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 dihydr