Biochimie, 1990 Feb-Mar, 72(2-3), 177 - 82 In vitro trimerization of outer membrane protein PhoE; de Cock H et al.; The folding of outer membrane protein PhoE of E coli into its native trimeric structure was studied in vitro by using monoclonal antibodies, which recognize cell-surface exposed, conformational epitopes of the protein . These antibodies were able to precipitate the in vitro synthesized PhoE protein, showing that the conformational epitopes are formed in vitro . From analysis by SDS--polyacrylamide gel electrophoresis, it appeared that the precipitated protein represents a folded monomer . The signal sequence interferes with the formation of the conformational epitopes . Outer membranes were required to induce the formation of the stable trimeric form of the protein . This trimerization was not accompanied by insertion into the outer membranes. Biochimie, 1990 Feb-Mar, 72(2-3), 169 - 76 Immunological approach of assembly and topology of OmpF, an outer membrane protein of Escherichia coli; Pages JM et al.; Various monoclonal antibodies (MoF) directed against cell-surface-exposed epitopes of OmpF, one major outer membrane pore protein of Escherichia coli B and K-12, have been used to study the assembly and the topology of the protein . This paper firstly describes the characterization of the OmpF epitopes recognized by the various monoclonal antibodies . A comparison between OmpC, OmpF and PhoE porins with respect to their primary amino acid sequence and their cell-surface exposed regions allows us to propose a rough model including 2 antigenic sites . The second part is focused on the assembly of the OmpF protein in the outer membrane . Various forms, precursor, unassembled monomer, metastable oligomer (pre-trimer) and trimer are detected with immunological probes directed against OmpF during a kinetic analysis of the process . The requirement for a concomitant lipid synthesis during the trimerization has been demonstrated by investigating the presence of a specific native epitope . The role of lipopolysaccharide during the stabilization of the conformation is discussed with regard to the various steps of assembly. Vaccine, 1990 Feb, 8(1), 85 - 91 Outer membrane PhoE protein of Escherichia coli as a carrier for foreign antigenic determinants: immunogenicity of epitopes of foot-and-mouth disease virus; Agterberg M et al.; Outer membrane protein PhoE of Escherichia coli was used for the expression of antigenic determinants of foot-and-mouth disease virus . Five hybrid PhoE proteins were constructed containing different combinations of two antigenic determinants of VP1 protein of the virus . The hybrid proteins were expressed in two E . coli strains and the proteins were correctly assembled into the outer membrane . The inserted epitopes were exposed at the surface of the cell and were antigenic in this PhoE-associated conformation . Immunization experiments, performed with partially purified protein, resulted in all cases in a significant anti-peptide antibody titre . In one case in which the hybrid protein with the largest insert was used, a neutralizing antibody response was detected. Mutat Res, 1990 Feb, 243(2), 159 - 64 Participation of rec genes of Escherichia coli K 12 in W-reactivation of UV-irradiated phage lambda; Simic D et al.; The effect of the recombinational deficiency on W-reactivation of UV-damaged phage lambda was explored . In this paper we show that W-reactivation is reduced by the recB21 and recF143 mutations after bleomycin (BM) and UV treatment . Combination of these mutations in the recB21recF143 double mutant blocks W-reactivation completely after BM induction, but leaves residual W-reactivation ability after UV-irradiation, which is abolished by the introduction of uvrB deficiency (delta(uvrB-chlA} . W-reactivation has been rendered constitutive in recB21C22sbcB15, but the efficiency of reactivation remained virtually constant over the range of BM and UV doses, indicating the role of the RecBC(D) enzyme in W-reactivation. Mutat Res, 1990 Feb, 240(2), 93 - 100 Effect of in vitro cleavage of apurinic/apyrimidinic sites on bleomycin-induced mutagenesis of repackaged lambda phage; Steighner RJ et al.; Previous studies have revealed bleomycin to be a potent base-substitution mutagen in repackaged phage lambda . In order to assess the role of apurinic/apyrimidinic (AP) sites in bleomycin-induced mutagenesis, bleomycin-damaged lambda DNA was treated with putrescine or endonuclease IV to effect cleavage of bleomycin-induced AP sites . The DNA was then packaged, the phage grown in SOS-induced E . coli, and the frequency of clear-plaque mutants in the progeny was determined . Bleomycin-induced mutagenesis was decreased approx . 2-fold by treating the DNA with putrescine, but was unaffected by endonuclease IV . The results are consistent with the production of bleomycin-induced mutation at certain AP sites having a closely opposed single-strand break, since such sites are cleaved by putrescine but not by endonuclease IV. J Bacteriol, 1990 Feb, 172(2), 1151 - 4 Loss of 4.5S RNA induces the heat shock response and lambda prophage in Escherichia coli; Bourgaize DB et al.; During depletion of 4.5S RNA, cells of Escherichia coli displayed a heat shock response that was simultaneous with the first detectable effect on ribosome function and before major effects on cell growth . Either 4.5S RNA is involved directly in regulating the heat shock response, or this particular impairment of protein synthesis uniquely induces the heat shock response . Several hours later, lambda prophage was induced and the cells lysed. J Bacteriol, 1990 Feb, 172(2), 1145 - 7 Detection of lipopolysaccharides by ethidium bromide staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Kido N et al.; A rapid and easy method for staining lipopolysaccharides with ethidium bromide is described . Lipopolysaccharides could be visualized by ethidium bromide with almost the same sensitivity as found with the silver-staining method in less than 30 min . The ethidium bromide-staining method was particularly suitable for staining lipopolysaccharides possessing acidic O-specific polysaccharides, which were poorly visualized by silver staining. J Virol, 1990 Feb, 64(2), 592 - 7 Specificities involved in the initiation of retroviral plus-strand DNA; Luo GX et al.; Reverse transcription of the retroviral RNA genome begins with tRNA-primed synthesis of a minus-strand DNA, which subsequently acts as the template for the synthesis of plus-strand DNA . This plus-strand DNA is initiated at a unique location and makes use of a purine-rich RNA oligonucleotide derived by RNase H action on the viral RNA . To determine the variables that are relevant to successful specific initiation of plus-strand DNA synthesis, we have used nucleic acid sequences from the genome of Rous sarcoma virus along with three different sources of RNase H: avian myeloblastosis virus DNA polymerase, murine leukemia virus DNA polymerase, and the RNase H of Escherichia coli . Our findings include evidence that specificity is controlled not only by the nucleic acid sequences but also by the RNase H . For example, while the avian reverse transcriptase efficiently and specifically initiates on the sequences of the avian retrovirus, the murine reverse transcriptase initiates specifically but at a location 4 bases upstream of the correct site. J Immunol, 1990 Feb 1, 144(3), 1015 - 22 Heterologous expression and epitope mapping of a human small nuclear ribonucleoprotein-associated Sm-B'/B autoantigen; Rokeach LA et al.; The U snRNP associated B'/B polypeptides are primary targets of Sm autoantibodies in patients with systemic lupus erythematosus . We have bacterially expressed a Sm-B'/B autoantigen from Raji cells as a fusion with the anthranilate synthase protein from Escherichia coli . The recombinant Sm-B'/B fusion displays comparable immunologic reactivity to the native protein when tested with both monoclonal and polyclonal antibodies . To map Sm-B'/B epitopes, we constructed a series of 12 anthranilate synthase fusions spanning different regions of Sm-B'/B and tested such fusions on immunoblots against a panel of characterized sera . In this manner, we have identified six epitopes, five of which overlap the proline-rich carboxyl-terminus of the protein . Some of these epitopes appear to be conformational . The human sera tested can be divided, according to the epitopes they recognize, into six groups . Finally, we have shown that anti-Sm recognition of the (U1)RNP-specific A protein is attributable to cross-reactivity between the Sm-B'/B and A autoantigens. Agric Biol Chem, 1990 Feb, 54(2), 519 - 25 Specificity of nucleotide sequence in DNA cleavage induced by D-glucosamine and D-glucosamine-6-phosphate in the presence of Cu2+; Watanabe K et al.; 32P-End-labeled restriction fragments derived from pBR322 and pUC9 DNAs were reacted with D-glucosamine or D-glucosamine-6-phosphate in the presence of Cu2+, and, after being heated at 90 degrees C in aqueous piperidine, the DNA products were analyzed on polyacrylamide gels for the sequence-specificity of alkali-labile cleavaged sites . The intensity of oligonucleotide bands of cleavaged sites was directly proportional to the concentration of aminosugars, indicating that the DNA cleavage was caused by the action of aminosugars themselves . The preferred DNA cleavage sites induced by these aminosugars were identical, both at pyrimidine-purine (5'----3') sequences, especially at thymineguanine ones, and to some extent at pyrimidine-pyrimidine (5'----3') sequences . The 6-phosphate moiety of D-glucosamine did not affect the specificity of DNA cleavage. Gene, 1990 Jan 31, 86(1), 19 - 25 Mutational analysis of the phage T4 morphogenetic 31 gene, whose product interacts with the Escherichia coli GroEL protein; Keppel F et al.; The phage T4 morphogenetic gene 31 has been sequenced . Its deduced gene product is a polypeptide of 111 aa, with a predicted Mr of 12064 and a pI of 4.88 . The proof that the assigned open reading frame (ORF) encodes Gp31 rests on the sequencing of two known gene 31 amber mutations, amN54 and NG71, demonstrating that these mutations result in translational termination within the assigned ORF . Furthermore, the sequencing of four different T4 epsilon mutations, isolated on the basis of allowing the phage to propagate on Escherichia coli groEL- hosts, showed that they are either missense mutations or 3-bp deletions in the gene 31 reading frame . The sequencing of neighboring DNA revealed the presence of five other ORFs, one of which overlaps gene 31 substantially, but in the opposite orientation. Gene, 1990 Jan 31, 86(1), 35 - 43 Factors affecting expression of the recF gene of Escherichia coli K-12; Sandler SJ et al.; This report describes four factors which affect expression of the recF gene from strong upstream lambda promoters under temperature-sensitive cIAt2-encoded repressor control . The first factor was the long mRNA leader sequence consisting of the Escherichia coli dnaN gene and 95% of the dnaA gene and lambda bet, N (double amber) and 40% of the exo gene . When most of this DNA was deleted, RecF became detectable in maxicells . The second factor was the vector, pBEU28, a runaway replication plasmid . When we substituted pUC118 for pBEU28, RecF became detectable in whole cells by the Coomassie blue staining technique . The third factor was the efficiency of initiation of translation . We used site-directed mutagenesis to change the mRNA leader, ribosome-binding site and the 3 bp before and after the translational start codon . Monitoring the effect of these mutational changes by translational fusion to lacZ, we discovered that the efficiency of initiation of translation was increased 30-fold . Only an estimated two- or threefold increase in accumulated levels of RecF occurred, however . This led us to discover the fourth factor, namely sequences in the recF gene itself . These sequences reduce expression of the recF-lacZ fusion genes 100-fold . The sequences responsible for this decrease in expression occur in four regions in the N-terminal half of recF . Expression is reduced by some sequences at the transcriptional level and by others at the translational level. Gene, 1990 Jan 31, 86(1), 27 - 33 Cyclic AMP synthesis in Escherichia coli strains bearing known deletions in the pts phosphotransferase operon; Levy S et al.; A series of isogenic strains harboring known deletions in the pts operon of Escherichia coli have been constructed by reverse genetics . Strains bearing deletions for the whole pts operon failed to grow on maltose or on carbon sources of the same class . In these strains the total cAMP synthesis was significantly lower than in a strain deleted only for the crr gene . This indicated that enzyme I or phosphorylated histidine-containing phosphotransferase protein in addition to its role in phosphorylating enzyme IIIGlc, is involved in adenylate cyclase (AC) activation or cAMP excretion . It was further shown that deletions in the pts operon do not affect synthesis of AC. Gene, 1990 Jan 31, 86(1), 11 - 7 Two domains in the terminal inverted-repeat sequence of transposon Tn3; Ichikawa H et al.; Tn3 and related transposons have terminal inverted repeats (IR) of about 38 bp that are needed as sites for transposition . We made mini-Tn3 derivatives which had a wild-type IR of Tn3 at one end and either the divergent IR of the Tn3-related transposon, gamma delta or IS101, or a mutant IR of Tn3 at the other end . We then examined both in vivo transposition (cointegration between transposition donor and target molecules) of these mini-Tn3 elements and in vitro binding of Tn3-encoded transposase to their IRs . None of the elements with an IR of gamma delta or IS101 mediated cointegration efficiently . This was due to inefficient binding of transposase to these IR . Most mutant IR also interfered with cointegration, even though transposase bound to some mutant IR as efficiently as it did to wild type . This permitted the Tn3 IR sequence to be divided into two domains, named A and B, with respect to transposase binding . Domain B, at positions 13-38, was involved in transposase binding, whereas domain A, at positions 1-10, was not . The A domain may contain the sequence recognized by some other (e.g., host) factor(s) to precede the actual cointegration event. Clin Chim Acta, 1990 Jan 31, 187(1), 11 - 20 Sensitive enzyme immunoassay for human Cu/Zn superoxide dismutase; Kurobe N et al.; A highly sensitive enzyme immunoassay method for measurement of human Cu/Zn superoxide dismutase (SOD) was established . Antisera were raised in rabbits by injecting SOD purified from human erythrocytes, and antibodies to SOD were purified by the use of Cu/Zn SOD-coupled Sepharose 4B . The assay system consisted of polystyrene balls with immobilized monospecific antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli . The assay was highly sensitive, and the minimum detection limit was 3 pg or 0.1 fmol/assay tube . Serum Cu/Zn SOD levels of normal healthy subjects (36.3 +/- 15.6, n = 120, 16-64 years old) were not related to age and sex . It was confirmed that Cu/Zn SOD levels in erythrocytes and blood plasma were significantly enhanced in patients with Down syndrome . Immunoreactive Cu/Zn SOD was detectable in all the tissues examined and was present at high concentrations in brain, adrenal gland, liver, heart and kidney. Gene, 1990 Jan 31, 86(1), 95 - 8 Cloning and expression of draTG genes from Azospirillum lipoferum; Fu HA et al.; A genomic library of Azospirillum lipoferum was constructed with phage lambda EMBL4 as vector . From this library, the genes encoding dinitrogenase reductase ADP-ribosyltransferase (DRAT), draT, and dinitrogenase reductase-activating glycohydrolase (DRAG), draG, were cloned by hybridization with the heterologous probes of Rhodospirillum rubrum . As in R . rubrum, draT is located between draG and nifH, the gene encoding dinitrogenase reductase (a substrate for the DRAG/DRAT system) . In the crude extract of Escherichia coli harboring the expression vector for this region, DRAT and DRAG enzyme activities were detected, confirming the identity of the cloned genes . Southern hybridization with genomic DNA from different Azospirillum spp., demonstrated a correlation between observable draTG hybridization and the biochemical demonstration of this covalent modification system. Biochem Biophys Res Commun, 1990 Jan 30, 166(2), 673 - 80 The chiral synthesis and biochemical properties of electron rich phenolic sulfoxide analogs of sparsomycin; Flynn GA et al.; A novel route to activated phenolic sulfoxide analogs of sparsomycin has been developed . These analogs display an enhanced "preincubation effect" as inhibitors of peptide-bond formation . This time-dependent component of inhibition, which is postulated to result from an enzyme-mediated Pummerer rearrangement, is the dominant route to inhibition in these activated analogs. Biochemistry, 1990 Jan 30, 29(4), 862 - 5 Intrinsic fluorescence of succinyl-CoA synthetase and four tryptophan mutants . Tryptophan 76 and tryptophan 248 of the beta-subunit are responsive to CoA binding; Nishimura JS et al.; Previous studies showed that modification of an average of one of the three tryptophan residues of succinyl-CoA synthetase of Escherichia coli abolished enzyme activity, but did not prevent phosphorylation of the enzyme by ATP {Ybarra, J., Prasad, A . R . S., & Nishimura, J.S . (1986) Biochemistry 25, 7174-7178} . In the present study, single mutations in which each of the three tryptophans (beta-Trp43, beta-Trp76, and beta-Trp248) has been changed to phenylalanine (designated W43F, W76F, and W248F) have been accomplished by the technique of site-directed mutagenesis and the mutant proteins isolated . In addition, a double mutant in which beta-Trp43 and beta-Trp248 were changed to phenylalanines (W43,248F) has also been isolated . Each of the mutant enzymes was practically as active as wild type . Since the emission spectrum of beta-Trp76 reflected a low fluorescence intensity for this residue, it was possible to obtain the emission spectrum of each tryptophan residue by using W43F, W248F, and W43,248F . From the positions of the emission maxima and the results of iodide quenching of fluorescence, it was deduced that beta-Trp248 is a surface residue, beta-Trp43 is buried, and beta-Trp76 is intermediate in location . Coenzyme A, but no other substrate, protected the fluorescence of beta-Trp76 and beta-Trp248, but not of beta-Trp43, against quenching by acrylamide . These results are consistent with an interaction between beta-Trp76 and beta-Trp248 and the binding site for CoA. Biochemistry, 1990 Jan 30, 29(4), 1016 - 24 Effect of the diaminocyclohexane carrier ligand on platinum adduct formation, repair, and lethality; Page JD et al.; Platinum compounds with the diaminocyclohexane (dach) carrier ligand are of particular interest because cell lines that have developed resistance to platinum compounds in general often retain sensitivity to dach-platinum compounds, suggesting that the dach carrier ligand affects the formation, repair, or lethality of platinum-DNA adducts . The effect of the dach ligand on platinum adduct formation was assessed by using the (HaeIII-HindIII)146 fragment of pBR322 treated to give equal amounts of dach- or ethylene-diamine-platinum adducts . The sites of adduct formation were mapped by digestion with Escherichia coli ABC excinuclease . There were no significant effects of the dach carrier ligand on the types or sites of platinum adduct formation . The effect of the dach ligand on platinum adduct repair was determined by using synthetic oligomers designed to have single, specific platinum adducts (G monoadduct; GG, AG, or GNG diadduct) with either the dach or ethylenediamine (en) carrier ligand . These adducts differed significantly in their ability to serve as substrates for ABC excinuclease with GNG greater than or equal to G greater than AG greater than GG . The dach carrier ligand had little effect on the recognition of AG and GG adducts by ABC excinuclease, but significantly improved the ability of ABC excinuclease to excise G monoadducts and GNG diadducts . These data suggest that if the carrier ligand has any effect on the repair of platinum adducts, it is more likely to exert that effect on the repair of platinum monoadducts or GNG diadducts rather than on the more abundant AG or GG diadducts . {14C}Thiourea incorporation was used to quantitate the rate of monoadduct to diadduct conversion.(ABSTRACT TRUNCATED AT 250 WORDS) Philos Trans R Soc Lond B Biol Sci, 1990 Jan 30, 326(1236), 505 - 13 Fructose transport by Escherichia coli; Kornberg HL; The utilization of fructose by Escherichia coli involves, as first step, the uptake of the sugar, normally via the phosphoenolpyruvate-dependent phosphotransferase system (PTS) . This fructose-specific PTS differs in several ways from that effecting the uptake of other sugars that also possess the 3,4,5-D-arabino-hexose configuration: these differences are discussed . Mutants that lack the genes ptsI and ptsH, which specify components of the PTS common to most PT-sugars, can mutate further to regain the ability to utilize fructose when this is present in relatively high concentration (i.e . greater than 2 mM) in the medium . Some of the properties of this unusual uptake system is discussed. Philos Trans R Soc Lond B Biol Sci, 1990 Jan 30, 326(1236), 455 - 63 Molecular analysis of an ATP-dependent anion pump; Rosen BP et al.; The plasmid-borne arsenical resistance operon encodes an ATP-driven oxyanion pump for the extrusion of the oxyanions arsenite, antimonite and arsenate from bacterial cells . The catalytic component of the pump, the 63 kDa ArsA protein, hydrolyses ATP in the presence of its anionic substrate antimonite (SbO2-) . The ATP analogue 5'-p-fluorosulphonylbenzoyladenosine was used to modify the ATP binding site(s) of the ArsA protein . From sequence analysis there are two potential nucleotide binding sites . Mutations were introduced into the N-terminal site . Purified mutant proteins were catalytically inactive and incapable of binding nucleotides . Conformational changes produced upon binding of substrates to the ArsA protein were investigated by measuring the effects of substrates on trypsin inactivation . The hydrophobic 45.5 kDa ArsB protein forms the membrane anchor for the ArsA protein . The presence of the ArsA protein on purified inner membrane can be detected immunologically . In the absence of the arsB gene no ArsA is found on the membrane . Synthesis of the ArsB protein is limiting for formation of the pump . Analysis of mRNA structure suggests a potential translational block to synthesis of the ArsB protein . Northern analysis of the ars message demonstrates rapid degradation of the mRNA in the arsB region. Philos Trans R Soc Lond B Biol Sci, 1990 Jan 30, 326(1236), 425 - 36 Lac permease of Escherichia coli: on the path of the proton; Kaback HR; Lactose/H+ symport in Escherichia coli is catalysed by a hydrophobic transmembrane protein encoded by the lacY gene that has been purified to homogeneity, reconstituted into proteoliposomes and shown to be completely functional as a monomer . Circular dichroic studies and hydropathy profiling of the amino-acid sequence of this 'lac' permease suggest a secondary structure in which the polypeptide consists of 12 hydrophobic segments in alpha-helical conformation that traverse the membrane in zig-zag fashion connected by shorter, hydrophilic domains with most of the charged residues and many of the residues commonly found in beta-turns . Support for certain general aspects of the model has been obtained from other biophysical studies, as well as biochemical, immunological and genetic approaches . Oligonucleotide-directed, site-specific mutagenesis is currently being utilized to probe the structure and function of the permease . Application of the technique provides an indication that Arg302 (putative helix IX), His322 (putative helix X) and Glu325 (putative helix X) may be sufficiently close to hydrogen-bond and that these residues play a critical role in lactose-coupled H+ translocation, possibly as components of a charge-relay type of mechanism . In contrast, Cys residues, which were long thought to play a central role in the mechanism of lactose/H+ symport, do not appear to be involved in either substrate binding or H+ translocation. Philos Trans R Soc Lond B Biol Sci, 1990 Jan 30, 326(1236), 411 - 23 The melibiose/Na+ symporter of Escherichia coli: kinetic and molecular properties; Pourcher T et al.; The role of the co-transported cation in the coupling mechanism of the melibiose permease of Escherichia coli has been investigated by analysing its sugar-binding activity, facilitated diffusion reactions and energy-dependent transport reactions catalysed by the carrier functioning either as an H+, Na+ or Li(+)-sugar symporter . The results suggest that the coupling cation not only acts as an activator for sugar-binding on the carrier but also regulates the rate of dissociation of the co-substrates in the cytoplasm by controlling the stability of the ternary complex cation-sugar-carrier facing the cell interior . Furthermore, there is some evidence that the membrane potential enhances the rate of symport activity by increasing the rate of dissociation of the co-substrates from the carrier in the cellular compartment . Identification of the melibiose permease as a membrane protein of 39 kDa by using a T7 RNA polymerase/promoter expression system is described . Site-directed mutagenesis has been used to replace individual carrier histidine residues by arginine to probe the functional contribution of each of the seven histidine residues to the symport mechanism . Only substitution of arginine for His94 greatly interferes with the carrier function . It is finally shown that mutations affecting the glutamate residue in position 361 inactivate translocation of the co-substrates but not their recognition by the permease. FEBS Lett, 1990 Jan 29, 260(2), 233 - 5 Ultracentrifugal analysis of the quaternary structure of the raf repressor from Escherichia coli; Jaenicke R et al.; The raf repressor from Escherichia coli regulates the expression of the plasmid-borne raf operon by switching between active and inactive conformational states . Ultracentrifugal analysis of the largely purified repressor proves the DNA-free protein to undergo concentration-dependent dissociation-association . High-speed sedimentation equilibria show that the 72 kDa dimer prevails under meniscus depletion conditions . At intracellular concentrations the 144 kDa dimer-of-dimers is the dominating species . It is suggested that the tetrameric structure of the raf repressor is involved in the recognition of the 18-basepair operator DNA. FEBS Lett, 1990 Jan 29, 260(2), 176 - 8 Thrombopoietic activity of human interleukin-6; Nagasawa T et al.; Thrombopoietin (TPO), a regulatory factor in platelet production, was purified from the conditioned medium of TNK-01 cells cultured in the presence of human interleukin-1 . The N-terminal sequence of purified TPO was determined to be VPPGEDSKDVAAPHRQPLT, identical to that of the N-terminal region of human interleukin-6 (IL-6) . Two forms of TPO with molecular masses of 24 and 27 kDa were identified as IL-6 by Western analysis using an anti-IL-6 antibody . Commercial recombinant human IL-6 produced in Escherichia coli, stimulated megakaryocyte colony formation in the presence of mouse interleukin-3 and increased the number of peripheral platelets in mice in a dose-dependent manner . From these results, it is concluded that human IL-6 has thrombopoietic activity. FEBS Lett, 1990 Jan 29, 260(2), 206 - 8 Phosphorus-containing inhibitors of aspartate transcarbamoylase from Escherichia coli; Laing NM et al.; A tetrahedral intermediate is the prominent feature of the generally accepted mechanism for aspartate transcarbamoylase . We have synthesized N-pyrophosphoryl-L-aspartate as a charged analogue of the postulated intermediate . Surprisingly, its affinity for the enzyme from Escherichia coli was substantially lower than that of the previously known inhibitor phosphonoacetyl-L-aspartate which contained a trigonal carbonyl group . Similar results were obtained with the corresponding mercaptosuccinate derivatives . We also tested a number of new pyrophosphate analogues as inhibitors . Our results cast doubt on some aspects of the current model for the mechanism of this enzyme. Biochim Biophys Acta, 1990 Jan 29, 1021(2), 175 - 81 Polarity-dependent voltage-gated porin channels from Escherichia coli in lipid bilayer membranes; Morgan H et al.; A porin preparation from Escherichia coli 0111:B4 consisting of Omp F and Omp C (with Omp F in excess) was purified by salt extraction procedures and investigated in bilayer lipid membranes formed according to the Montal-Mueller technique . The porin preparation was added to the KCl electrolyte compartment of the Montal-Mueller cell which was connected to the voltage source . As the porin incorporated into the membrane, asymmetric, voltage-gated ion channels were formed . Transmembrane voltages greater than +50 mV (measured with respect to the side of porin addition) caused channel closing, while negative voltages, on the other hand, had no effect on channel behaviour but did increase the rate of porin incorporation at higher voltages . With porin added to both compartments voltage gating no longer occurred . Single-channel conductances corresponded to effective pore diameters of 1.5 nm for opening events and 1.18 nm for channel closing events . The number of charges involved in gating was approximately 2. Eur J Biochem, 1990 Jan 26, 187(2), 455 - 60 Biophysical studies of engineered mutant proteins based on calbindin D9k modified in the pseudo EF-hand; Johansson C et al.; The genes for four mutant proteins from calbindin D9k, all with mutations in the N-terminal Ca2+-binding domain (pseudo EF-hand) have been synthesized and expressed in Escherichia coli . The purification scheme has been modified to minimize the formation of deamidated proteins . The set of modifications in the pseudo EF-hand is an attempt to turn this site into a structure resembling an archetypal EF-hand, with its characteristic 113Cd-NMR shift (-80 to -110 ppm) and high calcium-binding constants, whereas the C-terminal Ca2(+)-binding site (EF-hand) is kept intact in all mutant proteins . The mutant proteins studied here all have pseudo EF-hands with a lower calcium-binding constant and a higher calcium off-rate to the pseudo EF-hand than the wild-type protein . From the results obtained it is obvious that proline 20 in the pseudo EF-hand, which has been deleted or replaced by glycine in three of the mutants, has a stabilizing effect on calcium binding to that site . Furthermore, the modifications in the pseudo EF-hand seem to have only a local effect, leaving the tertiary structure of the protein and the calcium-binding properties of the unmodified site virtually unchanged. Eur J Biochem, 1990 Jan 26, 187(2), 409 - 16 Structural studies of the lysozyme coded by the pneumococcal phage Cp-1 . Conformational changes induced by choline; Sanz JM et al.; The CPL-1 lysozyme coded by the pneumococcal phage Cp-1 has been overproduced in Escherichia coli under the control of a modified lipoprotein lactose promoter . This result has provided the conditions to analyse the CPL-1 secondary structure by circular dichroism (CD) . The CD spectra recorded in the far-ultraviolet region showed, at neutral pH, two minima at 210 nm and 230 nm and a shoulder at 217 nm, whereas two bands at 260 nm and 295 nm were observed in the near-ultraviolet region . It has been estimated, by using the CDPROT program, that the protein is composed of 19% alpha-helix, 32% beta-sheet, 28% beta-turn and 21% random coil . Minor changes in the CD spectra were detected either when the pH was varied over 6-10 or when the ionic strength was increased to 1 M NaCl . Choline, a well known modulator of the enzyme activity that is present in the pneumococcal cell wall, induced remarkable changes in the intensities of the bands at 210, 230 and 295 nm, with the appearance of an unusual positive band at 225 nm . The conformational change was reversible and correlated with the competitive inhibitory effect of choline on the lysozyme activity, supporting, by a new and direct experimental approach, the basic role of choline in the recognition of the cell wall substrate . The analyses of the secondary structure prediction and the CD data reported here are compatible with the two-domain structure of CPL-1 reinforce our hypothesis that the C-terminal region is directly involved in the binding of the enzyme to the pneumococcal teichoic and lipoteichoic acids. Eur J Biochem, 1990 Jan 26, 187(2), 373 - 9 Autoregulation of PurR repressor synthesis and involvement of purR in the regulation of purB, purC, purL, purMN and guaBA expression in Escherichia coli; Meng LM et al.; The purR gene encodes a repressor (PurR) controlling the synthesis of the enzymes of purine biosynthesis . The subunit of PurR was identified as a 38-kDa polypeptide by SDS/polyacrylamide gel electrophoresis . Analysis of a purR-lacZ transcriptional fusion indicated that purR expression is autoregulated . This was confirmed by gel retardation and DNaseI footprinting experiments, where two PurR-binding sites were identified in the transcribed part of purR . Introduction of a purR mutation in wild-type and pur-lac fusion strains was found to abolish purine repression of all genes of the purine biosynthetic pathway except for purA. Cell, 1990 Jan 26, 60(2), 271 - 80 The ATPase activity of SecA is regulated by acidic phospholipids, SecY, and the leader and mature domains of precursor proteins; Lill R et al.; The ATPase activity of SecA is stimulated by E . coli plasma membrane vesicles bearing SecY protein and a precursor protein such as proOmpA . This activity is termed "translocation ATPase" . Liposomes alone can also stimulate SecA ATPase, but membrane proteins block this stimulation in native inner membranes . We define the stimulation of SecA ATPase by lipid as "SecA/lipid ATPase" . SecA/lipid ATPase, translocation ATPase, and translocation into inner membrane vesicles require acidic phospholipids, suggesting an underlying unity of mechanism . ProOmpA and ATP stabilize liposome-bound SecA . Full SecA/lipid ATPase activity and stability are also seen when a mixture of a leader peptide and either OmpA or maltose binding protein (MBP) are added instead of proOmpA, while neither the leader peptide alone nor OmpA or MBP suffice . Cytosolic proteins in conjuction with a leader peptide are less active in this reaction, indicating that liposome-bound SecA protein recognizes both leader and mature domains. Cell, 1990 Jan 26, 60(2), 247 - 57 The progesterone receptor stimulates cell-free transcription by enhancing the formation of a stable preinitiation complex; Klein-Hitpass L et al.; Highly purified chicken progesterone receptor (cPR) is shown to stimulate RNA synthesis directly in an in vitro transcription assay . Stimulation of transcription by cPR requires the presence of progesterone response elements (PREs) in the template and can be specifically inhibited by addition of competitor oligonucleotides containing PREs . Binding of receptor to two PREs is cooperative and leads to synergistic (27-fold) stimulation of transcription . A purified fusion protein containing the DNA binding domain of cPR linked to yeast ubiquitin was produced in E . coli and also functions in the transcription assay . Using this in vitro transcription system, we demonstrate that hormone-free cPR activated by salt treatment induces transcription of a test gene in a hormone-independent manner . Finally, we present evidence that the progesterone receptor acts by facilitating the formation of a stable preinitiation complex at the target gene promoter and thus augments the initiation of transcription by RNA polymerase II. Cell, 1990 Jan 26, 60(2), 329 - 36 Enzymatic formation and resolution of Holliday junctions in vitro; Muller B et al.; E . coli RecA protein promotes homologous pairing and reciprocal strand exchange reactions between duplex DNA molecules in vitro . Reaction intermediates contain Holliday junctions that are driven along the DNA at a maximal rate approaching 1000 bases per minute . T4 endonuclease VII cleaves Holliday junctions in vitro, and its inclusion in RecA-mediated reactions leads to the rapid formation of heteroduplex products . Product analysis indicates patch and splice recombinant molecules similar to those expected from in vivo recombination events . The combined formation and resolution of Holliday junctions has led us to propose a model for resolution based on the structure of RecA-DNA helices . One feature of this model is that resolution, which gives rise to the two types of recombinant product, may occur without need for isomerization of the junction. Eur J Biochem, 1990 Jan 26, 187(2), 417 - 24 Effect of the N-terminal hydrophobic sequence of hepatitis B virus surface antigen on the folding and assembly of hybrid beta-galactosidase in Escherichia coli; Lee SC et al.; To investigate the mechanism of inclusion body formation and the effect of a hydrophobic sequence on the in vivo polypeptide folding, the aggregation caused by recombinant fusion beta-galactosidase in Escherichia coli was examined . Two plasmids were constructed: pTBG(H-) carried only the preS2 sequence of the hepatitis B virus surface antigen (HBsAg) in front of the beta-galactosidase gene (lacZ) while pTBG(H+) carried an additional sequence encoding the amino-terminal hydrophobic sequence of the S region of HBsAg between preS2 and lacZ . Unlike cells expressing the fusion protein not containing the hydrophobic sequence, E . coli JM109/pTBG(H+) exhibited temperature-sensitive production of beta-galactosidase . As the culture temperature increased the activity decreased dramatically . This decrease in activity was not due to a decrease in fusion polypeptide production, but rather the fusion polypeptides containing the hydrophobic sequence aggregated within the cells at high temperature . However once the fusion polypeptides folded into proper conformation at low temperature, they maintained the activity even at high temperature . The results indicate that aggregation is a consequence of incorrect folding and assembly of the polypeptides, and is not derived from the native structure . The aggregates of the pTBG(H+)-encoded fusion polypeptides did not revert to active form when the culture temperature was lowered. Eur J Biochem, 1990 Jan 26, 187(2), 329 - 33 Aspartate aminotransferase with the pyridoxal-5'-phosphate-binding lysine residue replaced by histidine retains partial catalytic competence; Ziak M et al.; The active site residue lysine 258 of chicken mitochondrial aspartate aminotransferase was replaced with a histidine residue by means of site-directed mutagenesis . The mutant protein was expressed in Escherichia coli and purified to homogeneity . Addition of 2-oxoglutarate to its pyridoxamine form changed the coenzyme absorption spectrum (lambda max = 330 nm) to that of the pyridoxal form (lambda max = 330/392 nm) . The rate of this half-reaction of transamination (kcat = 4.0 x 10(-4)s-1) is five orders of magnitude slower than that of the wild-type enzyme . However, the reverse half-reaction, initiated by addition of aspartate or glutamate to the pyridoxal form of the mutant enzyme, is only three orders of magnitude slower than that of the wild-type enzyme, kmax of the observable rate-limiting elementary step, i.e . the conversion of the external aldimine to the pyridoxamine form, being 7.0 x 10(-2)s-1 . Aspartate aminotransferase (Lys258----His) thus represents a pyridoxal-5'-phosphate-dependent enzyme with significant catalytic competence without an active site lysine residue . Apparently, covalent binding of the coenzyme, i.e . the internal aldimine linkage, is not essential for the enzymic transamination reaction, and a histidine residue can to some extent substitute for lysine 258 which is assumed to act as proton donor/acceptor in the aldimine-ketimine tautomerization. Eur J Biochem, 1990 Jan 26, 187(2), 307 - 14 Rapid purification of homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography; Le Grice SF et al.; We have modified an Escherichia coli vector expressing 66-kDa HIV-1 reverse transcriptase (p66) so that it simultaneously expresses this and the pol-coded protease . The twin expression cassette yields high quantities of both reverse transcriptase and protease; however, under these conditions, 50% of the over-expressed p66 reverse transcriptase is processed, resulting in accumulation of large quantities of p66/p51 enzyme . Furthermore, addition of a poly(histidine) affinity label at the amino terminus of the reverse-transcriptase-coding sequence (His-p66) permits a simple, rapid purification of milligram quantities of either p66 or p66/p51 enzyme from a crude lysate by metal chelate affinity chromatography . Purified His-p66 and His-p66/His-p51 reverse transcriptase exhibit both reverse transcriptase and RNase H activity . Purification by metal chelate chromatography of a p66/p51 enzyme wherein only the p66 component is labelled strengthens the argument for the existence of a heterodimer. J Biol Chem, 1990 Jan 25, 265(3), 1578 - 87 Biosynthesis of the polysialic acid capsule in Escherichia coli K1 . The endogenous acceptor of polysialic acid is a membrane protein of 20 kDa; Weisgerber C et al.; The nature of endogenous acceptor molecules implicated in the membrane-directed synthesis of the polysialic acid (polySia) capsule in Escherichia coli K1 serotypes is not known . The capsule contains at least 200 sialic acid (Sia) residues that are elongated by the addition of new Sia residues to the nonreducing termini of growing nascent chains (Rohr, T . E., and Troy, F . A . (1980) J . Biol . Chem . 255, 2332-2342) . Presumably, chain growth starts when activated Sia residues are transferred to acceptors that are not already sialylated . In the present study, we used an acapsular mutant defective in synthesis of CMP-NeuAc to label acceptors with {14C}NeuAc and an anti-polySia-specific antibody (H.46) to identify the molecules to which the polySia was attached . {14C}Sia-labeled acceptors were solubilized with 2% Triton X-100, immunoprecipitated with H.46, and partially depolymerized with poly-alpha-2,8-endo-N-acetylneuraminidase . Approximately 5% of the {14C}Sia incorporated remained attached to endogenous acceptors . Double-labeling experiments were used to show that the non-Sia moiety of the acceptor was labeled in vivo with {14C}leucine and elongated in vitro with CMP-{3H}NeuAc . Concomitant with desialylation of the {3H}polySia-{14C}Leu acceptor was the appearance of a new {14C}Leu-labeled protein at 20 kDa . After strong acid hydrolysis, the 20-kDa labeled protein was shown to contain {14C}Leu . The acceptor molecules were not labeled metabolically with D-{3H}GlcN, 35SO4, or 32PO4, indicating that they do not appear to contain lipopolysaccharide, peptidoglycan, phosphatidic acid, or phospholipid . Based on these results, we conclude that the endogenous acceptor molecule is a membrane protein of about 20 kDa . The nature of attachment of polySia to acceptor is unknown . There are only 400-500 acceptor molecules/cell, which is about 100-fold fewer than the 50,000 polySia chains/cell . This suggests that each acceptor molecule may participate in the shuttling of about 100 polySia chains/cell . We hypothesize that the acceptor protein may function to translocate polySia chains from their site of synthesis on the cytoplasmic surface of the inner membrane to the periplasm. Nucleic Acids Res, 1990 Jan 25, 18(2), 229 - 33 Ribosomal protein S14 transcripts are edited in Oenothera mitochondria; Schuster W et al.; The gene encoding ribosomal protein S14 (rps14) in Oenothera mitochondria is located upstream of the cytochrome b gene (cob) . Sequence analysis of independently derived cDNA clones covering the entire rps14 coding region shows two nucleotides edited from the genomic DNA to the mRNA derived sequences by C to U modifications . A third editing event occurs four nucleotides upstream of the AUG initiation codon and improves a potential ribosome binding site . A CGG codon specifying arginine in a position conserved in evolution between chloroplasts and E . coli as a UGG tryptophan codon is not edited in any of the cDNAs analysed . An inverted repeat 3' of an unidentified open reading frame is located upstream of the rps14 gene . The inverted repeat sequence is highly conserved at analogous regions in other Oenothera mitochondrial loci. Nucleic Acids Res, 1990 Jan 25, 18(2), 337 - 43 Requirement of protein co-factor for the DNA-binding function of the human ski proto-oncogene product; Nagase T et al.; We identified the human c-ski gene product (c-Ski) as a protein with the apparent molecular weight of 100,000, p100c-ski, by using a c-Ski-specific polyclonal antibody . p100c-ski was a nuclear protein and p100c-ski in nuclear extracts of Molt4 cells bound to calf thymus DNA cellulose, but the bacterially synthesized c-Ski did not, suggesting that Ski was associated with another protein(s) and that the Ski complex had DNA-binding activity . This hypothesis was supported by the finding that the bacterially synthesized Ski bounds to DNA cellulose after being mixed with a nuclear extract of Molt4 cells . By use of a series of deletion mutants of Ski synthesized in an in vitro translation system, two portions in Ski were found to be necessary for the DNA binding of the Ski complex: the N-proximal portion containing a cystein/histidine-rich domain and the C-terminal portion including a region rich in basic amino acids. Nucleic Acids Res, 1990 Jan 25, 18(2), 313 - 21 Alignment of Escherichia coli K12 DNA sequences to a genomic restriction map; Rudd KE et al.; We use the extensive published information describing the genome of Escherichia coli and new restriction map alignment software to align DNA sequence, genetic, and physical maps . Restriction map alignment software is used which considers restriction maps as strings analogous to DNA or protein sequences except that two values, enzyme name and DNA base address, are associated with each position on the string . The resulting alignments reveal a nearly linear relationship between the physical and genetic maps of the E . coli chromosome . Physical map comparisons with the 1976, 1980, and 1983 genetic maps demonstrate a better fit with the more recent maps . The results of these alignments are genomic kilobase coordinates, orientation and rank of the alignment that best fits the genetic data . A statistical measure based on extreme value distribution is applied to the alignments . Additional computer analyses allow us to estimate the accuracy of the published E . coli genomic restriction map, simulate rearrangements of the bacterial chromosome, and search for repetitive DNA . The procedures we used are general enough to be applicable to other genome mapping projects. Nucleic Acids Res, 1990 Jan 25, 18(2), 305 - 12 Homology of lysS and lysU, the two Escherichia coli genes encoding distinct lysyl-tRNA synthetase species; Leveque F et al.; In Escherichia coli, two distinct lysyl-tRNA synthetase species are encoded by two genes: the constitutive lysS gene and the thermoinducible lysU gene . These two genes have been isolated and sequenced . Their nucleotide and deduced amino acid sequences show 79% and 88% identity, respectively . Codon usage analysis indicates the lysS product being more efficiently translated than the lysU one . In addition, the lysS sequence exactly coincides with the sequence of herC, a gene which is part of the prfB-herC operon . In contrast to the recent proposal of Gampel and Tzagoloff (1989, Proc . Natl . Acad . Sci . USA 86, 6023-6027), the lysU sequence is distinct from the open reading frame located adjacent to frdA, although large homologies are shared by these two genes. J Biol Chem, 1990 Jan 25, 265(3), 1419 - 24 High level expression of a truncated chicken progesterone receptor in Escherichia coli; Power RF et al.; Using a novel Escherichia coli system we have successfully overexpressed a region of the chicken progesterone receptor which encodes both the DNA- and hormone-binding domains . The expression system produces the truncated receptor fragment as an in-frame fusion with ubiquitin . This strategy greatly enhances both the solubility and stability of fusion proteins expressed in E . coli . Synthesis has been further improved by induction of the lambda PL promoter with nalidixic acid at low growth temperatures (less than or equal to 30 degrees C) rather than use of conventional heat induction protocols . We can produce 10 mg of receptor fragment/liter of cells using this system, and we estimate that at least 0.3 mg of this receptor material is biologically active, as assessed by DNA-binding and hormone-binding assays . Receptor produced in this manner is almost indistinguishable from authentic oviduct progesterone receptor using the criteria of hormone-binding specificity and affinity and binding to a progesterone response element . This expression system offers a cheap convenient method for the production of mg amounts of biologically active derivatives of progesterone receptor for biochemical studies. J Biol Chem, 1990 Jan 25, 265(3), 1282 - 5 Fate of the DnaA initiator protein in replication at the origin of the Escherichia coli chromosome in vitro; Yung BY et al.; The dnaA initiator protein binds specific sequences in the 245-base pair Escherichia coli origin (oriC) to form a series of complexes which eventually are opened enough to admit dnaB helicase into a prepriming complex (Bramhill, D., and Kornberg, A . (1988) Cell 52, 743-755) . ATP bound to a high-affinity site on dnaA protein is the preferred form for one or more of the early stages, but an elevated level of ATP is needed for a later stage; further evidence for a low-affinity site has now been obtained . We find that at limiting levels of dnaA protein only the ATP form produces an active initial complex; neither the ADP nor the non-nucleotide forms are effective . Augmentation of the activity of a limiting level of the ATP form of dnaA protein by the otherwise inert ADP form implies that at some stage of initiation both forms are active . The dnaA protein is essential up to the stage of forming the prepriming complex; upon salt dissociation from an oriC complex, the protein can be recycled to function at a fresh origin . Distinctive conformational states of the ATP form are implied by interactions with oriC DNA, by the influence of phospholipids on accelerating nucleotide exchange, and by the susceptibility to proteolytic cleavage. Nature, 1990 Jan 25, 343(6256), 341 - 6 Primary structure and functional expression from complementary DNA of a human interleukin-1 receptor antagonist; Eisenberg SP et al.; Human monocytes induced with adherent IgG secrete an interleukin-1 receptor antagonist which could be important for the in vivo regulation of IL-1 activity . A complementary DNA for this molecule has been isolated from a human monocyte library . Analysis of monocyte RNA indicates that the gene is transcriptionally regulated . The sequence of the receptor antagonist indicates that it is structurally similar to IL-1 beta . Expression of the cDNA in Escherichia coli yields IL-1 receptor antagonist activity. J Biol Chem, 1990 Jan 25, 265(3), 1787 - 93 Mutant of Escherichia coli K-12 with defective phosphorylation of two periplasmic transport proteins; Celis RT; Bacterial periplasmic transport systems require the function of a specific substrate-binding protein, located in the periplasm, and several cytoplasmic membrane transport components . In Escherichia coli K-12, the arginine-ornithine transport system requires an arginine-ornithine-binding protein and the lysine-arginine-ornithine (LAO) transport system includes a LAO-binding protein . Both periplasmic proteins can be phosphorylated by a single kinase . The enzyme exhibits a kinase activity and an ATPase activity . A mutant, defective in the phosphorylation of the arginine-ornithine and the LAO periplasmic proteins, was isolated and characterized . The defective enzymatic activity was reflected in substantially reduced levels of transport activity of the periplasmic transport systems that include each of the binding proteins . The binding proteins, extracted from the mutant, showed no detectable alterations in terms of quantity, electrophoretic mobility, or affinity constants . An apparent Km value of 1.0 mM was calculated for the ATPase activity of the defective enzyme . The ATPase activity of the wild-type enzyme yielded an apparent Km value of 50 microM . The amount of inorganic phosphate incorporated in vivo and in vitro into the binding proteins by the activity of the defective kinase was reduced to very low levels . A structural gene for the phosphorylating enzyme was located near the serA marker on the linkage map of E . coli . These results indicate that phosphorylation of the periplasmic transport protein is obligatorily linked to the normal function of the periplasmic transport system. J Biol Chem, 1990 Jan 25, 265(3), 1484 - 9 Identification of the binding site for activated protein C on the light chain of factors V and VIII; Walker FJ et al.; Activated protein C has been observed to bind to the light chains of factor Va and factor VIII . Fragments of the factor VIII light chain were produced by recombinant DNA techniques and expressed in Escherichia coli . Three fragments of the light chain were studied; L4 (residues 1974-2332), L3.2 (residues 1560-1829 and 2046-2332), and L3.3 (residues 1560-2052) . Two fragments, L4 and L3.3, which overlapped sequences between residues 1974-2052, inhibited the anticoagulant activity of activated protein C . Comparison of the sequences of factors V and VIII in this region revealed that residues 2005-2018 in the factor VIII sequence were homologous with residues 1861-1874 in the factor V sequence . The peptides Arg-Ala-Gly-Met-Gln-Thr-Phe-Leu-Ile (RAGMQTPFLI; residues 1865-1874) from the factor V sequence and His-Ala-Gly-Met-Ser-Thr-Leu-Phe-Ile-Val (HAGMSTLFIV; residues 2009-2018) from the factor VIII sequence were synthesized . Both peptides were observed to inhibit the anticoagulant activity of activated protein C and its inactivation of factors Va and VIII . Furthermore RAGMQTPFLI quenched the fluorescence of the dansyl-Glu-Gly-Arg-modified protease . Polyclonal antibodies against RAGMQTPFLI bound to factor Va and inhibited the anticoagulant activity of activated protein C and the inactivation of factor Va . These results indicate that a portion of the binding sites for activated protein C on the light chains of factors V and VIII are contained in the sequences RAGMQTPFLI or HAGMSTLFIV, respectively. Nucleic Acids Res, 1990 Jan 25, 18(2), 323 - 9 Characterization of the promoter region of Tetrahymena genes; Brunk CF et al.; The regions between adjacent histone H3 and H4 genes, as well as portions of the genes, from 22 species of Tetrahymena have been amplified using the polymerase chain reaction and sequenced . Both histone genes are transcribed divergently with initiation occurring within the intergenic region, thus 2 sets of 22 homologous Tetrahymena promoters can be compared . A sequence comparison of these regions reveals a single putative promoter element, with a consensus sequence TATCCAATTCARA, present in front of each gene . This sequence contains a 'CCAAT' box, which also occurs at 8 locations preceding other ciliate genes . No other putative promoter sequences are found in front of these sets of histone genes . Sequences searched for include 'TATA' boxes, 'GC' boxes and other sequences suggested as putative promoter elements for ciliate genes . The coding strand immediately preceding ciliate genes is very high in A content and the consensus sequence at the site of protein synthesis is AAAATGG. J Biol Chem, 1990 Jan 25, 265(3), 1783 - 6 Purification and properties of a kinase from Escherichia coli K-12 that phosphorylates two periplasmic transport proteins; Urban C et al.; The phosphorylation in vivo and in vitro of the arginine-ornithine and the lysine-arginine-ornithine (LAO) periplasmic transport proteins of Escherichia coli K-12 was previously reported (Celis, R . T . F . (1984) Eur . J . Biochem . 145, 403-411) . The phosphorylative reaction required ATP (as a direct energy donor), Mg2+, and a kinase that can be released by osmotic shock treatment of the cells . The enzyme was purified to electrophoretic homogeneity . The enzyme exhibited an ATPase activity and a kinase activity . Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gave an apparent molecular weight of 43,000 for the enzyme . The native protein showed the same molecular weight, suggesting that the protein is a monomer . The protein showed an apparent isoelectric point of 4.8 on isoelectric focusing . The two enzymatic reactions required a divalent cation and the apparent Km value for Mg2+ for the kinase activity was 0.5 mM . Mn2+ and Co2+ served as well as Mg2+, whereas Zn2+ and Ca2+ did not support activity . The ATPase activity of the enzyme yielded an apparent Km value for ATP of 50 microM . A similar value, Km of 100 microM, was calculated for the kinase activity with different concentrations of ATP . The enzyme showed a pH optimum of 7.3. Nucleic Acids Res, 1990 Jan 25, 18(2), 261 - 5 Molecular cloning, structure and expression of the yeast proliferating cell nuclear antigen gene; Bauer GA et al.; The budding yeast Saccharomyces cerevisiae is proving to be an useful and accurate model for eukaryotic DNA replication . It contains both DNA polymerase alpha (I) and delta (III) . Recently, proliferating cell nuclear antigen (PCNA), which in mammalian cells is an auxiliary subunit of DNA polymerase delta and is essential for in vitro leading strand SV40 DNA replication, was purified from yeast . We have now cloned the gene for yeast PCNA (POL30) . The gene codes for an essential protein of 29 kDa, which shows 35% homology with human PCNA . Cell cycle expression studies, using synchronized cells, show that expression of both the PCNA (POL30) and the DNA polymerase delta (POL3, or CDC2) genes of yeast are regulated in an identical fashion to that of the DNA polymerase alpha (POL1) gene . Thus, steady state mRNA levels increase 10-100-fold in late G1 phase, peak in early S-phase, and decrease to low levels in late S-phase . In addition, in meiosis mRNA levels increase prior to initiation of premeiotic DNA synthesis. J Biol Chem, 1990 Jan 25, 265(3), 1608 - 14 A cDNA encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Solanum tuberosum L; Dyer WE et al.; A cDNA encoding potato (Solanum tuberosum L.) 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway, was cloned into phage lambda gt11 . The clone represents the first cDNA for this enzyme from any eukaryotic source . The nucleotide sequence of the cDNA was determined, and its identity was confirmed through partial amino acid sequence analysis of the encoded enzyme . The cDNA contains a 1527-base pair open reading frame that encodes a polypeptide with a calculated molecular weight of 56,153 . The amino terminus of the deduced polypeptide resembles a chloroplast transit sequence . Amino acid sequence identities between the mature potato enzyme and the homologous isoenzymes from Escherichia coli are only about 22% . The potato cDNA hybridized to various plant mRNAs that are all about 2 kilobases in size. J Biol Chem, 1990 Jan 25, 265(3), 1588 - 93 Amino acid sequence of rat kidney gamma-glutamylcysteine synthetase; Yan N et al.; gamma-Glutamylcysteine synthetase catalyzes the first step in the synthesis of glutathione . The enzyme isolated from rat kidney has two subunits (heavy, Mr 73,000; and light, Mr 27,700) which may be dissociated by treatment with dithiothreitol . The heavy subunit exhibits all of the catalytic activity of the isolated enzyme and also feedback inhibition by glutathione . The light subunit has no known function and may not be an integral part of the enzyme . cDNA clones encoding rat kidney gamma-glutamylcysteine synthetase were isolated from a lambda gt11 cDNA library by immunoscreening with antibody against the isolated enzyme and further screening with oligonucleotide probes derived from several peptides whose sequences were determined by the Edman method . The nucleotide sequence of the mRNA for the heavy subunit was deduced from the sequences of the cDNA of three such clones . The sequence, which codes for 637 residues (Mr 72,614), contains all four of the independently determined peptide sequences (approximately 100 residues) . This amino acid sequence shows extremely low overall similarity to that of gamma-glutamylcysteine synthetase isolated from Escherichia coli. Biochemistry, 1990 Jan 23, 29(3), 837 - 43 Stabilization of recA protein-ssDNA complexes by the single-stranded DNA binding protein of Escherichia coli; Morrical SW et al.; In vitro recombination reactions promoted by the recA protein of Escherichia coli are enhanced by the single-stranded DNA binding protein (SSB) . SSB affects the assembly of the filamentous complexes between recA protein and ssDNA that are the active form of the recA protein . Here, we present evidence that SSB plays a complex role in maintaining the stability and activity of recA-ssDNA filaments . Results of ATPase, nuclease protection, and DNA strand exchange assays suggest that the continuous presence of SSB is required to maintain the stability of recA-ssDNA complexes under reaction conditions that support their recombination activity . We also report data that indicate that there is a functional distinction between the species of SSB present at 10 mM magnesium chloride, which enhances recA-ssDNA binding, and a species present at 1 mM magnesium chloride, which displaces recA protein from ssDNA . These results are discussed in the context of current models of SSB conformation and of SSB action in recombination activities of the recA protein. Biochemistry, 1990 Jan 23, 29(3), 690 - 6 Melibiose permease and alpha-galactosidase of Escherichia coli: identification by selective labeling using a T7 RNA polymerase/promoter expression system; Pourcher T et al.; Identification and selective labeling of the melibiose permease and alpha-galactosidase in Escherichia coli, which are encoded by the melB and melA genes, respectively, have been accomplished by selectively labeling the two gene products with a T7 RNA polymerase expression system {Tabor, S., & Richardson, C . C . (1985) Proc . Natl . Acad . Sci . U.S.A . 82, 1074} . Following generation of a novel EcoRI restriction site in the intergenic sequence between the two genes of the mel operon by oligonucleotide-directed, site-specific mutagenesis, melA and melB were separately inserted into plasmid pT7-6 of the T7 expression system . Expression of melB was markedly enhanced by placing a strong, synthetic ribosome binding site at an optimal distance upstream from the initiation codon of melB . Expression of cloned gene products was characterized functionally and by performing autoradiographic analysis on total cell, inner membrane, and cytoplasmic proteins from cells pulse labeled with (35S)methionine in the presence of rifampicin and resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis . The results first confirm that alpha-galactosidase is a cytoplasmic protein with an Mr of 50K; in contrast, the membrane-bound melibiose permease is identified as a protein with an apparent Mr of 39K, a value significantly higher than that of 30K previously suggested {Hanatani et al . (1984) J . Biol . Chem . 259, 1807}. J Mol Biol, 1990 Jan 20, 211(2), 415 - 26 Target site of Escherichia coli ribosomal protein S15 on its messenger RNA . Conformation and interaction with the protein; Philippe C et al.; The regulatory site of ribosomal protein S15 has been located in the 5' non-coding region of the messenger, overlapping with the ribosome loading site . The conformation of an in vitro synthesized mRNA fragment, covering the 105 nucleotides upstream from the initiation codon and the four first codons of protein S15, has been monitored using chemical probes and RNase V1 . Our results show that the RNA is organized into three domains . Domains I and II, located in the 5' part of the mRNA transcript, are folded into stable stem-loop structures . The 3'-terminal domain (III), which contains the Shine-Dalgarno sequence and the AUG initiation codon, appears to adopt alternative conformations . One of them corresponds to a rather unstable stem-loop structure in which the Shine-Dalgarno sequence is paired . An alternative potential structure involves a "pseudo-knot" interaction between bases of this domain and bases in the loop of domain II . The conformation of several RNA variants has also been investigated . The deletion of the 5'-proximal stem-loop structure (domain I), which has no effect on the regulation, does not perturb the conformation of the two other domains . The deletion of domain II, leading to a loss of regulatory control, prevents the formation of the potential helix involved in the pseudo-knot structure and results in a stabilization of the alternative stem-loop structure in domain III . The replacement of another base in domain III involved in pairing in the two alternative structures mentioned above should induce a destabilization of both structures and results in a loss of the translational control . However, the replacement of another base in domain III, which does not abolish the control, results in the loss of the conformational heterogeneity in this domain and yields a stable conformation corresponding to the pseudo-knot structure . Thus, it appears that any mutation that disrupts or alters the formation of the pseudo-knot impairs the regulatory mechanism . Footprinting experiments show that protein S15 is able to bind to the synthesized fragment and provide evidence that the protein triggers the formation of the pseudo-knot conformation . A mechanism can be postulated in which the regulatory protein stabilizes this particular structure, thus impeding ribosome initiation. J Mol Biol, 1990 Jan 20, 211(2), 407 - 14 Translational autocontrol of the Escherichia coli ribosomal protein S15; Portier C et al.; When rpsO, the gene encoding the ribosomal protein S15 in Escherichia coli, is carried by a multicopy plasmid, the mRNA synthesis rate of S15 increases with the gene dosage but the rate of synthesis of S15 does not rise . A translational fusion between S15 and beta-galactosidase was introduced on the chromosome in a delta lac strain and the expression of beta-galactosidase studied under different conditions . The presence of S15 in trans represses the beta-galactosidase level five- to sixfold, while the synthesis rate of the S15-beta-galactosidase mRNA decreases by only 30 to 50% . These data indicate that S15 is subject to autogenous translational control . Derepressed mutants were isolated and sequenced . All the point mutations map in the second codon of S15, suggesting a location for the operator site that is very near to the translation initiation codon . However, the creation of deletion mutations shows that the operator extends into the 5' non-coding part of the message, thus overlapping the ribosome loading site. J Mol Biol, 1990 Jan 20, 211(2), 359 - 72 N4 RNA polymerase II sites of transcription initiation; Abravaya K et al.; The RNA polymerase responsible for the synthesis of coliphage N4 middle RNAs, N4 RNA polymerase II, is composed of two subunits of 30,000 and 40,000 molecular weight . It is the smallest DNA-dependent RNA polymerase characterized to date . We have determined the sequences surrounding the sites of in vivo transcription initiation for this enzyme . Two regions of sequence homology are present: a box at +1, 3' AAAT 5', and a box, 3' TTCTGGAC 5' at a variable distance (16 to 24 base-pairs) upstream from +1 . Possible mechanisms for recognition of these sequences are discussed. J Mol Biol, 1990 Jan 20, 211(2), 351 - 8 Mutagenesis by proximity to the recA gene of Escherichia coli; Liu SK et al.; Escherichia coli recA (Prtc) strains, which produce protease constitutive RecA proteins in the absence of DNA-damaging treatments, display an increased frequency of spontaneous mutations . These mutations occurred preferentially in the neighborhood of the recA gene . This cis-like mutagenic effect was observed in the recA, rexAB, phoE and bio genes . The localized mutagenesis can be explained by the ease with which RecA(Prtc) proteins are activated to the protease state, which implies that there should be a relatively high concentration of activated RecA protein near the recA gene, where the protein is synthesized . The unusually high frequency of mutation in the recA gene is a novel example of an overactive gene preferentially turning itself down by mutation. J Mol Biol, 1990 Jan 20, 211(2), 427 - 45 In vitro secondary structure analysis of mRNA from lacZ translation initiation mutants; Schulz VP et al.; mRNA secondary structure can be an important determinant of the efficiency of translation initiation . To study the effect of secondary structure on translation initiation, in vitro secondary structure analysis was performed on 32 lacZ RNA transcripts that differ in their in vivo translation initiation efficiencies because of mutations . We have shown that well-translated RNA has a relatively unstructured translation initiation region in vitro . In contrast, the translation initiation region of many of the poorly translated RNA transcripts is involved in a stem-loop structure . Mutations that decrease the in vitro stability of the stem-loop increase the frequency of translation initiation . The sequences responsible for forming this stem-loop structure were localized to a small region of RNA . The results confirm some of the previous predictions of the RNA secondary structure of the mutant RNAs based on computer modeling, but they disagree with some of the predicted long-range interactions. Science, 1990 Jan 19, 247(4940), 332 - 6 Specific expression of a tyrosine kinase gene, blk, in B lymphoid cells; Dymecki SM et al.; Several pathways of transmembrane signaling in lymphocytes involve protein-tyrosine phosphorylation . With the exception of p56lck, a tyrosine kinase specific to T lymphoid cells that associates with the T cell transmembrane proteins CD4 and CD8, the kinases that function in these pathways are unknown . A murine lymphocyte complementary DNA that represents a new member of the src family has now been isolated and characterized . This complementary DNA, termed blk (for B lymphoid kinase), specifies a polypeptide of 55 kilodaltons that is related to, but distinct from, previously identified retroviral or cellular tyrosine kinases . The protein encoded by blk exhibits tyrosine kinase activity when expressed in bacterial cells . In the mouse and among cell lines, blk is specifically expressed in the B cell lineage . The tyrosine kinase encoded by blk may function in a signal transduction pathway that is restricted to B lymphoid cells. Science, 1990 Jan 19, 247(4940), 324 - 7 Cloning of an interleukin-3 receptor gene: a member of a distinct receptor gene family; Itoh N et al.; Interleukin-3 (IL-3) binds to its receptor with high and low affinities, induces tyrosine phosphorylation, and promotes the proliferation and differentiation of hematopoietic cells . A binding component of the IL-3 receptor was cloned . Fibroblasts transfected with the complementary DNA bound IL-3 with a low affinity {dissociation constant (Kd) of 17.9 +/- 3.6 nM} . No consensus sequence for a tyrosine kinase was present in the cytoplasmic domain . Thus, additional components are required for a functional high affinity IL-3 receptor . A sequence comparison of the IL-3 receptor with other cytokine receptors (erythropoietin, IL-4, IL-6, and the beta chain IL-2 receptor) revealed a common motif of a distinct receptor gene family. Biochim Biophys Acta, 1990 Jan 19, 1037(1), 16 - 23 Expression of human plasminogen activator inhibitor type-1 (PAI-1) in Escherichia coli as a soluble protein comprised of active and latent forms . Isolation and crystallization of latent PAI-1; Franke AE et al.; Expression of human recombinant plasminogen activator inhibitor type-1 (PAI-1) in Escherichia coli has led to crystallization of 'latent' PAI-1 . Cleavage with restriction endonucleases of a cDNA clone encoding PAI-1 yielded an 1127 base pair fragment encoding residues 2-376 of the 379 amino acid serpin . Synthetic DNA linkers were ligated to the 5' and 3' ends of the subclone to add an initiation codon and restore the full coding sequence, and the resulting semisynthetic gene was incorporated into an expression plasmid, pPAIST-7, under the control of the E . coli trp promoter . Transformation of E . coli GE81 with pPAIST-7 led to expression of unglycosylated PAI-1 . Lysates of expression cultures contained PAI-1 activity and PAI-1 protein with the predicted Mr . Unglycosylated PAI-1 from E . coli exhibited characteristic properties of authentic PAI-1: (1) it was recovered in both active and inactive (latent) forms; (2) its activity declined during incubation at 37 degrees C; (3) latent PAI-1 was activated by treatment with 4 M guanidine hydrochloride; (4) reactivated PAI-1 formed a detergent-stable complex with tissue plasminogen activator . Latent PAI-1 accounted for more than 85% of PAI-1 in cell lysates and was purified by ammonium sulfate fractionation, anion-exchange chromatography and hydrophobic interaction chromatography . The purified latent PAI-1 was crystallized. J Chromatogr, 1990 Jan 19, 499, 153 - 63 High-performance adsorption chromatography of transfer ribonucleic acids and proteins on 2-microns spherical beads of hydroxyapatite . Influence of sodium chloride and magnesium ions on the resolution; Lindeberg J et al.; The influence of sodium chloride and magnesium chloride on the adsorption of tRNA and proteins on a high-performance liquid chromatographic column of 2-microns spherical hydroxyapatite beads was investigated . The resolution of 14C-labelled aminoacyl-tRNA isoacceptors was improved in the presence of sodium chloride . Inclusion of magnesium chloride in the buffers led to a separation of two tRNA species that could not be fractionated with or without sodium chloride in the eluting buffers (the original properties of the column were lost, however, and could not be regenerated by simply returning to magnesium chloride-free phosphate buffer) . Also, the adsorption of some proteins was affected when salt was included in the buffers . For instance, the elution order of proteins could be changed by choosing an appropriate concentration of sodium chloride . This finding might be utilized to facilitate the purification of certain proteins. Biochim Biophys Acta, 1990 Jan 19, 1037(1), 30 - 8 The sulfhydryl content of L-threonine dehydrogenase from Escherichia coli K-12: relation to catalytic activity and Mn2+ activation; Craig PA et al.; When oxidized to cysteic acid by performic acid or converted to carboxymethylcysteine by alkylation of the reduced enzyme with iodoacetate, a total of six half-cystine residues/subunit are found in L-threonine dehydrogenase (L-threonine: NAD+ oxidoreductase, EC 1.1.1.103; L-threonine + NAD(+)----2-amino-3-oxobutyrate + NADH) from Escherichia coli K-12 . Of this total, two exist in disulfide linkage, whereas four are titratable under denaturing conditions by dithiodipyridine, 5,5'-dithiobis(2-nitrobenzoic acid), or p-mercuribenzoate . The kinetics of enzyme inactivation and of modification by the latter two reagents indicate that threonine dehydrogenase has no free thiols that selectively react with bulky compounds . While incubation of the enzyme with a large excess of iodoacetamide causes less than 10% loss of activity, the native dehydrogenase is uniquely reactive with and completely inactivated by iodoacetate . The rate of carboxymethylation by iodoacetate of one -SH group/subunit is identical with the rate of inactivation and the carboxymethylated enzyme is no longer able to bind Mn2+ . NADH (0.5 mM) provides 40% protection against this inactivation; 60 to 70% protection is seen in the presence of saturating levels of NADH plus L-threonine . Such results coupled with an analysis of the kinetics of inactivation caused by iodoacetate are interpreted as indicating the inhibitor first forms a reversible complex with a positively charged moiety in or near the microenvironment of a reactive -SH group in the enzyme before irreversible alkylation occurs . Specific alkylation of one -SH group/enzyme subunit apparently causes protein conformational changes that entail a loss of catalytic activity and the ability to bind Mn2+. Biochim Biophys Acta, 1990 Jan 19, 1037(1), 24 - 9 2-Amino-3-ketobutyrate CoA ligase of Escherichia coli: stoichiometry of pyridoxal phosphate binding and location of the pyridoxyllysine peptide in the primary structure of the enzyme; Mukherjee JJ et al.; Pure 2-amino-3-ketobutyrate CoA ligase from Escherichia coli, which catalyzes the cleavage/condensation reaction between 2-amino-3-ketobutyrate (the presumed product of the L-threonine dehydrogenase-catalyzed reaction) and glycine + acetyl-CoA, is a dimeric enzyme (Mr = 84,000) that requires pyridoxal 5'-phosphate as coenzyme for catalytic activity . Reduction of the hololigase with tritiated NaBH4 yields an inactive, radioactive enzyme adduct; acid hydrolysis of this adduct allowed for the isolation and identification of epsilon-N-pyridoxyllysine . Quantitative determinations established that 2 mol of pyridoxal 5'-phosphate are bound per mol of dimeric enzyme . After the inactive, tritiated enzyme adduct was digested with trypsin, a single radioactive peptide containing 23 amino acids was isolated and found to have the following primary structure: Val-Asp-Ile-Ile-Thr-Gly-Thr-Leu-Gly-Lys*-Ala-Leu-Gly-Gly-Ala-Ser-Gly-Gly -Tyr-Thr-Ala-Ala-Arg (where * = the lysine residue in azomethine linkage with pyridoxal 5'-phosphate) . This peptide corresponds to residues 235-257 in the intact protein; 10 residues around the lysine residue have a high level of homology with a segment of the primary structure of 5-aminolevulinate synthase from chicken liver. Biochemistry, 1990 Jan 16, 29(2), 552 - 61 Chromophore function and interaction in Escherichia coli DNA photolyase: reconstitution of the apoenzyme with pterin and/or flavin derivatives; Jorns MS et al.; Native DNA photolyase, as isolated from Escherichia coli, contains a neutral flavin radical (FADH.) plus a pterin chromophore (5,10-methenyltetrahydropteroylpolyglutamate) and can be converted to its physiologically significant form by reduction of FADH . to fully reduced flavin (FADH2) with dithionite or by photoreduction . Either FADH2 or the pterin chromophore in dithionite-reduced native enzyme can function as a sensitizer in catalysis . Various enzyme forms (EFADox, EFADH., EFADH2, EPteFADox, EPteFADH., EPteFADH2, EPte) containing stoichiometric amounts of FAD in either of its three oxidation states and/or 5,10-methenyltetrahydrofolate (Pte) have been prepared in reconstitution experiments . Studies with EFADox and EPte showed that these preparations retained the ability to bind the missing chromophore . The results suggest that there could be considerable flexibility in the biological assembly of holoenzyme since the order of binding of the enzyme's chromophores is apparently unimportant, the binding of FAD is unaffected by its redox state, and enzyme preparations containing only one chromophore are reasonably stable . The same catalytic properties are observed with dithionite-reduced native enzyme or EFADH2 . These preparations do not exhibit a lag in catalytic assays whereas lags are observed with preparations containing FADox or FADH . in the presence or absence of pterin . Photochemical studies show that these lags can be attributed to enzyme activation under assay conditions in a reaction involving photoreduction of enzyme-bound FADox or FADH . to FADH2 . EPte is catalytically inactive, but catalytic activity is restored upon reconstitution of EPte with FADox . The results show that pterin is not required for dimer repair when FADH2 acts as the sensitizer but that FADH2 is required when dimer repair is initiated by excitation of the pterin chromophore . The relative intensity of pterin fluorescence in EPte, EPteFADH., EPteFADox, or EPteFADH2 has been used to estimate the efficiency of pterin singlet quenching by FADH . (93%), FADox (90%), or FADH2 (58%) . Energy transfer from the excited pterin to flavin is energetically feasible and may account for the observed quenching of pterin fluorescence and also explain why photoreduction of FADox or FADH . is accelerated by the pterin chromophore . An irreversible photobleaching of the pterin chromophore is accelerated by FADH2 in a reaction that is accompanied by a transient oxidation of FADH2 to FADH. . Both pterin bleaching and FADH2 oxidation are inhibited by substrate. Biochemistry, 1990 Jan 16, 29(2), 496 - 504 Mechanisms of mutagenesis by the vinyl chloride metabolite chloroacetaldehyde . Effect of gene-targeted in vitro adduction of M13 DNA on DNA template activity in vivo and in vitro; Jacobsen JS et al.; 2-Chloroacetaldehyde (CAA), a metabolite of the carcinogenic industrial chemical vinyl chloride, reacts with single-stranded DNA to form the cyclic etheno lesions predominantly at adenine and cytosine . In both ethenoadenine and ethenocytosine, normal Watson-Crick hydrogen-bonding atoms are compromised . We have recently shown that CAA adduction leads to efficient mutagenesis in Escherichia coli predominantly at cytosines, and less efficiently at adenines . About 80% of the mutations at cytosines were C-to-T transitions, and the remainder were C-to-A transversions, a result similar to that of many noninstructional DNA lesions opposite which adenine residues are preferentially incorporated . It is widely believed that noninstructional lesions stop replication and depend on SOS functions for efficient mutagenesis . We have examined the effects of in vitro CAA adduction of the lacZ alpha gene of phage M13AB28 on in vivo mutagenesis in SOS-(UV)-induced E . coli . CAA adduction was specifically directed to a part of the lacZ sequence within M13 replicative form DNA by a simple experimental strategy, and the DNA was transfected into appropriate unirradiated or UV-irradiated cells . Mutant progeny were defined by DNA sequencing . In parallel in vitro experiments, the effects of CAA adduction on DNA replication by E . coli DNA polymerase I large (Klenow) fragment were examined . Our data do not suggest a strong SOS dependence for mutagenesis at cytosine lesions . While adenine lesions remain much less mutagenic than cytosine lesions, mutation frequency at adenines is increased by SOS . SOS induction does not significantly alter the specificity of base changes at cytosines or adenines.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1990 Jan 16, 29(2), 442 - 51 Reaction mechanism of Escherichia coli cystathionine gamma-synthase: direct evidence for a pyridoxamine derivative of vinylglyoxylate as a key intermediate in pyridoxal phosphate dependent gamma-elimination and gamma-replacement reactions; Brzovic P et al.; Cystathionine gamma-synthase catalyzes a pyridoxal phosphate dependent synthesis of cystathionine from O-succinyl-L-homoserine (OSHS) and L-cysteine via a gamma-replacement reaction . In the absence of L-cysteine, OSHS undergoes an enzyme-catalyzed, gamma-elimination reaction to form succinate, alpha-ketobutyrate, and ammonia . Since elimination of the gamma-substituent is necessary for both reactions, it is reasonable to assume that the replacement and elimination reaction pathways diverge from a common intermediate . Previously, this partitioning intermediate has been assigned to a highly conjugated alpha-iminovinylglycine quininoid (Johnston et al., 1979a) . The experiments reported herein support an alternative assignment for the partitioning intermediate . We have examined the gamma-replacement and gamma-elimination reactions of cystathionine gamma-synthase via rapid-scanning stopped-flow and single-wavelength stopped-flow UV-visible spectroscopy . The gamma-elimination reaction is characterized by a rapid decrease in the amplitude of the enzyme internal aldimine spectral band at 422 nm with a concomitant appearance of a new species which absorbs in the 300-nm region . A 485-nm species subsequently accumulates in a much slower relaxation . The gamma-replacement reaction shows a red shift of the 422-nm peak to 425 nm which occurs in the experiment dead time (approximately 3 ms) . This relaxation is followed by a decrease in absorbance at 425 nm that is tightly coupled to the appearance of a species which absorbs in the 300-nm region . Reaction of the substrate analogues L-alanine and L-allylglycine with cystathionine gamma-synthase results in bleaching of the 422-nm absorbance and the appearance of a 300-nm species . In the absence of L-cysteine, L-allylglycine undergoes facile proton exchange; in the presence of L-cysteine, L-allylglycine undergoes a gamma-replacement reaction to form a new amino acid, gamma-methylcystathionine . No long-wavelength-absorbing species accumulate during either of these reactions . These results establish that the partitioning intermediate is an alpha-imino beta,gamma-unsaturated pyridoxamine derivative with lambda max congruent to 300 nm and that the 485-nm species which accumulates in the elimination reaction is not on the replacement pathway. Biochemistry, 1990 Jan 16, 29(2), 435 - 42 Purification and properties of cystathionine gamma-synthase from overproducing strains of Escherichia coli; Holbrook EL et al.; To characterize the methionine biosynthetic enzyme cystathionine gamma-synthase from Escherichia coli, we have constructed high copy number plasmids containing the metB structural gene but lacking the closely linked metJ regulatory gene . When cloned into an appropriate strain, these plasmids can direct the overproduction of cystathionine gamma-synthase such that about 10% of the soluble protein is this enzyme . An efficient purification scheme has been developed that has allowed us to obtain gram quantities of enzyme . The active form is a tetramer with subunits of about 40,000 daltons and one pyridoxal phosphate cofactor per monomer . The kinetic constants for several enzyme-catalyzed reactions were determined at 25 degrees C . The Km value for the elimination reaction with O-succinyl-L-homoserine was calculated to be 0.33 mM with maximal velocity of 460 min-1 . The Km for the elimination (deamination) reaction with vinylglycine was 5.6 mM with maximal velocity of 900 min-1 . The Km values for the replacement reaction were calculated to be 1.0 mM for O-succinyl-L-homoserine and 0.05 mM for L-cysteine with maximal velocity of 700 min-1 . The enzyme shows an absorption band at 422 nm (epsilon = 8463 M-1 cm-1) attributable to the Schiff base form of the pyridoxal phosphate cofactor . Steady-state spectra of reaction complexes show appearance of new longer wavelength absorbing materials during reaction with O-succinyl-L-homoserine, vinylglycine, or vinylglycine and L-cysteine . Reaction with O-succinyl-L-homoserine and L-cysteine produces only a red shift and slight reduction of the band at 422 nm. Biochemistry, 1990 Jan 16, 29(2), 389 - 402 Crystal structures of phosphonoacetamide ligated T and phosphonoacetamide and malonate ligated R states of aspartate carbamoyltransferase at 2.8-A resolution and neutral pH; Gouaux JE et al.; The T----R transition of the cooperative enzyme aspartate carbamoyltransferase occurs at pH 7 in single crystals without visibly cracking many of the crystals and leaving those uncracked suitable for single-crystal X-ray analysis . To promote the T----R transition, we employ the competitive inhibitors of carbamoyl phosphate and aspartate, which are phosphonoacetamide (PAM) and malonate, respectively . In response to PAM binding to the T-state crystals, residues Thr 53-Thr 55 and Pro 266-Pro 268 move to their R-state positions to bind to the phosphonate and amino group of PAM . These changes induce a conformation that can bind tightly the aspartate analogue malonate, which thereby effects the allosteric transition . We prove this by showing that PAM-ligated T-state crystals (Tpam), space group P321 (a = 122.2 A, c = 142.2 A), when transferred to a solution containing 20 mM PAM and 8 mM malonate at pH 7, isomerize to R-state crystals (Rpam,mal,soak), space group also P321 (a = 122.2 A, c = 156.4 A) . The R-state structure in which the T----R transition occurs within the crystal at pH 7 compares very well (rms = 0.19 A for all atoms) with an R-state structure determined at pH 7 in which the crystals were initially grown in a solution of PAM and malonate at pH 5.9 and subsequently transferred to a buffer containing the ligands at pH 7 (Rpam,mal,crys) . In fact, both of the PAM and malonate ligated R-state structures are very similar to both the carbamoyl phosphate and succinate or the N-(phosphonoacetyl)-L-aspartate ligated structures, even though the R-state structures reported here were determined at pH 7 . Crystallographic residuals refined to 0.16-0.18 at 2.8-A resolution for the three structures. Biochemistry, 1990 Jan 16, 29(2), 317 - 22 Resonance energy transfer study on the proximity relationship between the GTP binding site and the rifampicin binding site of Escherichia coli RNA polymerase; Kumar KP et al.; Terbium (III) upon complexation with guanosine 5'-triphosphate showed remarkable enhancement of fluorescence emission at 488 and 545 nm when excited at 295 nm . Analysis of the binding data yielded a value for the mean Kd between Tb(III) and GTP of 0.2 microM, with three binding sites for Tb(III) on GTP . 31P and 1H NMR measurements revealed that Tb(III) mainly binds the phosphate moiety of GTP . Fluorescence titration of the emission signals of the TbGTP complex with varying concentrations of Escherichia coli RNA polymerase resulted in a Kd value of 4 microM between the TbGTP and the enzyme . It was observed that TbGTP can be incorporated in the place of GTP during E . coli RNA polymerase catalyzed abortive synthesis of dinucleotide tetraphosphate at T7A2 promoter . Both the substrate TbGTP and the inhibitor of the initiation of transcription rifampicin bind to the beta-subunit of E . coli RNA polymerase . This allows the measurement of the fluorescence excited-state energy transfer from the donor TbGTP-RNA polymerase to the acceptor rifampicin . Both emission bands of Tb(III) overlap with the rifampicin absorption, and the distances at 50% efficiency of energy transfer were calculated to be 28 and 24 A for the 488- and 545-nm emission bands, respectively . The distance between the substrate binding site and the rifampicin binding site on the beta-subunit of E . coli RNA polymerase was measured to be around 30 A . This suggests that the nature of inhibition of transcription by rifampicin is essentially noncompetitive with the substrate. Biochemistry, 1990 Jan 16, 29(2), 366 - 72 Inhibition of Escherichia coli glutamine synthetase by alpha- and gamma-substituted phosphinothricins; Logusch EW et al.; We have investigated the inhibition of Escherichia coli glutamine synthetase (GS) with alpha- and gamma-substituted analogues of phosphinothricin {L-2-amino-4-(hydroxymethylphosphinyl)butanoic acid (PPT)}, a naturally occurring inhibitor of GS . These compounds display inhibition of bacterial GS that is competitive vs L-glutamate, with Ki values in the low micromolar range . At concentrations greater than Ki the phosphinothricins caused time-dependent loss of enzyme activity, while dilution after enzyme inactivation resulted in recovery of enzyme activity . ATP was required for inactivation; the nonhydrolyzable ATP analogue AMP-PCP failed to support inhibition of GS by the phosphinothricins . The binding of these inhibitors to the enzyme was also characterized by measurement of changes in protein fluorescence, which provided similar inactivation rate constants k1 and k2 for the entire series of compounds . Rate constants koff for recovery were also determined by fluorescence measurement and were comparable for both PPT and the gamma-hydroxylated analogue GHPPT and significantly greater for the alpha- and gamma-alkyl-substituted compounds . Electron paramagnetic resonance spectra provided information on the interaction of the phosphinothricins with the manganese form of the enzyme in the absence of ATP, and significant binding was observed for PPT and GHPPT . 31P NMR experiments confirmed that enzyme inactivation is accompanied by hydrolysis of ATP, although phosphorylated phosphinothricins could not be detected in solution . The kinetic behavior of these compounds is consistent with a mechanism involving inhibitor phosphorylation, followed by release from the active site and simultaneous hydrolysis to form Pi and free inhibitor. Biochem Biophys Res Commun, 1990 Jan 15, 166(1), 50 - 60 Expression of human parathyroid hormone in Escherichia coli; Hogset A et al.; Human parathyroid hormone (hPTH) is a peptide hormone consisting of 84 amino acids . Using the expression plasmid pKK223-3 with the strong tacpromoter, we have produced a variant of hPTH in E . coli . From the expression plasmid construct the expected product was hPTH with an N-terminal extension of Met-Gly . The peptide was extracted from E . coli cells and purified by high performance liquid chromatography . In two different gel electrophoresis systems including identification by immunoblotting the product behaved exactly as an hPTH standard . N-terminal amino acid sequence analysis of the purified product showed traces of Gly-hPTH . At least 90% of the expressed product was N-terminally blocked, suggesting the presence of N-formyl-methionine . This variant of hPTH did not stimulate adenylate cyclase activity in rat osteosarcoma cell membranes. Biochem J, 1990 Jan 15, 265(2), 337 - 42 Selective overexpression of the QUTE gene encoding catabolic 3-dehydroquinase in multicopy transformants of Aspergillus nidulans; Beri RK et al.; The three enzymes necessary to catabolize quinate to protocatechuate are inducible by quinic acid, and transcription of their corresponding genes is controlled by the action of a positively acting activator gene and a negatively acting repressor gene . Transformed strains of Aspergillus nidulans containing multiple copies of the activator gene (QUTA) but single copies of the other QUT genes retain normal regulation of the gene cluster and do not show any overexpression of the three quinic acid catabolic enzymes . Transformed strains containing equal multiple copies of the activator gene (QUTA) and QUTE (encoding catabolic 3-dehydroquinase), but single copies of the other QUT genes, retain normal regulation of the QUT gene cluster, but selectively overexpress the QUTE gene upon quinic acid induction . Data are presented that strongly suggested that the gene QUTG, which is physically located within the QUT gene cluster and for which no function has been identified, is not required for expression of the gene cluster and does not encode a chlorogenic acid esterase. FEBS Lett, 1990 Jan 15, 260(1), 6 - 9 PAPS-reductase of Escherichia coli . Correlating the N-terminal amino acid sequence with the DNA of gene cys H; Krone FA et al.; The DNA of the gene complementing a PAPS-reductase-deficient strain of Escherichia coli was sequenced . The N-terminal amino acid sequence of the purified PAPS-reductase confirmed that cys H is the structural gene for this enzyme . The open reading frame extends 732 bases and encodes for a peptide of Mr = 27927 . The gene product is functionally active when supplemented with thioredoxin and immunologically related with the wild type enzyme. FEBS Lett, 1990 Jan 15, 260(1), 127 - 30 Structure of the gene encoding concanavalin A from Canavalia gladiata and its expression in Escherichia coli cells; Yamauchi D et al.; We have cloned and sequenced the gene encoding concanavalin A (Con A) from Canavalia gladiata . The sequence covers the whole transcribed region as well as the 5'- and 3'-untranscribed sequences . The coding sequence lacks introns . Con A expressed in Escherichia coli cells was purified by Sephadex G-50 affinity chromatography . The precursor of Con A expressed in E . coli undergoes a peptide cleavage and ligation in the same way as that synthesized during seed maturation. J Biol Chem, 1990 Jan 15, 265(2), 838 - 43 Analysis of fibrinogen A alpha-fusion proteins . Mutants which inhibit thrombin equivalently are not equally good substrates; Lord ST et al.; We have examined the interaction of thrombin with fibrinogen A alpha chain residues 7-16 . Using genetically engineered constructions, we have synthesized in Escherichia coli a fibrinogen A alpha 1-50 fusion protein and seven mutant proteins with single amino acid substitutions . These are: Asp7----Ala, Phe8----Tyr, Glu11----Ala, Gly12----Val, Gly13----Val, Gly14----Val, and Arg16----Leu . Competitive immunoassay of cell lysates showed that all the mutations but one, Arg16----Leu, altered the structure of the protein such that cross-reactivity with the A alpha-specific monoclonal antibody, Y18, was significantly reduced . The fusion proteins were purified and analyzed as thrombin inhibitors and substrates . All the fusion proteins are competitive inhibitors of the amidolytic hydrolysis of Spectrozyme TH, a thrombin-specific chromogenic substrate, with inhibition constants corresponding to that for fibrinogen . We conclude that these 7 amino acid substitutions do not alter thrombin binding to the fusion proteins . The fusion proteins were tested as substrates by monitoring thrombin-dependent peptide release . The natural sequence and three mutants, Asp7----Ala, Glu11----Ala, and Gly14----Val, are good substrates . The other mutants are either poor substrates or are not cleaved by thrombin within A alpha 1-50 . These results indicate that residues between Asp7 and Arg16 are critical to efficient peptide hydrolysis, whereas residues outside this region are critical to thrombin binding. J Biol Chem, 1990 Jan 15, 265(2), 1199 - 207 Cloning, expression, purification, and biological activity of recombinant native and variant human alpha 1-antichymotrypsins; Rubin H et al.; Human alpha 1-antichymotrypsin has been cloned, sequenced and expressed in Escherichia coli and recombinant protein as well as point-specific mutants have been purified and characterized . The corrected gene-deduced amino acid sequence has 45% overall identity with alpha 1-protease inhibitor, which is higher than the 42% previously reported (Chandra, T., Stackhouse, R., Kidd, V . J., Robson, J . H., and Woo, S . L . C . (1983) Biochemistry 22, 5055-5060) . Recombinant antichymotrypsin (rACT) is similar to natural antichymotrypsin with respect to the specificity of its interactions with proteases . Its second-order rate constant for association with bovine chymotrypsin is 6-8 x 10(5) M-1 s-1, which is identical to that of the serum-derived inhibitor . Site-specific mutagenesis has been used to produce two variants of rACT in which the P1 position has been changed from leucine to either methionine (L358M-rACT) or arginine (L358R-rACT) . L358M-rACT has a specificity of inhibitory activity toward serine proteases closely similar to that of native rACT . By contrast, the specificity of L358R-rACT is quite different from that of native rACT, most notably in efficiently inhibiting trypsin and human thrombin while showing a decreased ability to inhibit chymotrypsin. J Biol Chem, 1990 Jan 15, 265(2), 1179 - 87 Total reconstitution of DNA polymerase III holoenzyme reveals dual accessory protein clamps; O'Donnell M et al.; DNA polymerase III holoenzyme (holoenzyme) is the 10-subunit replicase of the Escherichia coli chromosome . In this report, pure preparations of delta, delta', and a gamma chi psi complex are resolved from the five protein gamma complex subassembly . Using these subunits and other holoenzyme subunits isolated from overproducing plasmid strains of E . coli, the rapid and highly processive holoenzyme has been reconstituted from only five pure single subunits: alpha, epsilon, gamma, delta, and beta . The preceding report showed that of the three subunits in the core polymerase, only a complex of alpha (DNA polymerase) and epsilon (3'-5' exonuclease) are required to assemble a processive holoenzyme on a template containing a preinitiation complex (Studwell, P.S., and O'Donnell, M . (1990) J . Biol . Chem . 265, 1171-1178) . This report shows that of the five proteins in the gamma complex only a heterodimer of gamma and delta is required with the beta subunit to form the ATP-activated preinitiation complex with a primed template . Surprisingly, the delta' subunit does not form an active complex with gamma but forms a fully active heterodimer complex with the tau subunit (as does delta) . Hence, the tau delta' and gamma delta heterodimers are fully active in the preinitiation complex reaction with beta and primed DNA . Holoenzymes reconstituted using the alpha epsilon complex, beta subunit, and either gamma delta or tau delta' are fully processive in DNA synthesis, and upon completing the template they rapidly cycle to a new primed template endowed with a preinitiation complex clamp . Since the holoenzyme molecule contains all of these accessory subunits (gamma, delta, tau, delta', and beta) in all likelihood it has the capacity to form two preinitiation complex clamps simultaneously at two primer termini . Two primer binding components within one holoenzyme may mediate its rapid cycling to multiple primers on the lagging strand and also provides functional evidence for the hypothesis of holoenzyme as a dimeric polymerase capable of simultaneous replication of both leading and lagging strands of a replication fork. J Biol Chem, 1990 Jan 15, 265(2), 1000 - 4 Deletions in the large (beta) subunit of a hetero-oligomeric aminoacyl-tRNA synthetase; Toth MJ et al.; Glycyl-tRNA synthetase is one of two aminoacyl-tRNA synthetases in Escherichia coli that is comprised of heterologous subunits which are organized in an alpha 2 beta 2 quaternary structure . The two subunits are encoded by a single mRNA with the region for alpha (303 codons) subunit followed by that for beta (689 codons) subunit . Five COOH-terminal deletions in the beta subunit coding region have been created . Each deletion protein has been investigated for its synthesis and stability in vivo, adenylate synthesis activity in vitro, and aminoacylation activity in vivo and in vitro . This has been done in the presence of free alpha subunit and, additionally, with alpha subunit that is fused by its carboxyl terminus to the amino terminus of each of the beta subunit deletion proteins . With a fused or unfused alpha chain, over 100 amino acids can be deleted from the carboxyl terminus of the beta chain without loss of in vivo complementation of a delta glyS (deletion) strain . Further analysis shows that the alpha subunit and approximately the amino-terminal half of the beta subunit are sufficient for the adenylate synthesis activity . In particular, a deletion of 306 amino acids from the COOH terminus of the beta subunit has little effect on the Km parameter for ATP or glycine in the pyrophosphate exchange reaction . The tRNA-dependent step in aminoacylation requires additional beta subunit sequences on the COOH-terminal side of those needed for adenylate synthesis . In these respects, the functional organization of the beta chain parallels that of several aminoacyl-tRNA synthetases which have only homologous subunits . In the case of the glycine enzyme, however, the heterologous alpha subunit is required for the elucidation of activities encoded by functional determinants of the beta chain. Harefuah, 1990 Jan 15, 118(2), 57 - 61 {Treatment of acute myeloid leukemia in a special hematology unit}; Douer D et al.; Since 1984, 47 patients with untreated acute myeloid leukemia (AML) were hospitalized in a special hematology unit for aggressive chemotherapy . Complete remission was obtained in 68%, 15% died of complications of treatment (infections and bleeding) and 15% had refractory leukemia . The actuarial survival after 3 years for patients in remission was 43% . No patients with refractory leukemia lived more than 1 year . The actuarial remission at 3 years of 21 patients who received additional courses of aggressive chemotherapy (consolidation treatment) was 42%, as opposed to 11% in 11 patients who received maintenance treatment . The 47 patients received 108 courses of aggressive chemotherapy including 47 for induction of remission . During 86 courses (80%) the patients developed fever and in 33 blood cultures were positive; during 16 courses a fungal infection developed . The most common bacterial infection was by E . coli . During the first induction treatment 5 patients died of sepsis and 1 of cerebral hemorrhage . None died during consolidation therapy . During the year preceding the opening of the unit, 12 AML patients were treated on regular medical wards, and five (42%) achieved a complete remission, while 6 died of complications during the first course of induction chemotherapy . Our findings are in line with those of similar units, which indicates the importance of special nursing units for the treatment of acute leukemia. Clin Chim Acta, 1990 Jan 15, 186(2), 175 - 87 Structure of alkaline phosphatases; Kim EE et al.; The crystal structure of alkaline phosphatase (AP) from Escherichia coli, which is a prototype for mammalian APs, has been refined to a crystallographic R-factor of 0.184 at 2.0 A resolution . During the course of the refinement residues 380 to 410 were retraced and 190 to 200 were shifted by one residue, and substantial changes in the active site of the enzyme were made . Based on the refined structure and the sequences of mammalian enzymes (25-30% strict homology) we have modelled the core of the three dimensional structures of the mammalian alkaline phosphatases . Considerable circumstantial evidence suggests that this is valid despite the fact that the mammalian enzymes are larger, contain carbohydrate and are membrane associated through a phosphatidylinositol moiety . The active site of the molecule is highly conserved but specific changes in the secondary ligands to bound phosphate and the Mg metal are observed. J Biol Chem, 1990 Jan 15, 265(2), 1171 - 8 Processive replication is contingent on the exonuclease subunit of DNA polymerase III holoenzyme; Studwell PS et al.; In this report we have taken the reconstitution approach to study which subunits of the heterotrimer core polymerase (alpha, epsilon, theta) participate in the highly processive replication of long DNA templates by DNA polymerase III holoenzyme (holoenzyme) . Comparison of the core and the alpha epsilon complex (the DNA polymerase and 3'-5' exonuclease subunits, respectively) shows they are both rapid and highly processive polymerases when they are reconstituted into a holoenzyme with the gamma complex (gamma delta delta' chi psi) and beta accessory proteins of holoenzyme . Specifically, holoenzyme reconstituted using either core or alpha epsilon completely replicates a uniquely primed 5.4-kilobase (kb) single-stranded DNA within 12 s in one binding event . Hence the theta subunit of core is not required for the processivity or speed of the holoenzyme . In contrast, when only the alpha subunit is reconstituted into the holoenzyme it is unable to replicate the entire 5.4-kb circle in one binding event but still retains a fairly high processivity of 1-3 kb and when given sufficient time for multiple binding events it finally finishes the entire circle . Therefore, highly processive DNA synthesis by holoenzyme is contingent on the epsilon exonuclease subunit . In light of these results the significance of the polymerase and exonuclease activities residing in two separate polypeptides is discussed. J Biol Chem, 1990 Jan 15, 265(2), 1067 - 76 DNA and nucleotide-induced conformational changes in the Escherichia coli Rep and helicase II (UvrD) proteins; Chao K et al.; The domain structures of the Escherichia coli Rep and Helicase II proteins and their ligand-dependent conformational changes have been examined by monitoring the sensitivity of these helicases to proteolysis by trypsin and chymotrypsin . Limited treatment of unliganded Rep protein (73 kDa) with trypsin results in cleavage at a single site in its carboxyl-terminal region, producing a 68-kDa polypeptide which is stabilized in the presence of ATP, ADP, or adenosine 5'-O-thiotriphosphate) (ATP gamma S) . The purified 68-kDa Rep tryptic polypeptide retains single-stranded (ss) DNA binding, DNA unwinding (helicase), and full ATPase activities . When bound to ssDNA, the Rep protein can be cleaved by trypsin at an additional site in its carboxyl-terminal region, producing a 58-kDa polypeptide that also retains ssDNA binding and ATPase activities . This 58-kDa Rep tryptic polypeptide can also be produced by further tryptic treatment of the 68-kDa Rep tryptic polypeptide when the latter is bound to ssDNA . This 58-kDa polypeptide displays a lower affinity for ssDNA indicating that the 10-kDa carboxyl-terminal peptide facilitates Rep protein binding to ssDNA . The 58-kDa Rep tryptic polypeptide is also stabilized in the presence of nucleotides . Based on these and previous studies that showed that the 68-kDa Rep tryptic polypeptide cannot support DNA replication in a system that is dependent upon the phi X174 cis-A protein (Arai, N . & Kornberg, A . (1981) J . Biol . Chem . 256, 5294-5298), we conclude that the carboxyl-terminal end (approximately 5 kDa) of the Rep protein is not required for its helicase or ATPase activities . However, we suggest that this region of the Rep protein is important for its interactions with the phi X174 cis-A protein . Limited treatment of unliganded Helicase II protein (82 kDa) with chymotrypsin results in cleavage after Tyr254, producing a 29-kDa amino-terminal polypeptide and a 53-kDa carboxyl-terminal polypeptide, which remain associated under nondenaturing conditions . This chymotrypsin cleavage reduces the ssDNA binding activity and eliminates the ssDNA-dependent ATPase and helicase activities of the Helicase II protein . The binding of ATP, ADP, ATP gamma S, and/or DNA to Helicase II protein results in protection of this site (Tyr254) from cleavage by chymotrypsin . Limited treatment of Helicase II protein with trypsin results in cleavage near its carboxyl-terminal end producing two polypeptides with apparent Mr = 72,000, in a manner similar to that observed with the Rep protein; these polypeptides are also stabilized by binding ATP or single-stranded DNA.(ABSTRACT TRUNCATED AT 400 WORDS) Biochem Biophys Res Commun, 1990 Jan 15, 166(1), 431 - 5 Role of minor subunits in the structural asymmetry of the Escherichia coli F1-ATPase; Bragg PD et al.; The beta subunits of the Escherichia coli F1-ATPase react independently with chemical reagents (Stan-Lotter, H . and Bragg, P.D . (1986) Arch . Biochem . Biophys . 248, 116-120) . Thus, one beta subunit is readily crosslinked to the epsilon subunit, another reacts with N-N'-dicyclohexylcarbodiimide (DCCD), and a third one is modified by 4-chloro-7-nitrobenzofurazan (NbfCl) . This asymmetric behaviour is not due to the association of the delta and epsilon subunits of the ATPase molecule with specific beta subunits since it is maintained in a delta, epsilon-deficient form of the enzyme. FEBS Lett, 1990 Jan 15, 260(1), 31 - 4 The transmembrane topology of the a {corrected} subunit from the ATPase in Escherichia coli analyzed by PhoA protein fusions; Bjorbaek C et al.; The atpB encodes the a {corrected} subunit of the H(+)-ATPase of E . coli . The topology of this membrane protein has been analyzed by PhoA fusions . The results support an