Proc Natl Acad Sci U S A, 1987 Jun, 84(12), 4007 - 11 cDNA and deduced amino acid sequence of human pulmonary surfactant-associated proteolipid SPL(Phe); Glasser SW et al.; Hydrophobic surfactant-associated protein of Mr 6000-14,000 was isolated from ether/ethanol or chloroform/methanol extracts of mammalian pulmonary surfactant . Automated Edman degradation in a gas-phase sequencer showed the major N-terminus of the human low molecular weight protein to be Phe-Pro-Ile-Pro-Leu-Pro-Tyr-Cys-Trp-Leu-Cys-Arg-Ala-Leu- . Because of the N-terminal phenylalanine, the surfactant protein was designated SPL(Phe) . Antiserum generated against hydrophobic surfactant protein(s) from bovine pulmonary surfactant recognized protein of Mr 6000-14,000 in immunoblot analysis and was used to screen a lambda gt11 expression library constructed from adult human lung poly(A)+ RNA . This resulted in identification of a 1.4-kilobase cDNA clone that was shown to encode the N-terminus of the surfactant polypeptide SPL(Phe) (Phe-Pro-Ile-Pro-Leu-Pro-) within an open reading frame for a larger protein . Expression of a fused beta-galactosidase-SPL(Phe) gene in Escherichia coli yielded an immunoreactive Mr 34,000 fusion peptide . Hybrid-arrested translation with this cDNA and immunoprecipitation of {35S}methionine-labeled in vitro translation products of human poly(A)+ RNA with a surfactant polyclonal antibody resulted in identification of a Mr 40,000 precursor protein . Blot hybridization analysis of electrophoretically fractionated RNA from human lung detected a 2.0-kilobase RNA that was more abundant in adult lung than in fetal lung . The larger RNA and translation product indicates that SPL(Phe) is derived by proteolysis of a large polypeptide precursor . The amino acid sequence of the predicted protein, beginning Phe-Pro-Ile-Pro-Leu-Pro-Try-, comprises a hydrophobic peptide that is a major protein component of surfactant lipid extracts used successfully to treat hyaline membrane disease in newborn infants . These proteins, and specifically SPL(Phe), may therefore be useful for synthesis of replacement surfactants for treatment of hyaline membrane disease in newborn infants or of other surfactant-deficient states. Proc Natl Acad Sci U S A, 1987 Jun, 84(12), 4002 - 6 Poliovirus polypeptide precursors: expression in vitro and processing by exogenous 3C and 2A proteinases; Nicklin MJ et al.; Plasmids have been constructed to generate substrates for the study of proteinases 2A and 3C of poliovirus . They contain the P1 (capsomer precursor) region of the poliovirus genome or P1 and part of P2 (a nonstructural precursor), which can be transcribed and translated in vitro . A transcript containing the entire 5' nontranslated region and the P1 region of the viral RNA gave poor translation in a reticulocyte translation system . Truncation of the 5' nontranslated region to its 3'-most segment gave acceptably good yields of radiolabeled P1 . P1 was specifically processed to yield capsomer proteins by enzymes supplied in a postmitochondrial supernatant from poliovirus-infected cells . Thus, proteinase 3C can be supplied exogenously (in trans) and effect processing . This system may be used to provide P1 for the assay of proteinase 3C . Precursors that lacked either the 1A or 1D regions were poor substrates for proteinase 3C--observations that demonstrated a stringent structural requirement in processing by 3C . The translation product of a transcript encoding P1 and part of P2 was rapidly cleaved at the P1-P2 site in the absence of infected-cell extract . A transcript that contained a mutated 2A region gave a stable P1-P2 precursor that could be processed specifically by exogenous proteinase from infected-cell fractions . Processing of P1 appeared to require cleavage of the P1-P2 bond . These results support our previous data that 2A is the second polioviral proteinase and also provides a means of assaying proteinase 2A in vitro. Proc Natl Acad Sci U S A, 1987 Jun, 84(11), 3901 - 5 The narL gene product activates the nitrate reductase operon and represses the fumarate reductase and trimethylamine N-oxide reductase operons in Escherichia coli; Iuchi S et al.; Escherichia coli, which can utilize O2, nitrate, fumarate, or trimethylamine N-oxide (Me3NO) as terminal electron acceptor, preferentially utilizes the one with the highest redox potential . Thus O2 prevents induction of nitrate, fumarate, and Me3NO reductases, and nitrate curtails the induction of fumarate and Me3NO reductases . Under anaerobic conditions the narL gene product, in the presence of nitrate, is known to activate transcription of the narC operon, which encodes nitrate reductase . This study shows that the same product plays a role in the repression by nitrate of the operons (frd and tor) that encode fumarate and Me3NO reductases . In contrast, the anaerobic repression of ethanol dehydrogenase by nitrate does not require the narL product . Expression of narL does not require the fnr gene product, a pleiotropic activator that is required for full expression of narC, frd, and tor. Proc Natl Acad Sci U S A, 1987 Jun, 84(11), 3668 - 72 Replication of mini-P1 plasmid DNA in vitro requires two initiation proteins, encoded by the repA gene of phage P1 and the dnaA gene of Escherichia coli; Wickner SH et al.; We have developed an in vitro DNA-replication system that replicates exogenously added mini-P1 plasmid DNA . The system consists of purified P1 RepA protein and a partially purified mixture of Escherichia coli replication proteins . It is essentially the same as that described for the replication of oriC plasmid DNA {Fuller, R.S., Kaguni, J.M . & Kornberg, A . (1981) Proc . Natl . Acad . Sci . USA 78, 7370-7374} . Mini-P1 DNA replication requires the E . coli DnaA initiation protein in addition to the P1 RepA initiation protein . The reaction is inhibited by rifampicin, novobiocin, and antibody to DnaB, suggesting the involvement of RNA polymerase, DNA gyrase, and DnaB protein . Replication is initiated in the region of the P1 origin of replication and proceeds unidirectionally as determined by electron microscopy . Thus, the in vitro system mimics the essential features of mini-P1 replication as suggested by genetic studies. Proc Natl Acad Sci U S A, 1987 Jun, 84(11), 3643 - 7 Initiation of simian virus 40 DNA replication in vitro: large-tumor-antigen- and origin-dependent unwinding of the template; Wold MS et al.; Analysis of the kinetics of simian virus 40 (SV40) DNA replication in vitro demonstrated the existence of a slow presynthesis reaction that occurs prior to onset of extensive chain elongation and is dependent on a subset of the cellular proteins required for the complete replication reaction . When the presynthesis reaction is carried out in the presence of topoisomerase I, it is possible to detect extensive unwinding of the template DNA . This unwinding reaction is specific for templates that contain the wild-type SV40 origin of DNA replication and requires SV40 large tumor antigen (T antigen), ATP, and a protein fraction derived from HeLa cells . The required cellular protein may be a eukaryotic single-stranded-DNA-binding protein (SSB), since unwinding of the template is also observed when Escherichia coli SSB is substituted for the HeLa protein fraction . These observations suggest that during the initial stages of SV40 DNA replication, T antigen binds specifically to the viral origin and locally unwinds the DNA . This origin-dependent unwinding reaction is presumably a prerequisite for subsequent priming and elongation steps. Proc Natl Acad Sci U S A, 1987 Jun, 84(11), 3638 - 42 Helicase properties of the Escherichia coli UvrAB protein complex; Oh EY et al.; The Escherichia coli UvrA protein has an associated ATPase activity with a turnover number affected by the presence of UvrB protein as well as by DNA . Specifically, the structure of DNA significantly influences the turnover rate of the UvrAB ATPase activity . Double-stranded DNA maximally activates the turnover rate 10-fold whereas single-stranded DNA maximally activates the turnover rate 20-fold, suggesting that the mode of interaction of UvrAB protein with different DNAs is distinctive . We have previously shown that the UvrAB protein complex, driven by the binding energy of ATP, can locally unwind supercoiled DNA . The nature of the DNA unwinding activity and single-stranded DNA activation of ATPase activity suggests potential helicase activity . In the presence of a number of helicase substrates, the UvrAB complex, indeed, manifests a strand-displacement activity--unwinding short duplexes and D-loop DNA, thereby generating component DNA structures . The energy for the activity is derived from ATP or dATP hydrolysis . Unlike the E . coli DnaB, the UvrAB helicase is sensitive to UV-induced photoproducts. Proc Natl Acad Sci U S A, 1987 Jun, 84(11), 3560 - 4 Nucleoside triphosphate-dependent DNA-binding properties of mos protein; Seth A et al.; We have previously shown that the mos gene product, p40mos, produced in Escherichia coli binds ATP and has ATPase activity . In the present study, we investigated the DNA-binding properties of p40mos and two mos deletion mutant proteins . Nitrocellulose blot protein-DNA binding assays showed that p40mos binds DNA in the presence of Mg2+-ATP and certain other nucleoside triphosphates . Ninety percent of the p40mos-bound DNA is dissociated if the complex is washed in the presence of 1 M NaCl or in the absence of ATP . p40mos-DNA binding is not observed in the presence of AMP or the nonhydrolyzable ATP analog adenosine 5'-{beta, gamma-methylene}-triphosphate; however, in the presence of ADP, p40mos binds DNA at 20% of the level that is observed with ATP . An N-terminal-deletion mutant protein, p19mos, has no DNA-binding activity, whereas a C-terminal-deletion mutant protein, p25mos, does . p25mos contains the ATP-binding domain, binds DNA in the presence of either ADP or ATP, and shows 5% and 45% binding (relative to that in the presence of ATP) in the presence of AMP and adenosine 5'-{beta, gamma-methylene}triphosphate, respectively . These results suggest that the N-terminal domain of p40mos is responsible for nucleoside triphosphate-mediated DNA binding . We also observed differential histone-DNA binding in the presence and absence of ATP. J Bacteriol, 1987 Jun, 169(6), 2650 - 8 Mode of initiation of constitutive stable DNA replication in RNase H-defective mutants of Escherichia coli K-12; von Meyenburg K et al.; The alternative pathway of DNA replication in rnh mutants of Escherichia coli can be continuously initiated in the presence of chloramphenicol, giving rise to constitutive stable DNA replication (cSDR) . We conducted a physiological analysis of cSDR in rnh-224 mutants in the presence or absence of the normal DNA replication system . The following results were obtained . cSDR allowed the cells to grow in the absence of the normal replication system at a 30 to 40% reduced growth rate and with an approximately twofold-decreased DNA content . cSDR initiation was random with respect to time in the cell cycle as well as choice of origins . cSDR initiation continued to increase exponentially for more than one doubling time when protein synthesis was inhibited by chloramphenicol . cSDR initiation was inhibited during amino acid starvation in stringent (relA+) but not in relaxed (relA1) strains, indicating its sensitivity to ppGpp . cSDR initiation was rifampin sensitive, demonstrating that RNA polymerase was involved . cSDR functioned in dnaA+ rnh-224 strains parallel to the normal oriC+ dnaA+-dependent chromosome replication system. J Bacteriol, 1987 Jun, 169(6), 2639 - 42 Identification, mapping, and cloning of the gene encoding cyanase in Escherichia coli K-12; Sung YC et al.; The gene in Escherichia coli for cyanase, designated cynS, was localized to a BglII restriction site approximately 1.7 kilobases from the lacA end of the lac operon . The gene was cloned into the pUC13 vector . Maxicell analysis of plasmid-encoded proteins confirmed that the BglII site is in the region encoding the structural gene for cyanase . Cyanase-deficient strains had increased sensitivity to cyanate and were not able to use cyanate as a nitrogen source. J Bacteriol, 1987 Jun, 169(6), 2516 - 22 Role of oxygen radicals in the phototoxicity of tetracyclines toward Escherichia coli B; Martin JP Jr et al.; Photoillumination of tetracycline derivatives with low-intensity (320- to 400-nm) light and visible light generated superoxide, observed as the reduction of ferricytochrome c . The rate of reduction was dependent on the tetracycline concentration and on the derivative being examined, with doxycycline greater than or equal to demeclocycline greater than tetracycline greater than oxytetracycline . Tetracycline-mediated cytochrome c reduction was oxygen dependent and inhibited up to 70% by superoxide dismutase . Illuminated tetracyclines were lethal to Escherichia coli B incubated in a glucose minimal medium containing chloramphenicol . This lethality was light dependent, oxygen dependent, and dependent on the concentration of tetracycline . Kill rates also varied according to the derivative under study, with doxycycline greater than or equal to demeclocycline greater than tetracycline greater than oxytetracycline . The addition of superoxide dismutase and catalase to the incubation medium partially protected E . coli B against the light-dependent lethality . Preinduction of intracellular superoxide dismutase and catalase substantially protected E . coli B against the phototoxicity of tetracyclines . Iron EDTA augmented the phototoxicity of tetracyclines, while diethylenetriaminepentaacetic acid protected against their lethality . Hydroxyl radical scavengers also conferred protection against tetracycline phototoxicity . The extent of protection was in order of the in vitro reactivity of the scavengers with the hydroxyl radical . These results indicate that superoxide, hydrogen peroxide, and the hydroxyl radical are generated by illuminated tetracyclines and are molecular agents of tetracycline phototoxicity in E . coli B. Inflammation, 1987 Jun, 11(2), 131 - 42 Influence of neutrophil cationic proteins on generation of superoxide by human polymorphonuclear cells during phagocytosis; Alam M et al.; The cationic proteins from neutrophil lysosomes have been shown to modulate phagocytic activity of granulocytes . The present study reports the effects of the cationic protein fractions on the generation of O2- by human PMNs during phagocytosis . Human PMNs were reacted with different phagocytic stimuli in the presence and absence of lysosomal cationic proteins and the amount of O2- generated was determined by superoxide dismutase inhibitable reduction of cytochrome c . Total cationic protein extract from neutrophil lysosomes enhanced O2- generated by PMNs during the phagocytosis of IgG-coated latex beads and opsonized zymosan particles . The analysis of the fractions of cationic proteins obtained from a Sephadex G-75 column showed that the O2- generation-enhancing activity was associated with the proteins eluted in fractions III and IV . A protein fraction mainly eluted in void volume inhibited the cytochrome c reduction by O2- formed during phagocytosis . This was due to the presence of superoxide dismutase-like activity since O2- generated by the xanthine-xanthine oxidase system was also inhibited by this fraction . The cationic protein fractions III and IV from the Sephadex G-75 column were further subfractionated . Although the O2(-)-enhancing activity was eluted in the same fractions as chymotrypsin activity, there was no quantitative correlation between the amount of O2- generation and chymotrypsin activity . Moreover, commercial chymotrypsin did not enhance O2- generation . Electrophoretic analysis of the isolated protein fractions suggests that O2- generation enhancing protein (SGEP) is different from lysozyme or chymotrypsin and probably represents previously undescribed protein. FEBS Lett, 1987 Jun 1, 216(2), 207 - 10 In situ distribution of EcoRI methylase and restriction endonuclease in cells of Escherichia coli Bs 5; Kohring GW et al.; Specific IgG antibodies were raised in rabbits against purified EcoRI methylase and restriction endonuclease . Post embedding labeling experiments, using the protein A-gold technique, were made with paraformaldehyde-glutaraldehyde fixed cells, embedded in Lowicryl K4M resin at low temperatures . Labeling with methylase-specific antibodies showed 60-70% of gold particles in the cytoplasm and 30-40% at the cell envelope, whereas the use of restriction enzyme-specific antibodies led to a distribution of 10-30% in the cytoplasm and 70-90% in the cell envelope . The results coincide with the proposed function of the enzymes: in the cytoplasm methylase protects the cells' own DNA from self-destruction, and the restriction endonuclease cuts foreign DNA when entering the cell. Exp Parasitol, 1987 Jun, 63(3), 288 - 94 Taenia hydatigena: isolation of mitochondrial DNA, molecular cloning, and physical mitochondrial genome mapping; Yap KW et al.; Mitochondrial DNA was isolated from Taenia hydatigena, T . crassiceps, and Echinococcus granulosus using a cetyltrimethylammonium bromide precipitation technique . The technique is simple, rapid, reproducible, and does not require extensive high speed ultracentrifugation . The advantage of using mitochondrial DNA from taeniid cestodes for comparative restriction analysis was demonstrated . Mitochondrial DNA of T . hydatigena was isolated as covalently closed circular molecules . These were linearized by single digestion with BamHI and the molecular weight was estimated from the linear form of 17.6 kb . The mitochondrial DNA of T . hydatigena is therefore similar in size and structure to that of many other animal species . The entire mitochondrial genome was cloned into pBR322 in Escherichia coli and a restriction map of the recombinant molecule was constructed . The potential of using the cloned mitochondrial genome as a probe in speciation studies as well as for providing functional information on the role of the cestode mitochondrion is discussed. J Virol, 1987 Jun, 61(6), 1957 - 63 Efficient resolution of replicated poxvirus telomeres to native hairpin structures requires two inverted symmetrical copies of a core target DNA sequence; DeLange AM et al.; The terminal hairpin sequences of the linear double-stranded DNA genome of the leporipoxvirus Shope fibroma virus (SFV) has been cloned in Saccharomyces cerevisiae and in recombination-deficient Escherichia coli as a palindromic insert within circular plasmid vectors . This sequence configuration is equivalent to the inverted repeat structure detected as a telomeric replicative intermediate during poxvirus replication in vivo . Previously, it has been shown that when circular plasmids containing this palindromic insert were transfected into SFV-infected cells, efficient replication and resolution generated linear minichromosomes with bona fide viral hairpin termini (A . M . DeLange, M . Reddy, D . Scraba, C . Upton, and G . McFadden, J . Virol . 59:249-259, 1986) . To localize the minimal target DNA sequence required for efficient resolution, a series of staggered unidirectional deletions were constructed at both ends of the inverted repeat . Analyses of the resolution efficiencies of the various clones indicate that up to 240 base pairs (bp) centered at the symmetry axis were required for maximal resolution to minichromosomes . To investigate the role of the AT-rich central axis sequences, which in SFV include 8 nonpalindromic bp, a unique AflII site at the symmetry axis was exploited . Bidirectional deletions extending from this AflII site and insertions of synthetic oligonucleotides into one of the deletion derivatives were constructed and tested in vivo . The efficiency with which these plasmids resolved to linear minichromosomes with hairpin termini has enabled us to define the minimal target DNA sequence as two inverted copies of an identical DNA sequence between 58 and 76 bp in length . The nonpalindromic nucleotides, which, after resolution, constitute the extrahelical residues characteristic of native poxviral telomeres, were not required for resolution . The close resemblance of the SFV core target sequence to the analogous region from the orthopoxvirus vaccinia virus is consistent with a conserved mechanism for poxviral telomere resolution. J Virol, 1987 Jun, 61(6), 1855 - 60 Binding of the pseudorabies virus immediate-early protein to single-stranded DNA; Chlan CA et al.; In an attempt to correlate the ability to activate transcription with affinity for single-stranded DNA, both wild-type and temperature-sensitive pseudorabies virus immediate-early proteins were tested for the ability to bind to single-stranded DNA columns . Wild-type and temperature-sensitive immediate-early proteins bound to nonspecific single-stranded DNA columns with similar affinities at both 0 and 40 degrees C . There did not seem to be a direct correlation between the ability to activate transcription and the ability to bind to single-stranded DNA . To study further the interactions that are involved in binding to single-stranded DNA, we expressed the immediate-early protein in an Escherichia coli expression vector . In this system the expressed immediate-early protein was not phosphorylated, nor could it be complexed with mammalian cell factors . The first trp construct did not express a soluble form of the immediate-early protein, presumably due to the insoluble nature of the trp leader . We deleted a large segment of the trpE gene and found that the immediate-early fusion protein was soluble . We tested this protein for its affinity for single-stranded DNA by passage over single-stranded DNA cellulose columns . The bacterially expressed immediate-early protein bound single-stranded DNA at least as well as did the wild-type protein . Affinity for single-stranded DNA did not appear to be dependent on the phosphorylation state nor on the presence of mammalian cell factors. J Virol, 1987 Jun, 61(6), 1821 - 7 Cellular mutation mediates T-antigen-positive revertant cells resistant to simian virus 40 transformation but not to retransformation by polyomavirus and adenovirus type 2; Bauer M et al.; T-antigen-positive transformation revertant cell lines were isolated from fully simian virus 40 (SV40)-transformed Fisher rat embryo fibroblast cells (REF 52 cells) by methionine starvation . Reversion of the transformed cells (SV-52 cells) was caused by a mutation within the cellular genome . To demonstrate this, we isolated SV40 DNA from the host genome, inserted it into plasmid pSPT18 DNA, cloned it in Escherichia coli, and microinjected it into the nuclei of the REF 52 cells . Fully transformed cells were obtained with the same efficiency (20 to 25%) as after microinjection of wild-type SV40 DNA I . Furthermore, the revertant cells were resistant to retransformation by SV40 . Following microinjection of wild-type SV40 DNA I, 42 independent cell lines were isolated . Cells of all analyzed lines acquired additional SV40 DNA copies, but changes in the cell morphology or growth characteristic were not demonstrable . However, the revertants were retransformable with a high efficiency after polyomavirus and adenovirus type 2 infections or microinjection . Also, fusion of the revertant cells with the grandparental REF 52 cells led to restoration of the transformed state. Anal Biochem, 1987 Jun, 163(2), 398 - 407 Use of conversion adaptors to clone antigen genes in lambda gt11; Stover CK et al.; A strategy has been devised and tested to employ EcoRI conversion adaptors for cloning 5' cohesive-ended restriction fragments into the unique EcoRI site of the lambda gt11 expression vector . Five lambda gt11 chromosomal libraries were constructed with Rickettsia tsutsugamushi genomic DNA digested with restriction enzymes generating five different 5' cohesive ends . Recombinant phage yields as high as 10(7) plaque forming units were achieved without amplification of the five libraries . Sequences encoding epitopes of all eight R . tsutsugamushi polypeptide antigens, previously identified by Western blot analysis, were obtained in the five lambda gt11 expression libraries . Recombinant antigen expression was dependent on lambda gt11 lac promoter induction in 39% of the recombinants assayed . This method significantly improves the efficiency of genomic lambda gt11 library construction by eliminating blunt-ended ligation and simplifying the removal of unligated EcoRI-ended oligonucleotides. Mol Gen Genet, 1987 Jun, 208(1-2), 10 - 4 Partial purification of an activity from human cells that promotes homologous pairing and the formation of heteroduplex DNA in the presence of ATP; Cassuto E et al.; An activity that can promote homologous pairing and strand transfer between suitable DNA substrates has been partially purified from human skin fibroblasts and from HeLa cells . The strand transfer reaction was investigated with DNA substrates consisting of single-stranded circular and duplex linear phage DNA . It requires ATP, and under optimal conditions yields heteroduplex molecules containing one strand from each parental DNA substrate . The reactions appears to be of the same general nature as those mediated by RecA proteins of Escherichia coli and the Rec1 protein of Ustilago maydis. Can Assoc Radiol J, 1987 Jun, 38(2), 129 - 30 Pyogenic spondylodiscitis without disc space collapse; Fontaine S et al.; An unusual patient with pyogenic spondylodiscitis and preservation of the disc space anteriorly is reported . The diagnosis of pyogenic spondylodiscitis may be difficult when there is underlying spinal disease . We emphasize the need for early systematic imaging studies and intervention in such patients, including careful evaluation of vertebral endplates on plain films, early use of radionuclide studies, and prompt needle aspiration. Virology, 1987 Jun, 158(2), 294 - 9 Reinitiation at the lambda DNA origin accompanies the host SOS response; Schnos M et al.; Abnormal reinitiation of replication from lambda origins has previously been found during infection in the presence of caffeine or cis-diamminedichloroplatinum II (cis-Pt) or when lambda infects a P2 lysogen . It was further shown that the reinitiations arising from cis-Pt treatment took place during the SOS response induced by the template damage caused by the drug . It is now shown that SOS induction by uv irradiation of the host also results in reinitiation events and that it is the SOS response itself rather than some other direct effect of the damaged host template that is responsible for the phenomenon . Parental sections of lambda replicative intermediates can supercoil, whereas daughter segments cannot . To explain the control that prevents reinitiation, it is proposed that normally the origin sequence has to be under superhelical tension to be a suitable substrate for the initiating machinery; once a round is in progress, the daughter origin sequences would not be under such tension and would therefore be inactive . It is shown that in an SOS environment the proposed requirement for a superhelical origin sequence is relaxed and consequently the control against reinitiation lost . Under such conditions, primary growing points that have encountered template lesions terminate and a new wave of replication initiates. Proc Natl Acad Sci U S A, 1987 Jun, 84(12), 4022 - 5 AUU-to-AUG mutation in the initiator codon of the translation initiation factor IF3 abolishes translational autocontrol of its own gene (infC) in vivo; Butler JS et al.; We previously showed that Escherichia coli translation initiation factor IF3 regulates the expression of its own gene infC at the translational level in vivo . Here we create two alterations in the infC gene and test their effects on translational autocontrol of infC expression in vivo by measuring beta-galactosidase activity expressed from infC-lacZ gene fusions under conditions of up to 4-fold derepression or 3-fold repression of infC expression . Replacement of the infC promoter with the trp promoter deletes 120 nucleotides of the infC mRNA 5' to the translation initiation site without affecting autogenous translational control . Mutation of the unusual AUU initiator codon of infC to the more common AUG initiator codon abolishes translation initiation factor IF3-dependent repression and derepression of infC expression in vivo . These results establish the AUU initiator codon of infC as an essential cis-acting element in autogenous translational control of translation initiation factor IF3 expression in vivo. Mutat Res, 1987 Jun, 178(2), 157 - 65 Relative mutagenic and prophage-inducing effects of mono- and di-alkyl nitrosoureas; Lijinsky W et al.; Mutagenic capacity, prophage-inducing ability, and decomposition rates of mono- and di-alkylnitrosoureas with the same alkyl groups were studied in aqueous systems using S . typhimurium strain TA1535 and E . coli strain BR339 (lambda) . Slower decomposition rates of nitrosodialkylureas compared with monoalkylnitrosoureas were not reflected in reduced mutagenicity, with the exception of the methylating agents . With the monoalkylnitrosoureas, mutagenicity varied with length of the alkyl chain, and was greatly increased by oxygen substituents . Among the dialkylnitrosoureas, mutagenesis and decomposition rates were dependent on the substitutent in the N-1 position, adjacent to the nitroso group . Prophage induction, on the other hand, was dependent on a substituent in the N-3 position . The results suggested the existence of two distinct mechanisms for DNA damage, one SOS-dependent and the other SOS-independent, by which different dialkylnitrosoureas acted . Neither in vitro assay system was a reasonable model for the carcinogenic activity of these compounds. Clin Immunol Immunopathol, 1987 Jun, 43(3), 289 - 300 Eosinophils of human colonic mucosa: C3b and Fc gamma receptor expression and phagocytic capabilities; Beeken WL et al.; Because little is known about eosinophils of the human intestine, we measured their C3b and Fc gamma receptor expression and phagocytic activity in mucosal suspensions from colon resections for large bowel neoplasms . Enzymatically dissociated suspensions were enriched for eosinophils by countercurrent centrifugation . C3b and Fc gamma receptors were measured by immunofluorescent assays with flow cytometry . Phagocytosis of Escherichia coli ON2 was determined by an in vitro microscopic method . Suspensions of normal tissue from neoplasm resections yielded 1.8 X 10(6) eosinophils/g mucosa, and these cells were more numerous than either macrophages or neutrophils . Fivefold enrichment was achieved by countercurrent centrifugation, and 75% of these cells expressed C3b receptors and 90% expressed Fc gamma receptors . Sixty-seven percent of mucosal eosinophils were phagocytic for E . coli ON2 and ingested a mean of 4.7 bacteria per cell . Eosinophils accounted for more overall phagocytic activity than either neutrophils or macrophages. EMBO J, 1987 Jun, 6(6), 1805 - 8 The role of lysine-132 and arginine-136 in the receptor-binding domain of the K99 fibrillar subunit; Jacobs AA et al.; The gene encoding the K99 fibrillar adhesin of Escherichia coli has been modified by oligonucleotide-directed, site-specific, mutagenesis . The tryptophan-67, lysine-132, lysine-133 or arginine-136 were replaced by leucine, threonine, threonine and serine, respectively . The threonine-133 mutant fibrillae were indistinguishable from wild-type fibrillae . In contrast, replacement of lysine-132 or arginine-136 by threonine or serine, respectively, resulted in mutant fibrillae which had completely lost adhesive capacity, suggesting that the positive charges of these residues are essential for the interaction with the negatively charged sialic acid residue of the receptor molecules . After the replacement of tryptophan-67 with leucine neither fibrillae nor subunits were detectable, indicating that the mutant product is unstable and that tryptophan-67 has an essential structural role in the K99 subunit. Pediatr Res, 1987 Jun, 21(6), 551 - 5 Small and large intestinal guanylate cyclase activity in children: effect of age and stimulation by Escherichia coli heat-stable enterotoxin; Guarino A et al.; Heat-stable enterotoxin (ST) producing Escherichia coli are a common cause of diarrhea in infants . ST acts through the stimulation of the guanylate cyclase-cGMP system . The effect of ST on the human intestine has not been investigated nor is any information available on the activity, distribution, or development of guanylate cyclase activity in the human intestine . The purpose of this study, therefore, was to characterize, these aspects of guanylate cyclase activity and to study the effect of ST on the activity and responsiveness of guanylate cyclase in the intestine of infants and children of various ages . We measured guanylate cyclase activity in 35 intestinal specimens, obtained operatively, from children aged 1 day to 16 yr . Guanylate cyclase activity was linear with protein concentration and time . Basal activity was similar in small intestine and in colon . In the small intestine, however, basal guanylate cyclase activity varied with age . It was maximal in children 1 day of age, and although somewhat variable, decreased with age thereafter . In colon, an age-related pattern was not found . E . coli ST stimulated guanylate cyclase activity in all specimens in a dose-related manner . In the small intestine ST-stimulation of guanylate cyclase was twice that found in colon . Furthermore, age affected the response of small intestinal guanylate cyclase to ST . Maximal response to ST was observed in children 1 day of age and ST stimulation was significantly greater in children less than 1 yr of age than in older children . In the colon, the response of guanylate cyclase to ST did not change with age.(ABSTRACT TRUNCATED AT 250 WORDS) J Clin Microbiol, 1987 Jun, 25(6), 1048 - 51 Multiplicity of serogroups and adhesins in enteropathogenic and enterotoxigenic Escherichia coli isolated from acute diarrhea in Senegal; Darfeuille-Michaud A et al.; Escherichia coli strains were isolated from 228 children with diarrhea in Senegal from 1982 to 1984 . Among these E . coli involved in cases of diarrhea, we found that 20.3% were enteropathogenic E . coli . Only 3.9% of the strains adhered to the brush borders of human intestinal enterocytes, and they belonged to different serotypes . All these adhesion-positive strains possessed genes encoding for the heat-stable enterotoxin, but their adhesive factors were different regarding serology with anti-colonization factor sera, hemagglutination patterns, electron microscopy structures, or major surface protein subunits. Eur J Biochem, 1987 Jun 1, 165(2), 437 - 42 Molecular cloning and nucleotide sequence of full-length cDNA for sweet potato catalase mRNA; Sakajo S et al.; A nearly full-length cDNA clone for catalase (pCAS01) was obtained through immunological screening of cDNA expression library constructed from size-fractionated poly(A)-rich RNA of wounded sweet potato tuberous roots by Escherichia coli expression vector-primed cDNA synthesis . Two additional catalase cDNA clones (pCAS10 and pCAS13), which contained cDNA inserts slightly longer than that of pCAS01 at their 5'-termini, were identified by colony hybridization of another cDNA library . Those three catalase cDNAs contained primary structures not identical, but closely related, to one another based on their restriction enzyme and RNase cleavage mapping analyses, suggesting that microheterogeneity exists in catalase mRNAs . The cDNA insert of pCAS13 carried the entire catalase coding capacity, since the RNA transcribed in vitro from the cDNA under the SP6 phage promoter directed the synthesis of a catalase polypeptide in the wheat germ in vitro translation assay . The nucleotide sequencing of these catalase cDNAs indicated that 1900-base catalase mRNA contained a coding region of 1476 bases . The amino acid sequence of sweet potato catalase deduced from the nucleotide sequence was 35 amino acids shorter than rat liver catalase {Furuta, S., Hayashi, H., Hijikata, M., Miyazawa, S., Osumi, T . & Hashimoto, T . (1986) Proc . Natl Acad . Sci . USA 83, 313-317} . Although these two sequences showed only 38% homology, the sequences around the amino acid residues implicated in catalytic function, heme ligand or heme contact had been well conserved during evolution. Arch Biochem Biophys, 1987 Jun, 255(2), 309 - 15 The defective proton-ATPase of uncA mutants of Escherichia coli: ATP-binding and ATP-induced conformational change in mutant alpha-subunits; Rao R et al.; Mutations in the uncA gene of Escherichia coli cause loss of both oxidative phosphorylation and ATP-driven generation of the transmembrane proton gradient . The uncA gene encodes the alpha-subunit of the F1-sector of the E . coli membrane proton-ATPase . F1-alpha-subunit from normal (unc+) E . coli binds ATP tightly (KD = 0.1 microM) and undergoes a large ATP-induced conformational change, but the functional role of the ATP-binding site is currently unknown . There is disagreement in the literature as to whether the ATP-binding site is present or lacking in F1-alpha-subunit from uncA mutant strains . One obstacle in studying this question is the difficulty of purifying mutant alpha-subunits in native form . In order to circumvent this difficulty we have studied ATP binding and ATP-induced conformational changes in mixtures of F1 subunits obtained by dissociating uncA mutant F1 . Anti-alpha antibody was used in conjunction with immunoblotting to identify the alpha-subunits in the mixtures . Retention of native conformation by the alpha-subunits was demonstrated by the fact that the dissociated alpha-subunits were fully competent to repolymerize with other F1 subunits to yield intact F1 aggregate . The results show that, contrary to previous reports, alpha-subunits from three catalytically defective uncA mutants do indeed bind ATP and do undergo an ATP-induced conformational change . The binding affinity of alpha-subunit for ATP was lower than normal in each of the three mutants, but this is not likely to be a significant factor under physiological conditions. J Bacteriol, 1987 Jun, 169(6), 2471 - 5 Regulation of expression of glutamine synthetase in a symbiotic Nostoc strain associated with Anthoceros punctatus; Joseph CM et al.; A characteristic of N2-fixing cyanobacteria in symbiotic associations appears to be release of N2-derived NH4+ . The specific activity of the primary ammonium-assimilating enzyme, glutamine synthetase (GS), was found to be three- to fourfold lower in Nostoc sp . strain 7801 grown in symbiotic association with the bryophyte Anthoceros punctatus than in free-living Nostoc sp . strain 7801 . Quantitative immunological assays with antisera against GS purified from Nostoc sp . strain 7801 and from Escherichia coli indicated that similar amounts of the GS protein were present in symbiotic (50 micrograms mg-1) and free-living (68 micrograms mg-1) cultures . The conclusion from these experiments is that GS is regulated by a posttranslational mechanism in Anthoceros-associated Nostoc sp . strain 7801 . However, the results of comparative catalytic and immunological experiments between N2- and NH4+-grown free-living Nostoc sp . strain 7801 implied control of GS synthesis . A correlation was not observed between the level of GS expression and the extent of symbiotic heterocyst differentiation in Nostoc sp . strain 7801 associated with A . punctatus. J Bacteriol, 1987 Jun, 169(6), 2460 - 5 Assembly of a chemically synthesized peptide of Escherichia coli type 1 fimbriae into fimbria-like antigenic structures; Abraham SN et al.; Escherichia coli type 1 fimbriae are composed of subunits, each of which comprises 158 amino acids . We synthesized a copy of a 13-residue peptide, located near the NH2 terminus of the fimbrial subunit, that assumed some of the properties of type 1 fimbriae . At pH 5.5 the synthetic peptide autoassembled into fibrillar structures that resembled type 1 fimbriae except that they appeared less rigid and rodlike . A quaternary structure-specific monoclonal antibody against type 1 fimbriae recognized the synthetic peptide in the assembled but not the unassembled state . Furthermore, when the synthetic peptide was injected in its fimbrial conformation into rabbits, it evoked antibodies that reacted with type 1 fimbriae isolated from E . coli. Biosci Rep, 1987 Jun, 7(6), 475 - 83 Nuclear accumulation of a heat-shock 70-like protein during herpes simplex virus replication; LaThangue NB et al.; A monoclonal antibody defines an antigen, p68, related to hsp70, which is located in nuclei of uninfected exponential cells . Nuclear p68 is released by DNase but not RNase treatment suggesting an association with DNA . Lytic productive infection of confluent quiescent BHK 21 cells with herpes simplex virus type-2 causes p68 to accumulate in nuclei . The effect is specific for HSV-2, and does not occur in HSV-1 infected cells . Maximum nuclear accumulation of p68 requires virus DNA synthesis although a significant accumulation occurs in the absence of such synthesis . It is suggested that the nuclear accumulation of p68 is an aspect of a cellular stress response to lytic infection with HSV-2. Microb Pathog, 1987 Jun, 2(6), 465 - 72 K88 fimbrial antigens: identification of antigenic determinants by the use of synthetic peptides; Krogfelt KA et al.; Identification of antigenic determinants of K88 fimbriae was approached by immunization of rabbits with BSA conjugated synthetic peptides mimicking either a predicted common determinant or type specific determinants . Anti-peptide sera were assayed by ELISA and sandwich ELISA . It was shown that sera raised against the peptides corresponding to the K88ab and K88ad variants of the 213-219 segment were able to recognize the corresponding antigenic variant of the native fimbriae whereas the rest of the antisera did not to any significant degree react with intact fimbriae . In Western blots all anti-peptide sera were able to recognize the K88 proteins . Similarly in ELISA assays all raised sera showed affinity to denatured K88 proteins. Microb Pathog, 1987 Jun, 2(6), 425 - 34 Serological response to type 1-like somatic fimbriae in diarrheal infection due to classical enteropathogenic Escherichia coli; Karch H et al.; Fimbriae from enteropathogenic Escherichia coli strain E2349/69 (0127:H6) and its plasmid-minus derivative, MAR20, were purified and characterized as type 1-like by their physicochemical and hemagglutination patterns . Sera from adult volunteers challenged with the diarrheagenic parent strain and the attenuated plasmid-minus derivative were examined to detect an immune response, using the purified fimbriae as antigens in an enzyme linked immunosorbent assay (ELISA) and immunoblot assay . An anti-fimbrial response was evident in sera of 7 of 10 volunteers fed the diarrheagenic parent strain E2348 but also in 8 of 9 individuals fed the attenuated, plasmid-cured, derivative MAR20 . The antibody response appeared specific in that the sera failed to react in an ELISA and by immunoblot assay with type 1 fimbriae from other E . coli . These findings suggest that the type 1 fimbriae of this representative EPEC strain are antigenically distinct . The results of this investigation provide the first evidence of seroconversion to type 1-like fimbriae in infections caused by diarrheagenic E . coli. Isr J Med Sci, 1987 Jun, 23(6), 759 - 62 Cloning of Mycoplasma pneumoniae DNA and expression of P1-epitopes in Escherichia coli; Frydenberg J et al.; A genomic library of Mycoplasma pneumoniae was constructed by cloning partial Sau3A-digested genomic DNA into the expression plasmids pEX1 to pEX3 . The recombinant clones were screened for production of M . pneumoniae P1-antigen by an in situ colony enzyme-linked immunosorbent assay (ELISA) blot method with a monospecific rabbit antiserum raised against the surface protein P1 . The length of the translated P1-sequence and the size of the inserted DNA were determined . By comparison it was shown that six clones contained DNA fragments coding for an internal part of the P1-protein and eight clones code for the C-terminal part of terminated P1-protein . In reactions against sera from patients suffering from M . pneumoniae infection and sera from healthy persons, one of the internal clones and five of the C-terminal clones reacted with one or two of the patient sera, but only one clone reacted with all patient sera. Isr J Med Sci, 1987 Jun, 23(6), 585 - 90 Transcription control elements of the Mycoplasma pneumoniae rRNA operon; Hyman HC et al.; The single RNA operon of Mycoplasma pneumoniae was cloned into a lambda vector and subcloned into pBR322 . This was carried out in order to enable the analysis of the transcription control regions of this operon . S1 nuclease mapping was used to locate the 5' ends of RNA transcripts synthesized from the operon . The 5' ends of the 23S, 16S, and a precursor RNA synthesized in vivo in M . pneumoniae were mapped on the DNA template . Preliminary in vitro transcription experiments using RNA polymerase of Escherichia coli led to the conclusion that E . coli recognizes one promoter in the 5' region of the M . pneumoniae rRNA operon . The startsite of the in vitro transcript seems to lie downstream from the 5' end of the M . pneumoniae precursor transcript . Preliminary sequencing of the 5' regions of the M . pneumoniae rRNA operon and of the M . capricolum rRNA B operon enabled their comparison to each other and to known sequences from other organisms. Arch Microbiol, 1987 Jun, 148(1), 44 - 51 Factors affecting transcriptional regulation of the formate-hydrogen-lyase pathway of Escherichia coli; Birkmann A et al.; The regulatory elements involved in expression of the gene (fdhF) for the selenopolypeptide of formate dehydrogenase and of a gene (or transcriptional unit) (hyd) specifically responsible for the formation of the gas-evolving hydrogenase (hydrogenase 3) in Escherichia coli were investigated . Formate (or a product of it) is required for expression of both systems since in a pyruvate-formate-lyase deficient mutant induction occurs only when formate is supplemented externally . Under this condition, formate can partially overcome repression by nitrate . The transcription of both the fdhF gene and the hydrogenase-3-encoding systems is independent of the presence of a wild-type fnr gene when formate is present, supporting the view that the Fnr effect on the formation of the formate-hydrogen-lyase pathway is indirect . Mutations blocking the synthesis of a functional molybdenum cofactor also had no major affect on fdhF and hyd expression . The nucleotide sequence of the 5' flanking region of the fdhF gene was determined and the transcription start point of the fdhF gene was localized by nuclease S1 mapping . Nuclease Bal31 generated deletion clones were constructed and the regulation of their expression was studied . Anaerobic expression and induction by formate depended on the presence of a stretch of approximately 185 nucleotides upstream of the translation start . Elements mediating formate induction and oxygen or nitrate repression could not be separated physically . The regulatory features of the fdhF upstream region bear striking resemblance to systems whose expression are dependent upon upstream activating elements. Nature, 1987 Jun 25-Jul 1, 327(6124), 716 - 7 Site-specific mutagenesis of AIDS virus reverse transcriptase; Larder BA et al.; Human immunodeficiency virus (HIV) is the causative agent of AIDS (acquired immune deficiency syndrome) a disease which poses a serious challenge to modern medicine . If we are to conquer this disease we will need a protective vaccine or effective drugs able to block the life cycle of the virus . An early stage in the invasion of the host cell is the conversion of the RNA genome of the virus to a double-stranded DNA intermediate which subsequently becomes integrated into the host cell chromosome . The enzyme reverse transcriptase is crucial in this process and is thus an obvious chemotherapeutic target . In this study we have used site-directed mutagenesis of this enzyme expressed in Escherichia coli to reveal several important functional regions of the protein including putative components of the triphosphate binding site and pyrophosphate exchange sites. J Bacteriol, 1987 Jun, 169(6), 2843 - 6 Promoter of the pertussis toxin operon and production of pertussis toxin; Nicosia A et al.; Pertussis toxin (PT), the major virulence factor of Bordetella pertussis, is composed of five different subunits whose genes are organized as an operon . We report the mapping of the promoter region of the PT operon and show that this promoter is only weakly active in Escherichia coli . Bordetella parapertussis and Bordetella bronchiseptica, which do not produce any PT, are shown to have a weaker promoter sequence for this operon and not to produce any detectable PT mRNA . We show that transcription of the PT operon in B . pertussis was constant throughout until the late stationary phase, when transcription significantly decreased . Analysis of the transposon Tn5 mutant BP347 showed that the product of the vir locus was required for transcription of the PT operon . Characterization of the Tn5 mutant BP356 showed that subunit S3 was required for the release of PT into the extracellular medium. J Bacteriol, 1987 Jun, 169(6), 2697 - 701 Posttranscriptional regulation of ribosomal protein S20 and stability of the S20 mRNA species; Mackie GA; I have tested whether selective degradation of mRNA for ribosomal protein S20 of Escherichia coli occurs under conditions for which the expression of S20 is regulated posttranscriptionally . Blot hybridization of total RNA extracted from cultures at different times after addition of rifampin has permitted the estimation of relative levels of the two S20 mRNA species and their half-lives . In a strain harboring a plasmid containing the complete gene for S20, including the transcriptional terminator, moderate posttranscriptional repression of S20 synthesis is accompanied by a substantial increase in the half-lives of both S20 mRNAs relative to those in the haploid parental strain . In an otherwise identical strain in which the transcriptional terminator is deleted from the plasmid-borne S20 genes, the half-life of total S20 mRNA declines more than twofold relative to that in the haploid parent . Thus accelerated decay of the mRNAs for ribosomal protein S20 appears to be an artifact of deletion of the transcriptional terminator, rather than a physiologically significant consequence of translational repression. Biochem Biophys Res Commun, 1987 May 29, 145(1), 40 - 5 DAPI-pUC8 complex: a tool to investigate biological effects of nucleic acid-drug interaction; Palu G et al.; A complex consisting of pUC8, a 1.8 Md plasmid, and 4'-6-diamidino-2-phenylindole, a DNA-binding agent, has been performed in vitro under different conditions of ionic strength, and used to transform competent cells . A strong interference with the plasmid-coded activities, related to the P/D ratio where at the DNA-drug complex was formed, was shown to occur . Since the compound does not inhibit the uptake process neither affects plasmid activity once dissociated at high ionic strength, it is likely to be acting from inside the cell while still in the form of a DNA-adduct . This system is proposed as a useful tool to investigate the effects on target genes of drugs endowed with DNA sequence specificity. Biochem Biophys Res Commun, 1987 May 29, 145(1), 190 - 5 Retention of enzymatic activity by N-terminal domain (1-78) T4-lysozyme: expression of synthetic DNA in Escherichia coli; Phipps J et al.; DNA of 235 b.p . coding for N-terminal domain (1-78) T4-lysozyme was synthesized and cloned by ligating twelve synthetic fragments with a linearized plasmid pUCE8 followed by transformation . On expression in E . coli strain JM103 cells, colonies containing the synthetic DNA were found to be lytic . On purification, clone ptly . 23-5 was found to contain polypeptide (M.W . 10,500), corresponding to N-terminal domain, its dimeric and aggregate form . It was identified by amino acid sequence analysis of the dimeric form. Biochem Biophys Res Commun, 1987 May 29, 145(1), 291 - 7 Inhibition of viral reverse transcriptase by 2',5'-oligoadenylates; Liu DK et al.; Viral reverse transcriptase activity was inhibited in a concentration dependent manner by 2',5'-oligoadenylate . Kinetically this inhibition was of a mixed type where 2',5'-oligoadenylate was not strictly competitive with dTTP . The potency of inhibition was more marked in the absence than in the presence of sulfhydryl agents . 2',5'-oligoadenylate had no effect on DNA-dependent E . coli DNA polymerase and was much less active against mammalian DNA polymerases . This is the first report of reverse transcriptase inhibition by an inducible constitutive natural ligand. Biochim Biophys Acta, 1987 May 27, 913(1), 60 - 5 Reevaluation of citrate lyase from Escherichia coli; Quentmeier A et al.; The subunit structure of citrate lyase from Escherichia coli was shown to be similar to that of all other lyases investigated so far . The three different subunits with molecular masses of 55.5 kDa, (large subunit) 35 kDa (medium-sized subunit) and 12.5 kDa (small subunit, acyl carrier protein) occurred in a ratio of 1:1:1 . Using high-pressure liquid chromatography, it was possible to demonstrate that the reported large acyl carrier protein, with a molecular mass of 85 kDa was a contaminating protein associated with citrate lyase multienzyme complex; it could be removed by anion-exchange chromatography with Q-Sepharose . The typical two configurations of citrate lyase, the 'star' form and the 'ring' form with a diameter of 14.3 nm and 15.4 nm, respectively, could be detected by electron microscopy. Nucleic Acids Res, 1987 May 26, 15(10), 4085 - 97 Cleavage of single-stranded DNA by plasmid pT181-encoded RepC protein; Koepsel RR et al.; RepC protein encoded by plasmid pT181 has single-stranded endonuclease and topoisomerase-like activities . These activities may be involved in the initiation (and termination) of pT181 replication by a rolling circle mechanism . RepC protein cleaves the bottom strand of DNA within the origin of replication at a single, specific site when the DNA is in the supercoiled or linear (double or single-stranded) form . We have found that RepC protein will also cleave single-stranded DNA at sites other than the origin of replication . We have mapped the secondary cleavage sites on pT181 DNA . When the DNA is in the supercoiled, or linear, double-stranded form, only the primary site within the origin is cleaved . However, when the DNA is present in the single-stranded form, several strong and weak cleavage sites are observed . The DNA sequence at these cleavage sites shows a strong similarity with the primary cleavage site . The presence of Escherichia coli SSB protein inhibited cleavage at all of the secondary nick sites while the primary nick site remained susceptible to cleavage. Nucleic Acids Res, 1987 May 26, 15(10), 4273 - 89 Regulation of the Escherichia coli excision repair gene uvrC . Overlap between the uvrC structural gene and the region coding for a 24 kD protein; Moolenaar GF et al.; The UvrA, UvrB and UvrC proteins of E . coli are subunits of a DNA repair enzyme, the ABC exonuclease . In this paper we study the uvrC regulatory region . The uvrC structural gene is preceded by an open reading frame encoding a 24 kD protein . A uvrC promoter has been mapped within this gene . The transcription start of a second promoter located 5' of the 24 kD gene is mapped in vivo . We show that transcription from both promoters on the chromosome is not inducible by UV damage . The possible translation start codons of the UvrC and of the 24 kD protein are determined . Sequences encoding the N-terminal part of the UvrC protein overlap with sequences encoding the C-terminal part of the 24 kD protein . To examine a possible function of the 24 kD gene in repair, a 24 kD insertion mutant was created in the chromosome . The mutant however only slightly affects the UV sensitivity of the cell . Transcription of P3 alone provides sufficient UvrC protein for the normal repair of UV lesions. Nucleic Acids Res, 1987 May 26, 15(10), 4145 - 62 Stereospecificity of nucleases towards phosphorothioate-substituted RNA: stereochemistry of transcription by T7 RNA polymerase; Griffiths AD et al.; Transcription by T7 RNA polymerase has been studied using a chiral ATP analogue . The Sp diastereoisomer of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) was incorporated into RNA with an apparent KM of approximately 15 microM, similar to that for ATP; the Rp diastereoisomer was neither a substrate nor a competitive inhibitor . The configuration of the phosphodiester link in the RNA produced was analyzed with stereospecific nucleases . The rate of nuclease digestion was compared with the rate of digestion of phosphorothioate-substituted RNA of known stereochemistry synthesized by E . coli RNA polymerase . Surprisingly, the nucleases exhibited reduced discrimination compared with their activity on dinucleotides . The results show that phosphorothioate-substituted RNA transcribed by T7 RNA polymerase has the same configuration as that transcribed by E . coli RNA polymerase, ie . Rp . Thus, the reaction proceeds with inversion of configuration at phosphorus. FEBS Lett, 1987 May 25, 216(1), 73 - 8 Expression of ricin A chain in Escherichia coli; O'Hare M et al.; DNA encoding ricin A chain was derived from preproricin cDNA and ligated into the expression vector pDS5/3 . Transcription is controlled from the coliphage promoter PN25 fused with the lac operator of E.coli . When induced, E.coli 71.18 cells transformed with the recombinant plasmid express ricin A chain which is soluble and has full biological activity. J Biol Chem, 1987 May 25, 262(15), 7412 - 7 Role of amino-terminal positive charge on signal peptide in staphylokinase export across the cytoplasmic membrane of Escherichia coli; Iino T et al.; Staphylokinase mutants having amino acid substitutions within the amino-terminal charged segment of the signal peptide have been produced by in vitro oligonucleotide-directed mutagenesis . When the processing of the gene products was analyzed in Escherichia coli cells, the rate of processing of the mutant staphylokinase precursor decreased as the net charge became more negative . A net positive charge, but not specific amino acid residues, was required on the amino-terminal segment for efficient processing . Staphylokinase precursor having a net negative charge accumulated in the cytoplasm, tending to bind to the cytoplasmic membrane as determined by subcellular fractionation and immunoelectron microscopy . Although a mutant carrying an amino acid substitution in the hydrophobic segment and wild-type staphylokinases had an interfering effect on the processing of other normal secreted proteins, this effect was lost when they also contained charge-altering substitutions in the amino-terminal region . From these results, we concluded that a positive charge on the amino-terminal segment of the staphylokinase signal peptide is required for entrance into the protein export process. J Biol Chem, 1987 May 25, 262(15), 7391 - 7 Identification of the genes in the Escherichia coli ileS-lsp operon . Analysis of multiple polycistronic mRNAs made in vivo; Miller KW et al.; The genes encoding isoleucyl-tRNA synthetase (ileS) and prolipoprotein signal peptidase (lsp) of Escherichia coli were previously shown to be co-transcribed (Miller, K . W., and Wu, H . C . (1987) J . Biol . Chem . 262, 389-393) . However, the boundaries of this transcriptional unit have not been established . In this regard, DNA sequence determination has shown that ileS and lsp are closely flanked by four open reading frames, i.e . x-ileS-lsp-orf149-orf316-orf304 . To define the boundaries of the operon, we applied Northern blotting hybridization and mRNA 5'-end mapping to analyze mRNA from a wild-type strain (SM31) and a mutant strain (SM31-2B4) that exhibits an increased expression of prolipoprotein signal peptidase . Four ileS-lsp co-transcripts were detected in RNA from the strain SM31 . In addition to these four mRNAs, two new, highly abundant co-transcripts were also detected in RNA from the mutant . Based upon the determination of the 5'-ends of the mRNAs and analysis of their coding sequences, we conclude that the six mRNAs actually are comprised of three pairs of related mRNAs . The two mRNAs in a given pair have the same 5'-termini (all located upstream of or within gene x), but vary with respect to the identity of their 3'-terminal coding sequence (lsp or orf316) . In conclusion, the ileS-lsp operon contains five genes, x-ileS-lsp-orf149-orf316, whose transcription probably is dependent upon promoter(s) located upstream of or within gene x . The next gene downstream, orf304, apparently does not reside in the operon. J Biol Chem, 1987 May 25, 262(15), 7189 - 94 Structure of the yeast valyl-tRNA synthetase gene (VASI) and the homology of its translated amino acid sequence with Escherichia coli isoleucyl-tRNA synthetase; Jordana X et al.; The VASI gene encoding the valyl-tRNA synthetase from yeast was isolated and sequenced . The gene-derived amino acid sequence of yeast valyl-tRNA synthetase was found to be 23% homologous to the Escherichia coli isoleucyl-tRNA synthetase . This is the highest level of homology reported so far between two distinct aminoacyl-tRNA synthetases and is indicative of an evolutionary relationship between these two molecules . Within these homologous sequences, two functional regions could be recognized: the HIGH region which forms part of the binding site of ATP and the KMSKS region which is recognized as the consensus sequence for the binding of the 3'-end of tRNA (Hountondji, C., Dessen, Ph., and Blanquet, S . (1986) Biochemie (Paris) 68, 1071-1078) . Secondary structure predictions as well as the presence of both HIGH and KMSKS regions, delineating the nucleotide-binding domain and the COOH-terminal helical domain in aminoacyl-tRNA synthetases of known three-dimensional structure, suggest that the yeast valyl-tRNA synthetase polypeptide chain can be folded into three domains: an NH2-terminal alpha-helical region followed by a nucleotide-binding topology and a COOH-terminal domain composed of alpha-helices which probably carries major sites in tRNA binding. J Biol Chem, 1987 May 25, 262(15), 7264 - 72 RNA polymerase pauses in vitro within the Escherichia coli origin of replication at the same sites where termination occurs in vivo; Rokeach LA et al.; An in vitro transcription system able to distinguish initiation at the 16-kDa promoter from elongation events was used to identify factors that might participate in transcription termination within oriC . Pausing in the oriC region occurs at the same sites where termination occurs in vivo . Ten of these sites overlap RNA:DNA junctions in oriC . The pausing that occurs in vitro was not converted to efficient termination by guanosine 5'-diphosphate 3'-diphosphate, NusA, and Rho alone or in combination, or by DnaA suggesting that in vivo other or additional factors contribute to termination at oriC . Transcription from the 16-kDa promoter was 90% inhibited by the nucleotides guanosine 5'-diphosphate 3'-diphosphate and guanosine 5'-triphosphate 3'-diphosphate in agreement with previous observations that this promoter is stringently regulated. J Biol Chem, 1987 May 25, 262(15), 7213 - 9 The role of oxygen radicals in dye-mediated photodynamic effects in Escherichia coli B; Martin JP et al.; Photosensitive dyes representative of the thiazines, xanthenes, acridines, and phenazines mediated phototoxicity in Escherichia coli B . The observed phototoxicity was sensitizer-, light-, and oxygen-dependent and is therefore a photodynamic effect . Hydroxyl radical scavengers conferred protection against the photodynamic action of all of the representative dyes . The extent of protection was dependent on the concentration of scavenger and on the in vitro reactivity of the scavenger with the hydroxyl radical . Exogenous superoxide dismutase and catalase partially protected the cells against the dye-mediated phototoxicity, and prior induction of intracellular superoxide dismutase and catalase by growth in glucose minimal medium containing manganese and paraquat substantially protected E . coli B against the photodynamic action of all of the dyes examined . Combinations of protective treatments against the phototoxicity of all four classes of dyes, including superoxide dismutase and catalase preinduction and addition of extracellular superoxide dismutase and catalase or the addition of hydroxyl radical scavengers, provided nearly complete protection against the oxygen-dependent component of dye-mediated lethality . E . coli B grown in glucose minimal medium containing manganese and photosensitive dyes induced manganese superoxide dismutase . The extent of induction was correlated with the dyes' ability to photooxidize NADH in vitro . Thus, oxygen radicals are primarily responsible for the oxygen-dependent toxicity of the photosensitive dyes examined, and one adaptive response of E . coli B to a dye-mediated oxidative threat is to induce superoxide dismutase. FEBS Lett, 1987 May 25, 216(1), 45 - 50 Lipocortin-like anti-phospholipase A2 activity of endonexin; Fauvel J et al.; Endonexin (protein II, 32.5 kDa) has been purified to homogeneity from bovine liver in the following steps: selective extraction by EGTA from membranes precipitated with Triton X-100/calcium; chromatography on DEAF-TSK 545 at pH 7.0, endonexin being eluted at 0.1 M NaCl; affinity chromatography on polyacrylamide-immobilized phosphatidylserine; gel filtration on TSK 3000 . The amino acid composition was essentially similar to that previously reported . Using {3H}oleic acid-labelled Escherichia coli membranes as substrate, endonexin inhibited phospholipase A2 from pig pancreas . Maximal inhibition was 55 and 70%, whereas 50% inhibition occurred at 480 and 120 nM endonexin and lipocortin II, respectively . These data could be related to common features shared by both lipocortins/calpactins and endonexin, i.e . the presence of a consensus sequence and the ability to bind to anionic phospholipids in a calcium-dependent manner. J Biol Chem, 1987 May 25, 262(15), 7135 - 41 Probing the catalytic subunit of the tonoplast H+-ATPase from oat roots . Binding of 7-chloro-4-nitrobenzo-2-oxa-1,3,-diazole to the 72-kilodalton polypeptide; Randall SK et al.; The purified tonoplast H+-ATPase from oat roots (Avena sativa L . var . Lang) consists of at least three different polypeptides with masses 72, 60, and 16 kDa . We have used covalent modifiers (inhibitors) and polyclonal antibodies to identify the catalytic subunit of the H+-pumping ATPase . The inactivation of ATPase activity by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (Nbd-Cl, an adenine analog) was protected by MgATP or MgADP, and showed kinetic properties consistent with active site-directed inhibition . Under similar conditions, {14C}Nbd-Cl preferentially labeled the 72-kDa polypeptide of the purified ATPase . This binding was reduced by MgATP or 2' (3')-)O-(2,4,6-trinitrophenyl) ATP . Nbd-Cl probably modified cysteinyl--SH or tyrosyl--OH groups, as dithiothreitol reversed both ATPase inactivation and {14C}Nbd-Cl binding to the 72-kDa subunit . The finding that N-ethylmaleimide inhibition of ATPase activity was protectable by nucleotides is consistent with the idea of sulfhydryl groups in the ATP-binding site . Polyclonal antibody made to the 72-kDa polypeptide specifically reacted (Western blot) with a 72-kDa polypeptide from both tonoplast-enriched membranes and the purified tonoplast ATPase, but it did not cross-react with the mitochondrial or Escherichia coli F1-ATPase . The antibody inhibited tonoplast ATPase and H+-pumping activities . We conclude from these results that the 72-kDa polypeptide of the tonoplast H+-ATPase contains an ATP- (or nucleotide-) binding site that may constitute the catalytic domain. J Mol Biol, 1987 May 20, 195(2), 289 - 97 The use of transposon TnphoA to detect genes for cell envelope proteins subject to a common regulatory stimulus . Analysis of osmotically regulated genes in Escherichia coli; Gutierrez C et al.; The transposon TnphoA can be used specifically to detect bacterial genes that code for cell envelope proteins . We have used TnphoA to search for genes regulated by osmolarity in Escherichia coli . Among approximately 30,000 random insertions of TnphoA into the chromosome, we have found 700 independent fusions that produce hybrid proteins with alkaline phosphatase activity . Of these, 37 were induced after growth in a medium of high osmolarity and none was repressed . Osmo-inducible fusions of phoA were found to ompC and to a gene that is probably proU . These two genes were already known to be transcriptionally induced by osmolarity . In addition, eight other genes, designated osm, were identified and mapped on the bacterial chromosome . The expression of these genes is induced by solutes that are unable to decrease the turgor pressure applied to the envelope . One of the osm genes, osmI, is also specifically induced by glycerol, which does diffuse across the cytoplasmic membrane . The expression and osmoregulation of all the osm genes were shown to occur independently of ompR and envZ, which control the expression and osmoregulation of the ompC and ompF genes in E . coli. J Mol Biol, 1987 May 20, 195(2), 261 - 72 Escherichia coli integration host factor binds specifically to the ends of the insertion sequence IS1 and to its major insertion hot-spot in pBR322; Gamas P et al.; We report here that the ends of IS1 are bound and protected in vitro by the heterodimeric protein integration host factor (IHF) . Under identical conditions, RNA polymerase binds to one of these ends (IRL) and protects a region that includes the sequences protected by IHF . Other potential sites within IS1, identified by their homology to the apparent consensus sequence, are not protected . Footprinting analysis of deletion derivatives of the ends demonstrates a correspondence between the ability of the end sequence to bind IHF and its ability to function as an end in transposition . Nonetheless, some transposition occurs in IHF- cells, indicating that IHF is not an essential component of the transposition apparatus . IHF also binds and protects four closely spaced regions within the major hot-spot for insertion of IS1 in the plasmid pBR322 . This striking correlation of hot-spot and IHF-binding sites suggests a possible role for IHF in IS1 insertion specificity. Biochemistry, 1987 May 19, 26(10), 2706 - 11 Fourier transform infrared investigation of the Escherichia coli methionine aporepressor; Yang PW et al.; This study represents the first physicochemical analysis of the recently cloned methionine repressor protein (Met aporepressor) from Escherichia coli . Infrared spectrometry was used to investigate the secondary structure and the hydrogen-deuterium exchange behavior of the E . coli Met aporepressor . The secondary structure of the native bacterial protein was derived by analysis of the amide I mode . The amide I band contour was found to consist of five major component bands (at 1625, 1639, 1653, 1665, and 1676 cm-1) which reflect the presence of various substructures . The relative areas of these component bands are consistent with a high alpha-helical content of the peptide chain secondary structure in solution (43%) and a small amount of beta-sheet structure (7%) . The remaining substructure is assigned to turns (10%) and to unordered (or less ordered) structures (40%) . The temperature dependence of the infrared spectra of native Met aporepressor in D2O medium over the temperature interval 20-80 degrees C indicates that there are two discrete thermal events: the first thermal event, centered at 42 degrees C, is associated with the hydrogen-deuterium exchange of the hard-to-exchange alpha-helical peptide bonds accompanied by a partial denaturation of the protein, while the second event, centered around 50 degrees C, represents the irreversible thermal denaturation of the protein. Biochemistry, 1987 May 19, 26(10), 2690 - 6 Kinetic analysis of T7 RNA polymerase-promoter interactions with small synthetic promoters; Martin CT et al.; Specific interactions between T7 RNA polymerase and its promoter have been studied by a simple steady-state kinetic assay using synthetic oligonucleotide promoters that produce a short five-base message . A series of promoters with upstream lengths extending to promoter positions -19, -17, -14, and -12 show that promoters extending to -19 and -17 produce very specific transcripts with initiation rate constant Kcat = 50 min-1 and a Michaelis constant Km = 0.02 microM, indicating that the consensus sequence to position -17 is sufficient for maximum promoter usage . Shortening the upstream region of the promoter to -14 substantially increases Km (0.3 microM) but does not significantly reduce the maximum velocity (kcat = 30 min-1) . Finally, truncation of the promoter at position -12 results in extremely low levels of specific transcription . The coding and noncoding strands appear to make different contributions to promoter recognition . Although the double-stranded promoter of upstream length -12 is very poor as a transcription template, extension of only the noncoding strand to -17 very significantly improves both Kcat and Km . In contrast, extension of only the coding strand results in no significant improvement . Substitution of an AT base pair at position -10 by CG (as found in T3 RNA polymerase promoters) produces a 10-fold increase in Km, with little effect on Kcat . Comparison of two promoters containing a base pair mismatch at this site (AG or CT) demonstrates that promoter recognition is very sensitive to the nature of the base on the noncoding strand and is only slightly affected by the presence of a mismatch created by a wrong base in the coding strands.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1987 May 19, 26(10), 2674 - 82 Effect of single amino acid replacements on the folding and stability of dihydrofolate reductase from Escherichia coli; Perry KM et al.; The role of the secondary structure in the folding mechanism of dihydrofolate reductase from Escherichia coli was probed by studying the effects of amino acid replacements in two alpha helices and two strands of the central beta sheet on the folding and stability . The effects on stability could be qualitatively understood in terms of the X-ray structure for the wild-type protein by invoking electrostatic, hydrophobic, or hydrogen-bonding interactions . Kinetic studies focused on the two slow reactions that are thought to reflect the unfolding/refolding of two stable native conformers to/from their respective folding intermediates {Touchette, N . A., Perry, K . M., & Matthews, C . R . (1986) Biochemistry 25, 5445-5452} . Replacements at three different positions in helix alpha B selectively alter the relaxation time for unfolding while a single replacement in helix alpha C selectively alters the relaxation time for refolding . This behavior is characteristic of mutations that change the stability of the protein but do not affect the rate-limiting step . In striking contrast, replacements in strands beta F and beta G can affect both unfolding and refolding relaxation times . This behavior shows that these mutations alter the rate-limiting step in these native-to-intermediate folding reactions . It is proposed that the intermediates have an incorrectly formed beta sheet whose maturation to the structure found in the native conformation is one of the slow steps in folding. J Biol Chem, 1987 May 15, 262(14), 6778 - 84 Interaction of RNA with transformed glucocorticoid receptor . II . Identification of the RNA as transfer RNA; Ali M et al.; An endogenous RNA (designated as PIVB RNA), which is capable of associating with the 4 S glucocorticoid receptor (GR) to generate the 6 S form, has been purified from AtT-20 cells (Ali, M., and Vedeckis, W . V . (1987) J . Biol . Chem., 262, 6771-6777) . We describe here the physiochemical properties, GR-RNA interaction characteristics, and the chemical identification of PIVB RNA . 32P-Labeled PIVB RNA was similar to transfer RNA (tRNA) in its sedimentation coefficient (4 S) on sucrose gradients, electrophoretic mobility on formaldehyde-agarose gels, and receptor binding characteristics . The amino acid acceptor activity of PIVB RNA displayed a typical tRNA-dependent saturation curve and was 2-3-fold higher than that of homologous rabbit liver tRNA when tested using rabbit liver aminoacyl-tRNA synthetase . The purified {3H} aminoacyl-PIVB complex was also capable of binding to the 4 S GR to generate the 6 S form . The analysis of PIVB RNA on an acrylamide-urea sequencing gel revealed that it contained a major tRNA of 76 nucleotides and other minor tRNA species of 74 and 78 nucleotides . The identity of the tRNA present in the PIVB RNA was indirectly deduced by analyzing the 3H-amino acids, liberated from the {3H}aminoacyl-PIVB RNA (tRNA) complex, and subsequent analysis on an amino acid analyzer . PIVB RNA mainly contained tRNAArg (51.8%), tRNALys (17.1%), and tRNAHis (9.2%) which together accounted for 78% of the total PIVB tRNA . The remaining 22% of tRNA was contributed by threonine, valine, aspartic acid, alanine, and phenylalanine tRNAs . The GR displayed no species specificity, and tRNA samples from mouse, cow, rabbit, yeast, and Escherichia coli can bind to the mouse 4 S GR to generate the 6 S form . However, PIVB RNA did not affect the sedimentation profiles of albumin, chymotrypsinogen, and histone, indicating that PIVB RNA does not bind to all proteins . Thus, there may exist some specificity both at the level of protein (GR) and the selection of RNA (tRNA) . The GR binding to PIVB RNA occurred at low (nM) receptor concentration, and PIVB RNA showed limited capacity to shift 4 S GR to the 6 S form . 22.4 X 10(-11) mol of PIVB RNA can completely shift 4.8 X 10(-13) mol of 4 S GR to 6 S . That is, PIVB RNA has to be in a 500-600-fold excess over the amounts of GR to observe a stable 6 S GR X RNA complex on sucrose gradients . These results conclusively demonstrate that the transformed GR specifically binds to endogenous tRNA. J Biol Chem, 1987 May 15, 262(14), 6595 - 604 Properties of an Escherichia coli rhodanese; Alexander K et al.; A rhodanese enzyme of less than 20,000 molecular weight has been purified from Escherichia coli . The enzyme is accessible to substrates upon addition of whole cells to standard assay mixtures . This rhodanese has a Stokes radius of 17 A which for a globular protein corresponds to a molecular weight close to 14,000 . It undergoes autoxidation to a polymeric form which is probably an inert dimer . Enzyme inactivated by oxidation can be reactivated by millimolar concentrations of cysteine . Steady-state initial velocity measurements indicate that the enzyme catalyzes the transfer of sulfane sulfur by way of a double displacement mechanism with formation of a covalent enzyme-sulfur intermediate . The turnover number for the enzyme-catalyzed reaction, with thiosulfate as donor substrate and cyanide ion as the sulfur acceptor, is 260 s-1 . This value corresponds to a catalytic efficiency 60% of that measured for a previously characterized bovine liver enzyme of more than twice the molecular weight . Furthermore, KmCN is 24 mM which is 2 orders of magnitude higher than the value observed previously for the bovine enzyme . Evidence from chemical inactivation studies implicates an essential sulfhydryl group in the enzyme activity . It is proposed that this group is the site of substrate-sulfur binding in the obligatory enzyme-sulfur intermediate . Furthermore, a cationic site important for binding of the donor thiosulfate is tentatively identified from anion inhibition studies . Tests of alternate acceptor substrates indicate that the physiological dithiol, dihydrolipoate, is a more efficient acceptor than cyanide ion for the enzyme-bound sulfur . Of possibly greater physiological significance, it has been found that the enzyme catalyzes the formation of iron-sulfur centers . Other work indicates the E . coli rhodanese is subject to catabolite repression and suggests a physiological role for the enzyme in aerobic energy metabolism. Biochem J, 1987 May 15, 244(1), 151 - 7 Effect of low nucleotide concentrations on abortive elongation catalysed by wheat-germ RNA polymerase II; Job C et al.; A kinetic study of the effect of elongating nucleotide concentration on the reactions of abortive elongation catalysed by wheat-germ RNA polymerase II on a poly{d(A-T)} template suggests that the shift from abortive to productive elongation may involve the participation of at least two nucleotides, according to a mechanism very similar to that reported for Escherichia coli RNA polymerase . Experiments performed with non-complementary nucleotides with respect to the DNA template, and with substrate derivatives, allow an analysis of the substrate specificity during these reactions . Similar experiments performed with poly{d(A-A-T)}.poly{d(T-T-A)} as template provide a starting point for a better understanding of the effect of DNA sequence on the rates of abortive and productive elongation catalysed by the plant enzyme. J Immunol, 1987 May 15, 138(10), 3468 - 74 Development and regulation of chlamydia-responsive murine B lymphocytes; Levitt D et al.; We have examined characteristics of chlamydia-stimulated mouse B cells as well as cells that regulate polyclonal responses in vitro . B lymphocyte proliferation stimulated by chlamydia arises at a similar time as Escherichia coli lipopolysaccharide (LPS)-induced proliferative responses during ontogeny . In contrast, development of immunoglobulin (Ig)-secreting cells after chlamydia stimulation is delayed by several weeks relative to ontogeny of LPS-inducible plaque-forming cells (PFC) . The lack of Ig secretion by immature B cells is not due to a deficiency of Lyb5+ B lymphocytes, since X-linked immunodeficient (xid) NBF1 mice that lack this B lymphocyte population respond well to chlamydia stimulation . Adherent cells are important for chlamydia-stimulated B lymphocyte differentiation, but are not as necessary for their proliferation . Neither adult adherent cells nor T cells can correct the inability of immature spleen cells to develop into Ig-secreting cells; spleen cells from 2-wk-old mice (i.e., immature B cells) will not suppress adult B lymphocyte responses to chlamydia . When B lymphocytes are separated according to their buoyant densities, chlamydia stimulates low density (activated) B cells to proliferate and differentiate better than high density (resting) cells . Proliferative responses to chlamydia arise earlier during ontogeny, do not require adherent cells, and can proceed to a relatively greater extent in resting B cell population (compared with activated B cells) than induction of Ig-secreting cells. Anal Biochem, 1987 May 15, 163(1), 79 - 87 In vitro sodium bisulfite mutagenesis of restriction endonuclease recognition sites; Merlo DJ et al.; Sodium bisulfite treatment of single-stranded DNA deaminates exposed cytosine residues to form uracil, resulting in cytosine-to-thymidine transition mutations following DNA replication . We have used this reaction in vitro to destroy the recognition sequences for the restriction endonucleases HindIII and XmaI in the aminoglycoside 3'-phosphotransferase I coding region of plasmid pUC4K . This procedure should be applicable to the mutation of any recognition sequence of restriction endonucleases which generate cytosine-containing single-stranded ends . The possibility of mutagenesis of restriction sites to generate stop codons in coding regions is discussed. Anal Biochem, 1987 May 15, 163(1), 188 - 95 Hybridization as a technique for studying interchain interactions in the catalytic trimers of aspartate transcarbamoylase; Yang YR et al.; Since subunit interactions in regulatory enzymes mediate the ligand-promoted conformational changes responsible for their allosteric properties, it is necessary to have techniques for determining the effects of ligands and mutational alterations on the strength of the interchain interactions . In aspartate transcarbamoylase from Escherichia coli, the multiple interchain interactions are so linked that it is difficult to study them separately . Therefore, we have focused on the nonallosteric catalytic trimers isolated from the holoenzyme and have used the rate of hybrid formation between native and succinylated protein as a measure of the dissociation of the trimers into single polypeptide chains . Although catalytic trimers exhibit no evident dissociation in sedimentation studies at 10(-8) M, incubation of mixtures of native and succinylated trimers for long periods of time (days) yielded hybrids which are readily detected by polyacrylamide gel electrophoresis . This sensitive technique was used to demonstrate that the substrate, carbamoylphosphate, and the bisubstrate analog, N-(phosphonacetyl)-L-aspartate, cause a marked strengthening of the interchain interactions, whereas the inhibitor, sodium pyrophosphate, at concentrations as low as 10 mM, promotes dissociation of the trimers . This weakening of the interchain interactions by pyrophosphate facilitated the isolation and purification of functionally competent hybrid trimers by a technique which was much more convenient and provided higher yields than previous, more drastic methods which employed urea or guanidine hydrochloride to cause dissociation of the trimers . The hybridization technique was useful in studying the effects of mutational alterations on the strength of the interchain interactions and the ability of active and inactive mutants to bind pyrophosphate. J Biol Chem, 1987 May 15, 262(14), 6921 - 30 Characterization of lipocortin I and an immunologically unrelated 33-kDa protein as epidermal growth factor receptor/kinase substrates and phospholipase A2 inhibitors; Haigler HT et al.; Reversible calcium-dependent association with a particulate fraction from human placenta was used as the first step in the purification of substrates for the epidermal growth factor-stimulated protein kinase . A protein with apparent Mr of 35,000 was purified to homogeneity, and the sequence was determined for approximately one-fourth of the protein . These residues could be aligned exactly with the previously published sequence of lipocortin I derived from the cDNA from a human lymphoma . Two other proteins that appear to be formed by proteolytic removal of 12 or 26 of the amino acids from the NH2 terminus of the protein also were isolated . Placental lipocortin I was phosphorylated in Tyr-21 in an epidermal growth factor-dependent manner by the kinase activity in a particulate fraction from A431 cells; half-maximal phosphorylation occurred at 50 nM lipocortin I . Lipocortin I phosphorylated on Tyr-21 was approximately 10-fold more sensitive to tryptic cleavage at Lys-26 than was the native protein . Placental lipocortin I and its two truncated forms were potent inhibitors of pancreatic phospholipase A2 activity . Another 33-kDa protein that was not related immunologically to lipocortin I or lipocortin II (calpactin I) also was purified from the EGTA extract of placenta . The unidentified protein inhibited phospholipase A2 but was not a substrate for the epidermal growth factor-stimulated kinase . The mechanism by which these proteins inhibit phospholipase A2 activity was investigated . Attempts to detect direct interaction between these proteins and the enzyme were unsuccessful . However, both the unidentified protein, lipocortin I, and 32P-labeled lipocortin I bound in a Ca2+-dependent manner to the {3H}oleic acid-labeled Escherichia coli membranes used as substrate in the phospholipase A2 assay . Heparin, which is known to block lipocortin I inhibition of phospholipase A2, also blocked binding of lipocortin I to E . coli membranes . The results of these and other experiments raise the possibility that placental lipocortin I inhibits phospholipase A2 activity in this assay by coating the phospholipid and thereby blocking interaction of enzyme and substrate. J Biol Chem, 1987 May 15, 262(14), 6877 - 85 Helicase action of dnaB protein during replication from the Escherichia coli chromosomal origin in vitro; Baker TA et al.; Initiation of bidirectional replication from the origin of the Escherichia coli chromosome (oriC) proceeds through stages in which the components of the two replication forks are assembled . From a complex containing proteins dnaA, dnaB, and dnaC bound at oriC, the dnaB helicase moves in both directions to unwind the duplex . In the absence of replication, this unwinding generates a bubble at oriC coated by single strand binding protein . Addition of gyrase allows unwinding to proceed extensively in both directions from oriC at 60 base pairs/s/fork at 37 degrees C . This rate is sharply dependent on temperature and also stimulated by both primase and DNA polymerase III holoenzyme, even in the absence of DNA synthesis . Primer and DNA synthesis are efficient when coupled to template unwinding . DNA synthesis proceeds bidirectionally from oriC at a rate limited by unwinding . With extensive unwinding preceding DNA synthesis, initiations are not limited to oriC. Biochem Biophys Res Commun, 1987 May 14, 144(3), 1143 - 6 Recombinant human granulocyte colony-stimulating factor enhances superoxide release in human granulocytes stimulated by the chemotactic peptide; Kitagawa S et al.; Recombinant human granulocyte colony-stimulating factor (G-CSF) by itself was not an effective stimulus for inducing the release of superoxide (O-2) in human granulocytes . However, G-CSF was able to prime human granulocytes, and enhanced O-2 release stimulated by the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP) . The preincubation with G-CSF for 5-10 min at 37 degrees C was sufficient for priming the cells . The optimal enhancing effect was obtained at 25 ng/ml of G-CSF . The enhancement of O-2 release by G-CSF was observed over the complete range of effective concentrations of FMLP (10(-8)-10(-6) M) . These findings indicate that G-CSF is a potent activator of mature granulocyte functions. FEBS Lett, 1987 May 11, 215(2), 261 - 5 The amphiphilicity of ACP helices: a means of macromolecular interaction? Ernst-Fonberg ML, Tucker MM, Fonberg IB. ACP interacts with diverse proteins in an unknown way . Possibly there is a similar mode of interaction between ACP and all ACP-binding proteins, the amphiphilic helix . The hydrophobicities of helices from 4 different ACPs were compared . Hydrophobic moment plots were prepared for ACP helices and those of many EF hand calcium-binding proteins . Both groups of proteins occupied the same region of the plot. Nucleic Acids Res, 1987 May 11, 15(9), 3743 - 59 Expression of porcine pancreatic phospholipase A2 . Generation of active enzyme by sequence-specific cleavage of a hybrid protein from Escherichia coli; de Geus P et al.; The cDNA coding for the porcine pancreatic prophospholipase A2 (proPLA) has been cloned and expressed in E . coli . Expression of proPLA could only be obtained in the form of intracellular aggregates after fusing the 15 kDa proPLA to a large (greater than or equal to 45 kDa) bacterial peptide . The fusion protein was readily purified from cell lysates, and specifically cleaved . Cleavage of the fusion protein was achieved with either hydroxylamine (at Asn/Gly sequences in the denatured protein), or trypsin (between the pro- and the mature PLA in the renatured fusion protein) . The former method releases a proPLA-like enzyme, while the latter directly yields PLA . Renaturation of the fusion protein was made possible by the use of a recently reported new S-sulphonation method . The released (pro)PLA was purified (yields of 2-3 mg/ltr of culture medium), and showed identical properties compared to native (pro)PLA. FEBS Lett, 1987 May 11, 215(2), 274 - 8 A fragment of an endogenous inhibitor produced in Escherichia coli for calcium-activated neutral protease (CANP) retains an inhibitory activity; Imajoh S et al.; A C-terminal fragment of an endogenous rabbit liver inhibitor for calcium-activated neutral protease (CANP) was produced in Escherichia coli and its inhibitory activity was examined after purification . The truncated inhibitor (373 amino acid residues), which contains two internal repeat structures, inhibits 2 mol CANP whereas the native liver inhibitor (639 residues), containing four internal repeat structures, inhibits 4 mol CANP . This supports the hypothesis that the repeating unit is the functional unit of inhibition . The results also indicate that post-translational modification of the inhibitor is not essential for inhibition. Nucleic Acids Res, 1987 May 11, 15(9), 3653 - 70 Protein binding sites on Escherichia coli 16S RNA; RNA regions that are protected by proteins S7, S14 and S19 in the presence or absence of protein S9; Wiener L et al.; 14C-labelled proteins from E . coli 30S ribosomal subunits were isolated by HPLC, and selected groups of these proteins were reconstituted with 32P-labelled 16S RNA . The isolated reconstituted particles were partially digested with ribonuclease A, and the RNA fragments protected by the proteins were separated by gel electrophoresis and subjected to sequence analysis . Protein S7 alone gave no protected fragments, but S7 together with S14 and S19 protected an RNA region comprising the sequences 936-965, 972-1030, 1208-1262 and 1285-1379 of the 16S RNA . Addition of increasing amounts of protein S9 to the S7/S14/S19 particle resulted in a parallel increase in the protection of the hairpin loop between bases 1262 and 1285 . The results are discussed in terms of the three-dimensional folding of 16S RNA in the 30S subunit. Biophys Chem, 1987 May 9, 26(2-3), 171 - 9 Computer simulation of T3/T7 phage infection using lag times; Buchholtz F et al.; A minimal mechanism is proposed which describes the transcriptional and translational processes for four phage proteins (RNA polymerase, DNase, primase and DNA polymerase) involved in T3/T7 DNA replication . Phage DNA replication is also included . It is shown how lag times may be incorporated into a kinetic mechanism . The distinct three-stage transport of phage DNA into the bacterial host (E . coli) is considered . DNA transport is assumed to be rate-determining for the transcription of class I and II proteins . Transcriptional and translational lag times have been calculated on the basis of available gene mapping of T7 phages . The kinetic behavior of T7 and T3 phage infection is practically identical . The hydrolysis of bacterial DNA by phage DNase (endonculease and exonuclease) as well as the subsequent phosphorylation to the deoxymononucleoside triphosphates are assumed to be rate-determining in phage DNA replication . Good agreement with experiment is obtained in our computer simulations. Biophys Chem, 1987 May 9, 26(2-3), 149 - 61 Complexes of the single-stranded DNA-binding protein from Escherichia coli (Eco SSB) with poly(dT) . An investigation of their structure and internal dynamics by means of electron microscopy and NMR; Greipel J et al.; Based on electron microscopy and NMR spectroscopy it is deduced that Eco SSB binds with moderate cooperativity to polynucleotides . Evidence is provided that the protein binds in its tetrameric form to the nucleic acid forming a nucleosome-like structure . NMR-spectroscopic analysis of the complexes shows that the carboxy-terminal region of the Eco SSB maintains a high flexibility even when the protein is immobilized in large protein-protein clusters. Biophys Chem, 1987 May 9, 26(2-3), 235 - 46 Temperature-gradient gel electrophoresis . Thermodynamic analysis of nucleic acids and proteins in purified form and in cellular extracts; Rosenbaum V et al.; A temperature-gradient gel electrophoresis technique and its application to the study of structural transitions of nucleic acids and protein-nucleic acid complexes are described . The temperature gradient is established in a slab gel by means of a simple ancillary device for a commercial horizontal gel apparatus . The gradient may be freely selected between 10 and 80 degrees C, and is highly reproducible and linear . In a normal application the biopolymers migrate perpendicular to the temperature gradient so that every individual molecule is at constant temperature throughout electrophoresis . The structural transition of a biopolymer is seen as a continuous band which is retarded or speeded up in the temperature range of the transition . Dissociation processes are mostly irreversible under the conditions of electrophoresis and, therefore, show up as discontinuous transitions from a slow-moving to fast-moving band . As examples the conformational transitions of viroids, double-stranded RNA from reovirus, double-stranded satellite RNA from cucumber mosaic virus and repressor-operator complexes have been studied . It could be shown that by this method dsRNA molecules may be differentiated which differ only in one base-pair, or proteins differing in one amino acid only . As a particular advantage, temperature-gradient gel electrophoresis allows the study of conformational transitions of biopolymers which have not been purified . The biopolymer may either be identified by silver staining as a specific band among many others or, if the study is carried out on nucleic acids, these may be recorded by hybridization with a radioactive probe. Cell, 1987 May 8, 49(3), 329 - 36 Parental DNA strands segregate randomly during embryonic development of Caenorhabditis elegans; Ito K et al.; The fate of gamete DNA was followed in the next generation embryos of the nematode C . elegans . Either male worms or spermless hermaphrodites were grown on bromodeoxyuridine-containing E . coli in order to label germ-line DNA . Matings then produced embryos in which only the DNA strands provided by the gametes contained label . This original gamete DNA could be detected during embryonic development by using a fluorescently labeled monoclonal antibody specific to bromodeoxyuridine . Both the number and position of fluorescent spots in the embryo indicate that gamete DNA strands segregate randomly during development . Random segregation of parental DNA strands rules out models of development that invoke chromosome imprinting or immortal DNA strands. Nature, 1987 May 7-13, 327(6117), 77 - 9 Misreading of DNA templates containing 8-hydroxydeoxyguanosine at the modified base and at adjacent residues; Kuchino Y et al.; It has been shown previously that deoxyguanosine residues in DNA are hydroxylated at the C-8 position both in vitro and in vivo to produce 8-hydroxydeoxyguanosine (8-OH-dG) by various agents that produce oxygen radicals such as reducing reagents-O2, metal ions-O2, polyphenol-H2O2-Fe3+, asbestos-H2O2 or ionizing radiation . These agents are mostly either mutagenic or carcinogenic; therefore, the formation of 8-OH-dG can also be considered a likely cause of mutation or carcinogenesis by oxygen radicals . It is of interest to know whether the 8-OH-dG residue in DNA is misread during DNA replication . To answer this question, we have examined the effect of the 8-OH-dG residue in DNA on the fidelity of DNA replication using a DNA synthesis system in vitro with Escherichia coli DNA polymerase I (Klenow fragment) . The synthetic oligodeoxynucleotides, with or without an 8-OH-dG residue in a specified position, were chemically synthesized and used as templates for DNA synthesis under the conditions of the dideoxy chain termination sequencing method . Surprisingly, in addition to misreading of the 8-OH-dG residue itself, pyrimidines next to the 8-OH-dG residue (G has not yet been tested) were also misread. J Biol Chem, 1987 May 5, 262(13), 6082 - 8 Left-handed Z-DNA binding by the recA protein of Escherichia coli; Blaho JA et al.; recA binding to left-handed Z-DNA was measured using nitrocellulose filter binding assays with four DNA polymers with defined nucleotide sequences and four recombinant plasmids . Two to 7-fold preferential binding of recA to Z-DNA polymers was observed . Left-handed Z-DNA polymer binding by recA required ATP or its nonhydrolyzable analog, ATP(gamma S), while ADP inhibited binding . Complex formation with both B- and Z-forms was influenced by polymer length; recA bound longer DNAs better . recA binding to recombinant plasmids containing supercoil-stabilized Z-DNA was essentially similar to that found for the control vector; thus, no preferential binding of recA to the Z-form was observed . Comparative experiments with the rec1 protein of Ustilago maydis and the Escherichia coli recA protein were performed . In our hands, recA and rec1 have a similar capacity for binding left-handed Z-DNA polymers and for binding recombinant plasmids containing B- and/or Z-regions . recA contains a left-handed Z-DNA-stimulated ATPase activity . This activity differs from the right-handed B-DNA-stimulated activity since it is less sensitive to increasing pH . The kinetics of ATP hydrolysis in B-DNA/Z-DNA mixing experiments showed that the turnover of the Z-DNA recA complex was slower than for B-DNA suggesting that left-handed Z-DNA is more stably bound by recA . Our results are consistent with the postulate that left-handed Z-DNA is involved in genetic recombination. J Mol Biol, 1987 May 5, 195(1), 89 - 97 All three elements of the lac ps promoter mediate its transcriptional response to DNA supercoiling; Borowiec JA et al.; The supercoiling response of four closely related promoters was examined in vitro . It was found that changes in all three elements of the lac ps promoter, i.e . the -10 sequence, the -35 sequence, and the spacer length, alter the transcriptional response to DNA supercoiling . Thus, the promoter as a whole, not just the melted region, mediates the supercoiling response . It is proposed that DNA supercoiling changes the structure of the promoter DNA to a form that can be recognized by RNA polymerase and then easily melted . All four promoter variants tested show the same qualitative response to the introduction of DNA supercoiling; that is, transcription is increased compared to relaxed DNA . However, for three of the four promoters, the rate peaked at intermediate levels of supercoiling and declined at higher superhelicities . Each mutation was found to alter both the extent of stimulation that can be achieved and the amount of superhelicity associated with maximal stimulation . The trend is that the stronger promoters are stimulated less, and this maximal stimulation occurs on templates containing fewer superhelical turns . At the level of supercoiling that may pertain in vivo, changes in superhelicity would result in considerable differential changes in the strengths of these promoters. Biochemistry, 1987 May 5, 26(9), 2616 - 23 Selective alteration of substrate specificity by replacement of aspartic acid-189 with lysine in the binding pocket of trypsin; Graf L et al.; To test the role of Asp-189 which is located at the base of the substrate binding pocket in determining the specificity of trypsin toward basic substrates, this residue was replaced with a lysine residue by site-directed mutagenesis . Both rat trypsinogen and Lys-189 trypsinogen were expressed and secreted into the periplasmic space of Escherichia coli . The proteins were purified to homogeneity and activated by porcine enterokinase, and their catalytic activities were determined on natural and synthetic substrates . Lys-189 trypsin displayed no catalytic activity toward arginyl and lysyl substrates . Further, there was no compensatory change in specificity toward acidic substrates; no cleavage of aspartyl or glutamyl bonds was detected . Additional studies of substrate specificity involving gas-phase sequence analyses of digested natural substrates revealed an inherent but low chymotrypsin-like activity of trypsin . This activity was retained but modified by the Asp to Lys change at position 189 . In addition to hydrolyzing phenylalanyl and tyrosyl peptide bonds, the mutant enzyme has the unique property of cleaving leucyl bonds . On the basis of computer graphic modeling studies of the Lys-189 side chain, it appears that the positively charged NH2 group is directed outside the substrate binding pocket . The resulting hydrophobic cavity may explain the altered substrate specificity of the mutant enzyme . The relatively low chymotrypsin-like activity of both recombinant enzymes may be due to distorted positioning of the scissile bond with respect to the catalytic triad rather than to the lack of sufficient interaction between the hydrophobic side chains and the substrate binding pocket of the enzyme. J Biol Chem, 1987 May 5, 262(13), 6357 - 64 Early steps initiating a degradation pathway in Escherichia coli . Characterization of the first intermediate; Wang SS et al.; Our previous studies demonstrated that a site-specific cleavage event initiates the degradation of large premature termination polypeptides of beta-galactosidase in Escherichia coli . We have isolated the first cleavage intermediate, the "B" polypeptide, by elution from sodium dodecyl sulfate-polyacrylamide gels . The NH2 terminus of this protein, determined by automated Edman degradation, was that of the wild-type molecule and thus established that the first cleavage event was at the COOH-terminal end . The sequence of the COOH-terminal end of the B polypeptide was determined by using the enzyme carboxypeptidase Y . Direct assignment of COOH-terminal residues was made by using o-phthaldialdehyde derivatization and the stoichiometry confirmed by a double-label analysis . The COOH-terminal end of the B polypeptide is at position 837 in the beta-galactosidase sequence . If a single endoproteolytic cleavage event was responsible, the cleavage would have occurred between 2 threonine residues (at positions 837 and 838) that are located within a hydrophobic domain . We have observed other covalent modifications that precede the appearance of the B polypeptide, but these do not appear to participate in signaling the first cleavage event . The structure of the COOH-terminal end of B suggests a high degree of specificity by the initial cleavage enzyme . We propose that this unique site serves as a specific signal and that exposure of this site to the specific cleavage enzyme controls the event initiating the degradation pathway. J Biol Chem, 1987 May 5, 262(13), 6389 - 95 Constitutive function of a positively regulated promoter reveals new sequences essential for activity; Keilty S et al.; A consensus "-10" recognition sequence for RNA polymerase was created at the positively regulated lambda Pre promoter by introducing three single base pair mutations . This altered promoter, Pre*, functions constitutively in vivo and in vitro at high efficiency despite very poor consensus "-35" region sequence homology . We examined the influence of the -35 region sequence information on promoter function by shifting the wild type -35 region +/- 2 base pairs relative to the -10 region consensus sequence and by completely replacing it with alternative DNA sequences . In every case, the altered Pre* promoters retained transcriptional activity although differences in their transcriptional efficiencies were observed . Apparently the Pre* promoter does not require specific -35 region sequences for constitutive promoter activity, although the -35 region sequences can modulate overall promoter strength . In addition, by point mutation analysis we have identified bases immediately upstream of the -10 hexamer which are essential for constitutive function of the Pre* promoter . We propose that these mutants define an extended -10 region at Pre* that compensates for its poor -35 region sequence information by providing critical contacts that stabilize productive RNA polymerase binding. J Biol Chem, 1987 May 5, 262(13), 6039 - 45 Sites of covalent modification in Trg, a sensory transducer of Escherichia coli; Nowlin DM et al.; The Trg protein mediates chemotactic response of Escherichia coli to the attractants ribose and galactose . Like other transducers, Trg is a transmembrane protein that undergoes post-translational covalent modification . The modifications are hydrolysis (deamidation) of certain glutamine side chains to create glutamate residues and methylation of specific glutamates to form carboxyl methyl esters . Analysis of radiolabeled, tryptic peptides by high performance liquid chromatography and gas-phase sequencing allowed direct identification of the modified residues of Trg . The protein has 5 methyl-accepting residues . Four, at positions 304, 310, 311, and 318, are contained in a 23-residue tryptic peptide ending in lysine . The fifth, at position 500, is within a 25-residue tryptic peptide ending in arginine . At two sites, 311 and 318, glutamines are deamidated to create methyl-accepting glutamates . There is not a required order of modification among the sites . However, there is a substantial preference for methylation on the arginine peptide and, among sites on the lysine peptide, for the middle pair . Comparison of sequences surrounding modified residues identified in this work for Trg and previously for Tsr and Tar suggests a consensus sequence for methyl-accepting sites of Ala/Ser-Xaa-Xaa-Glu-Glu*-Xaa-Ala/OH-Ala-OH/Ala, where OH signifies Ser or Thr and the asterick marks the site of modification. J Mol Biol, 1987 May 5, 195(1), 75 - 87 Double-chain-cut sites are recombination hotspots in the Red pathway of phage lambda; Thaler DS et al.; The Red recombination pathway of phage lambda is shown to target recombination to double-chain ends of DNA . A double-chain cut, delivered in vivo to only one of two parents participating in a lambda lytic cross by a type II restriction endonuclease, increases the proportion of crossing over in the interval containing the cut compared with other intervals . The stimulating effect of a cut is evident whether replication is inhibited or permitted . Cut stimulation can move away from the initial cut-site, presumably by double-chain degradation . Movement of the stimulating effect of a cut is dependent on the Escherichia coli gene recA when the cross is carried out under conditions that inhibit phage replication . When replication is permitted, all aspects of cut-stimulated recombination are independent of recA . Evidence is presented to show that the reaction that is stimulated by cutting is often non-reciprocal at the molecular level. J Biol Chem, 1987 May 5, 262(13), 6301 - 7 The defective proton-ATPase of uncD mutants of Escherichia coli . Identification by DNA sequencing of residues in the beta-subunit which are essential for catalysis or normal assembly; Parsonage D et al.; Six mutant uncD alleles, affecting essential residues of the beta-subunit of Escherichia coli proton-ATPase, have been identified by intragenic complementation mapping, cloning, and DNA sequencing . Five of the mutations impair catalysis but do not cause structural perturbation of F1-ATPase . The amino acid substitutions found were as follows: uncD412, Gly-142----Ser; uncD430 and uncD431, both Arg-246----Cys; uncD478, Ser-174----Phe; and uncD484, Met-209----Ile . Kinetic characteristics of each corresponding mutant F1-ATPase are described or reviewed . In each case, the major determinant of impaired catalysis appears to be an attenuation of positive catalytic site cooperativity . Additionally, each mutation affects intrinsic properties of the catalytic site, including affinity for ATP, the ratio between unisite-bound substrate and products, and the rate of release of product inorganic phosphate under unisite ATP hydrolysis conditions . These effects are discussed in terms of a structural model of the catalytic nucleotide-binding domain of beta-subunit proposed recently (Duncan, T.M., Parsonage, D., and Senior, A.E . (1986) FEBS Lett . 208, 1-6) . Each of the mutations lies within that domain . The uncD409 allele abolishes normal assembly of F1-ATPase . The amino acid substitution is Gly-214----Arg, which is suggested to affect a beta-turn connecting a beta-strand and an alpha-helix in the predicted nucleotide-binding domain of the beta-subunit. J Mol Biol, 1987 May 5, 195(1), 215 - 8 Increased production of a knotted form of plasmid