into membranes from wild-type yeast . Invertase assembly is only partly affected by the sec62 mutation in vivo and is nearly normal with mutant membranes in vitro . A potential membrane location for the SEC62 gene product is supported by evaluation of the molecular clone . DNA sequence analysis reveals a 32-kD protein with no obvious NH2-terminal signal sequence but with two domains of sufficient length and hydrophobicity to span a lipid bilayer . Sec62p is predicted to display significant NH2- and COOH-terminal hydrophilic domains on the cytoplasmic surface of the ER membrane . The last 30 amino acids of the COOH terminus may form an alpha-helix with 14 lysine and arginine residues arranged uniformly about the helix . This domain may allow Sec62p to interact with other proteins of the putative translocation complex. J Cell Biol, 1989 Dec, 109(6 Pt 1), 2641 - 52 Multiple genes are required for proper insertion of secretory proteins into the endoplasmic reticulum in yeast; Rothblatt JA et al.; Genes that function in translocation of secretory protein precursors into the ER have been identified by a genetic selection for mutant yeast cells that fail to translocate a signal peptide-cytosolic enzyme hybrid protein . The new mutants, sec62 and sec63, are thermosensitive for growth and accumulate a variety of soluble secretory and vacuolar precursors whose electrophoretic mobilities coincide with those of the corresponding in vitro translated polypeptides . Proteolytic sensitivity of precursor molecules in extracts of mutant cells confirms that polypeptide translocation is blocked . Some form of interaction among the SEC61 (Deshaies, R . J., and R . Schekman . 1987 . J . Cell Biol . 105:633-645), SEC62 and SEC63 gene products is suggested by the observation that haploid cells containing any pair of the mutations are inviable at 24 degrees C and show a marked enhancement of the translocation defect . The translocation defects of two mutants (sec62 and sec63) have been reproduced in vitro . sec63 microsomes display low and thermolabile translocation activity for prepro-alpha-factor (pp alpha F) synthesized with a cytosol fraction from wild type yeast . These gene products may constitute part of the polypeptide recognition or translocation apparatus of the ER membrane . Pulse-chase analysis of the translocation-defective mutants demonstrates that insertion of pp alpha F into the ER can proceed posttranslationally. J Bacteriol, 1989 Dec, 171(12), 6580 - 5 Purification of a new dihydrolipoamide dehydrogenase from Escherichia coli; Richarme G; I purified a new dihydrolipoamide dehydrogenase from a lpd mutant of Escherichia coli deficient in the lipoamide dehydrogenase (EC 1.6.4.3) common to the pyruvate dehydrogenase (EC 1.2.4.1) and 2-oxoglutarate dehydrogenase complexes . The occurrence of the new lipoamide dehydrogenase in lpd mutants, including a lpd deletion mutant and the immunological properties of the enzyme, showed that it is different from the lpd gene product . The new dihydrolipoamide dehydrogenase had a molecular weight of 46,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . It was expressed in low amounts . It catalyzed the NAD+-dependent reduction of dihydrolipoamide with a maximal activity of 20 mumol/min per mg of protein and exhibited a hyperbolic dependence of catalytic activity on the concentration of both dihydrolipoamide and NAD+ . The possible implication of the new dihydrolipoamide in the function of 2-oxo acid dehydrogenase complexes is discussed, as is its relation to binding protein-dependent transport. J Bacteriol, 1989 Dec, 171(12), 6511 - 6 New mre genes mreC and mreD, responsible for formation of the rod shape of Escherichia coli cells; Wachi M et al.; New shape-determining genes in the mre cluster at 71 min on the Escherichia coli chromosome map, named mreC and mreD, were identified by complementation experiments using delta mre-678 mutant cells, which have a 5-kilobase-pair deletion encompassing the mre region, and by DNA sequencing . The delta mre-678 mutant cells required three genes, the previously reported mreB gene and the two new genes, to restore the normal rod shape of the cells and normal sensitivity of growth to mecillinam . The mreC gene is preceded by the mreB gene and by a 65-base-pair spacing sequence containing a palindrome sequence and a possible Shine-Dalgarno sequence . The deduced amino acid sequence of the MreC protein consists of 367 amino acid residues with a molecular weight of 39,530 . The initiation codon of the mreD gene overlaps the termination codon of the mreC gene by one nucleotide residue . The deduced amino acid sequence of the MreD protein consists of 162 amino acid residues with a molecular weight of 18,755 . In vitro, the coding frames of mreC and mreD produced proteins with Mrs of 40,000 and 15,000, respectively, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J Bacteriol, 1989 Dec, 171(12), 6493 - 502 Effect of variation of charged and uncharged tRNA(Trp) levels on ppGpp synthesis in Escherichia coli; Rojiani MV et al.; We introduced into a stringent Escherichia coli tryptophan auxotroph a plasmid bearing the tRNA(Trp) gene under the control of an inducible promoter . This allows us to manipulate the total concentration of tRNA(Trp) in the cell according to whether and when inducer is added to the culture . We also manipulated the concentration of Trp-tRNA(Trp) in vivo since the strain used bears a mutation in the Trp-tRNA synthetase affecting the Km for tryptophan, such that varying the exogenous concentration of tryptophan led to variation in the level of Trp-tRNA(Trp) in the cell . With this system, we found that the signal eliciting ppGpp synthesis during a stringent response triggered by tryptophan limitation did not depend on the absolute concentration of either charged or uncharged tRNA(Trp) but rather depended on a decline in the ratio of charged/uncharged tRNA(Trp) . In addition, we found that the amplitude of the response, once triggered by tryptophan limitation, was determined by the total concentration of tRNA(Trp) present in the cell (which is mostly uncharged at that point in time) . However, excess uncharged tRNA(Trp) did not amplify ppGpp synthesis triggered by limitation of a different amino acid . These data provide in vivo support for the in vitro-derived model of ppGpp synthesis on ribosomes. J Bacteriol, 1989 Dec, 171(12), 6473 - 81 Methyl-directed repair of frameshift heteroduplexes in cell extracts from Escherichia coli; Learn BA et al.; The methyl-directed DNA repair efficiency of a series of M13mp9 frameshift heteroduplexes 1, 2, or 3 unpaired bases was determined by using an in vitro DNA mismatch repair assay . Repair of hemimethylated frameshift heteroduplexes in vitro was directed to the unmethylated strand; was dependent on MutH, MutL, and MutS; and was equally efficient on base insertions and deletions . However, fully methylated frameshift heteroduplexes were resistant to repair, while totally unmethylated substrates were repaired with no strand bias . Hemimethylated 1-, 2-, or 3-base insertion and deletion heteroduplexes were repaired by the methyl-directed mismatch repair pathway as efficiently as the G.T mismatch . These results are consistent with earlier in vivo studies and demonstrate the involvement of methyl-directed DNA repair in the efficient prevention of frameshift mutations. J Bacteriol, 1989 Dec, 171(12), 6430 - 6 Molecular structure and immunity specificity of colicin E6, an evolutionary intermediate between E-group colicins and cloacin DF13; Akutsu A et al.; The primary structure of a 3.1-kilobase E6 or E3 segment carrying colicin and related genes was determined . Plasmid ColE6-CT14 showed striking homology to ColE3-CA38 throughout this segment, including homology to the secondary immunity gene, immE8, downstream of the E6 or E3 immunity gene . The ColE3-CA38 and ColE6-CT14 sequences, however, contained an exceptional hot spot region encoding both the colicin-active domain (RNase region) and the immunity protein, reflecting their different immunity specificities . On the other hand, some chimeric plasmids were constructed through homologous recombination between colicin E3 and cloacin DF13 operons . The resulting plasmids were deduced to produce chimeric colicins with a colicin E3-type N-terminal part, a cloacin DF13-type C-terminal-active domain, and the DF13 immunity protein . The killing spectra of the chimeric colicins and the immunities of the plasmids were identical to those of colicin E6 and ColE6-CT14, respectively, showing that the colicin E6 immunity specificity is completely equivalent to that of cloacin DF13 . Nevertheless, colicin E6 has been found to show a sequence diversity from cloacin DF13 almost to the same extent as that from colicin E3 in their RNase and immunity regions, indicating that only a small number of amino acids defines the immunity specificity for discrimination between colicins E3 and E6 (or cloacin DF13). Crit Care Med, 1989 Dec, 17(12), 1314 - 9 Contribution of peripheral blood pooling to central hemodynamic disturbances during endotoxin insult in intact dogs; D'Orio V et al.; The aim of the present study was to determine possible effects of Escherichia coli endotoxin on peripheral vascular compliance and relate them to concomitant central hemodynamic disturbances . Endotoxin was infused at 0.25 micrograms/kg.min during 2 h in six anesthetized dogs, while six additional animals served as controls . Vascular compliance of the systemic circulation was calculated in intact animals from the changes in CVP after known changes in systemic blood volume . In control dogs, vascular compliance averaged 2.3 ml/mm Hg.kg body weight . During slow endotoxin infusion, cardiovascular effects were measurable only after a certain period of time had elapsed from the start of endotoxin insult and consisted of hypotension associated with systemic vasodilation . Systemic BP decreased gradually from 124 to 68 mm Hg while vascular compliance was finally increased by 100%, when compared to control values . This latter rise was responsible for a reduction in the cardiac preloads . Pulmonary wedge pressure and CVP were decreased from 7.1 to 3.4 and from 4.5 to 2.6 mm Hg, respectively . However, parallel to the decrease in left ventricular preload, endotoxin induced a progressive decrease in left ventricular afterload . Because of the balance in ventricular loading, cardiac output remained almost unchanged . After volume loading (dextran 30 ml/kg), cardiac output was remarkably increased from 3.28 to 6.24 L/min.m2 while peripheral vasodilation was not affected by this maneuver . It is concluded that low dose endotoxin infusion induces in dogs a hemodynamic pattern similar to human sepsis . The left ventricular loading changes are related to an enhanced systemic vascular compliance from 2.3 to 4.5 ml/mm Hg.kg . High flow shock state is encountered provided peripheral blood pooling is compensated by adequate volume replacement. Carcinogenesis, 1989 Dec, 10(12), 2261 - 7 N-ethyl-N-nitrosourea induces A:T to C:G transversion mutations as well as transition mutations in SOS-induced Escherichia coli; Eckert KA et al.; The fixation of DNA lesions induced in Escherichia coli by N-ethyl-N-nitrosourea (ENU) occurs by both SOS-dependent and SOS-independent pathways . To determine whether these pathways result in differential processing of ENU-induced lesions, we have analyzed the DNA sequence changes of mutations induced at a plasmid-encoded herpes simplex virus type 1 thymidine kinase gene by ENU treatment of plasmid-bearing RecA- and RecA+ bacteria, and by transformation of RecA-, RecA+ and SOS-induced RecA+ bacteria with ENU-modified plasmid DNA . Transition mutations were the predominant types of base substitution mutations observed for wild-type and RecA- E . coli, consistent with the SOS-independent mispairing of O6-ethylguanine and O4-ethylthymine adducts during DNA replication . Under conditions of SOS processing of ENU lesions, however, we observed the frequent induction of A:T----C:G transversion mutations . The proportion of A:T----C:G transversion mutations (42%) observed after transformation of SOS-induced bacteria with ENU modified DNA was approximately equal to that of the G:C----A:T transitions (46%) . The frequencies of these mutations were increased 20- and 5-fold respectively over that observed for non-induced RecA+ cells . We suggest that ethylated DNA lesions which normally block DNA replication can be processed to yield A:T----C:G transversion mutations in SOS-induced E . coli. Cell, 1989 Dec 1, 59(5), 927 - 37 Regulation of intracellular pH by a neuronal homolog of the erythrocyte anion exchanger; Kopito RR et al.; We have isolated AE3, a novel gene expressed primarily in brain neurons and in heart . The predicted AE3 polypeptide shares a high degree of identity with the anion exchange and cytoskeletal binding domains of the erythrocyte band 3 protein . Expression of AE3 cDNA in COS cells leads to chronic cytoplasmic acidification and to chloride- and bicarbonate-dependent changes in intracellular pH, confirming that this gene product is an anion exchanger . Characterization of an AE3 mutant lacking the NH2-terminal 645 amino acids demonstrates that the COOH-terminal half of the polypeptide is both necessary and sufficient for correct insertion into the plasma membrane and for anion exchange activity . The NH2-terminal domain may play a role in regulating the activity of the exchanger and may be involved in the structural organization of the cytoskeleton in neurons. Am J Epidemiol, 1989 Dec, 130(6), 1176 - 86 Bacteriuria in representative population samples of persons aged 72-79 years; Nordenstam G et al.; Screening for bacteriuria was performed between 1984 and 1988 in persons aged 72-79 years representative of the general population in Goteborg, Sweden . The frequency of bacteriuria (greater than or equal to 10(5)/ml) at a single screening was 6% and 16% at age 72 years and 6% and 14% at age 79 years for the screened men (n = 235 and 259) and women (n = 259 and 297), respectively . By repeated screening after one month and 30 months of those previously negative at age 72 years, an additional 4% and 3% of men and 3% and 7% of women with bacteriuria were detected . Bacteriuric persons were excluded from further screening and controlled by frequent cultures during several years, with careful monitoring of clinical interventions . The persistence of untreated bacteriuria was analyzed in relation to bacterial species and number in the untreated subgroup of bacteriuric individuals . Nine of 10 Escherichia coli (E . coli) with less than 10(6)/ml and 22/22 non-E . coli strains disappeared spontaneously . In contrast, 20/26 (77%, p less than 0.01) with greater than or equal to 10(6) E . coli/ml persisted . Of 17 persons with bacteriuria persisting at least 12 months, 16 were women and 16 had E . coli . Of 201 E . coli cultures obtained from this group, 94% had greater than or equal to 10(6)/ml, and 99% had greater than or equal to 5 x 10(5)/ml . The results indicate that screening for high counts (greater than 10(6)/ml) of E . coli most effectively detects persisting bacteriuria in the general elderly population. Mutat Res, 1989 Dec, 227(4), 247 - 50 The recB gene product is essential for exonuclease V-dependent DNA degradation in vivo; Brcic-Kostic K et al.; The recB21 mutation abolishes the exonuclease activity of the RecBCD enzyme (exonuclease V) of Escherichia coli . This might be due to the polar effect of recB21 on expression of the recD gene, the product of which is an essential component of the RecBCD enzyme . To achieve synthesis of the recD gene product, the recD+ plasmid was introduced into the recB21 mutant . Degradation of the endogenous DNA damaged by gamma-rays and degradation of the DNA of a phage T4 gene 2 mutant were nevertheless abnormally small in this strain . Thus, the functional recB gene product is required for the degradative function of the RecBCD enzyme. Mutat Res, 1989 Dec, 227(4), 199 - 205 Mutagenic action of heavy ions on Escherichia coli cells; Tokarova B et al.; Induction of direct mutations in the lactose operon of E . coli cells by gamma-radiation and accelerated heavy ions with different LET was studied . The experiments were performed with the wild-type PolA and LexA strains . A quadratic dependence of the mutation rate on the dose of different radiations for the wild-type strain and the PolA mutant was observed . However, different types of radiation showed different relative genetic effectivenesses (RGE) . The dependence of RGE on LET for the wild-type and PolA strain has a maximum . A LexA strain showed much reduced mutation rates and a linear dose response . The RGE decreased with increasing LET of ionizing radiation. Mol Cell Biol, 1989 Dec, 9(12), 5464 - 72 Interaction of the H4 autonomously replicating sequence core consensus sequence and its 3'-flanking domain; Holmes SG et al.; Yeast autonomously replicating sequence (ARS) elements are composed of a conserved 11-base-pair (bp) core consensus sequence and a less well defined 3'-flanking region . We have investigated the relationship between the H4 ARS core consensus sequence and its 3'-flanking domain . The minimal sequences necessary and sufficient for function were determined by combining external 3' and 5' deletions to produce a nested set of ARS fragments . Sequences 5' of the core consensus were dispensable for function, but at least 66 bp of 3'-flanking domain DNA was required for full ARS function . The importance of the relative orientation of the core consensus element with respect to the 3'-flanking domain was tested by precisely inverting 14 bp of DNA including the core consensus sequence by oligonucleotide mutagenesis . This core inversion mutant was defective for all ARS function, showing that a fixed relative orientation of the core consensus and 3'-flanking domain is required for function . The 3'-flanking domain of the minimal functional H4 ARS fragment contains three sequences with a 9-of-11-bp match to the core consensus . The role of these near-match sequences was tested by directed mutagenesis . When all near-match sequences with an 8-of-11-bp match or better were simultaneously disrupted by point mutations, the resulting ARS construct retained full replication function . Therefore, multiple copies of a sequence closely related to the core consensus element are not required for H4 ARS function. Int J Lepr Other Mycobact Dis, 1989 Dec, 57(4), 817 - 24 Overproduction, affinity purification and characterization of 65-kDa protein of Mycobacterium leprae in Escherichia coli; Nomaguchi H et al.; The 65-kDa protein of Mycobacterium leprae was produced in an Escherichia coli strain carrying a plasmid harboring the recloned gene coding for the protein . The protein was purified through affinity chromatography prepared with the IgG fraction of a monoclonal antibody which was prepared against the 65-kDa protein . The purified 65-kDa protein also reacted immunologically with the monoclonal antibody IIIE9, which recognizes the epitope for M . leprae, prepared by Buchanan, et al . BALB/c mice were inoculated with M . leprae and 4 months later were skin tested with the purified 65-kDa protein . Gross changes were observed at the skin-test site . The role of the protein in protective immunity against M . leprae foot pad infection in mice was also studied. Infect Immun, 1989 Dec, 57(12), 3765 - 9 Development of the human immune response against the major surface protein (gp190) of Plasmodium falciparum; Muller HM et al.; The 190-kilodalton glycoprotein (gp190) of Plasmodium falciparum, the precursor of the major surface proteins of merozoites, is considered a promising candidate for a blood stage malaria vaccine . DNA sequences specific for the gp190 of the two isolates K1 and MAD20 were subcloned and expressed in Escherichia coli . The panel of fusion proteins obtained represents about 80% of the polymorphic sequences observed so far within various isolates of P . falciparum . Sera from individuals living in a malaria-endemic area of West Africa were tested in immunoblots against the gp190 fusion proteins, and antibody reactivity was mapped to defined regions of the gp190 . Depending on the age of the individual and on the presence of parasites in the blood, distinct regions of gp190 were differentially recognized by the respective antibodies . Similarly, the analysis of sera from German patients with acute malaria revealed a distinct pattern . When grouped according to age and to parasitemia, the reactivity of the sera of people living in malaria-endemic areas may indicate a correlation between certain gp190 regions and protective immune response. Infect Immun, 1989 Dec, 57(12), 3727 - 34 Adhesion of colonization factor antigen II-positive enterotoxigenic Escherichia coli strains to human enterocytelike differentiated HT-29 cells: a basis for host-pathogen interactions in the gut; Neeser JR et al.; Enterotoxigenic Escherichia coli are the most common cause of travelers' and infant diarrhea in less-developed countries . In the present work, among several metabolically labeled human diarrheagenic E . coli strains, enterotoxigenic strains expressing colonization factor antigen II were shown to bind to HT-29 intestinal cell monolayers when these cells were grown in conditions promoting their enterocytic differentiation . Indirect immunofluorescence with fimbrial antisera revealed that pathogen attachment was associated with the production of a specific bacterial adhesin, the E . coli surface antigen CS3 . Scanning and transmission electron micrographs showed an apical pattern of colonization, characteristic of enterotoxigenic E . coli infections . The above data were consistent with all observations previously made with human enterocytes obtained from intestinal biopsies . The lectin-carbohydrate nature of this cell-cell recognition mechanism was also established . Bacterial binding to differentiated HT-29 cells was inhibited by a mixture of newborn meconium glycopeptides . By coating the cell layers with the plant agglutinin from Evonymus europaea, pathogen attachment was also prevented . Binding of 125I-labeled CS3 adhesin and E . europaea agglutinin to brush border membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose revealed three bands of about 30, 20, and 13 kilodaltons, which acted as receptors for both bacterial and plant lectins . These data suggest that the sugar units to which the bacterial colonization factor CS3 binds are synthesized as carbohydrate chains of three brush border membrane glycoproteins in HT-29 cells by a differentiation-specific pathway. Infect Immun, 1989 Dec, 57(12), 3708 - 14 Molecular cloning and characterization of the 15-kilodalton major immunogen of Treponema pallidum; Purcell BK et al.; Pathogen-specific membrane immunogens of Treponema pallidum subsp . pallidum (T . pallidum) have been identified previously by phase partitioning with the nonionic detergent Triton X-114 . One of these antigens, a 15-kilodalton (kDa) polypeptide, is expressed in relatively small quantities in T . pallidum but is highly immunogenic in both human and experimental syphilis . The native T . pallidum antigen was purified to homogeneity from the mixture of Triton X-114 detergent-phase proteins by chromatofocusing . Recombinant Escherichia coli clones were selected from a T . pallidum genomic DNA library by using monoclonal antibodies specific to the 15-kDa antigen; immunoblotting and minicell analyses confirmed expression of the 15-kDa protein in the transformants . Southern hybridization with a 1.1-kilobase fragment of DNA encoding the 15-kDa-antigen gene indicated that the gene is probably present in a single copy within the genomes of both T . pallidum and T . pallidum subsp . pertenue (the agent of yaws), while it is absent from the genome of the nonpathogenic Treponema phagedenis biotype Reiter . Cell fractionation studies with Triton X-114 demonstrated that the recombinant polypeptide possesses hydrophobic properties similar to those of the native antigen and localized the cloned 15-kDa antigen to the inner membrane of E . coli . Protein processing experiments in minicells revealed that a precursor appears to be processed to the mature 15-kDa polypeptide. Vet Immunol Immunopathol, 1989 Dec, 23(3-4), 201 - 11 Production and purification of bovine monocyte-derived interleukin 1; Lederer JA et al.; Few studies have addressed the biological and molecular nature of bovine interleukin 1 (IL-1) . In an effort to increase our understanding of the role of bovine IL-1 in bovine immunology, we investigated various parameters of its production by LPS-stimulated monocytes in vitro . Bovine monocytes isolated by our methods constitutively released IL-1 activity, as measured by the murine thymocyte IL-1 assay . Monocyte release of IL-1 activity was further augmented when the cells were incubated with 0.005-10 micrograms per ml of Escherichia coli lipopolysaccharide (LPS) . The presence of 1, 5, or 10 percent heat-inactivated fetal bovine serum (FBS) enhanced LPS-stimulated bovine monocyte release of IL-1 activity as compared with monocytes cultured under serum-free conditions . We used a combination of size-exclusion and reverse-phase high-performance liquid chromatography (HPLC) to purify bovine IL-1 from serum-free monocyte culture supernatants . Size-exclusion HPLC resulted in a single peak of biological activity with an approximate molecular weight of 18,000 daltons . Further purification by reverse-phase HPLC demonstrated at least three major molecular species with IL-1 activity . Besides providing information about production of IL-1 by bovine monocytes in vitro, this study also describes a protocol to purify bovine IL-1 for future studies addressing its biological functions. Shi Yan Sheng Wu Xue Bao, 1989 Dec, 22(4), 433 - 44 A study of the chromatin structure of human beta-like hemoglobin genes in K562 cell line with a modified assay of nick-translation of nuclei; Zhu JD; A modified assay of nick-translation of nuclei has been developed to study the chromatin structure of human beta-like globin genes in nuclei of K 562 cell line . Nuclei were gently digested with DNase I and nick-translated with E . coli DNA polymerase I in the presence of 32P-triphosphate nucleotides . The total DNA from the labelled nuclei was used as probes to hybridize restricted fragments of beta-like globin genes which have been immobilized on Diazobenzyloxymethyl (DBM) paper . Using this approach we have observed that in K 562 nuclei all beta-like globin genes, including epsilon, gamma, delta, and beta-globin genes and human 18 S ribosomal genes are preferentially labelled in comparison to alpha-lactalbumin and c-sis genes which do not express in K 562 cells, but the total DNA from nick-translated nuclei of a nonerythroid cell line hybridized none of those genes except for 18 S ribosomal gene . This assay is a simple and fast method for surveying chromatin structure of any individual DNA sequence in nuclei once the corresponding clone is available. Poult Sci, 1989 Dec, 68(12), 1643 - 52 Genetically engineered antigen confers partial protection against avian coccidial parasites; Danforth HD et al.; A fusion protein of beta-galactosidase and Eimeria tenella produced in a recombinant Escherichia coli strain was injected into chickens and elicited partial protection against an oral challenge with Eim . tenella parasites . The fusion protein contained a 31 kilodalton (kD) coccidial antigen designated as 5401 . The DNA sequencing of the 5401 antigen-coding sequence revealed that this protein segment was highly negatively charged and strongly hydrophilic, and contained an amino-acid sequence repeated five times . A dose-titration study showed that immunizing chickens with a single subcutaneous injection of the 5401 antigen at 1,200 to 4,800 nanograms (ng)/bird in Freund's complete adjuvant decreased lesion scores, mortality, and feed conversions compared to unimmunized, challenged controls . Using the 1,200 and 2,400 ng/bird of the 5401 antigen, group weight gains were higher than for the unimmunized, challenged birds . In three other trials using the 5401 antigen at 2,400 ng/bird with light, medium, and heavy coccidial infections, significant protection was evidenced by reduced lesion scores, increased individual weight gains, or both . In addition, feed conversions were reduced when compared with unimmunized controls or birds immunized with a noncoccidial protein E . coli extract . Western blot analysis of sporozoite preparations with serum from 5401-immunized birds labeled two antigenic bands of 66 and less than 200 kD . These results indicate that the coccidial proteins produced in E . coli are potentially effective immunogens for protecting chickens against avian coccidiosis. Hinyokika Kiyo, 1989 Dec, 35(12), 2049 - 56 {Benzidine dyes and risk of bladder cancer}; Miyakawa M et al.; Until the early 1970's there was little concern about dyes which contain benzidine as an integral part of their chemical structure . Furthermore, use of the finished dyes was not considered dangerous . To ascertain whether azo dyes are associated with risk of development of bladder tumors in workers who handpaint Yuzen-type silk kimonos in Kyoto, we investigated the disintegration of dyes to benzidine . In these studies, we found that in rats and mice benzidine-based dyes are metabolized to benzidine and that the azo linkage of benzidine dyes is reduced by Escherichia coli and soil bacteria . These experimental findings were reported previously . In this report, we outline an approach to these studies . Many of the dyes used to color paper, textiles, lipstick, bait used by fishermen, as well as hair dyes, and dyes used in research, for pharmaceutical products, and by defence personnel for the detection of liquid chemical warfare agents, have been shown to be potentially mutagenic or carcinogenic . We review the literature on these dyes. J Pathol, 1989 Dec, 159(4), 323 - 7 Adriamycin-induced DNA strand breaks in HeLa and in P388 leukaemia cells detected using in situ nick translation; Maehara Y et al.; DNA strand breaks produced by adriamycin (ADR) were measured in HeLa cells and ADR-sensitive and -resistant P388 leukaemia cells, using the in situ nick translation method . The break sites in the DNA were translated artificially in the presence of Escherichia coli DNA polymerase I and 3H-labelled dTTP, and were visualized by autoradiographic observation of the grains . The DNA strand breaks in the HeLa cells increased in a dose-dependent manner, compared with findings in the untreated control cells, i.e., 15.2 fold at 20 micrograms/ml of ADR for 1 h . This level correlated with DNA single-strand breaks detected by the alkaline elution method . DNA breaks were also noted in the ADR-sensitive P388 cells, but in the ADR-resistant cells the level of DNA strand breaks was low . The enhanced cytotoxicity is apparently the consequence of the enhanced potential of ADR to cause breaks in the DNA strands . Our findings show that the survival response of the cells decreases and the level of DNA strand breaks increases following exposure to ADR . ADR resistance may be mediated by a reduction in the level of DNA strand breaks. J Cell Biol, 1989 Dec, 109(6 Pt 1), 3063 - 71 Neurite extension and neuronal survival activities of recombinant S100 beta proteins that differ in the content and position of cysteine residues; Winningham-Major F et al.; S100 beta produced in Escherichia coli from a synthetic gene (Van Eldik, L . J., J . L . Staecker, and F . Winningham-Major . 1988 . J . Biol . Chem . 263:7830-7837) stimulates neurite outgrowth and enhances cell maintenance in cultures of embryonic chick cerebral cortex neurons . In control experiments, the neurite extension activity is reduced by preincubation with antibodies made against bovine brain S100 beta . When either of the two cysteines in S100 beta are altered by site-directed mutagenesis, the resultant proteins maintain the overall biochemical properties of S100 beta, but lose both the neurite extension and neuronal survival activities . However, another S100 beta mutant, in which the relative position of one of the two cysteines was changed, had neurotrophic activity similar to that of the unmodified protein . These and other results indicate that (a) specific neurite extension activity and neuronal survival activity are two related activities inherent to the S100 beta molecule; (b) a disulfide-linked form of S100 beta is required for full biological activity, and (c) the relative position of the cysteines can be modified . These data suggest potential in vivo roles for S100 beta in the development and maintenance of neuronal function in the central nervous system, and demonstrate the feasibility of the longer term development of selective pharmacological agents based on the S100 beta structure. Eur J Cell Biol, 1989 Dec, 50(2), 435 - 41 The polymorphic human chaperonin protein HuCha60 is a mitochondrial protein sensitive to heat shock and cell transformation; Waldinger D et al.; The HuCha60 protein, a polymorphic protein on two-dimensional gels of human lymphocytes, is found to be structurally and functionally related to the Escherichia coli groEL gene product: The structural homology is evident from the N-terminal amino-acid sequence analysis and from the immunological cross-reactivity with an antiserum against the E . coli groEL gene product . The functional homology is suggested by the heat sensitivity and the growth dependence of this protein . Both genetic variants of the HuCha60 occurring on the two-dimensional protein pattern of lymphocytes, the common "a" variant and the rare "b" variant, are strongly enhanced after heat shock . The expression of the HuCha60 in resting or normally growing cultures human cells is in general low, whereas in mitogen-stimulated cells or transformed cell lines the synthesis of the HuCha60 is strongly enhanced . After cell fractionation and subsequent two-dimensional gel electrophoresis and immunoblotting, the HuCha60 has been found to be mainly expressed in mitochondria . In the cytosol fraction two different molecular weight forms of the HuCha60 have been observed with low expression . Also in the nuclear fraction, HuCha60 is present in low concentration. Mol Microbiol, 1989 Dec, 3(12), 1735 - 44 Analysis of genes coding for the sialic acid-binding adhesin and two other minor fimbrial subunits of the S-fimbrial adhesin determinant of Escherichia coli; Schmoll T et al.; The S fimbrial adhesin (Sfa) enables Escherichia coli to attach to sialic acid-containing receptor molecules of eukaryotic cells . As previously reported, the genetic determinant coding for the Sfa of an E . coli O6 strain was cloned, the gene coding for the major fimbrial subunit was identified and sequenced and the S specific adhesin was detected . Here we present evidence that in addition to the major subunit protein SfaA three other minor subunit proteins, SfaG (17 kD), SfaS (14 kD) and SfaH (31 kD) can be isolated from the S-specific fimbrial adhesin complex . The genes coding for these minor subunits were identified, mutagenized separately and sequenced . Using haemagglutination tests, electron-microscopy and quantitative ELISA assays with monoclonal anti-SfaA and anti-SfaS antibodies the functions of the minor subunits were determined . It was determined that SfaS is identical to the S-specific adhesin, which also plays a role in determination of the degree of fimbriation of the cell . The minor subunit SfaH also had some influence on the level of fimbriation of the cell, while SfaG is necessary for full expression of S-specific binding . It was further shown that the amino-terminal protein sequence of the isolated SfaS protein was identical to the protein sequence calculated from the DNA sequence of the sfaS gene locus. Mol Microbiol, 1989 Dec, 3(12), 1685 - 95 Genes for biosynthesis and assembly of CS3 pili of CFA/II enterotoxigenic Escherichia coli: novel regulation of pilus production by bypassing an amber codon; Jalajakumari MB et al.; The complete nucleotide sequence of the 4746bp HindIII fragment encoding the genes for the biosynthesis and assembly of CS3 pili has been determined . By site-directed mutagenesis in conjunction with analysis of the plasmid-encoded proteins in minicells, the actual reading frames for the various products have been determined . This demonstrated that the genes for four of the proteins (63 kD, 48 kD, 33 kD, and 20 kD in size) are encoded entirely within the same open reading frame as a fifth protein (104 kD) . However, for synthesis of this latter protein, suppression or readthrough of an internal amber codon is required . Termination at this codon is also necessary for synthesis of the former proteins . Two further proteins are also encoded within the HindIII fragment: a 27 kD precursor of a periplasmic protein and the 17.5kD precursor of the major CS3 fimbrial subunit. J Clin Microbiol, 1989 Dec, 27(12), 2724 - 9 Relationship among selected Leptospira interrogans serogroups as determined by nucleic acid hybridization; Nielsen JN et al.; Leptospiral DNAs from a variety of Leptospira interrogans serogroups of veterinary significance, as well as a nonpathogenic leptospira, were compared by Southern blot hybridization of EcoRI-digested genomic DNA . The serogroups examined could be assigned to one of three groups on the basis of the degree of cross-hybridization between genomic DNAs . Only a few restriction fragments hybridized between the three groups, and most of these were shown to contain ribosomal DNA . The restriction fragment length polymorphism observed among the intergroup hybridizations allowed differentiation among serogroups and, in some cases, serovars . Under the hybridization conditions used, no hybridization was observed between leptospiral DNA and Leptonema, Escherichia coli, or porcine DNA. Mol Gen Mikrobiol Virusol, 1989 Dec, (12), 3 - 12 {Escherichia coli phage receptors . Minor porins and proteins participating in the specific transport as phage receptors}; Likhacheva NA et al.; Except for the main porin proteins OmpC and OmpF there exist the membrane proteins participating in the transport of specific substrates: phosphates, nucleosides, iron, vitamin B12, maltose and maltodextrins, that also play the role of phage receptors . Some phages use as receptors the porins determined by the genes of lambdoid prophages . LamB protein that serves receptor for phage lambda exposes the amino acids sequence on the outer surface of membranes that participates in phage adsorption . The sequence is similar to tetrapeptide of fibronectin responsible for binding with the surface of cellular receptor in eucaryotes. Jpn J Genet, 1989 Dec, 64(6), 417 - 34 The transposable element Tn3 promotes general recombination at the neighboring regions; Kondo K et al.; Transposon Tn3 was inserted into a tRNA operon of the amber suppressor Su+2 on a transducing phage (lambda hcI857nin5pSu+2) by selecting phages with ampicillin resistance and Su- phenotypes . In a strain thus obtained, Tn3 was inserted between the promoter and the first tRNA gene of the operon, which was determined by DNA sequencing . The Su+2 tRNA operon on the transducing phage consisted of two tRNA genes for tRNA(Met) and Su+2 tRNA(2Gln), which was a deletion derivative of the supB-E tRNA operon of E . coli containing seven tRNA genes in the order of promoter-Met-Leu-Gln1-Gln1-Met-Gln2-Gln2 . Proliferating the lambda hcI857nin5pSu+2::Tn3 in E . coli cells, a number of phages which had lost Tn3 were isolated, and their tRNA gene compositions as well as the DNA structures of the tRNA operon were analyzed . In many cases the tRNA genes which had been deleted from the original transducing phage were regained from the chromosomal supB-E operon . Thus the loss of Tn3 from the phages was not due to excision of the transposon but due to the replacement of a portion of the tRNA operon, including Tn3, with the host homologous region that did not contain Tn3 . This type of replacement takes place rather efficiently as a consequence of Tn3 insertion, owing to the general recombination occurring between homologous tRNA genes of phage and host chromosomes in the presence of either host recA or phage red . No such enhanced recombination in a similar cross between phage and host chromosomes was observed with the Tn3 present in the trans position on an independent plasmid . We conclude that inserting Tn3 in cis promotes general recombination in the neighboring regions . Possible mechanisms for this new type of genetic effect of Tn3 are discussed . During the course of this study, a natural defective mutation (T11) was also detected in one of the duplicated tRNA(2Gln) genes in an E . coli K12 strain we used. J Med Virol, 1989 Dec, 29(4), 273 - 83 Detection by antibody probes of human papillomavirus type 6 E5 proteins in respiratory papillomata; Chen SL et al.; We have demonstrated the expression of proteins arising from the E5a and E5b open reading frames (ORFs) of human papillomavirus type 6c (HPV-6c) in respiratory tract papillomata . Recombinant plasmids were constructed to express the ORFs in the bacterial vectors pATH and pRIT2T . Fusion proteins were purified and injected into rabbits to produce polyclonal antibodies . Characterized antibodies generated against these fusion proteins were used in immunoperoxidase assays to identify the presence and distribution of HPV-6 E5 proteins in biopsy specimens of respiratory tract papillomata . The results showed that the E5a and E5b proteins were distributed throughout the thickness of the epithelium in the papillomata but not in the basal layer . The proteins were found in nuclei and in the cytoplasm of koilocytotic cells . Positive reactivity with a similar distribution in the epithelium and subcellular location was obtained in papillomata induced by other HPV-6 subtypes . This cross-reactivity was not unexpected, since nucleotide and amino acid (aa) sequence comparisons between HPV-6c and -6e demonstrated 79% sequence identity with 15 aa substitutions in the 91 aa of E5a . The E5b ORF of HPV-6c has the potential to encode a protein of 74 aa that differed at 28 positions compared with the 72 aa of HPV-6e. FEMS Microbiol Lett, 1989 Dec, 53(3), 253 - 7 Identification of the rhaA, rhaB and rhaD gene products from Escherichia coli K-12; Badia J et al.; The three structural genes rhaA, rhaB and rhaD, that specify the enzymes rhamnose isomerase, rhamnulose kinase and rhamnulose 1-phosphate aldolase respectively, have been cloned from Escherichia coli K-12 . The precise location of the genes has been determined by gene complementation analysis and by enzymatic assays of strains transformed with recombinant plasmids containing different parts of the cloned region . The corresponding gene products have been studied by their expression in maxicells . Protein products of 47 kDa, 52-54 kDa and 32 kDa have been assigned to rhamnose isomerase, rhamnulose kinase and rhamnulose 1-phosphate aldolase respectively. DNA, 1989 Dec, 8(10), 759 - 77 Superpolylinkers in cloning and expression vectors; Brosius J; Versatile DNA polylinkers of more than 300 bp were constructed . They contain the recognition sequences of all restriction enzymes--whether known or still to be discovered--that recognize palindromic hexamers . In addition to these 64 uninterrupted hexameric recognition sites, a number of sites containing interrupted palindromes and nonpalindromic sequences and two recognition sequences with 8 bp are present . Polylinkers (in several variants) were inserted into frequently utilized Escherichia coli cloning vectors such as pBluescript (yielding pSLJ10, pSL250, pSL260, pSL270, and pSL300), pUC18/pUC19 (yielding pSL180 and pSL190, respectively), or pUC118/pUC119 (yielding pSL1180 and pSL1190, respectively) . A subtle color discrimination between presence and absence of insert in pSL300 (mid-blue to light-blue or white) was seen in a number of test ligations . The mid-blue color that is generated by pSL300 is presumably due to translational restarts . A different intergenic region for translational restarts was used in plasmids pSL251, pSL261, pSL271, and pSL301 . The polylinker was also inserted into expression vector pUC120, yielding pSE1200, and into expression vector pKK233-2, yielding pSE220 and a shortened version thereof, pSE280 . Finally, the polylinker was inserted into pTrc99A, resulting in pSE380, which carries a lac repressor gene . This expands the use of the expression system beyond lacIq strains to other bacterial hosts . These versatile vectors have broad applications in genetic engineering. Am J Vet Res, 1989 Dec, 50(12), 2093 - 100 Induction of Escherichia coli mastitis in cows fed selenium-deficient or selenium-supplemented diets; Erskine RJ et al.; Ten Holstein heifers were fed a selenium-deficient (SeD) diet (0.04 mg of Se/kg on a total ration dry-matter basis) 3 months before calving and throughout their first lactation . A selenium-supplemented (SeS) diet (2 mg of Se/head/d) was fed to a group of 10 heifers . In about the 14th week of lactation, the cows were challenge-exposed to Escherichia coli by administering 15 to 40 colony-forming units (CFU) into 1 mammary gland . Selenium concentration (microgram/ml) in blood around the time of challenge exposure was 0.033 +/- 0.002 (mean +/- SEM) in SeD and 0.132 +/- 0.006 in SeS cows . Infections were established in all challenge-exposed quarters . The frequency of quarter atrophy and agalactia, and reduction in whole-udder milk yield in the first 4 days after challenge exposure, were greater (P less than 0.05) in the SeD cows . Log10 peak bacterial concentrations in milk were higher (P less than 0.05) in SeD (7.63 +/- 0.34 CFU/ml) than in SeS cows (5.57 +/- 0.66 CFU/ml) . Mean log bacterial concentration was significantly higher (P less than 0.05) from 12 to 20 hours after challenge exposure in SeD than in SeS cows . Duration of infection was significantly greater (P less than 0.05) in SeD (162.0 +/- 12.0) than in SeS cows (114.4 +/- 18.0 hours) . Milk somatic cell counts increased significantly more slowly (P less than 0.05) in SeD than in SeS cows from 8 to 16 hours after challenge exposure . Ratios of milk somatic cells to bacteria in milk were significantly lower (P less than 0.05) in SeD than in SeS cows at 12 and 16 hours after challenge exposure. Mol Gen Genet, 1989 Dec, 220(1), 147 - 53 Complete maps of IS1, IS2, IS3, IS4, IS5, IS30 and IS150 locations in Escherichia coli K12; Birkenbihl RP et al.; In this paper complete distribution maps are presented of the seven IS elements 1, 2, 3, 4, 5, 30 and 150 . These maps were obtained during the construction of an almost complete restriction map of the Escherichia coli genome of K12 strain BHB2600 . The positions of IS elements were correlated to this map . The distribution of integration sites of all IS types is nonrandom . Besides a large gap from 79 min to 96 min, there is a pronounced IS cluster at 6 min and another at 97 min, map locations that have low gene incidences on the classical map . One cluster coincides with a region of IS induced rearrangements . The IS distribution pattern was compared to patterns of strains W3110 and HB101. J Gen Virol, 1989 Dec, 70 ( Pt 12), 3269 - 80 The nucleotide sequence of coxsackievirus A9; implications for receptor binding and enterovirus classification; Chang KH et al.; The complete nucleotide sequence of the genome of coxsackievirus A9 (CAV-9) has been determined from cDNA cloned in Escherichia coli . Excluding the 3' poly(A) stretch, the RNA genome is 7452 nucleotides long and encodes a single polyprotein of 2201 amino acids . Comparison of the nucleotide and predicted amino acid sequences with those of the coxsackieviruses B1, B3 and B4 reveals a surprising degree of homology, with overall amino acid homologies of 86.9%, 86.2% and 87.0%, respectively . In contrast, there is much less homology to another coxsackie A virus, CAV-21, 60.4% overall amino acid homology . This demonstrates the high degree of diversity within the CAV group and indicates that the current classification does not directly correlate with molecular genetic properties . One major feature of CAV-9 is an insertion, relative to all other enteroviruses sequenced to date, which is located at the C terminus of VP1, and includes an arginine-glycine-aspartic acid tripeptide . Such sequences in a number of other proteins are known to have activity in promoting attachment to cell receptors and the implications for CAV-9 receptor binding are discussed. Biochem J, 1989 Dec 1, 264(2), 397 - 402 Purification and properties of uroporphyrinogen III synthase (co-synthase) from an overproducing recombinant strain of Escherichia coli K-12; Alwan AF et al.; The Escherichia coli hemD gene, encoding the enzyme uroporphyrinogen III synthase (co-synthase), was cloned into multi-copy plasmids in E . coli cells that were used to generate strains producing up to 1000 times the concentration of the synthase in the wild-type . The enzyme was purified to homogeneity from these strains in milligram amounts . The enzyme is a monomer of Mr 28,000 with an isoelectric point of 5.2 and a pH optimum of 7.8 . The specific activity of the purified synthase is 1500 units/mg and the Km for the substrate, pre-uroporphyrinogen, is 5 microM . The N-terminal sequence of the enzyme is Ser-Ile-Leu-Val-Thr-Arg-Pro-Ser-Pro-Ala-Gly-, in agreement with the gene-derived protein sequence . The enzyme contains four 5,5'-dithiobis-(2-nitrobenzoic acid)-titratable groups, one reacting rapidly with the reagent and three further groups having lower reactivity . The enzyme is heat-sensitive, and during heat inactivation all four thiol groups become equally available for reaction. Proc Natl Acad Sci U S A, 1989 Dec, 86(24), 9861 - 5 A ubiquitin carrier protein from wheat germ is structurally and functionally similar to the yeast DNA repair enzyme encoded by RAD6; Sullivan ML et al.; The RAD6 gene from the yeast Saccharomyces cerevisiae encodes a ubiquitin carrier protein (E2) required for a variety of cellular processes including DNA repair, induced mutagenesis, and sporulation . Here we identify an E2 from a higher plant, wheat, that is similar to RAD6 with respect to both structure and in vitro substrate specificity . The protein was purified from wheat germ by a combination of ubiquitin covalent affinity chromatography and anion-exchange HPLC and has an apparent molecular mass of 23 kDa {referred to as E2(23 kDa)} . E2(23 kDa) was capable of binding ubiquitin by means of a thiol ester linkage in an ATP-dependent and ubiquitin-activating enzyme-dependent reaction . In the presence of a variety of target proteins, E2(23 kDa), like the RAD6 gene product, formed covalent ubiquitin-protein conjugates in vitro only with histones in a ubiquitin protein ligase-independent reaction . E2(23 kDa) recognized both core and linker histones with an apparent order of preference of H2A greater than or equal to H1 greater than H2B greater than H3 greater than H4 . This E2 protein was approximately 17-fold more effective at conjugating ubiquitin to histones than three other purified wheat germ E2 proteins tested . Mouse anti-E2(23 kDa) antibodies were used to isolate E2(23 kDa) DNA sequences from a wheat cDNA expression library . Antibody-positive clones were confirmed by amino acid identity of the sequence deduced from the cDNA to the peptide sequence of an E2(23 kDa) tryptic fragment . Protein expressed in Escherichia coli by the E2(23 kDa) cDNA was capable of both thiol ester adduct formation and conjugation of ubiquitin to histones . Analysis of the E2(23 kDa) cDNA shows that it encodes a protein with considerable amino acid sequence similarity to the yeast RAD6 gene product . Similarities exist at the amino terminus, the region surrounding the putative ubiquitin binding site, and at the carboxyl terminus, which is unusually acidic . Based on both the structural and enzymatic similarities to the RAD6 gene product, E2(23 kDa) may represent the first DNA repair enzyme identified in higher plants. Proc Natl Acad Sci U S A, 1989 Dec, 86(24), 9793 - 7 Mapping the active-site tyrosine of vaccinia virus DNA topoisomerase I; Shuman S et al.; Site-directed mutagenesis of the vaccinia virus gene encoding a type I DNA topoisomerase implicates Tyr-274 as the active-site residue that forms a covalent adduct with DNA during cycles of DNA-strand breakage and reunion . Replacement of Tyr-274 by phenylalanine results in loss of the ability of the enzyme to relax negatively supercoiled DNA as well as to form the covalent DNA-protein intermediate . Substitution of phenylalanine for tyrosine at nine other sites in the protein has no apparent effect on enzyme activity . Amino acid sequence alignment reveals Tyr-274 to be homologous to Tyr-727 and Tyr-771, respectively, of the type I topoisomerases from Saccharomyces cerevisiae and Saccharomyces pombe; Tyr-727 and Tyr-771 have been shown to represent the active-site tyrosines of those enzymes . Sequence comparison of the active-site regions defines a motif Ser-Lys-Xaa-Xaa-Tyr common to the viral and cellular type I topoisomerases, including the human enzyme. Mutat Res, 1989 Dec, 215(2), 161 - 5 Mutations by near-ultraviolet radiation in Escherichia coli strains lacking superoxide dismutase; Hoerter J et al.; In wild-type Escherichia coli, near-ultraviolet radiation (NUV) was only weakly mutagenic . However, in an allelic mutant strain (sodA sodB) that lacks both Mn- and Fe-superoxide dismutase (SOD) and assumed to have excess superoxide anion (O2-), NUV induced a 9-fold increase in mutation above the level that normally occurs in this double mutant . When a sodA sodB double mutant contained a plasmid carrying katG+ (excess HP-I catalase), mutation by NUV was reduced to wild-type (sodA+ sodB+) levels . Also, in the sodA sodB xthA triple mutant, which lacks exonuclease III (exoIII) in addition to SOD, the mutational frequency by NUV was reduced to wild-type levels . This synergistic action of NUV and O2- suggested that pre-mutational lesions occur, with exoIII converting these lesions to stable mutants . Exposure to H2O2 induced a 2.8-fold increase in mutations in sodA sodB double mutants, but was reduced to control levels when a plasmid carrying katG+ was introduced . These results suggest that NUV, in addition to its other effects on cells, increases mutations indirectly by increasing the flux of OH . radicals, possibly by generating excess H2O2. Proc Natl Acad Sci U S A, 1989 Dec, 86(23), 9385 - 8 Sequences near the termini are required for transposition of the maize transposon Ac in transgenic tobacco plants; Coupland G et al.; Deletion derivatives of the maize transposable element Activator (Ac) were constructed in vitro and inserted into a kanamycin resistance gene . These constructions were then introduced into tobacco protoplasts derived from plants previously transformed with Ac . The ability of each deletion derivative to excise was measured by whether or not kanamycin-resistant tobacco calli were recovered . This allowed us to determine the length of DNA present at each terminus that is required to respond to the products expressed by the Ac element present in the genome . We show that around 200 base pairs (bp) are required at both ends for excision to occur at wild-type levels . When between 100 and 200 bp were retained at one of the ends, reduced frequencies of excision were detected . With less than 100 bp remaining at either end, no excision was detected . In addition, we show that although similar lengths of DNA are required at each terminus, the termini are not interchangeable . The significance of these data is discussed with respect to the protein(s) which interact(s) with the termini of Ac. Proc Natl Acad Sci U S A, 1989 Dec, 86(23), 9228 - 32 Aspartic acid-96 is the internal proton donor in the reprotonation of the Schiff base of bacteriorhodopsin; Otto H et al.; Above pH 8 the decay of the photocycle intermediate M of bacteriorhodopsin splits into two components: the usual millisecond pH-independent component and an additional slower component with a rate constant proportional to the molar concentration of H+, {H+} . In parallel, the charge translocation signal associated with the reprotonation of the Schiff base develops a similar slow component . These observations are explained by a two-step reprotonation mechanism . An internal donor first reprotonates the Schiff base in the decay of M to N and is then reprotonated from the cytoplasm in the N----O transition . The decay rate of N is proportional to {H+} . By postulating a back reaction from N to M, the M decay splits up into two components, with the slower one having the same pH dependence as the decay of N . Photocycle, photovoltage, and pH-indicator experiments with mutants in which aspartic acid-96 is replaced by asparagine or alanine, which we call D96N and D96A, suggest that Asp-96 is the internal proton donor involved in the re-uptake pathway . In both mutants the stoichiometry of proton pumping is the same as in wild type . However, the M decay is monophasic, with the logarithm of the decay time {log (tau)} linearly dependent on pH, suggesting that the internal donor is absent and that the Schiff base is directly reprotonated from the cytoplasm . Like H+, azide increases the M decay rate in D96N . The rate constant is proportional to the azide concentration and can become greater than 100 times greater than in wild type . Thus, azide functions as a mobile proton donor directly reprotonating the Schiff base in a bimolecular reaction . Both the proton and azide effects, which are absent in wild type, indicate that the internal donor is removed and that the reprotonation pathway is different from wild type in these mutants. Proc Natl Acad Sci U S A, 1989 Dec, 86(23), 9104 - 8 Escherichia coli replication termination protein impedes the action of helicases; Lee EH et al.; Identification of the consensus sequence for termination of replication (ter) in Escherichia coli and the isolation of the ter-binding protein (TBP) allowed us to test their effects on replication forks initiated at the unique origin of the E . coli chromosome (oriC) in a purified enzyme system . Replication was severely impeded by ter in a unique orientation when purified TBP was supplied to bind it . The target for blockage within the replication complex can now be ascribed to the inability of dnaB helicase to separate the duplex strands when it encounters ter bound by TBP . Other helicases, such as rep and uvrD proteins, that translocate on DNA and displace strands in the direction opposite to that of dnaB protein are also blocked, but only when the TBP-bound ter is oriented in the other direction . From these results, we infer that the orientation of ter confers a particular polarity on the TBP seated on it, such that a helicase is blocked when it confronts TBP from one side, but can act, presumably by displacing TBP, when facing its other side . Thus, the intrinsic nature of the oriented TBP-ter complex is responsible for impeding the helicases, rather than any protein-protein interactions. J Cell Biol, 1989 Dec, 109(6 Pt 1), 2665 - 75 A yeast gene important for protein assembly into the endoplasmic reticulum and the nucleus has homology to DnaJ, an Escherichia coli heat shock protein; Sadler I et al.; When nuclear localization sequences (termed NLS) are placed at the N terminus of cytochrome c1, a mitochondrial inner membrane protein, the resulting hybrid proteins do not assemble into mitochondria when synthesized in the yeast Saccharomyces cerevisiae . Cells lacking mitochondrial cytochrome c1, but expressing the hybrid NLS-cytochrome c1 proteins, are unable to grow on glycerol since the hybrid proteins are associated primarily with the nucleus . A similar hybrid protein with a mutant NLS is transported to and assembled into the mitochondria . To identify proteins that might be involved in recognition of nuclear localization signals, we isolated conditional-lethal mutants (npl, for nuclear protein localization) that missorted NLS-cytochrome c1 to the mitochondria, allowing growth on glycerol . The gene corresponding to one complementation group (NPL1) encodes a protein with homology to DnaJ, an Escherichia coli heat shock protein . npl1-1 is allelic to sec63, a gene that affects transit of nascent secretory proteins across the endoplasmic reticulum . Rothblatt, J . A., R . J . Deshaies, S . L . Sanders, G . Daum, and R . Schekman . 1989 . J . Cell Biol . 109:2641-2652 . The npl1 mutants reported here also weakly affect translocation of preprocarboxypeptidaseY across the ER membrane . A normally nuclear hybrid protein containing a NLS fused to invertase and a nucleolar protein are not localized to the nucleus in npl1/sec63 cells at the nonpermissive temperature . Thus, NPL1/SEC63 may act at a very early common step in localization of proteins to the nucleus and the ER . Alternatively, by affecting ER and nuclear envelope assembly, npl1 may indirectly alter assembly of proteins into the nucleus. J Bacteriol, 1989 Dec, 171(12), 6867 - 9 Suppression of the lethal effect of acidic-phospholipid deficiency by defective formation of the major outer membrane lipoprotein in Escherichia coli; Asai Y et al.; The Escherichia coli pgsA3 allele encoding a defective phosphatidylglycerophosphate synthase is lethal for all but certain strains . Genetic analysis of such strains has revealed that the lethal effect is fully suppressed by the lack of the major outer membrane lipoprotein that consumes phosphatidylglycerol for its maturation. J Bacteriol, 1989 Dec, 171(12), 6753 - 63 Nucleotide sequences and operon structure of plasmid-borne genes mediating uptake and utilization of raffinose in Escherichia coli; Aslanidis C et al.; The plasmid-borne raf operon encodes functions required for inducible uptake and utilization of raffinose by Escherichia coli . Raf functions include active transport (Raf permease), alpha-galactosidase, and sucrose hydrolase, which are negatively controlled by the Raf repressor . We have defined the order and extent of the three structural genes, rafA, rafB, and rafD; these are contained in a 5,284-base-pair nucleotide sequence . By comparisons of derived primary structures with known subunit molecular weights and an N-terminal peptide sequence, rafA was assigned to alpha-galactosidase (708 amino acids), rafB was assigned to Raf permease (425 amino acids), and rafD was assigned to sucrose hydrolase (476 amino acids) . Transcription was shown to initiate 13 nucleotides upstream of rafA; a putative promoter, a ribosome-binding site, and a transcription termination signal were identified . Striking similarities between Raf permease and lacY-encoded lactose permease, revealed by high sequence conservation (76%), overlapping substrate specificities, and similar transport kinetics, suggest a common origin of these transport systems . alpha-Galactosidase and sucrose hydrolase are not related to host enzymes but have their counterparts in other species . We propose a modular origin of the raf operon and discuss selective forces that favored the given gene organization also found in the E . coli lac operon. J Bacteriol, 1989 Dec, 171(12), 6703 - 9 Hydrolysis of bis(5'-nucleosidyl) polyphosphates by Escherichia coli 5'-nucleotidase; Ruiz A et al.; Two enzymatic activities that split diadenosine triphosphate have been reported in Escherichia coli: a specific Mg-dependent bis(5'-adenosyl) triphosphatase (EC 3.6.1.29) and the bis(5'-adenosyl) tetraphosphatase (EC 3.6.1.41) . In addition to the activities of these two enzymes, a different enzyme activity that hydrolyzes dinucleoside polyphosphates is described . After purification and study of its molecular and kinetic properties, we concluded that it corresponded to the 5'-nucleotidase (EC 3.1.3.5) that has been described in E . coli . The enzyme was purified from sonic extracts and osmotic shock fluid . From sonic extracts, two isoforms were isolated by chromatography on ion-exchange Mono Q columns; they had a molecular mass of about 100 kilodaltons (kDa) . From the osmotic shock fluid, a unique form of 52 kDa was recovered . Mild heating transformed the 100-kDa isoform to a 52-kDa form, with an increase in activity of about threefold . The existence of a 5'-nucleotidase inhibitor described previously, which associates with the enzyme and is not liberated in the osmotic shock fluid, may have been responsible for these results . The kinetic properties and substrate specificities of both forms (52 and 100 kDa) were almost identical . The enzyme, which is known to hydrolyze AMP and uridine-(5')-diphospho-(1)-alpha-D-glucose, but not adenosine-(5')-diphospho-(1)-alpha-D-glucose, was also able to split adenosine-(5')-diphospho-(5)-beta-D-ribose, ribose-5-phosphate, and dinucleoside polyphosphates {diadenosine 5',5'''-P1,P2-diphosphate,diadenosine 5',5'''-P1,P3-triphosphate, diadenosine 5',5'''-P1,P4-tetraphosphate, and bis(5'-guanosyl) triphosphate} . The effects of divalent cations and pH on the rate of the reaction with different substrates were studied. J Bacteriol, 1989 Dec, 171(12), 6573 - 9 Gyrase inhibitors can increase gyrA expression and DNA supercoiling; Franco RJ et al.; Treatment of bacterial cells with inhibitors of gyrase at high concentration leads to relaxation of DNA supercoils, presumably through interference with the supercoiling activity of gyrase . Under certain conditions, however, the inhibitors can also increase supercoiling . In the case of coumermycin A1, this increase occurs at low drug concentrations . Oxolinic acid increases supercoiling in a partially resistant mutant . We found that increases in chromosomal DNA supercoiling, which were blocked by treatment with chloramphenicol, were accompanied by an increased expression rate of gyrA . This result is consistent with gyrase being responsible for the increase in supercoiling . In wild-type cells, increases in gyrA expression were transient, suggesting that when supercoiling reaches sufficiently high levels, gyrase expression declines . Oxolinic acid studies carried out with a delta topA strain showed that drug treatment also increased plasmid supercoiling . The levels of supercoiling and topoisomer heterogeneity were much higher when the plasmid contained one of several promoters fused to galK . Since oxolinic acid causes an increase in gyrA expression, it appears that gyrase levels may be important in transcription-mediated changes in supercoiling even when topoisomerase I is absent. J Bacteriol, 1989 Dec, 171(12), 6437 - 45 Isolation, characterization, and inactivation of the APA1 gene encoding yeast diadenosine 5',5'''-P1,P4-tetraphosphate phosphorylase; Plateau P et al.; The gene encoding diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) phosphorylase from yeast was isolated from a lambda gt11 library . The DNA sequence of the coding region was determined, and more than 90% of the deduced amino acid sequence was confirmed by peptide sequencing . The Ap4A phosphorylase gene (APA1) is unique in the yeast genome . Disruption experiments with this gene, first, supported the conclusion that, in vivo, Ap4A phosphorylase catabolizes the Ap4N nucleotides (where N is A, C, G, or U) and second, revealed the occurrence of a second Ap4A phosphorylase activity in yeast cells . Finally, evidence is provided that the APA1 gene product is responsible for most of the ADP sulfurylase activity in yeast extracts. J Virol, 1989 Dec, 63(12), 5037 - 45 Cleavage of small peptides in vitro by human rhinovirus 14 3C protease expressed in Escherichia coli; Cordingley MG et al.; The 3C region of human rhinovirus 14 was expressed in Escherichia coli . The microbially synthesized protease was functional, since the expressed precursor underwent autoproteolytic processing to generate mature molecules of the expected molecular weight and antigenicity . Mutation of the putative active-site Cys-146 residue to an alanine resulted in the synthesis of unprocessed precursor molecules . Large quantities of the 20-kilodalton protease were purified by a simple purification protocol, and the resulting molecule was shown to be biologically active in vitro against synthetic peptides corresponding to the 2C-3A cleavage site . This site was cleaved with high efficiency and fidelity and was used to generate kinetic data on the 3C protease . The protease exhibited sensitivity to Zn2+, was capable of cleaving five of seven rhinovirus cleavage site peptides tested with variable efficiency, and could distinguish authentic substrate peptides from control peptides containing the dipeptide cleavage sequence pair Gln-Gly. J Virol, 1989 Dec, 63(12), 5023 - 9 The essential 65-kilodalton DNA-binding protein of herpes simplex virus stimulates the virus-encoded DNA polymerase; Gallo ML et al.; The 65-kilodalton DNA-binding protein (65KDBP) of herpes simplex virus type 1 (HSV-1), the product of the UL42 gene, is required for DNA replication both in vitro and in vivo, yet its actual function is unknown . By two independent methods, it was shown that the 65KDBP stimulates the activity of the HSV-1-encoded DNA polymerase (Pol) . When Pol, purified from HSV-1-infected cells, was separated from the 65KDBP, much of its activity was lost . However, addition of the 65KDBP, purified from infected cells, stimulated the activity of Pol 4- to 10-fold . The ability of a monoclonal antibody to the 65KDBP to remove the Pol-stimulating activity from preparations of the 65KDBP confirmed that the activity was not due to a trace contaminant . Furthermore, the 65KDBP did not stimulate the activity of other DNA polymerases derived from T4, T7, or Escherichia coli . The 65KDBP gene transcribed in vitro from cloned DNA and translated in vitro in rabbit reticulocyte lysates also was capable of stimulating the product of the pol gene when the RNAs were cotranslated . The product of a mutant 65KDBP gene missing the carboxy-terminal 28 amino acids exhibited wild-type levels of Pol stimulation, while the products of two large deletion mutants of the gene could not stimulate Pol activity . These experiments suggest that the 65KDBP may be an accessory protein for the HSV-1 Pol. Endocrinology, 1989 Dec, 125(6), 2800 - 5 In the mouse, the activation of the hypothalamic-pituitary-adrenal axis by a lipopolysaccharide (endotoxin) is mediated through interleukin-1; Rivier C et al.; The mechanisms through which endotoxin stimulates the hypothalamic-pituitary-adrenal axis are not well understood . In the studies reported here we tested the hypothesis that endotoxin increases plasma ACTH levels by releasing interleukin-1 (Il-1) . Two experimental tools reported to interfere with the biological activity of IL-1 were used: antibodies directed against IL-1 receptors and alpha MSH . In a first series of experiments, adult male mice were injected with a lipopolysaccharide (LPS; 25 micrograms), antibodies against IL-1 receptor, alpha MSH (1-30 micrograms), or LPS and either IL-1 antibodies or alpha MSH . All treatments were administered ip . The endotoxin LPS caused a marked increase in plasma ACTH levels, measured 6 h later . Both alpha MSH and the Il-1 receptor antibodies, while having no effect by themselves, significantly (P less than or equal to 0.01) blocked LPS-induced ACTH release . In a second series of experiments, mice were injected ip with 500 ng recombinant human (rh) Il-1 alpha or 100 ng rhIl-1 beta in the presence or absence of alpha MSH (1-30 micrograms, ip) . While not altering ACTH secretion induced by rhIl-1 alpha, 10-30 micrograms alpha MSH significantly (P less than or equal to 0.01) interfered with the effect of rhIl-1 beta . These results suggest 1) that endotoxin activates the hypothalamic-pituitary-adrenal axis through a mechanism involving the activation of interleukin-1 receptors; and 2) that the effect of rhIl-1 beta, but not -alpha, on ACTH secretion by the mouse can be partially blocked by alpha MSH. J Trace Elem Electrolytes Health Dis, 1989 Dec, 3(4), 187 - 91 Mutagenic effects of lead (II) bromide; Maslat AO et al.; The mutagenicity of lead (II) bromide (a combustion product of the gasoline additives lead (IV) tetraethyl and 1,2-dibromoethane) was investigated using various strains of bacteria . Taking prodigiosin (the red pigment) production as a marker, lead (II) bromide was found to be mutagenic in S . marcescens, leading to the appearance of white mutant colonies that are unable to produce such a pigment . This compound was also found to be mutagenic in E . coli KMBL1851, resulting in the appearance of rifampicin-resistant mutants in addition to Met+ and His+ revertants . Some of the S . marcescens mutants were found to be reversible, able to resynthesize prodigiosin . Differences in the sensitivity to antibiotics as well as in the biochemical properties were detected between the mutants and their corresponding wild types . Lead (II) bromide gave positive results in the Ames test performed with strain TA 1535. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1989 Dec, 11(6), 438 - 41 {Site of endotoxic pulmonary vasoconstriction and the effect of anisodamine}; Sun RY; Using arterial and venous occlusion techniques in an in situ, isolated left goat lung preparation, the total arteriovenous pressure drop across the lung was partitioned vertically into pressure drops across the relatively indistensable arteries (delta Pa) and veins (delta Pv) and the middle distensible vessels (delta Pm) . Endotoxin primarily increased delta Pv and Pc . Anisodamine only showed a slight effect on pressure drops in different segment under normal conditions . It could attenuate the endotoxin effect however . Serotonin mainly increased delta Pa and showed no effect on delta Pv and delta Pm . Norepinephrine and histamine increased delta Pv and Pc while it showed almost no effect on delta Pa . It is possible that the norepinephrine and histamine mediate the pulmonary hypertension caused by endotoxin. Biochem J, 1989 Dec 1, 264(2), 533 - 8 The expression of canine cardiac phospholamban in heterologous systems; Cook EA et al.; A synthetic phospholamban gene has been cloned and expressed in Escherichia coli, producing both native phospholamban and a fusion protein with 81 amino acids of the influenza virus NS1 protein . Both the native phospholamban and fusion proteins produced extensive cell lysis upon induction of gene expression, but only the native protein underwent spontaneous pentamer formation in E . coli . Translation in vitro of mRNA produced by transcription in vitro of phospholamban cDNA was used to demonstrate the spontaneous aggregation of phospholamban to form pentamers in this system also, both in the presence and absence of exogenous microsomes from canine pancreas or heart . Phospholamban produced by translation in vitro was apparently susceptible to proteolysis by enzymes present in the particulate fractions in these experiments. Proc Natl Acad Sci U S A, 1989 Dec, 86(23), 9134 - 8 Maltose transport in membrane vesicles of Escherichia coli is linked to ATP hydrolysis; Dean DA et al.; We examined the energy requirement for maltose transport in right-side-out membrane vesicles derived from Escherichia coli . When membrane vesicles were made from strains producing tethered maltose-binding proteins by dilution of spheroplasts into phosphate buffer, those from an F0F1 ATPase-containing (unc+) strain transported maltose in the presence of an exogenous electron donor, such as ascorbate/phenazine methosulfate, at a rate of 1-5 nmol/min per mg of protein, whereas those from an isogenic unc- strain failed to transport maltose . Transport in vesicles obtained from the latter strain could be restored in the presence of electron donors if the vesicles were made to contain NAD+ and either ATP or an ATP-regenerating system . ATP hydrolysis was apparently required for transport, since nonhydrolyzable ATP analogues did not sustain transport . Maltose transport significantly increased ATP hydrolysis in ATP-containing vesicles from unc- cells . Finally, ATP-containing vesicles from unc- strains producing normal maltose-binding proteins could accumulate maltose in the absence of electron donors . These results provide convincing evidence that it is the hydrolysis of ATP that drives maltose transport, and probably also other periplasmic-binding-protein-dependent transport systems. J Bacteriol, 1989 Dec, 171(12), 6853 - 8 Overproduction of truncated subunit a of H+-ATPase causes growth inhibition of Escherichia coli; Eya S et al.; Genes (uncB) for wild-type and mutant a subunits of Escherichia coli H+-ATPase (F0F1) were cloned into recombinant plasmids . The subunits were expressed under the control of a weak promoter of the unc operon at 30 degrees C and strong promoters of lambda phage at 42 degrees C . At 30 degrees C, the wild type and a truncated (Glu-269----end) a subunit complemented the defect of the a subunit mutant KF24A (Trp-111----end), whereas the other mutant subunits (Trp-111----end, Trp-231----end, Gln-252----end, and a subunit with a deletion of residues 21 to 227) did not . Three mutant subunits (Trp-231----end, Gln-252----end, and Glu-269----end) and the wild-type a subunit caused growth inhibition associated with cell elongation, an uneven distribution of membrane proteins, and an altered septum structure when they were expressed at 42 degrees C . These phenomena were not observed with the other mutant subunits, suggesting that overproduction of the middle region (between residues 111 and 230) of the a subunit causes growth inhibition. Genetika, 1989 Dec, 25(12), 2138 - 50 {Interference of inducible repair processes in Escherichia coli}; Vasil'eva SV et al.; Radiation and the majority of chemical mutagens produce lesions in the DNA of cells which provoke the induction--as a reverse response--of some inducible repair processes . One of them is the adaptive response--highly specific in the repair of damages, induced by alkylating agents . This repair pathway decreases the toxic and mutagenic effects of many alkylating agents and can be induced in Escherichia coli cells exposed to sublethal concentrations of the same agents . By contrast, the SOS repair pathway in E . coli is non-specific and transient phenomenon which leads, among other things, to bacterial mutagenesis . It is controlled by the regulatory RecA protein which in its activated form promotes the cleavage of LexA repressor, allowing for the increased transcription of about 17 repressed genes--SOS regulon . The latter is not associated with the adaptive response . Nevertheless, there are experimental data indicating that the adaptive response is able to reduce some functions of the SOS repair activity--W-reactivation, W-mutagenesis and lambda phage induction . A relatively new bacterial short-term assay for genotoxicity, the SOS chromotest with E . coli PQ37 as an indicator organism, makes it possible to measure SOS induction indirectly, on the basis of a simple colorimetric assay . In the present study, the SOS chromotest in a completely automated system "Bioscreen C" was used to study interference between the adaptive and SOS responses in E . coli . Our data indicate that there is an inhibitory effect of the adaptive response on the SOS induction, as well as the negative interference between two successive SOS responses, at a transcriptional level of the SOS induction. In Vitro Cell Dev Biol, 1989 Dec, 25(12), 1147 - 54 A mouse hybrid cell line that supports gene expression from a variety of promoters in amplifiable vectors; Gopal TV et al.; A hybrid cell line was constructed by fusion of mouse L-cells with an NIH3T3 cell line derivative containing a hybrid gene consisting of the mouse immunoglobulin kappa (IgK) variable gene promoter linked to the Escherichia coli gpt gene . Such hybrids grew to a much higher density compared to either of the parental cell lines . The utility of this cell line as a host to express foreign genes was tested by the expression of TGF-beta cDNA using the cytomegalovirus promoter . The vector also contained the human dihydrofolate reductase (DHFR) gene driven by SV40 early promoter, to allow for the amplification of the transfected gene . Initial transformants, selected at 100 nM methotrexate (MTX), were subsequently selected for resistance to a higher concentration of MTX (2 microM) . Such clones expressed an increased level of TGF-beta when compared to the initial transformants . Both the initial transformants and the clones with the amplified DHFR gene produced TGF-beta in an acid-activatable precursor form . This mouse hybrid host cell line also allowed the expression of foreign genes cloned in an eukaryotic expression vector with the mouse IgK variable region promoter and human growth hormone as the reporter gene, whereas such vectors did not function in CHO cells . The mouse hybrid cell line was also found to be capable of being used with a broad range of promoters. Circ Shock, 1989 Dec, 29(4), 311 - 8 Effect of dietary linolenic acid on endotoxin-induced thromboxane and prostacyclin production by equine peritoneal macrophages; Morris DD et al.; In laboratory animals, the incorporation of alpha linolenic acid or other n-3 series fatty acids into the diet results in marked changes in cell membrane composition as well as arachidonic acid metabolism . The purpose of the present study was to determine whether endotoxin-induced thromboxane A2 (TxA2) and/or prostacyclin (PGI2) production by equine peritoneal macrophages was altered by feeding horses a diet containing 8% linseed oil as a source of alpha linolenic acid for 8 weeks . Peritoneal macrophages were cultured in vitro in the presence of endotoxin (LPS) (0.5-500 ng/ml) or calcium ionophore for 6 and 24 hours . After horses were fed the alpha linolenic acid-enriched diet, their peritoneal macrophage production of TxA2 was reduced in response to 0.5 ng/ml and 5 ng/ml LPS . compared to that before the diet (P less than .05) . The production of PGI2 during 6 hour incubation with 5 ng/ml and 50 ng/ml LPS and during 24 hour incubation with 5 ng/ml LPS were reduced, compared to that before the diet (P less than .05) . Peritoneal macrophage production of PGI2 during 24 hour incubation with nothing, LPS (0.5 ng/ml, 5 ng/ml and 500 ng/ml), and calcium ionophore was greater than during 6 hour incubation, after horses were fed the ALA-rich diet (P less than .05) . Results suggest that linseed oil supplementation may be an aid in prophylaxis of endotoxemia in horses. Proc Natl Acad Sci U S A, 1989 Dec, 86(23), 9465 - 9 Blocking of passive sensitization of human mast cells and basophil granulocytes with IgE antibodies by a recombinant human epsilon-chain fragment of 76 amino acids; Helm B et al.; The recombinant peptide corresponding to residues 301-376 at the junction of constant regions 2 and 3 of the human IgE epsilon chain blocked the in vivo passive sensitization of human skin mast cells and in vitro sensitization of human basophil granulocytes with human IgE antibodies . An injection of the recombinant peptide or E myeloma protein into normal skin sites 1 hr before sensitization with an allergic serum blocked passive sensitization . In this system, approximately 10-fold higher molar concentration of the recombinant peptide than E myeloma protein was required for 50% inhibition of Prausnitz-Kustner reactions . When the mononuclear cells of two normal individuals were preincubated with the recombinant peptide or E myeloma protein for 15 min before passive sensitization with the same allergic serum and the cells were challenged with an optimal concentration of an antigen, approximately 11- to 13-fold higher concentration of the recombinant peptide than E myeloma protein was required for 50% inhibition of antigen-induced histamine release . Further studies with several recombinant peptides indicated that amino acid resides 363-376 in the Fc epsilon-chain fragment are not essential for binding of the peptide to Fc epsilon-chain receptor I. J Cell Biol, 1989 Dec, 109(6 Pt 1), 2677 - 91 A novel GTP-binding protein, Sar1p, is involved in transport from the endoplasmic reticulum to the Golgi apparatus; Nakano A et al.; SAR1, a gene that has been isolated as a multicopy suppressor of the yeast ER-Golgi transport mutant sec12, encodes a novel GTP-binding protein . Its nucleotide sequence predicts a 21-kD polypeptide that contains amino acid sequences highly homologous to GTP-binding domains of many ras-related proteins . Gene disruption experiments show that SAR1 is essential for cell growth . To test its function further, SAR1 has been placed under control of the GAL1 promoter and introduced into a haploid cell that had its chromosomal SAR1 copy disrupted . This mutant grows normally in galactose medium but arrests growth 12-15 h after transfer to glucose medium . At the same time, mutant cells accumulate ER precursor forms of a secretory pheromone, alpha-mating factor, and a vacuolar enzyme, carboxypeptidase Y . We propose that Sec12p and Sarlp collaborate in directing ER-Golgi protein transport. J Bacteriol, 1989 Dec, 171(12), 6517 - 20 Genetic selection and DNA sequences of 4.5S RNA homologs; Brown S et al.; A general strategy for cloning the functional homologs of an Escherichia coli gene was used to clone homologs of 4.5S RNA from other bacteria . The genes encoding these homologs were selected by their ability to complement a deletion of the gene for 4.5S RNA . DNA sequences of the regions encoding the homologs were determined . Since this approach does not require that the homologous genes hybridize with probes generated from the E . coli sequence, the sequences of the homologs were not all similar to the sequence of the E . coli gene . Despite the dissimilarity of the primary sequences of some of the homologs, all could be folded to obtain a similar structure. Mutat Res, 1989 Dec, 227(4), 215 - 9 Antimutagenic effect of garlic (Allium sativum L.) on 4NQO-induced mutagenesis in Escherichia coli WP2; Zhang YS et al.; Aqueous extract prepared from garlic bulbs markedly suppressed the mutagenesis in both E . coli WP2 trp- and E . coli WP2 trp- uvrA- induced by 4-nitroquinoline 1-oxide (4NQO), but not that induced by UV . Cellular toxicity, inhibition of the expression of the Trp+ phenotype and delay of the first cell division after 4NQO treatment were not observed in the presence of the extract . Since the extract showed identical antimutagenic effects against 4NQO in both test strains but no effect on the mutagenesis of UV, it seems that the extract might act by inactivating the electrophilic group(s) of 4NQO or inhibiting its metabolic activation. J Immunol, 1989 Dec 1, 143(11), 3609 - 13 Polyspecific binding of Escherichia coli beta-galactosidase by murine antibodies to DNA; Pisetsky DS et al.; To characterize further polyspecific interactions of antibodies to DNA, the binding of sera from autoimmune MRL-lpr/lpr mice to Escherichia coli beta-galactosidase (beta-gal) was analyzed . This protein was selected for study because of preliminary observations that sera from autoimmune mice bound unexpectedly to cloned fusion protein constructions containing beta-gal . Using ELISA assays, sera from MRL-lpr/lpr mice demonstrated high levels of antibodies to both DNA and beta-gal, in titers significantly greater than those of BALB/c controls . Affinity chromatography using beta-gal-Sepharose demonstrated that antibodies enriched for anti-beta-gal activity bound both DNA as well as beta-gal, indicating the presence of a population of cross-reactive anti-DNA antibodies . Furthermore, anti-DNA mAb of MRL-lpr/lpr strain origin also bound beta-gal by ELISA, although these levels were lower than those to DNA . Together, these results extend the range of polyspecific binding of murine anti-DNA antibodies to bacterial proteins . They further suggest caution in the interpretation of immunoassays using fusion protein constructions containing beta-gal, especially with sera from autoimmune mice. Plant Mol Biol, 1989 Dec, 13(6), 673 - 84 The malate synthase gene of cucumber; Graham IA et al.; The complete sequences of a full-length cDNA clone and a genomic clone encoding the Cucumis sativus glyoxysomal enzyme malate synthase, have been determined . The sequences have enabled us to identify putative control regions at the 5' end of the gene, three introns, and possible alternative polyadenylation sites at the 3' end . The deduced amino acid sequence predicts a polypeptide of 64,961 molecular weight, which has 48% identity with that of Escherichia coli . Comparison of the sequence of malate synthase from cucumber with that from E . coli and with other glyoxysomal and peroxisomal enzymes, shows that a conserved C-terminal tripeptide is a common feature of those enzymes imported into microbodies. J S Afr Vet Assoc, 1989 Dec, 60(4), 201 - 5 {Clinico-pathological changes after intravenous administration of endotoxin in the horse}; Stadler P et al.; The results of a study conducted to determine the clinico-pathological changes in 4 experimentally-induced cases of endotoxaemia in the horse are reported on . Endotoxaemia was induced by injecting commercially available E . coli 055:B5 lipopolysaccharide intravenously at a dose of 1 microgram kg-1 . The haematocrit, red cell count, total and differential white cell counts, thrombocyte count, prothrombin time, partial thromboplastin time, fibrinogen level, level of fibrin degradation products, arterial acid-base status, serum lactate and blood glucose were determined repeatedly . Changes that occurred, include increases in the haematocrit and red cell count, a leucopaenia followed by a leucocytosis caused mainly by changes in the number of neutrophils, the development of disseminated intravascular coagulation, minor changes in the arterial acid base parameters, hyperglycaemia followed by hypoglycaemia and an increase in serum lactate. New Biol, 1989 Dec, 1(3), 285 - 96 Transcription of infectious yellow fever RNA from full-length cDNA templates produced by in vitro ligation; Rice CM et al.; Yellow fever (YF) virus is the prototype member of the flavivirus family, a diverse group of human and animal pathogens . A live-attenuated strain of YF virus, called 17D, has been used successfully for human vaccination for more than 50 years . In this report we describe the construction of full-length YF 17D cDNA templates that can be transcribed in vitro to yield infectious YF virus RNA . Because of the instability of full-length YF cDNA clones and their toxic effects on Escherichia coli, we developed a strategy in which full-length templates for transcription were constructed by in vitro ligation of appropriate restriction fragments . The YF virus recovered from cDNA was indistinguishable from the parental virus by several criteria . This system should facilitate the molecular genetic analysis of flavivirus replication and attenuation and may allow YF 17D to be used as a carrier for immunologically important epitopes from other disease agents. Biochem Int, 1989 Dec, 19(6), 1323 - 38 Structural properties of ribosomal protein L11 from Escherichia coli; Choli T; Protein L11 has been isolated from the large subunit of the E . coli ribosome under non-denaturing conditions and studied by proton magnetic resonance spectroscopy, limited proteolysis, and fluorescence and UV spectroscopy . The protein consists of two domains, a tightly-folded N-terminal part and a C-terminal half with an extended and loosely folded conformation . It is likely that the N-terminal domain is located on the surface of the subunit whereas the C-terminal part is buried within the ribosomal structure . The two tyrosines in the N-terminal region behave as solvent-exposed residues, in good agreement with iodination studies on L11 in situ . It appears probable that the central region of L11, in which the protease cleavages occur, plays an important part in structural and functional aspects. J Protein Chem, 1989 Dec, 8(6), 701 - 17 Epitopes of Escherichia coli ribosomal protein S13; Syu WJ et al.; To analyze the immunochemical structure of Escherichia coli ribosomal protein S13 and its organization in situ, we have generated and characterized 22 S13-specific monoclonal antibodies . We used a competitive enzyme-linked immunosorbent assay to divide them into groups based on their ability to inhibit binding of one another . The discovery of five groups with distinct binding properties suggested that a minimum of five distinct determinants on S13 are recognized by our monoclonal antibodies . The locations of the epitopes detected by these monoclonal antibodies have been mapped on S13 peptides . Three monoclonal antibodies bind a S13 C-terminal 34-residue segment . All the other 19 monoclonal antibodies bind a S13 N-terminal segment of about 80 residues . The binding sites of these 19 monoclonal antibodies have been further mapped to subfragments of peptides . Two monoclonal antibodies recognized S131-22; three monoclonal antibodies bound to S131-40; the binding sites of three other antibodies have been located in S1323-80, with epitopes possibly associated with residues 40-80 . The remaining 11 monoclonal antibodies did not bind to these subfragments . These data provide molecular basis to the structure of S13 epitopes, whose in situ accessibility may reveal the S13 organization on the ribosome. J Membr Biol, 1989 Dec, 112(3), 267 - 75 Voltage-sensitive ion channel of Escherichia coli; Delcour AH et al.; A voltage-sensitive, cation-selective ion channel of Escherichia coli has been reconstituted into liposomes and studied with the patch-clamp method . The single channel conductance was 91 pS in symmetric solutions of 150 mM KCl . Many channels were open most of the time, with frequent brief transitions to closed levels . Multiple conducting units could close and reopen simultaneously, and this apparent cooperativity in gating was increased with depolarizing voltages . Above a voltage threshold, the channels closed irreversibly, often in groups. Arch Biochem Biophys, 1989 Dec, 275(2), 344 - 53 Anti-idiotypic antibodies elicited by pterin recognize active site epitopes in dihydrofolate reductases and dihydropteridine reductase; Ratnam S et al.; Monoclonal antibodies (mAbs) against antipterin immunoglobulin and dihydropteridine reductase (DHPR) and also polyclonal antibodies against human dihydrofolate reductase (DHFR) were obtained . The anti-idiotypic mAbs and anti-DHPR mAbs bind specifically to human DHFR, Escherichia coli DHFR, soybean seedling DHFR, and human DHPR in solid-phase immunoassays . Further, the mAbs bind to the native but not to the denatured forms of DHFRs . The monoclonal antibodies also inhibit the enzymatic activity of human DHFR but not that of human DHPR . Competitive solid-phase immunoassays show stoichiometric inhibition by methotrexate and partial inhibition by NADPH of mAb binding to human DHFR . Cyanogen bromide fragments derived from human DHFR (residues 15-52 and 53-111), containing several active site residues, bind partially to some of the monoclonal antibodies . Accordingly, polyclonal antibodies to peptide 53-111 of human DHFR cross-react to some extent with human DHPR . Data from competitive immunoassays in which the binding of the various mAbs was tested singly and in combination with other mAbs suggest that these antibodies bind to a common region on human DHFR . The results also indicate that the mAbs display some heterogeneity with respect to specific epitopes . These data suggest that despite the absence of significant amino acid sequence homologies among the various DHFRs and DHPR, they have a fundamentally similar topography at the site of binding of the pterin moiety that is recognized by the anti-idiotypic mAbs generated by pterin . In the relatively simple structure of the pterin ring system there are different substituent groups at positions C4 and C6 in methotrexate, 7,8-dihydrofolate, and 7,8-dihydrobiopterin, suggesting that these antibodies are specific for regions on various proteins that interact with the remainder of the pterin moiety . These mAbs and similar mAbs specified by substituent groups on pterin may thus be used as specific probes or inhibitors of various folate-dependent enzymes and transport proteins . They should also provide insights into some of the general features of antibody recognition of protein antigens. J Bacteriol, 1989 Dec, 171(12), 6862 - 6 Characterization in vitro of the defect in a temperature-sensitive mutant of the protein subunit of RNase P from Escherichia coli; Baer MF et al.; We have studied the assembly of Escherichia coli RNase P from its catalytic RNA subunit (M1 RNA) and its protein subunit (C5 protein) . A mutant form of the protein subunit, C5A49, has been purified to apparent homogeneity from a strain of E . coli carrying a thermosensitive mutation in the rnpA gene . The heat inactivation kinetics of both wild-type and mutant holoenzymes are similar, an indication of equivalent thermal stability . However, when the catalytic efficiencies of the holoenzymes were compared, we found that the holoenzyme containing the mutant protein had a lower efficiency of cleavage than the wild-type holoenzyme at 33, 37, and 44 degrees C . We then explored the interaction of M1 RNA and C5 protein during the assembly of the holoenzyme . The yield of active holoenzyme obtained by reconstitution with wild-type M1 RNA and C5A49 protein in vitro can be considerably enhanced by the addition of excess M1 RNA, just as it can be in vivo . We concluded that the Arg-46----His-46 mutation in the C5A49 protein affects the ability of the protein to participate with M1 RNA in the normal assembly process of RNase P. Anesth Analg, 1989 Dec, 69(6), 714 - 20 Hypertonic saline solution-hetastarch for fluid resuscitation in experimental septic shock; Armistead CW Jr et al.; Hypertonic colloid solutions have been found efficacious in the resuscitation from hemorrhagic/traumatic shock . The present study investigated the hemodynamic, gasometric, and metabolic effects of hypertonic colloids in endotoxic shock in the dog . Thirty minutes after administration of 3 mg/kg normal body weight of Escherichia coli endotoxin, dogs were randomly assigned to receive 10 mL/kg hydroxyethylstarch (HES) either in 0.9% NaCl (HES, 10 dogs) or in 7.5% NaCl (HT-HES, 10 dogs) in 30 min . Thereafter, 0.9% NaCl solution was administered in volumes adequate to maintain pulmonary artery balloon-occluded pressure at baseline levels . Total fluid administered averaged 64 +/- 30 mL/kg (mean +/- SD) in the HES group and 73 +/- 34 mL/kg in the HT-HES group . As these differences were not statistically significant, total sodium load was higher in the HT-HES group . The persistent volume effect was associated with persistently lower hematocrit and protein levels in the HT-HES group . Initial fluid resuscitation with HT-HES resulted in arterial pressure, cardiac filling pressures, cardiac output, stroke volume, and rates of oxygen delivery and oxygen consumption that were greater than those with HES . Vascular resistances were similar . Analysis of left ventricular function curves also indicated an improvement in cardiac performance . However, these effects almost completely vanished during the remainder of the study . In the HT-HES group, serum sodium and osmolality levels increased to 167 +/- 4 mEq/L and 344 +/- 4 mOsm/kg H2O, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) Biochem Biophys Res Commun, 1989 Nov 30, 165(1), 474 - 80 Detection of total DNA with single-stranded DNA binding protein conjugates; Sheldon EL 3rd et al.; We have developed a rapid and sensitive method for total DNA measurement using single-stranded DNA binding protein from E coli conjugated with horseradish peroxidase or urease . To detect DNA, the sample is heated or alkali treated to denature the DNA and then filtered through nylon or nitrocellulose membranes . After the single-stranded DNA is bound to the membrane, single-stranded DNA binding protein enzyme-conjugate is incubated with the membrane . Next, the unbound conjugate is washed off the membrane and the bound conjugate detected colorimetrically . The assay can detect 10 pg of DNA in less than 3 hr . This method can be applied to the detection of DNA contamination in therapeutic proteins produced by recombinant DNA or hybridoma techniques. Gene, 1989 Nov 30, 83(2), 207 - 13 The Escherichia coli melR gene encodes a DNA-binding protein with affinity for specific sequences located in the melibiose-operon regulatory region; Webster C et al.; Crude extracts, made from Escherichia coli cells carrying a plasmid in which the melR gene was expressed from the galP2 promoter, were used as a source of MelR protein . Using DNase I footprinting and gel retardation assays, we show that MelR binds to two sites located from nucleotides (nt) -49 to -75 and -85 to -113, upstream from the melAB transcription start point . The two sites contain identical 18-bp sequences . Specific binding is unaltered by deletions that remove 1 or 6 amino acids (aa) from the C terminus of MelR, but is abolished by deletion of 16, 24 or more aa residues . Sequence homologies between MelR and other DNA-binding proteins are discussed. Vet Immunol Immunopathol, 1989 Nov 30, 23(1-2), 75 - 83 Polymorphonuclear leucocyte function: relationship between induced migration into the bovine mammary gland and in vitro cell activity; Grommers FJ et al.; Low doses of 10(-7) mg Escherichia coli endotoxin applied as intramammary infusion into single bovine quarters induced a rise in milk cell count without other inflammatory signs . Significantly fewer quarters responded in early lactation than in mid lactation . Maximum cell count was also somewhat later and less pronounced in early lactation . The rise in milk cell count after infusion of E . coli endotoxin was related to in vitro chemotactic activity of blood polymorphonuclear leucocytes (PMN) . PMN isolated from cows which did not respond with a rise in milk cell count upon endotoxin infusion showed a diminished chemotactic activity in vitro as compared to PMN isolated from animals which did respond to an intramammary endotoxin infusion with a rise in milk cell count . No differences in phagocytic and metabolic activity were observed in vitro between the PMN isolated from the two groups of animals. Biochim Biophys Acta, 1989 Nov 30, 999(2), 176 - 82 Interaction between catalytic and regulatory sites of the pyruvate dehydrogenase from Escherichia coli studied by the ESR technique; Graupe K et al.; The accessibility of sulfhydryl groups at the pyruvate dehydrogenase component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli was reinvestigated . Hydrophobic interactions appear to control the reactivity of an essential cysteine residue at the active site with thiol reagents . This explains why the essential cysteine residue reacts only with thiol reagents of minor polarity, like p-hydroxymercuribenzoate or phenylmercuric nitrate, but not with Ellman's reagent or jodoacetamide . The pyruvate dehydrogenase component was modified with a nitroxide derivative of p-hydroxymercuribenzoate . The ESR spectrum of the spin-labelled enzyme changed dramatically upon addition of the cofactors thiamine diphosphate and Mg2+ . Obviously spin-spin interaction occurs under these conditions caused by a transition of an inactive to an active state of the enzyme . The same conformational change is observed when the allosteric activator AMP instead of the cofactors was bound to the enzyme . The implications of these results for the allosteric regulation of the pyruvate dehydrogenase complex are discussed. Biochem Biophys Res Commun, 1989 Nov 30, 165(1), 164 - 7 Electron transfer facilitated by superoxide dismutase: a model for membrane redox systems? Peterson DA, Eaton JW. Membranes, which are an amalgam of proteins and lipids, effect electron transfer through largely unknown mechanisms . Using albumin with bound fatty acids as a model, we have investigated the possible role of these two membrane constituents in electron transfer . In the presence of albumin: fatty acid, there is substantial enhancement of the reduction of ferricytochrome C by ferrous iron . To assess the possible role of free superoxide in cytochrome C reduction, we added mammalian copper/zinc containing superoxide dismutase (Cu/Zn SOD), which catalyzes the transfer of electrons between superoxide anion radicals, forming oxygen and hydrogen peroxide . Surprisingly, in the presence of either albumin or fatty acid free albumin, Cu/Zn SOD actually accelerates electron transfer from ferrous iron to ferricytochrome C . By contrast, neither inactive Cu/Zn SOD nor active manganese SOD facilitates the ferrous iron-dependent reduction of cytochrome C . These results suggest that, in some circumstances, Cu/Zn SOD may transfer electrons to alternative acceptors and that such transfer depends upon the unique reduction/oxidation reaction mechanism of Cu/Zn SOD . If so, this ubiquitous enzyme could be involved in regulating cellular electron transfer reactions as well as acting as a superoxide 'detoxify-ing' agent. Biochim Biophys Acta, 1989 Nov 30, 999(2), 208 - 16 The inducible trimethylamine N-oxide reductase of Escherichia coli K12: its localization and inducers; Silvestro A et al.; We used an anti-trimethylamine-N-oxide reductase (EC 1.6.6.9) serum and different immunological techniques (Ouchterlony, rocket immunoelectrophoresis, immunoblotting) to show that dimethylsulphoxide (DMSO), tetrahydrothiophene 1-oxide (THTO) and pyridine N-oxide (PNO) were effective inducers of the inducible form of trimethylamine N-oxide reductase . We confirmed this genetically and biochemically using a strain in which phage MudII 1734 carrying lacZ was inserted into torA, the structural gene for inducible trimethylamine-N-oxide reductase . By subcellular fractionation and quantitation with rocket immunoelectrophoresis, we showed that the enzyme was principally localized in the periplasmic fraction . Constitutive trimethylamine-N-oxide reductase was localized in the membrane fraction and, like the inducible enzyme showed a broad specificity with respect to various compounds such as DMSO, THTO and PNO . Apart from their immunological properties, the two enzymes could be clearly differentiated by their temperature stability. Gene, 1989 Nov 30, 83(2), 321 - 9 A short N-proximal region of prochymosin inhibits the secretion of hybrid proteins from Escherichia coli; Little S et al.; A gene encoding bovine prochymosin (PC) was fused to the coding sequence (phoA) for the Escherichia coli alkaline phosphatase (AP) signal peptide and expressed in E . coli under the control of the phoA promoter . Upon induction, an AP-PC fusion protein was produced which was neither processed nor exported into the periplasm . We investigated this lack of secretion by constructing a series of gene fusions in which different regions of the PC gene were inserted between the coding regions of the AP leader and mature protein . Analysis of the cellular location of the proteins encoded by these fusions revealed that a region of PC (between amino acids 6 and 29) prevented processing and secretion of an AP-PC fusion when inserted near to the AP signal peptide . In contrast, when this 'blocking sequence' was inserted elsewhere in AP the hybrid proteins were efficiently processed and translocation was initiated. Gene, 1989 Nov 30, 83(2), 187 - 95 Effects of internal deletions on the priming activity of the phage phi 29 terminal protein; Zaballos A et al.; A series of internal deletions of gene 3, coding for the phage phi 29 DNA terminal protein, have been constructed and characterized . In addition, a substitution mutant in the sequence corresponding to amino acids (aa) 49-51 was obtained . The priming activity of the substitution mutant protein, in the formation of the protein p3-dAMP initiation complex, was drastically reduced suggesting that some of the aa present at position 49-51 are essential for p3 function . Deletions of 8 to 33 aa, from aa residue 48 towards the N terminus of the substitution mutant, further decreased the priming activity of the protein . The activity of deletion mutants lacking 15 or 21 aa from residue 57 towards the C terminus, and also containing a point mutation at position 56, was greatly reduced, and no activity was seen when 24 aa were lacking. Biochemistry, 1989 Nov 28, 28(24), 9423 - 30 Binding of pyrimidin-2-one ribonucleoside by cytidine deaminase as the transition-state analogue 3,4-dihydrouridine and the contribution of the 4-hydroxyl group to its binding affinity; Frick L et al.; Cytidine deaminase, purified to homogeneity from constitutive mutants of Escherichia coli, was found to bind the competitive inhibitors pyrimidin-2-one ribonucleoside (apparent Ki = 3.6 x 10(-7) M) and 5-fluoropyrimidin-2-one ribonucleoside (apparent Ki = 3.5 x 10(-8) M) . Enzyme binding resulted in a change of the lambda max of pyrimidin-2-one ribonucleoside from 303 nm for the free species to 239 nm for the bound species . The value for the bound species was identical with that of an oxygen adduct formed by combination of hydroxide ion with 1,3-dimethyl-2-oxopyrimidinium (239 nm), but lower than that of a sulfur adduct formed by combination of the thiolate anion of N-acetylcysteamine with 1,3-dimethyl-2-oxopyrimidinium (259 nm) . The results suggest that pyrimidin-2-one ribonucleoside is bound by cytidine deaminase as an oxygen adduct, probably the covalent hydrate 3,4-dihydrouridine, rather than intact or as an adduct involving a thiol group of the enzyme . In dilute solution at 25 degrees C, the equilibrium constant for formation of a single diastereomer of 3,4-dihydrouridine from pyrimidin-2-one ribonucleoside was estimated as approximately 4.7 x 10(-6), from equilibria of dissociation of water, protonation of 1-methylpyrimidin-2-one, and combination of the 1,3-dimethylpyrimidinium cation with the hydroxide ion.(ABSTRACT TRUNCATED AT 250 WORDS) Biochim Biophys Acta, 1989 Nov 28, 1006(2), 227 - 36 Molecular cloning and expression of cDNA for rat pancreatic cholesterol esterase; Kissel JA et al.; A full-length cDNA complementary to the rat pancreatic cholesterol esterase mRNA was isolated by screening a rat pancreatic cDNA expression library in lambda gt11 vector with antibodies against the porcine pancreatic cholesterol esterase . The isolated cholesterol esterase cDNA is 2050 bp in length and contains an open reading frame coding for a protein of 612 amino acids . A 20-amino acid hydrophobic leader sequence is predicted, based on the position of the first ATG initiation codon upstream from the sequenced amino terminus of the isolated cholesterol esterase . The cholesterol esterase cDNA was subcloned into a mammalian expression vector, pSVL, for transfection studies . Expression of the cDNA in COS cells resulted in the production of bile salt-stimulated cholesterol esterase . Comparison of the cholesterol esterase cDNA sequence with other proteins revealed that the pancreatic cholesterol esterase is identical to rat pancreatic lysophospholipase . The primary structure of cholesterol esterase displayed no significant homology with other lipases, although the putative lipid interfacial recognition site of G-X-S-X-G is present in the cholesterol esterase sequence . However, the cholesterol esterase sequence revealed a 63-amino-acid domain which is highly homologous to the active site domain of other serine esterases . These data suggest that cholesterol esterase may be a member of the serine esterase supergene family . Analysis of the cholesterol esterase structure also revealed a repetitive sequence enriched with Pro, Asp, Glu, Ser, and Thr residues at the C-terminal end of the protein . This sequence is reminiscent of the PEST-rich sequences in short-lived proteins, suggesting that cholesterol esterase may have a short half-life in vivo . Northern blot hybridization showed that the bile salt-stimulated cholesterol esterase mRNA is present in liver suggesting that this protein may also be synthesized by liver cells. Biochemistry, 1989 Nov 28, 28(24), 9256 - 63 Introduction of a cysteine protease active site into trypsin; Higaki JN et al.; Active site serine 195 of rat anionic trypsin was replaced with a cysteine by site-specific mutagenesis in order to determine if a thiol group could function as the catalytic nucleophile in serine protease active site environment . Two genetically modified rat thiol trypsins were generated; the first variant contained a single substitution of Ser195 with Cys (trypsin S195C) while the second variant contained the Ser195 to Cys as well as an Asp102 to Asn substitution (trypsin D102N,S195C) that more fully mimics the putative catalytic triad of papain . Both variants were expressed as his J signal peptide-trypsin fusion proteins to high levels under the control of the tac promoter . The mature forms of both variants were secreted into the periplasmic space of Escherichia coli . Trypsin S195C shows a low level of activity toward the activated ester substrate Z-Lys-pNP, while both trypsin S195C and trypsin D102N,S195C were active toward the fluorogenic tripeptide substrate Z-GPR-AMC . Esterase and peptidase activities of both thiol trypsin variants were inhibited by known Cys protease inhibitors as well as by specific trypsin inhibitors . The kcat of trypsin S195C was reduced by a factor of 6.4 x 10(5) relative to that of trypsin while the kcat of trypsin D102N,S195C was lowered by a factor of 3.4 x 10(7) with Z-GPR-AMC as substrate . Km values were unaffected . The loss of activity of trypsin D102N,S195C was partially attributed to an inappropriate Asn102-His57 interaction that precludes the formation of the catalytically competent His57-Cys195 ion pair although loss of the negative charge of D102 at the active site probably contributes to diminished activity.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1989 Nov 28, 28(24), 9350 - 60 1H NMR structural characterization of a recombinant kringle 2 domain from human tissue-type plasminogen activator; Byeon IJ et al.; The kringle 2 domain of human tissue-type plasminogen activator (t-PA) has been characterized via 1H NMR spectroscopy at 300 and 620 MHz . The experiments were performed on the isolated domain obtained by expression of the 174-263 portion of t-PA in Escherichia coli {Cleary et al . (1989) Biochemistry 28, 1884-1891} . The spectrum of t-PA kringle 2 is characteristic of a globular structure and shows overall similarity to that of the plasminogen (PGN) kringle 4 . Spectral comparison with human and bovine PGN kringle 4 identifies side-chain resonances from Leu46, which afford a fingerprint of kringle folding, and from most of the aromatic ring spin systems . Assignment of signals arising from the His13, His48a, and His64 side chains, which are unique to t-PA kringle 2, was assisted by the availability of a His64----Tyr mutant . Ligand-binding studies confirm that t-PA kringle 2 binds L-lysine with an association constant Ka approximately 11.9 mM-1 . The data indicate that homologous or conserved residues relative to those that compose the lysine-binding sites of PGN kringles 1 and 4 are involved in the binding of L-lysine to t-PA kringle 2 . These include Tyr36 and, within the kringle inner loop, Trp62, His64, Trp72, and Tyr74 . Acid/base titration of aromatic singlets in the presence of L-lysine yields pKa* approximately 6.25 and approximately 4.41 for His13 and His64, respectively, and shows that the His48a imidazole group does not protonate down to pH* approximately 4.3 . Thus, the His48a and His64 side chains are in solvent-shielded locations . As observed for the PGN kringles, the Trp62 indole group titrates with pKa* approximately 4.60, which indicates proximity of the side chain to a titratable carboxyl group, most likely that of Asp57 at the binding site . Several labile NH protons of t-PA kringle 2 exhibit retarded H-exchange kinetics, requiring more than a week in 2H2O for full deuteration in the presence of L-lysine at 37 degrees C . This reveals that kringle 2 is endowed with a compact, dynamically stable conformation . Proton Overhauser experiments in 1H2O, centered on well-resolved NH resonances between 9.8 and 12 ppm, identify signals arising from the His48a imidazole NH3 proton and the three Trp indole NH1 protons . A strong dipolar interaction was observed among the Trp25 indole NH1, the Tyr50 amide NH, and the His48a imidazole CH2 protons, which affords evidence for an aromatic cluster in t-PA kringle 2 similar to that found at the hydrophobic kernel of PGN kringles.(ABSTRACT TRUNCATED AT 400 WORDS) Biochim Biophys Acta, 1989 Nov 27, 986(2), 257 - 62 The F1F0-ATPase of Escherichia coli . The substitution of glycine by valine at position 29 in the c-subunit affects function but not assembly; Fimmel AL et al.; Site-directed mutagenesis has been used to construct two mutations within the uncE gene, coding for the c-subunit of the F1F0-ATPase, resulting in the substitution of Gly-29 by Val and Gly-18 by Leu . The strain carrying the Gly-29----Val substitution is unable to grow on succinate as sole carbon source and possesses an uncoupled growth yield, while the strain carrying the Gly-18----Leu substitution possesses a wild-type phenotype . Membranes prepared from the strain carrying the Gly-29----Val substitution possess low levels of ATPase activity and are proton-impermeable . The F1-ATPase activity of this strain was found to be inhibited by approx . 75% when bound to the membrane . These results are discussed in relation to a previously proposed model for the F0 (Cox, G.B., Fimmel, A.L., Gibson, F . and Hatch, L . (1986) Biochim . Biophys . Acta 849, 62-69). Nucleic Acids Res, 1989 Nov 25, 17(22), 9333 - 40 Restoration of a translational stop-start overlap reinstates translational coupling in a mutant trpB'-trpA gene pair of the Escherichia coli tryptophan operon; Das A et al.; The trpB and trpA coding regions of the polycistronic trp mRNA of Escherichia coli are separated by overlapping translation stop and start codons . Efficient translation of the trpA coding region is subject to translational coupling, i.e., maximal trpA expression is dependent on prior translation of the trpB coding region . Previous studies demonstrated that the trpA Shine-Dalgarno sequence (within trpB) and/or the location of the trpB stop codon influenced trpA expression . To examine the effect of stop codon location specifically, we constructed plasmids in which different nucleotide sequences preceding the trpA start codon were retained, and only the reading frame was changed . When trpB translation proceeded in the wild type reading frame and terminated at the normal trpB stop codon, trpA polypeptide levels were elevated over the levels observed when translation stopped before or after the natural trpB stop codon . The proximity of the trpB stop codon to the trpA start codon therefore markedly influences trpA expression. Nucleic Acids Res, 1989 Nov 25, 17(22), 9147 - 63 Sequence specificity of the P6 pairing for splicing of the group I td intron of phage T4; Ehrenman K et al.; Seventeen non-directed td- (thymidylate synthase-deficient) splicing-defective mutations isolated in phage T4 were localized within the catalytic core of the ribozyme . All of the mutations occur in conserved structural elements that form part of the td intron core secondary structure . Remarkably, seven of the seventeen independently isolated mutations clustered in the dinucleotide 5' element (P6{5'}) of the putative two-base-pair P6 stem . An analysis of this region was undertaken by site-directed mutagenesis of the plasmid-borne td gene, leading to the following findings: First, the short P6 pairing in the td secondary structure model was verified with appropriate P6{5'} and P6{3'} compensatory mutations . Second, all P6{5'} and P6{3'} mutants are defective in the first step of splicing, guanosine-dependent 5' splice site cleavage, whereas their activity at the 3' splice site is variable . Third, residual in vitro splicing activity of the mutants altered on only one side of the P6 pairing is correlated with the ability to form an alternative two-base-pair P6 stem . Fourth, the degree to which the compensatory mutants have their splicing activity restored is highly condition-dependent . Restoration of phenotype of the compensatory P6{5'}:{3'} constructs is weak under stringent in vitro conditions as well as in vivo . This sequence specificity is consistent with phylogenetic conservation of the P6 pairing elements in group I introns, and suggests either structural constraints on the P6 stem or a dual function of one or both pairing elements. J Biol Chem, 1989 Nov 25, 264(33), 20120 - 30 Mutagenesis by single site-specific arylamine-DNA adducts . Induction of mutations at multiple sites; Gupta PK et al.; Two related carcinogen adducts, N-(deoxyguanosin-8-yl)-2-aminofluorene (AF) or N-(deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene (AAF), were introduced into the lacZ' gene at base position 6253 of the minus strand of M13mp9 viral DNA . The construction of this site-specifically modified DNA was accomplished by first preparing a gapped heteroduplex missing 7 nucleotides at position 6251-6257 followed by ligation with an unmodified heptamer or with a heptamer containing either an AF or AAF adduct . These site-specifically modified templates were transfected into competent wild-type Escherichia coli cells (JM103) and a uvrA strain (SMH12) . The mutation spectrum was determined by phenotypic selection of colorless plaques indicating a defective beta-galactosidase marker enzyme and by an in situ hybridization procedure to detect single base pair mismatches in the adduct region . DNA sequencing was used to characterize 179 of the mutants obtained . We found that both adducts were capable of inducing base substitution mutations at the adduct site and in the local region of the adduct . A specific frameshift (+1G) was also observed at a displaced site . All of the frameshift mutations occurred at the ligation site of the modified oligonucleotide . Control experiments with an unmodified oligonucleotide did not show an enhancement of mutations at this site, indicating that the adducts may have been responsible for these frameshifts . The mutations spectra induced by these adducts suggest that mutagenesis depends not only on adduct structure but also the sequence in which the adduct is located and the host cell type used for mutation expression. J Biol Chem, 1989 Nov 25, 264(33), 20054 - 9 Molecular cloning and expression of ribosome releasing factor; Ichikawa S et al.; Ribosome releasing factor (RRF) is responsible for the release of ribosomes from messenger RNA at the termination of Escherichia coli protein biosynthesis (Hirashima, A., and Kaji, A . (1972) Biochemistry 11, 4037-4044) . RRF has been partially analyzed by Edman degradation to obtain its amino acid sequence . Based on this analysis, a 47-nucleotide probe was synthesized and used to screen clones from the Clarke and Carbon Gene Bank which carry sequences within the 0-10 min region of the E . coli gene map . The entire RRF cistron was detected on the plasmid pLC6-32 . The DNA sequence of RRF was determined, and it was deduced that RRF consists of 185 amino acids with a calculated molecular weight of 20,639 . A rho-independent transcriptional termination sequence was found immediately downstream of the RRF cistron . The RRF gene was subcloned into the vector pUC19, and the resulting plasmid was named pRR1 . E . coli harboring this plasmid expressed much more RRF than cells containing pUC19, and it was biologically active. J Biol Chem, 1989 Nov 25, 264(33), 19637 - 47 Substrate and DNA binding to a 50-residue peptide fragment of DNA polymerase I . Comparison with the enzyme; Mullen GP et al.; The fluorescent nucleotide 2',3'-trinitrophenyl-ATP (TNP-ATP) binds at the triphosphate substrate binding site of the large (Klenow) fragment of DNA polymerase I (Pol I) as detected by direct binding studies measuring the increase in fluorescence of this ligand (n = 1.0, KD = 0.07 microM) . The enzyme-TNP-ATP complex binds Mg2+ and Mn2+ tightly (KD = 0.05 microM) as measured by an increase in fluorescence on titrating with these metals . The substrate dGTP competitively displaces TNP-ATP from the enzyme (KD = 5.7 microM) de-enhancing the fluorescence . The polymerase reaction is half-maximally inhibited by 0.8 microM TNP-ATP in the presence of dATP (10 microM) as substrate . A region of the amino acid sequence of Pol I (peptide I) consisting of residues 728-777 has been synthesized and found to contain significant secondary structure by CD both in water and 50% methanol/water . In water at 3 degrees C, peptide I binds the substrate analog TNP-ATP (KD = 0.03 microM) with a stoichiometry of 0.2 . In 50% methanol at 3 degrees C, peptide I binds TNP-ATP with a higher stoichiometry than in water, consistent with a 1:1 complex, but biphasically (16% of the peptide, KD = 0.09 microM; 84% of the peptide, KD = 5.0 microM), and competitively binds the Pol I substrates dATP, TTP, and dGTP (KD = 230-570 microM) . Evidence from size exclusion high performance liquid chromatography suggests that these two forms of the peptide are monomer and dimer, respectively . Significantly, the peptide I-TNP-ATP complex binds duplex DNA, tightly (KD = 0.1-0.5 microM) and stoichiometrically, and single stranded DNA more weakly . The peptide I-duplex DNA complex binds both TNP-ATP (KD = 0.5-1.5 microM) and Pol I substrates (KD = 350-2100 microM) stoichiometrically . In a control experiment, a second peptide, peptide II, based on residues 840-888 of the Pol I sequence, retains secondary structure, as detected by CD, but displays no binding of TNP-ATP . The ability of peptide I, which represents only 8% of the large fragment of Pol I, to bind both substrates and duplex DNA indicates that residues 728-777 constitute a major portion of the substrate binding site of this enzyme. J Biol Chem, 1989 Nov 25, 264(33), 19564 - 72 Cloning of murine adipocyte lipid binding protein in Escherichia coli . Its purification, ligand binding properties, and phosphorylation by the adipocyte insulin receptor; Chinander LL et al.; The murine adipocyte lipid binding protein (ALBP) has been cloned into Escherichia coli, purified from expressing cultures, and its ligand binding and phosphorylation properties studied . In the cloning strategy, the recombinant, pT7-5 rALBP, was transformed into E . coli strain K38 harboring plasmid pGP1-2 which directs the synthesis of T7 RNA polymerase . Upon shifting the temperature from 30 to 42 degrees C to induce T7 RNA polymerase expression, the 14.6-kDa recombinant ALBP (rALBP) was expressed for approximately 2 h and accumulated to about 1% of total E . coli protein . The recombinant ALBP was soluble in E . coli extracts and resistant to bacterial proteolysis . A procedure for purifying rALBP was developed utilizing immuno-chemical detection based upon reactivity with anti-murine ALBP antiserum . A combination of acidic ammonium sulfate fractionation, gel permeation chromatography, and carboxymethyl ion-exchange high performance liquid chromatography separation was used to prepare homogeneous rALBP . Sequence analysis of rALBP indicated that the initiating methionine residue had been removed and the amino-terminal cysteine residue was not blocked . Purified rALBP exhibited stoichiometric, saturable binding of oleic acid (n = 1.0, K0.5 approximately 100 microM) and retinoic acid (n = 1.0, K0.5 approximately 170 microM) . Incubation of rALBP with wheat germ agglutinin-purified insulin receptor, ATP, and 100 nM insulin resulted in a 5-fold stimulation of rALBP phosphorylation above the basal state . Kinetic analysis of rALBP phosphorylation by the 3T3-L1 insulin receptor kinase yielded a Michaelis constant (Km) of 50 microM and a maximal velocity of 1 mol of rALBP phosphorylated/min/mol insulin binding sites . Phosphoamino acid analysis indicated that phosphorylation occurred upon tyrosine . These results indicate that murine ALBP has been cloned and expressed in E . coli, purified to homogeneity, and is a substrate for the insulin receptor tyrosyl kinase in vitro. J Biol Chem, 1989 Nov 25, 264(33), 19475 - 7 The N-terminal cysteine of human asparagine synthetase is essential for glutamine-dependent activity; Van Heeke G et al.; Site-specific mutagenesis was used to replace the N-terminal cysteine in human asparagine synthetase by an alanine . The mutant enzyme was expressed in the yeast Saccharomyces cerevisiae, and the asparagine synthetase activity was analyzed in vitro . The mutation resulted in the loss of the glutamine-dependent asparagine synthetase activity, while the ammonia-dependent activity remained unaffected . These results confirm the existence of a glutamine amidotransfer domain with an N-terminal cysteine essential for the glutamine-dependent asparagine synthetase activity. Nucleic Acids Res, 1989 Nov 25, 17(22), 9193 - 204 RNase H cleavage of RNA hybridized to oligonucleotides containing methylphosphonate, phosphorothioate and phosphodiester bonds; Furdon PJ et al.; Three types of 14-mer oligonucleotides were hybridized to human beta-globin pre-mRNA and the resultant duplexes were tested for susceptibility to cleavage by RNase H from E . coli or from HeLa cell nuclear extract . The oligonucleotides contained normal deoxynucleotides, phosphorothioate analogs alternating with normal deoxynucleotides, or one to six methylphosphonate deoxynucleosides . Duplexes formed with deoxyoligonucleotides or phosphorothioate analogs were susceptible to cleavage by RNase H from both sources, whereas a duplex formed with an oligonucleotide containing six methylphosphonate deoxynucleosides alternating with normal deoxynucleotides was resistant . Susceptibility to cleavage by RNase H increased parallel to a reduction in the number of methylphosphonate residues in the oligonucleotide . Stability of the oligonucleotides in the nuclear extract from HeLa cells was also tested . Whereas deoxyoligonucleotides were rapidly degraded, oligonucleotides containing alternating methylphosphonate residues remained unchanged after 70 minutes of incubation . Other oligonucleotides exhibited intermediate stability. Nucleic Acids Res, 1989 Nov 25, 17(22), 9063 - 73 Sequence and characteristics of IS900, an insertion element identified in a human Crohn's disease isolate of Mycobacterium paratuberculosis; Green EP et al.; The complete sequence of an insertion element IS900 in Mycobacterium paratuberculosis is reported . This is the first characterised example of a mycobacterial insertion element . IS900 consists of 1451bp of which 66% is G + C . It lacks terminal inverted and direct repeats, characteristic of Escherichia coli insertion elements but shows a degree of target sequence specificity . A single open reading frame (ORF 1197) coding for 399 amino acids is predicted . This amino acid sequence, and to a lesser extent the nucleotide sequence, show significant homologies to IS110, an insertion element of Streptomyces coelicolor A3(2) . It is proposed that IS900, IS110, and similar insertion elements recently identified in disease isolates of Mycobacterium avium are members of a phylogenetically related family . IS900 will provide highly specific markers for the precise identification of Mycobacterium paratuberculosis, useful in defining its relationship to animal and human diseases. J Biol Chem, 1989 Nov 25, 264(33), 19648 - 53 Tryptic fragments of the Escherichia coli DNA gyrase A protein; Reece RJ et al.; Treatment of the Escherichia coli DNA gyrase A protein with trypsin generates two large fragments which are stable to further digestion . The molecular masses of these fragments are 64 and 33 kDa, and they are shown to be derived from the N terminus and the C terminus of the A protein, respectively . These fragments could represent structural and/or functional domains within the A subunit of DNA gyrase . The trypsin-cleaved A protein (A'), in combination with the B subunit of gyrase, can support ATP-dependent supercoiling of relaxed DNA and other reactions of DNA gyrase . The isolated 64-kDa fragment will also catalyse DNA supercoiling in the presence of the B protein, but the 33-kDa fragment shows no enzymic activities . We conclude that the N-terminal 64-kDa fragment represents the DNA breakage/reunion domain of the A protein, while the 33-kDa fragment may contribute to the stability of the gyrase-DNA complex. Nature, 1989 Nov 23, 342(6248), 451 - 3 Demonstration by genetic suppression of interaction of GroE products with many proteins; Van Dyk TK et al.; The way in which proteins attain and maintain their final form is of fundamental importance . Recent work has focused on the role of a set of ubiquitous proteins, termed chaperonins, in the assembly of phage and multisubunit proteins . The range of chaperonin action is unknown; they could interact with most cellular polypeptides or have a limited subset of protein partners . Included in the chaperonin family is the essential heat-shock regulated Escherichia coli groEL gene product . Over-expression of the groE operon in E . coli causes enhanced assembly of heterologously expressed ribulose bisphosphate carboxylase subunits and suppresses the heat-sensitive mutant phenotype of several dnaA alleles . It has been inferred that suppression of heat-sensitive mutations is confined to dnaA alleles and that this confinement could reflect an interaction between the groE operon products and a dnaA protein aggregate at the replication origin . We now report that multiple copies of the groE operon suppress mutations in genes encoding several diverse proteins . Our data indicate a general role for the groE operon products, the GroEL and GroES proteins, in the folding-assembly pathways of many proteins. J Mol Biol, 1989 Nov 20, 210(2), 323 - 36 Protein-induced conformational changes in 16 S ribosomal RNA during the initial assembly steps of the Escherichia coli 30 S ribosomal subunit; Mandiyan V et al.; The mechanism of 16 S ribosomal RNA folding into its compact form in the native 30 S ribosomal subunit of Escherichia coli was studied by scanning transmission electron microscopy and circular dichroism spectroscopy . This approach made it possible to visualize and quantitatively analyze the conformational changes induced in 16 S rRNA under various ionic conditions and to characterize the interactions of ribosomal proteins S4, S8, S15, S20, S17 and S7, the six proteins known to bind to 16 S rRNA in the initial assembly steps . 16 S rRNA and the reconstituted RNA-protein core particles were characterized by their mass, morphology, radii of gyration (RG), and the extent and stability of 16 S rRNA secondary structure . The stepwise binding of S4, S8 and S15 led to a corresponding increase of mass and was accompanied by increased folding of 16 S rRNA in the core particles, as evident from the electron micrographs and from the decrease of RG values from 114 A and 91 A . Although the binding of S20, S17 and S7 continued the trend of mass increase, the RG values of these core particles showed a variable trend . While there was a slight increase in the RG value of the S20 core particles to 94 A, the RG value remained unchanged (94 A) with the further addition of S17 . With subsequent addition of S7 to the core particles, the RG values showed an increase to 108 A . Association with S7 led to the formation of a globular mass cluster with a diameter of about 115 A and a mass of about 300 kDa . The rest of the mass (about 330 kDa) remained loosely coiled, giving the core particle a "medusa-like" appearance . Morphology of the 16 S rRNA and 16 S rRNA-protein core particles, even those with all six proteins, does not resemble the native 30 S subunit, contrary to what has been reported by others . The circular dichroism spectra of the 16 S rRNA-protein complexes and of free 16 S rRNA indicate a similarity of RNA secondary structure in the core particles with the first four proteins, S4, S8, S15, S20 . The circular dichroism melting profiles of these core particles show only insignificant variations, implying no obvious changes in the distribution or the stability of the helical segments of 16 S rRNA . However, subsequent binding of proteins S17 and S7 affected both the extent and the thermal stability of 16 S rRNA secondary structure.(ABSTRACT TRUNCATED AT 400 WORDS) Eur J Biochem, 1989 Nov 20, 185(3), 573 - 9 Tryptophan phosphorescence as a monitor of the structural role of metal ions in alkaline phosphatase; Cioni P et al.; The phosphorescence properties of Trp109 in alkaline phosphatase from Escherichia coli have been utilized to probe the conformation of the polypeptide following the removal of metal ions, reconstitution with Zn2+ and Cd2+ and phosphorylation . The complete removal of metal ions induces a drastic loosening of the protein structure that extends to the inner core of the macromolecule . While binding of a single metal ion/subunit (A-site occupancy) restores the holoconformer, practically no structuring effect is observed upon B-site occupancy by the second incoming metal ion . An exception to this rule occurs at alkaline pH and when the adjacent subunit in the dimer is metal-free . Under these circumstances a conformation of the subunit more compact than that of the fully saturated dimer manifests some degree of communication across the subunit interface . The binding of more than two metal ions/monomer generally destabilizes the protein, the effect being more pronounced at acid pH . Finally, the binding of inorganic phosphate restores the native-like configuration abolishing any destabilization induced by excess metal ions and acid pH . If the negative cooperativity towards metal binding to A sites in doubly metalated forms at pH 8 is in substantial agreement with 113Cd-NMR data, the equivalence in conformation between Zn2+- and Cd2+-reconstituted alkaline phosphatase emphasizes that no serious structural changes are introduced by the metal replacement. FEBS Lett, 1989 Nov 20, 258(1), 22 - 6 Animal and plant mitochondria contain specific thioredoxins; Bodenstein-Lang J et al.; Thioredoxins have been purified from pig heart and potato tuber mitochondria which differ in chromatographic behaviour, enzyme activating capacity, and slightly higher molecular mass (Mr = 12,500) from the major thioredoxin(s) present in mitochondria-free fractions of the same tissue . Both mt-thioredoxins can serve as hydrogen donor for E . coli ribonucleotide reductase but only the plant protein activates spinach chloroplast NADP malate dehydrogenase in vitro . Mitochondrial target enzymes specifically activated by thioredoxin have not as yet been identified. J Mol Biol, 1989 Nov 20, 210(2), 281 - 92 Genetic analysis of the switch that controls porin gene expression in Escherichia coli K-12; Slauch JM et al.; The two-component regulatory system, OmpR and EnvZ, in Escherichia coli controls the differential expression of ompF and ompC in response to medium osmolarity . Previous studies suggest that EnvZ functions as a membrane sensor relaying information to the DNA-binding protein, OmpR, which in turn activates expression of the appropriate promoter . A strategy has been devised to isolate and characterize a collection of missense mutations in ompR that alter, but do not abolish protein function . Mutants were isolated using strains that contain the ompR and envZ genes in separate chromosomal locations yet maintain the production of both regulatory proteins at physiological levels . Such an arrangement facilitates ompR diploid analysis and tests of epistasis with known envZ mutations . The data obtained indicate that OmpR works in both a positive and negative fashion to control the transcription of ompF and this result forms the basis of a model for porin regulation that explains the switch from OmpF to OmpC production in response to increasing medium osmolarity. FEBS Lett, 1989 Nov 20, 258(1), 39 - 41 Interaction of the LexA repressor and the uvrC regulatory region; Stark T et al.; We have studied the in vitro interaction of the LexA repressor protein and the uvrC regulatory region . We find that there is specific binding to two regions, the region we have defined as lexA1 and the lexA2-lexA3 region . Our findings support the possibility of an inducible regulation for this complex operon. J Mol Biol, 1989 Nov 20, 210(2), 293 - 302 Cleavage by RNase III in the transcripts of the met Y-nus-A-infB operon of Escherichia coli releases the tRNA and initiates the decay of the downstream mRNA; Regnier P et al.; The metY gene coding for a minor form of the initiator tRNA is the first gene of a complex polycistronic operon also encoding the transcription termination factor NusA and the translation initiation factor IF2 . The mixed tRNA-mRNA polycistronic transcript is cleaved by RNase III in a hairpin structure downstream from the tRNA . This cleavage separates the tRNA from the mRNA and initiates the rapid degradation of the 5' extremity of the downstream mRNA . Dissociation of the structural (tRNA) and informational (mRNA) RNAs from this operon is also achieved by independent transcription in vivo . The presence of two transcription terminators located downstream from metY produces a small tRNAMetf2 precursor transcript, whereas an internal promoter situated between metY and the first open reading frame directs the transcription of only the protein-coding part of the operon. Science, 1989 Nov 17, 246(4932), 922 - 6 Cognate DNA binding specificity retained after leucine zipper exchange between GCN4 and C/EBP; Agre P et al.; Both C/EBP and GCN4 are sequence-specific DNA binding proteins that control gene expression . Recent evidence implicates C/EBP as a transcriptional regulator of genes involved in lipid and carbohydrate metabolism . The C/EBP protein binds avidly to the dyad symmetric sequence 5'-ATTGCGCAAT-3'; GCN4 regulates the transcription of genes that control amino acid biosynthesis in yeast, and binds avidly to the dyad symmetric sequence 5'-ATGA(G/C)TCAT-3' . Both C/EBP and GCN4 bind DNA via the same structural motif . This motif has been predicted to be bipartite, consisting of a dimerization interface termed the "leucine zipper" and a DNA contact surface termed the "basic region." Specificity of DNA binding has been predicted to be imparted by the basic region . As a test of this hypothesis, recombinant proteins were created wherein the basic regions and leucine zippers of GCN4 and C/EBP were reciprocally exchanged . In both of the recombinant polypeptides, DNA binding specificity is shown to track with the basic region. Cell, 1989 Nov 17, 59(4), 709 - 17 Biochemical analysis of transcriptional activation by Jun: differential activity of c- and v-Jun; Bohmann D et al.; The human proto-oncogene product, c-Jun, is a member of the AP-1 family of transcription factors, which mediate the regulation of gene expression in response to extracellular signaling . Comparison of c-Jun and v-Jun by in vitro transcription assays revealed that v-Jun has significantly greater transcriptional activity than c-Jun . Analysis of Jun mutants expressed in bacteria indicates that this difference in transcriptional activity is due to the presence of a regulatory domain located at the N-terminal region of c-Jun . Other Jun mutants identify an activation domain rich in acidic and proline residues toward the C-terminal end of the molecule, in a region near the DNA binding domain . These findings suggest that during retroviral transduction, a constitutively active Jun protein has been generated by deleting a negatively acting domain . This putative repressor domain may also play a role in the signal-dependent induction of c-Jun activity. Nature, 1989 Nov 16, 342(6247), 248 - 51 Crystal structure of chaperone protein PapD reveals an immunoglobulin fold; Holmgren A et al.; The chaperone protein PapD mediates assembly of pili in Escherichia coli . Its polypeptide chain folds into two immunoglobulin-type domains that are homologous in sequence to the human lymphocyte differentiation antigen Leu-1/CD5. Arch Biochem Biophys, 1989 Nov 15, 275(1), 92 - 7 Expression of the trichodiene synthase gene of Fusarium sporotrichioides in Escherichia coli results in sesquiterpene production; Hohn TM et al.; Trichodiene synthase is a sesquiterpene cyclase involved in the biosynthesis of trichothecene mycotoxins . We report that insertion of the unaltered trichodiene synthase gene of Fusarium sporotrichioides into the Escherichia coli expression vector pDR540 produced an inactive polypeptide with a molecular weight approximately 2000 greater than that of trichodiene synthase . This result is consistent with the presence of an intron in the trichodiene synthase gene, and prompted us to specifically delete a putative 60-nucleotide intron sequence . Insertion of the intron-deleted open reading frame into pDR540 resulted in the production of active enzyme . Trichodiene synthase activity in crude extracts from induced cultures was 0.07 nmol/min/mg of protein and represented 0.05-0.10% of the total cell protein . A cross-reactive protein was present with the same apparent molecular weight as the subunit of native trichodiene synthase . The recombinant enzyme was partially purified and shown to have properties closely resembling those of the native enzyme . Trichodiene was detected in ethyl acetate extracts from induced cultures at a concentration of 60 micrograms/liter after 4.5 h . These findings support the primary structure recently reported for trichodiene synthase and demonstrate that the expression of a sesquiterpene cyclase in E . coli results in sesquiterpene production.
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