Mutat Res, 1987 Mar, 183(2), 103 - 8 Induction of SOS responses in Escherichia coli by 5-fluorouracil; Oda Y; The inducibility of SOS responses by 5-fluorouracil (5-FU), which has been used as an antitumor drug, was studied in Escherichia coli cells which have different DNA repair capacities for UV lesions . Expression of the umuC gene was apparently induced by 5-FU in the wild-type and uvrA strains, but not in lexA and recA strains . The inducibility of the umuC gene by 5-FU, the metabolite of which inhibits thymidylate synthetase, was abolished in cultures containing deoxythymidine monophosphate which is converted from deoxyuridine monophosphate by thymidylate synthetase . These results suggest that 5-FU may exert its SOS inducibility by inhibiting thymidylate synthetase and then disturbing DNA metabolism but not by incorporating 5-FU residues into RNA . Further, 5-FU weakly induced mutations in E . coli. J Immunol, 1987 Mar 1, 138(5), 1542 - 5 Diacylglycerol mass measurements in stimulated HL-60 phagocytes; Preiss JE et al.; The mass of sn-1,2-diacylglycerol in crude lipid extracts from differentiated HL-60 phagocytes was measured by quantitative conversion of the diacylglycerol to {32P}-labeled phosphatidic acid catalyzed by E . coli diacylglycerol kinase . The chemotactic peptide N-formyl-Met-Leu-Phe caused a time- and concentration-dependent increase in diacylglycerol that was maximal at 4 min . Diacylglycerol returned toward basal levels by 15 min . The basal level of diacylglycerol was 290 +/- 25 pmol/10(7) cells (n = 36) . Maximally effective concentrations of N-formyl-Met-Leu-Phe and N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys increased diacylglycerol to 176% +/- 16 of basal (n = 8) and 198% +/- 15 of basal (n = 4), respectively . t-Boc-Phe-Leu-Phe-Leu-Phe, a competitive antagonist of formyl peptide receptor function, competitively inhibited the N-formyl-Met-Leu-Phe-induced diacylglycerol increase . Pretreatment of the cells with pertussis toxin abolished the stimulated rise in diacylglycerol, whereas depletion of extracellular Ca2+ markedly inhibited the increase . The Ca2+ ionophore A23187 stimulated a large (450% of basal) and persistent (greater than 30 min) increase in diacylglycerol . These data suggest that agents which raise intracellular Ca2+ levels in differentiated HL-60 cells produce a prolonged increase in cellular diacylglycerol which may activate protein kinase C. J Korean Med Sci, 1987 Mar, 2(1), 65 - 70 Purification of heat-labile enterotoxin from an enterotoxin from an enterotoxigenic Escherichia coli of human origin by monoclonal immunoaffinity chromatography; Cho MJ et al.; Heat-labile enterotoxin (LT) was purified from an enterotoxigenic Escherichia coli 015H11 of human origin . The purification steps included French pressure cell disruption of the bacteria, salting-out, DEAE-Sephacel on chromatography . Application of this procedure resulted in a 95.1-fold purification of LT with a yield of 19.9% as determined by rabbit ileal loop assay . The final LT preparation showed only one protein-staining band on polyacrylamide gel electrophoresis, indicating that the purified LT was homogeneous. Plasmid, 1987 Mar, 17(2), 117 - 36 Characterization of the maintenance functions of IncFIV plasmid R124; Campbell IG et al.; The genetic arrangement of the regions involved in R124 replication and incompatibility have been located and their homology to the IncFI basic replicons has been assessed . We show that R124 has homology with all three basic replicons, RepFIA, RepFIB, and RepFIC, and that these regions, FIVA, RepFIVB, and RepFIVC, are widely separated on the R124 genome . Cloning of autonomously replicating fragments has shown that RepFIVB and RepFIVC are functional in R124 and express incompatibility . The FIVA region was unable to form a functional replicon and when cloned into pUC8 lacked incompatibility activity . A fourth region of R124 was identified, which although not essential for replication stabilized mini-R124 plasmid replication and exhibited incompatibility with R124 . This region, designated IncIV, showed no homology to RepFIA, RepFIB, or RepFIC . Incompatibility expression of IncIV required only the EcoRI fragment E13 but the strength of the reaction was modified in the presence of other fragments . The replication and incompatibility properties of an R124 deletion derivative indicated that R124 can switch its replication to either RepFIVB or RepFIVC when in the presence of an incompatible plasmid . The ambiguous incompatibility reactions reported for R124 is a result of the expression of the two functional replicons, RepFIVB and RepFIVC, and that expressed by IncIV. Bioorg Khim, 1987 Mar, 13(3), 350 - 8 {Expressing plasmid vectors on the basis of Escherichia coli beta-galactosidase gene fragments}; Chakhmakhcheva OG et al.; With the use of synthetic DNA fragments, a set of new plasmid vectors has been obtained . The vectors provided high level expression of peptides and small proteins in E . coli as fusions with fragments of beta-galactosidase of various length . These vectors were used to achieve expression of a synthetic gene for a functionally active fragment of bacteriorhodopsin . The yields of hybrid proteins consisting of beta-galactosidase and bacteriorhodopsin fragments were in the range of 5-30% from the total amount of cellular protein. Biochem J, 1987 Mar 1, 242(2), 539 - 50 Galactoside-proton symport in a lacYUN mutant of Escherichia coli investigated by analysis of transport progress curves; Page MG; The kinetics of galactoside-proton symport catalysed by a wild-type strain and one carrying a mutation, previously reported to cause uncoupling of the symport reaction, have been examined . The mutation does not affect the stoichiometry during the initial period of uptake, when the internal concentration of galactoside is low, but it does result in much greater competition from the galactoside as it is accumulated . Simple methods for the analysis of the uptake progress curves have been developed and used to estimate the initial rate of uptake and affinity for internal galactoside . The maximum rate of uptake is decreased by a factor of 2 at most whereas the affinity for internal galactoside is increased up to 50-fold by the mutation . The pH-dependence of the galactoside efflux reaction is changed in a manner which suggests that the defect is in the interaction between proton-binding and galactoside-binding sites rather than in the structure of either site. Mol Gen Genet, 1987 Mar, 206(3), 519 - 21 Replication of mini-F plasmid in vitro promoted by purified E protein; Muraiso K et al.; An in vitro system for replication of mini-F plasmid DNA was constructed . This system consists of an ammonium sulfate fraction II (Fuller et al . 1981) from Escherichia coli extract, exogenously added purified E protein encoded by mini-F plasmid, and mini-F DNA in a closed circular form . Experiments with this system showed that the 217 bp DNA region which contains the A + T rich cluster and the four 19 bp direct repeats responsible for incB incompatibility is essential for mini-F DNA replication. Mol Gen Genet, 1987 Mar, 206(3), 505 - 9 Isolation and characterization of the regulatory HEX2 gene necessary for glucose repression in yeast; Niederacher D et al.; The HEX2 gene which is necessary for glucose repression and is involved in the regulation of hexokinase PII synthesis and maltose uptake, has been cloned by complementation of a hex2 mutant, and selection for restored growth on maltose . Glucose repression in the transformants was like that in the wild type . The HEX2 gene was localized within a 2.15 kb fragment . The restriction map was confirmed by Southern hybridization of genomic DNA . Based on 30 tetrads, the linkage between HEX2 and TRP1 was determined as 10 cM . Plasmid integration directed to the genomic site of the cloned gene also gave a similar linkage distance between the amino acid auxotroph plasmid marker and genomic TRP1 . Gene disruption of HEX2 yielded nonrepressible transformants with elevated hexokinase PII activity showing inhibition by maltose; this provides clear evidence that the HEX2 gene has been isolated. Mol Gen Genet, 1987 Mar, 206(3), 452 - 9 Cloning and characterization of the immunity region of phage phi 80; Coste G et al.; The immunity region of phage phi 80 has been localized . It codes for at least three proteins: a protein of 34 kDa which has the biological properties of the phage repressor, and two other proteins of 9 kDa and 18 kDa which are the first proteins on the rightward operon . These two proteins are negatively regulated by the 34 kDa protein at a divergent promoter site . By position analogy with phage lambda, but not by its biological activity, the 9 kDa protein could be the cro product . The 18 kDa protein is able to block totally UV induction of phage phi 80. Mol Gen Genet, 1987 Mar, 206(3), 419 - 27 Comparison of the organisation of the genomes of phenotypically diverse plasmids of incompatibility group P: members of the IncP beta sub-group are closely related; Smith CA et al.; Comparison of physical maps of the broad host range plasmids R751, R906 and R772, belonging to the IncP beta sub-group of the Escherichia coli incompatibility group P, reveals two large regions of similarity, separated by dissimilar regions which contain the majority of the cleavage sites for restriction endonucleases with hexanucleotide recognition sites . Mapping of the regions of these plasmids which show homology to probes specific for genetically characterised segments of the distantly related IncP alpha plasmid RK2, involved in plasmid maintenance or conjugal transfer, reveals that all four plasmids share a similar genetic organisation . In each case the homologous plasmid backbone is interrupted by heterologous segments both between the essential replication loci oriV and trfA, and between the conjugal transfer regions tra1 and tra2, although in the case of R772 the segment of the backbone carrying the trfA and tra2 regions is inverted relative to that of the other plasmids . However, in the case of pJP4, shown to be a fourth member of the IncP beta sub-group, the backbone is interrupted only by a single large segment adjacent to the trfA region . Mapping of the regions of the four IncP beta plasmids which show homology to Tn501 and nucleotide sequence determination at the ends of the homologous regions reveals that R906, R772 and pJP4 share a common mercury resistance region . This region, which appears to have been inactivated in R772, was probably inserted into a common ancestor of these plasmids by the transposition of an element related to an ancestor of Tn501 . R751 shows no trace of the mercury resistance region, but contains a short relict of Tn501, derived from an independent insertion event. Mol Gen Genet, 1987 Mar, 206(3), 368 - 76 Regulation of the ban gene containing operon of prophage P1; Heisig A et al.; A physical map of the ban gene of P1 and sites relevant to its regulation has been deduced from cloning of the appropriate regions of P1 wild-type and of P1 ban regulatory mutants . The cloning required the presence of P1 repressor in the cell confirming the existence of a repressible ban operon (Austin et al . 1978) . Evidence for additional member(s) of that operon is presented . Of particular interest for understanding the regulation of ban are the relative positions of a binding site for the P1 repressor and of the regulatory mutations bac and crr that render ban expression constitutive . The results reveal a repressible operon-like structure of about 4 kb within the P1 EcoRI-3 fragment that comprises a c1 repressor binding site/bac - additional gene(s) - crr/ban in the clockwise direction of the circular map of P1. EMBO J, 1987 Mar, 6(3), 737 - 42 Yeast DNA polymerase--DNA primase complex; cloning of PRI 1, a single essential gene related to DNA primase activity; Lucchini G et al.; The immunopurified yeast DNA polymerase--DNA primase complex is constituted by DNA polymerase I polypeptides and by three other protein species, called p74, p58 and p48, which we show to be immunologically unrelated . The gene encoding the p48 polypeptide has been identified by immunological screening of a lambda gt11 yeast genomic DNA library . Antiserum specific for p48 inhibits DNA primase, and immunoreactive, inhibitory antibodies are affinity-purified by the clone-encoded protein, thus relating the p48 polypeptide to DNA primase activity . The entire gene has been cloned, and the 1.45-kb p48 mRNA is overproduced in cells containing the gene in high copy number . Gene disruption and Southern hybridization experiments demonstrate that the p48 protein is encoded by a single gene and it performs an essential function. EMBO J, 1987 Mar, 6(3), 729 - 36 The product of the mei3+ gene, expressed under control of the mating-type locus, induces meiosis and sporulation in fission yeast; McLeod M et al.; In fission yeast the ability to undergo meiosis and sporulation is conferred by the matP+ and matM+ genes of the mating-type locus . Inactivation of ran1+, a negative regulator of meiosis, is thought to be an essential step in meiotic initiation . We have isolated a further meiotic control gene mei3+, and have shown the following: a null allele of mei3 totally inhibits meiosis; the mei3+ RNA transcript and its translational product are expressed only in matP+/matM+ diploids entering meiosis; forced expression of mei3+ in vegetative cells provokes haploid meiosis and sporulation . We suggest that the product of mei3+ gene, a protein of 21 kd, initiates meiosis by inactivating ran1+. EMBO J, 1987 Mar, 6(3), 713 - 21 Three suppressor mutations which cure a mitochondrial RNA maturase deficiency occur at the same codon in the open reading frame of the nuclear NAM2 gene; Labouesse M et al.; Dominant mutations of the nuclear NAM2 gene are able to compensate for a deficiency of the maturase encoded by the fourth intron of the mitochondrial cytochrome b gene . We have determined the complete nucleotide sequence of the NAM2-1 suppressor allele . The results of S1 nuclease protection experiments show that two overlapping poly(A)+ RNAs are transcribed from the gene using different promoters . The longer transcript contains two open reading frames (ORFs), a long ORF which could encode a protein of 894 amino acids, mol . wt 102,000 daltons, and a short ORF of 51 codons which is omitted from the shorter transcript . The wild-type nam2+ and two other suppressor alleles, NAM2-6 and NAM2-7, have been cloned . A comparison of the sequence of the wild-type and the three suppressor alleles shows that on three separate occasions the same codon specifying glycine was mutated (once to serine and twice to cysteine) . Finally sequence comparisons identified two regions in the long ORF, distinct from the position of the suppressor mutations, that could correspond to binding domains for a nucleotide and a nucleic acid. Anal Biochem, 1987 Mar, 161(2), 272 - 9 Direct mapping of rare messenger RNAs by means of oligomer-directed ribonuclease H cleavage; Berger SL; A method has been developed for characterizing rare messenger RNAs in the bulk population by using oligodeoxyribonucleotide: RNA hybrids as substrates for Escherichia coli ribonuclease H . Two 1.3-kb mRNAs in lymphocyte cytoplasm, interferon-gamma (0.002% of polyadenylated mRNA), and prothymosin-alpha, have been studied . Interferon-gamma mRNA was cut virtually completely into two fragments, each about 0.6 kb in length, by using an interferon-specific 24-mer to direct cleavage . Prothymosin-alpha mRNA in the same bulk population was unaffected by this treatment . When the 24-mer was replaced by a 12-mer, whose sequence was based on an incomplete cDNA clone for prothymosin-alpha, the products included two fragments of prothymosin-alpha mRNA . The sum of the fragment lengths equaled the length of the mRNA . Although the reaction directed by the smaller oligomer did not go to completion, the 12-mer, and hence the cDNA clone from which it was derived, could nevertheless be oriented with respect to prothymosin-alpha mRNA . With this technique, sequences in mRNA can be mapped without first isolating full-length cDNA clones. Mol Gen Mikrobiol Virusol, 1987 Mar, (3), 3 - 6 {Detailed mapping of the Hly-region of the plasmid pHly241 . Homology of the hemolysis determinants of plasmids pHly241 and pHly195}; Berezkina NE et al.; Data on the hemolysin synthesis determined by the plasmid pHly241, belonging to the incompatibility group I2, and its derivatives are presented in this paper . The restriction analysis of the pHly241 plasmid has resulted in the detailed mapping of the hly determinant region in the plasmid . The substantial homology has been demonstrated between the regions of the plasmids pHly241 and pHly195 coding for hemolysin synthesis and excretion from the bacterial cell. Genetika, 1987 Mar, 23(3), 397 - 404 {Plasmid vectors of "insertion inactivation" of the trimethoprim resistance marker}; Gorovits RL et al.; We demonstrate the possibility of using the T4 phage frd gene as an insertion inactivation marker within pBR322, in plasmids with changing copy number and expression of foreign genes under control . The structural part of the frd genes contains unique recognition sites for EcoRI and SalI endonucleases . Transformants with recombinant plasmids carrying the frd gene grow on media with up to 500 mkg/ml trimethoprim, whatever the gene dosage. Proc Natl Acad Sci U S A, 1987 Mar, 84(5), 1215 - 8 The E2 "gene" of bovine papillomavirus encodes an enhancer-binding protein; Moskaluk C et al.; The E2 early open reading frame (presumably gene) of bovine papillomavirus-1 was fused in frame with the collagen-beta-galactosidase-encoding region of the vector pJG200 and was expressed in and partially purified from Escherichia coli . The hybrid protein specifically bound to the enhancer region of bovine papillomavirus at several sites . DNase I-cleavage protection analysis of one such site revealed the protected sequence . A comparison of the protected sequence with the remainder of the DNA sequences that also have affinity for the protein revealed a consensus sequence having the motif AATTGGCGGNNCG, in which N is any nucleotide . The protected region also includes a sequence with 2-fold rotational symmetry--ATCGGTG/CACCGAT. Proc Natl Acad Sci U S A, 1987 Mar, 84(5), 1210 - 4 Regulation of c-myc and c-fos mRNA levels by polyomavirus: distinct roles for the capsid protein VP1 and the viral early proteins; Zullo J et al.; The levels of c-myc, c-fos, and JE mRNAs accumulate in a biphasic pattern following infection of quiescent BALB/c 3T3 mouse cells with polyomavirus . Maximal levels of c-myc and c-fos mRNAs were seen within 1 hr and were nearly undetectable at 6 hr after infection . At 12 hr after infection mRNA levels were again maximal and remained elevated thereafter . Empty virions (capsids) and recombinant VP1 protein, purified from Escherichia coli, induced the early but not the late phase of mRNA accumulation . Virions, capsids, and recombinant VP1 protein stimulated {3H}thymidine nuclear labeling and c-myc mRNA accumulation in a dose-responsive manner paralleling their affinity for the cell receptor for polyoma . The second phase of mRNA accumulation is regulated by the viral early gene products, as shown by polyomavirus early gene mutants and by a transfected cell line (336a) expressing middle tumor antigen upon glucocorticoid addition . These results suggest that polyomavirus interacts with the cell membrane at the onset of infection to increase the levels of mRNA for cellular genes associated with cell competence for DNA replication, and subsequently these levels are maintained by the action of the early viral proteins. Mutat Res, 1987 Mar, 177(1), 17 - 26 Induction of the SOS system in a dam-3 mutant: a diagnostic strain for chemicals causing DNA mismatches; Quillardet P et al.; We have developed a strain of E . coli in which expression of the SOS function sfiA, monitored by means of a sfiA::lacZ operon fusion, is efficiently triggered by the two base analogues 2-aminopurine and 5-bromo-2'-deoxyuridine . This strain resulted from introduction of a dam-3 mutation into a Uvr+, Rfa+ derivative of strain PQ37 used in the SOS chromotest, a bacterial colorimetric assay for genotoxins (Quillardet et al., 1982) . The dam-3 mutation affects the mismatch correction system in E . coli . We show that the SOS-inducing capacity of a weak SOS inducer such as the alkylating agent ethyl methanesulfonate was also increased in the dam-3 strain . We provide evidence that the increase in SOS inducibility due to the dam-3 mutation is specific for compounds causing DNA mismatches and propose the use of the dam-3 derivative of PQ37 as a diagnostic strain for such agents . This diagnostic strain can be a useful addition to the SOS chromotest. J Gen Virol, 1987 Mar, 68 ( Pt 3), 633 - 42 Synthesis in Escherichia coli and immunological characterization of a polypeptide containing the cleavage sites associated with trypsin enhancement of rotavirus SA11 infectivity; Arias CF et al.; About 45% of the rotavirus SA11 VP3 gene was inserted into a thermoinducible expression plasmid under the control of phage lambda PL promoter . The primary translation product predicted on the basis of the plasmid construction was a hybrid protein in which the 98 amino-terminal amino acids of phage MS2 polymerase were followed by amino acids 42 to 387 of the VP3 protein, which included the region containing the cleavage sites associated with trypsin enhancement of infectivity . On induction, a polypeptide that had the expected mol . wt . and contained VP3-related amino acid sequences as judged by immunological criteria, was synthesized to a level representing about 15% of the total bacterial protein . When a bacterial lysate enriched for the fusion polypeptide was injected into mice, it induced antibodies which inhibited haemagglutination and neutralized SA11 rotavirus infectivity. J Bacteriol, 1987 Mar, 169(3), 981 - 9 Capsule synthesis in Escherichia coli K-12 is regulated by proteolysis; Torres-Cabassa AS et al.; lon mutants of Escherichia coli K-12 are defective in an ATP-dependent protease, are UV sensitive, and overproduce the capsular polysaccharide colanic acid . Six structural genes needed for capsular polysaccharide synthesis (cps) are transcriptionally regulated by lon as well as by three other regulatory genes, rcsA, -B, and -C (S . Gottesman, P . Trisler, and A . S . Torres-Cabassa, J . Bacteriol . 162:1111-1119, 1985) . We have cloned rcsA, the gene for a positive regulator of capsule synthesis, onto multicopy plasmids and defined the gene by both insertions and deletions . The product of rcsA has been identified as an unstable protein of 27 kilodaltons . RcsA has a half-life of 5 min in lon+ cells and one of 20 min in lon cells . The availability of RcsA is the limiting factor for capsule synthesis; doubling the gene dosage of rcsA+ significantly increases expression of cps genes . Our results are consistent with a model in which the presence of a lon mutation increases the synthesis of capsular polysaccharide via stabilization of RcsA. J Bacteriol, 1987 Mar, 169(3), 1286 - 90 secD, a new gene involved in protein export in Escherichia coli; Gardel C et al.; New mutants of Escherichia coli altered in protein export were identified in phoA-lacZ and lamB-lacZ gene fusion strains by searching for mutants that showed an altered lactose phenotype . Several mutations mapped in a new gene, secD . These mutants were, in general, cold sensitive for growth, and the mutations led to an accumulation of precursor of exported proteins . The secD gene is closely linked to tsx on the E . coli chromosome, but separable from another gene proposed to be involved in export, ssaD, which maps nearby . A plasmid carrying secD+ was identified and used to show that the mutations are recessive . The secD gene may code for a component of the cellular export machinery. J Bacteriol, 1987 Mar, 169(3), 1272 - 8 Fusions of the Escherichia coli gyrA and gyrB control regions to the galactokinase gene are inducible by coumermycin treatment; Menzel R et al.; We have previously shown that the genes encoding the two subunits of Escherichia coli DNA gyrase are regulated in a manner which is dependent on DNA conformation . When the DNA encoding the gyrA and gyrB genes is relaxed, both genes are expressed at a high level; in negatively supercoiled DNA they are expressed at a low level . In this paper we describe fusions of both the gyrA and gyrB 5' sequences to the E . coli galactokinase gene . In such fusions we found that galactokinase can be induced by treating the cells with coumermycin A1, an inhibitor of DNA gyrase . Our results suggest that the regulation occurs at the transcriptional level and that only a small region of DNA is necessary for coumermycin-induced gene expression. J Bacteriol, 1987 Mar, 169(3), 1254 - 9 Influence of GATC sequences on Escherichia coli DNA mismatch repair in vitro; Lu AL; The effect of the number and position of DNA adenine methylation (dam) sites, i.e., d(GATC) sequences, on mismatch repair in Escherichia coli was investigated . The efficiency of repair was measured in an in vitro assay which used an f1 heteroduplex containing a G/T mismatch within the single EcoRI site . Both an increase in the number of dam sites and a shortened distance between dam site and mismatched site increased the efficiency of mismatch repair . The sequences adjacent to d(GATC) also affected the efficiency of methylation-directed mismatch repair . Furthermore, heteroduplexes with one extra dam site located close to either the 5' or 3' end of the excised base increased the repair efficiency to about the same extent . The findings suggest that the mismatch repair pathway has no preferred polarity. J Bacteriol, 1987 Mar, 169(3), 1246 - 53 Chemotaxis in Escherichia coli: construction and properties of lambda tsr transducing phage; Callahan AM et al.; The tsr gene of Escherichia coli, located at approximately 99 min on the chromosomal map, encodes a methyl-accepting protein that serves as the chemoreceptor and signal transducer for chemotactic responses to serine and several repellents . To determine whether any other chemotaxis or motility genes were located in the tsr region, we constructed and characterized two lambda tsr transducing phages that each contain about 12 kilobases of chromosomal material adjacent to tsr . lambda tsr70 carries sequences from the promoter-proximal side of tsr; lambda tsr72 carries sequences from the promoter-distal side of tsr . Restriction maps of the bacterial inserts in these phages and Southern hybridization analyses of the bacterial chromosome indicated that the tsr gene is transcribed in the counterclockwise direction on the genetic map . Insert deletions were isolated in lambda tsr70 and transferred into the host chromosome to examine the null phenotype of tsr . All such strains exhibited wild-type swimming patterns and chemotactic responses to a variety of stimuli, but were specifically defective in serine taxis and other Tsr-mediated responses . In addition, UV programming experiments demonstrated that Tsr and several of its presumptive degradation products were the only bacterial proteins encoded by lambda tsr70 and lambda tsr72 that required host FlbB/FlaI function for expression . These findings indicate that there are probably no other chemotaxis-related genes in the tsr region . A series of tsr point mutations were isolated by propagating lambda tsr70 on a mutD host and used to construct a fine-structure map of the tsr locus . These mutations should prove valuable in exploring structure-function relationships in the Tsr transducer. J Bacteriol, 1987 Mar, 169(3), 1174 - 81 Multiple SecA protein isoforms in Escherichia coli; Liebke HH; To define the anti-SecA-LacZ antiserum, immunoprecipitates produced with either whole anti-SecA-LacZ rabbit antiserum or affinity-purified antibodies were used to analyze nondenatured lysates of Escherichia coli . The antiserum contains antibodies that recognize different proteins . Antibody purified by preadsorption to the SecA-LacZ hybrid protein precipitated only the SecA protein from extracts . In contrast, antibody purified from the intact SecA protein precipitated several additional proteins with SecA protein . Ribosomal protein L7L12 is one of the polypeptides coprecipitated with SecA protein by antibody purified by immunoadsorption to the intact SecA protein as well as by unfractionated anti-SecA-LacZ antiserum . Two-dimensional gel electrophoresis of the SecA protein immunoprecipitated by either antiserum or purified antibody indicated that the SecA protein exists in at least two, and probably four, isoforms . Only one of the SecA isoforms is present in a ribosomal preparation. J Bacteriol, 1987 Mar, 169(3), 1080 - 8 The 65-kilodalton antigen of Mycobacterium tuberculosis; Shinnick TM; The immune response of the host to the antigens of Mycobacterium tuberculosis plays the key role in determining immunity from infection with as well as the pathogenicity of this organism . A 65-kilodalton (kDa) protein has been identified as one of the medically important antigens of M . tuberculosis . The gene encoding this antigen was isolated from a lambda gt11-M . tuberculosis recombinant DNA library using monoclonal antibodies directed against the 65-kDa antigen as the specific probes . The nucleotide sequence of this gene was determined, and a 540-amino-acid sequence was deduced . This sequence was shown to correspond to that of the 65-kDa antigen by constructing a plasmid in which this open reading frame was fused to the lacZ gene . The resulting fusion protein reacted specifically with the anti-65-kDa protein antibodies . A second long open reading frame was found downstream of the 65-kDa antigen gene which could encode a protein of 517 amino acids . This putative protein contained 29 tandemly arranged partial or complete matches to a pentapeptide sequence. J Bacteriol, 1987 Mar, 169(3), 1061 - 70 Structural analysis of the Escherichia coli K-12 hisT operon by using a kanamycin resistance cassette; Arps PJ et al.; We constructed a series of recombinant plasmids containing a kanamycin resistance (Kmr) cassette upstream from, within, and downstream from hisT, which encodes the tRNA modification enzyme pseudouridine synthase I . These Kmr insertions were then crossed directly into the bacterial chromosome . We determined growth characteristics, assayed in vivo hisT expression, and mapped in vivo hisT operon transcripts for the Kmr insertion mutants . We also analyzed polypeptides synthesized in minicells from plasmids containing Kmr cassettes . The combined results from these experiments demonstrate new features concerning the structure and expression of the complex operon that contains hisT . We show that the minimum size of the operon is approximately 3,500 base pairs and that it contains at least four genes, which are arranged in the order usg-2 (pdxB), usg-1, hisT, and dsg-1 and encode polypeptides with apparent molecular masses of 42,000, 45,000, 31,000, and 17,000 daltons, respectively . Of these genes, only the functions of usg-2 (pdxB) and hisT are known, and genetic evidence suggests that these two genes do not require usg-1 or dsg-1 for function, usg-2 (pdxB) is required for growth of bacteria on minimal medium at 37 degrees C . In contrast, the three genes at the end of the hisT operon are dispensable and form a transcription unit that is expressed from a relatively strong internal promoter . The phenotypes of the Kmr insertion mutants and results from gene expression experiments further confirm the position of the internal promoter and locate additional genetic signals in the DNA sequence around hisT . The experiments reported here also indicate several interesting properties of the Kmr cassette as a tool for probing complex operons. J Bacteriol, 1987 Mar, 169(3), 1056 - 60 In vivo D-serine deaminase transcription start sites in wild-type Escherichia coli and in dsdA promoter mutants; Bornstein-Forst SM et al.; The D-serine deaminase structural (dsdA) and regulatory (dsdC) genes are transcribed with opposite polarity from an intergenic region comprising more than 600 base pairs . The order of genes in the dsd region is supN-dsdA-dsdC-aroC---his . The DNA sequence of the intergenic region has been slightly revised from a previously published version (E . McFall and L . Runkel, J . Bacteriol . 154:1508-1512, 1983) . The dsdA gene is preceded by a long open reading frame . The dsdA in vivo transcription start sites for the wild type (base pair +1) and for three phenotypically distinct promoter constitutive mutants were determined by the S1 nuclease method . They are identical and are located about 81 base pairs upstream of the translation start site . D-Serine deaminase regulation is normal in rho mutants . Possible mechanisms for dsdA activation are discussed. J Bacteriol, 1987 Mar, 169(3), 1029 - 36 Molecular cloning and characterization of the Streptomyces hygroscopicus alpha-amylase gene; Hoshiko S et al.; We have isolated and sequenced a gene (amy) coding for alpha-amylase (EC 3.2.1.1.) from the Streptomyces hygroscopicus genome (H . Hidaka, Y . Koaze, K . Yoshida, T . Niwa, T . Shomura, and T . Niida, Die Starke 26:413-416, 1974) . Amylase was purified to obtain amino acid sequence information which was used to synthesize oligonucleotide probes . amy-containing Escherichia coli cosmids identified by hybridization did not express amylase activity . Subcloning experiments indicated that amy could be expressed from the lac promoter in E . coli or from its own promoter in S . lividans . The amy nucleotide sequence indicated that it coded for a protein of 52 kilodaltons (478 amino acids) . Secreted alpha-amylase contained amino- and carboxy-terminal as well as internal amino acid sequences which were consistent with the nucleotide sequence . The 30-residue leader sequence showed similarities to those found in other procaryotes . The DNA sequence 5' to the amy structural gene contained a sequence complementary to the 3'-terminal sequence of 16S rRNA of S . lividans (M . J . Bibb and S . N . Cohen, Mol . Gen . Genet . 187:265-277, 1982) . The transcriptional start points of amy were determined by mung bean nuclease mapping, but the promoter of amy was not similar to the consensus sequence found in other procaryotes. J Virol, 1987 Mar, 61(3), 866 - 75 Posttranslational processing of an Epstein-Barr virus-encoded membrane protein expressed in cells transformed by Epstein-Barr virus; Baichwal VR et al.; The BamHI Nhet fragment of the B958 strain of Epstein-Barr virus (EBV) encodes a membrane protein (BNLF-1) that is present in cells transformed by EBV . We made a hybrid protein in which a polypeptide sequence from the carboxyl-terminal part of BNLF-1 is fused to Escherichia coli beta-galactosidase . This hybrid protein was used to immunize rabbits, and the resulting antiserum was purified by immunoaffinity chromatography . The antiserum was able to immunoprecipitate BNLF-1 from cell lysates . We found that BNLF-1 is phosphorylated at serines in EBV genome-positive B-cell lines . Pulse-chase analyses with {35S}methionine indicated that BNLF-1 is turned over in lymphoblasts with a half-life of approximately 5 h . Protein immunoblots of EBV genome-positive B-cell lines revealed both a 62,000-molecular-mass band corresponding to BNLF-1 and a myriad of lower-molecular-mass bands . We postulate that these lower-molecular-mass bands are degradation products resulting from the turnover of BNLF-1 in cells . The BNLF-1 gene was expressed in COS cells, and the protein was both phosphorylated and turned over in these cells. Gene Anal Tech, 1987 Mar-Apr, 4(2), 23 - 6 Storage of spheroplasts at -70 degrees C for transfection with phi X174 RF DNA; Malling HV et al.; A method was developed for preparation of spheroplasts used for transfection with phi X174 RF DNA . This method had a high-level competence and retained the competence for up to one year of storage in 7% DMSO at -70 degrees C. Proc Natl Acad Sci U S A, 1987 Mar, 84(6), 1624 - 8 Large palindromes in the lambda phage genome are preserved in a rec+ host by inhibiting lambda DNA replication; Shurvinton CE et al.; A large palindrome carried by phage lambda has been shown to prevent growth of the phage on a rec+ strain of Escherichia coli . The phage do form plaques on recBC sbcB strains, but the palindrome is not stable--deletions that either destroy the palindrome or diminish its size overgrow the original engineered palindrome-containing phage . We have prepared stocks of lambda carrying a palindrome that is 2 X 4200 base pairs long . These phage stocks are produced by induction of a lysogen in which the two halves of the palindrome are stored at opposite ends of the prophage and are of sufficient titer (10(9) phage per ml) to enable one-step growth experiments with replication-blocked phage . We find that the large palindrome as well as a lesser palindrome of 2 X 265 base pairs are recovered intact among particles carrying unreplicated chromosomes following such an infection of a rec+ host . We propose that DNA replication drives the extrusion of palindromic sequences in vivo, forming secondary structures that are substrates for the recBC and sbcB gene products. Microb Pathog, 1987 Mar, 2(3), 195 - 209 Molecular cloning and characterization of the genetic determinant encoding CS3 fimbriae of enterotoxigenic Escherichia coli; Boylan M et al.; The genetic determinant encoding the synthesis and surface expression of CS3 fimbriae of colonization factor antigen II-(CFA/II-) positive enterotoxigenic Escherichia coli was cloned on a 5.1 kb HindIII DNA fragment in pBR322 from the wild-type plasmid pCS001 to yield the CS3+ plasmid pCS100 . Subcloning of EcoRI fragments of 1.8 kb and 2.5 kb into vector plasmid pACYC184 and the isolation of a series of pCS100::Tn5 insertion mutants revealed that more than one cistron was involved in the synthesis and expression of CS3 fimbriae . Polypeptides of 94, 26, 24, 17 and 15 kDa were detected in E . coli minicells harbouring pCS100 . In Western immunoblotting the 17 kDa and 15 kDa polypeptides reacted with specific anti-CS3 fimbriae serum . The 15 kDa polypeptide comigrated with the structural subunit of CS3 fimbriae . Inhibition of protein processing in minicells by ethanol confirmed that the 17 kDa polypeptide was the precursor form of the 15 kDa structural subunit . A physical map of the cloned DNA was constructed showing the location and direction of transcription of the genes for the 17 and 94 kDa polypeptides . Using the 5.1 kb HindIII fragment of pCS100 as a genetic probe for the CS3 determinant, Southern hybridization analysis of plasmid and total cellular DNA was performed in wild-type enterotoxigenic E . coli strains. Biochem Int, 1987 Mar, 14(3), 517 - 24 Antipili antibody affords protection against ascending pyelonephritis in rat: evaluated by renal brush border membrane enzymes; Garg UC et al.; Kinetic parameters (Km and Vmax) of renal brush border membrane (BBM) enzymes alkaline phosphatase, maltase, leucine aminopeptidase and gamma-glutamyltranspeptidase were worked out in control, infected and immunized-infected rats . There was no significant change in the Km of all the enzymes studied in three groups . The Vmax of all the enzymes studied decreased significantly (p less than 0.05) 3 or 4 days postinfection and onwards in the left obstructed kidney of infected and immunised-infected animals . However, in the right unobstructed kidney the Vmax of alkaline phosphatase and leucine aminopeptidase increased significantly (p less than 0.05) in the early stages and decreased (p less than 0.05) in later stages in both the experimental groups . The significant difference (p less than 0.05) in the Vmax of infected and immunized-infected groups at various stages of infection revealed the partial protective role of antipili antibody against ascending pyelonephritis. Tsitologiia, 1987 Mar, 29(3), 338 - 44 {Genetic transformation of somatic cells . XII . Mutations in human satellite DNA introduced into Chinese hamster cells}; Tomilin NV et al.; Data are presented about detailed restriction mapping, hybridization and sequence analysis of plasmid pRT301 rescued into E . coli 25-30 cell divisions after transfection of Chinese hamster cells . This plasmid contains an insert of human satellite III fragment (HS3), which was replicated many times in mammalian cells, and we found multiple point mutations in this non-selectable fragment, mainly single inserts of GC base pair . Specificity of mutagenesis of HS3 induced by transfection and replication in Chinese hamster cells differs from mutagenesis specificity found by other authors for selectable genes. EMBO J, 1987 Mar, 6(3), 749 - 59 Characterization and localization of the even-skipped protein of Drosophila; Frasch M et al.; On the basis of homeo box cross-homology we have isolated the pair-rule gene even-skipped (eve) of Drosophila . The eve transcription unit appears to be less than 1.5 kb in length, and encodes a single mRNA of approximately 1.4 kb . The nucleotide sequence of genomic and cDNA clones indicates that the eve protein is composed of 376 amino acid residues, and that its homeo domain shares only approximately 50% amino acid identity with the homeo domains of previously characterized genes . Using antibodies raised against a beta-galactosidase fusion protein we show that the eve protein is distributed in a series of seven transverse stripes at the cellular blastoderm stage, and is localized primarily within the nuclear regions of those embryonic cells that express the gene . After gastrulation, seven weakly stained stripes of eve expression appear, resulting in a transient pattern that consists of a total of 14 evenly spaced stripes . Both the original and new stripes gradually disappear during germ band elongation . A second expression pattern emerges during neurogenesis, whereby eve protein is detected in discrete subsets of neurons in each of the ventral ganglia. EMBO J, 1987 Mar, 6(3), 705 - 11 Independent mutations at the amino terminus of a protein act as surrogate signals for mitochondrial import; Vassarotti A et al.; Intracellular delivery of the mitochondrial F1-ATPase beta-subunit precursor from the cytoplasm into the matrix of mitochondria is prevented by deletion of its mitochondrial import signal, a basic amphipathic alpha-helix at its amino terminus . Using a complementation assay, we have selected spontaneous mutations which restore the correct in vivo localization of the protein containing the import signal deletion . Analysis of these mutations revealed that different functional surrogate mitochondrial targeting signals formed within a narrow region of the extreme amino terminus of the import signal deleted beta-subunit . These modifications specifically replace different acidic residues with neutral or basic residues to generate a less acidic amphipathic helix within a region of the protein which is accessible for interaction with the membrane surface . The observations of this study confirm the requirement for amphipathicity as part of the mitochondrial import signal and suggest how mitochondrial targeting signals may have evolved within the extreme amino terminus of mitochondrial proteins. J Hosp Infect, 1987 Mar, 9(2), 175 - 81 Role of R-plasmids in adherence and hydrophobicity in Escherichia coli; Sainz V et al.; Twenty-five R-plasmids from different incompatibility groups were conjugatively transferred to the laboratory strain Escherichia coli JE2571 and the variations in surface hydrophobicity and adherence to human buccal epithelial cells were investigated . It was found that some R-plasmids produce significant variations in adherence and/or hydrophobicity but that these variations show no quantitative or qualitative correlation. Proc Natl Acad Sci U S A, 1987 Mar, 84(6), 1575 - 9 Cloning and sequence of the human nuclear protein cyclin: homology with DNA-binding proteins; Almendral JM et al.; A full-length cDNA clone for the human nuclear protein cyclin has been isolated by using polyclonal antibodies and sequenced . The sequence predicts a protein of 261 amino acids (Mr 29,261) with a high content of acidic (41, aspartic and glutamic acids) versus basic (24, lysine and arginine) amino acids . The identity of the cDNA clone was confirmed by in vitro hybrid-arrested translation of cyclin mRNA . Blot-hybridization analysis of mouse 3T3 and human MOLT-4 cell RNA revealed a mRNA species of approximately the same size as the cDNA insert . Expression of cyclin mRNA was undetectable or very low in quiescent cells, increasing after 8-10 hr of serum stimulation . Inhibition of DNA synthesis by hydroxyurea in serum-stimulated cells did not affect the increase in cyclin mRNA but inhibited 90% the expression of H3 mRNA . These results suggest that expression of cyclin and histone mRNAs are controlled by different mechanisms . A region of the cyclin sequence shows a significant homology with the putative DNA binding site of several proteins, specially with the transcriptional-regulator cAMP-binding protein of Escherichia coli, suggesting that cyclin could play a similar role in eukaryotic cells. Infect Immun, 1987 Mar, 55(3), 555 - 8 F41 pili as protective antigens of enterotoxigenic Escherichia coli that produce F41, K99, or both pilus antigens; Runnels PL et al.; Pigs suckling dams that have been vaccinated with pilus antigen are protected against challenge with enterotoxigenic Escherichia coli (ETEC) strains that express the same pilus antigen . However, some ETEC strains express more than one pilus antigen . Pregnant swine were vaccinated either with E . coli HB101 that harbored a recombinant plasmid coding for F41 expression (F41+) or with the HB101 parent strain that carries the pHC79 vector (F41-) . Suckling pigs born to vaccinated dams were challenged with ETEC that expressed either K99, F41, or both pilus antigens . Production of F41 in vivo was demonstrated by immunofluorescence assay of sections of ileum and by seroconversion against F41 antigen by pigs challenged with F41+ and K99+ F41+ ETEC strains . The F41+ vaccine protected against challenge with an F41+ ETEC strain . In contrast, F41+ vaccination did not protect against challenge with K99+ or K99+ F41+ ETEC strains . The F41- vaccine did not protect against challenge with any strain used . The results indicate that K99+ F41+ ETEC strains produce F41 antigen in the small intestine during disease and that F41+ vaccination can be a protective antigen if the challenge strain expresses only F41 antigen, but that F41+ vaccination may not protect against strains that produce both K99 and F41 antigens. Infect Immun, 1987 Mar, 55(3), 534 - 40 Stimulation of human polymorphonuclear leukocyte oxidative metabolism by type 1 pili from Escherichia coli; Goetz MB et al.; We compared the degree to which Escherichia coli phase variants which do (T1P+ E . coli) or do not (T1P- E . coli) express type 1 pili (T1P) stimulate human polymorphonuclear leukocyte (PMN) oxidative activity . Unopsonized T1P+ E . coli stimulated the release of 0.20 to 0.24 nmol of H2O2 per 10(6) PMN per min and the consumption of 1.4 to 4.0 nmol of O2 per 10(6) PMN per min; no measurable PMN oxidative activity was stimulated by unopsonized T1P- E . coli . In the presence of serum opsonins, T1P+ E . coli stimulated the release of 1.12 to 1.16 nmol of H2O2 per 10(6) PMN per min and the consumption of 5.0 to 6.0 nmol of O2 per 10(6) PMN per min, whereas T1P- E . coli stimulated the release of 0.42 to 0.43 nmol of H2O2 per 10(6) PMN per min and the consumption of 0.6 to 2.0 nmol of O2 per 10(6) PMN per min . Although unaggregated T1P did not stimulate PMN, latex beads coated with T1P (T1P-latex) stimulated alpha-methylmannoside-inhibitable, opsonin-independent PMN oxidative activity . The activity stimulated by either T1P+ E . coli or T1P-latex was susceptible to inhibition by cytochalasin B . Latex particles coated with bovine serum albumin or mannose-resistant pili did not stimulate PMN . These data indicate that T1P+ E . coli stimulate PMN oxidative metabolism more effectively than do T1P- E . coli and that a similar PMN oxidative response follows cellular stimulation by either unopsonized T1P+ or opsonized T1P- E . coli . Furthermore, T1P-latex faithfully mimics the ability of T1P+ E . coli to stimulate PMN oxidative metabolism . Such particles may be useful in further analyses of cellular responses to T1P+ E . coli. J Immunol, 1987 Mar 1, 138(5), 1397 - 402 The localization of the antibody response in milk or bile depends on the nature of the antigen; Dahlgren UI et al.; Immunization in the Peyer's patches of rats with horse spleen ferritin or Escherichia coli 06 carrying type 1 pili resulted in an IgA antibody response detected in milk and bile and an IgG and IgM antibody response in serum, milk, and bile . The IgA antibody response to type 1 pili was as a mean 5.0-fold higher in milk than in bile . In contrast IgA antibody activity to 06 LPS was as a mean 6.3-fold higher in bile than in milk . The IgA antibodies to ferritin were randomly distributed between milk and bile . The IgG and IgM antibody activity to all three antigens studied were higher in the milk than in the bile . The secretory antibody response could be transferred from immunized rats to unimmunized rats with mesenteric lymph node cells (MLN) taken from donor rats 4 days after immunization in the Peyer's patches . IgA antibodies to pili and ferritin appeared solely in the milk of the recipients, whereas IgA antibodies to the 06 LPS only appeared in the bile . The ratios serum:milk and serum:bile for the IgG and IgM antibodies indicated an antigen-specific direction of homing with local production of these two isotypes primarily in the mammary gland . Antibody-forming cells of the IgA class could not be detected in the MLN on the day the cells were transferred . It is concluded that the difference seen in antibody distribution between milk and bile is not due to dissemination of antigen, but instead a result of different homing or expansion at the mucosal-glandular site dependent on the antigen specificity of the migrating cells. J Gen Microbiol, 1987 Mar, 133 ( Pt 3), 597 - 607 Investigation of Escherichia coli fumarate reductase subunit function using transposon Tn5; Latour DJ et al.; Seventy two Tn5 transposon insertions were isolated in the frd operon carried on the multicopy plasmid pFRD79 . The polar nature of these mutations permitted examination of the expression and localization of the frd polypeptides in novel subunit combinations . The minimal catalytic unit is the FRDA plus B dimer . A transposon within frdB (frdB::Tn5) produces inactive, soluble FRDA polypeptide which has covalently attached 8 alpha(N3-histidyl)flavin adenine dinucleotide cofactor . A transposon mutation within frdC (frdC::Tn5) produces soluble, catalytically active dimer . An insertion in frdD (frdD::Tn5) produces both a soluble trimer composed of FRDABC, and a tetramer of FRDABC and truncated FRDD bound to the inner membrane . Eighty percent of the activity is in the soluble form . Using this mutant, the requirement for FRDD both for optimal activity of the catalytic domain and for proper anchorage in the cytoplasmic membrane was demonstrated. Mol Biol (Mosk), 1987 Mar-Apr, 21(2), 506 - 14 {Comparison of the conformation of RNA from phage MS2 and 16S rRNA . Accessibility to nucleases S1 and SV specific to secondary structure and thermal stability}; Grechko VV et al.; The hydrolysis of E . coli 16S rRNA by nucleases specific to the secondary structure elements (S1 and SV), the melting of the RNA after partial hydrolysis by nuclease S1 and the electrophoretic mobility of hydrolysis products after denaturation-renaturation of RNA were studied . It was shown that the sensitivity of 16S rRNA to nuclease S1 depends on Zn or Mg ions concentration . The melting curves after partial hydrolysis by nuclease S1 were characterized by a decrease of the hyperchromic effect (by approximately 15%) and by a increase of Tm (by 3 degrees) . After RNA denaturation followed by slow or fast renaturation the electrophoretic patterns of the hydrolysis products were not changed, as in the case of phage MS2 RNA . It was supposed, that the rRNA molecule has a stable "nucleus" (or "nuclei"), which is organized as an intramolecular association of parallelly oriented double-stranded fragments of this RNA . Previously, such a mode of the spatial organization was proposed by us for phage MS2 RNA. Biochem J, 1987 Mar 1, 242(2), 565 - 72 Escherichia coli endonuclease III is not an endonuclease but a beta-elimination catalyst; Bailly V et al.; The oligonucleotide {5'-32P}pdT8d(-)dTn, containing an apurinic/apyrimidinic (AP) site {d(-)}, yields three radioactive products when incubated at alkaline pH: two of them, forming a doublet approximately at the level of pdT8dA when analysed by polyacrylamide-gel electrophoresis, are the result of the beta-elimination reaction, whereas the third is pdT8p resulting from beta delta-elimination . The incubation of {5'-32P}pdT8d(-)dTn, hybridized with poly(dA), with E . coli endonuclease III yields two radioactive products which have the same electrophoretic behaviour as the doublet obtained by alkaline beta-elimination . The oligonucleotide pdT8d(-) is degraded by the 3'-5' exonuclease activity of T4 DNA polymerase as well as pdT8dA, showing that a base-free deoxyribose at the 3' end is not an obstacle for this activity . The radioactive products from {5'-32P}pdT8d(-)dTn cleaved by alkaline beta-elimination or by E . coli endonuclease III are not degraded by the 3'-5' exonuclease activity of T4 DNA polymerase . When DNA containing AP sites labelled with 32P 5' to the base-free deoxyribose labelled with 3H in the 1' and 2' positions is degraded by E . coli endonuclease VI (exonuclease III) and snake venom phosphodiesterase, the two radionuclides are found exclusively in deoxyribose 5-phosphate and the 3H/32P ratio in this sugar phosphate is the same as in the substrate DNA . When DNA containing these doubly-labelled AP sites is degraded by alkaline treatment or with Lys-Trp-Lys, followed by E . coli endonuclease VI (exonuclease III), some 3H is found in a volatile compound (probably 3H2O) whereas the 3H/32P ratio is decreased in the resulting sugar phosphate which has a chromatographic behaviour different from that of deoxyribose 5-phosphate . Treatment of the DNA containing doubly-labelled AP sites with E . coli endonuclease III, then with E . coli endonuclease VI (exonuclease III), also results in the loss of 3H and the formation of a sugar phosphate with a lower 3H/32P ratio that behaves chromatographically as the beta-elimination product digested with E . coli endonuclease VI (exonuclease III) . From these data, we conclude that E . coli endonuclease III cleaves the phosphodiester bond 3' to the AP site, but that the cleavage is not a hydrolysis leaving a base-free deoxyribose at the 3' end as it has been so far assumed . The cleavage might be the result of a beta-elimination analogous to the one produced by an alkaline pH or Lys-Trp-Lys . Thus it would seem that E . coli 'endonuclease III' is, after all, not an endonuclease. Biokhimiia, 1987 Mar, 52(3), 484 - 92 {Effect of cAMP modifiers on the cytotoxic activity of macrophages}; Neskoromnyi AG et al.; The effects of various cAMP modifiers on the changes in the intracellular cAMP level and on the coupling of the cAMP system with realization of macrophage cytotoxicity depending on their functional activity were studied . Nonactivated and activated by E . coli polysaccharides peritoneal macrophages of BALB/c mice and macrophage-like cells of J744 mice were incubated in the presence of cAMP modifiers and further assayed for cytolytic and cytostatic activities . Cells of tetraploid strain of Ehrlich carcinoma G10 were used as target cells . Among other modifiers only dibutyryl-cAMP caused a steady increase of the intracellular nucleotide content, whereas methylisobutylxanthine and isoproterenol in combination with methylisobutylxanthine caused only a temporary increase of the cAMP level . Isoproterenol did not induce any appreciable changes in the intracellular cAMP level . All modifiers under study suppressed the cytotoxic activity of macrophages irrespective of the nature of changes in the intracellular cAMP content . It was assumed that cAMP accomplishes a triggering function in the regulation of the cytotoxic activity of macrophages and that the cAMP system is universal in the regulation of cytotoxicity at various functional states of macrophages. Biokhimiia, 1987 Mar, 52(3), 452 - 8 {Interpretation of the phenomenon of K+ transport activation in Escherichia coli during transition to anaerobiosis}; Puchkov EO et al.; Earlier it was demonstrated that the transition of E . coli K-12 cells to anaerobiosis is accompanied by the activation of K+ uptake . K+ that are additionally accumulated during the transition to anaerobiosis are released from the cells after the turning on of the respiratory chain . The K+ accumulation by the cells is potential-dependent both under aerobic and anaerobic conditions . A correlation was found between the degree of acidification of the cytoplasm and the rate of K+ uptake during the transition to anaerobiosis . It was assumed that under aerobic conditions the functioning of the electrogenic system of K+ uptake is concomitant with the operation of the K+ release system, K+/H+ antiporter, which is inactivated at the beginning of anaerobiosis, presumably as a result of cytoplasm acidification . This effect manifests itself as the activation of K+ uptake . The trigger function of the K+/H+ antiporter in E . coli cells was suggested to provide for the control of the intracellular pH as well as the switching from the aerobic to the anaerobic pathway of energy metabolism. Prikl Biokhim Mikrobiol, 1987 Mar-Apr, 23(2), 179 - 84 {Distribution of enzymes of nucleic acid biosynthesis and their precursors during fractionation of Escherichia coli MRE-600 extract}; Khomov VV et al.; Distribution of the enzymes of template-dependent and template-independent polynucleotide syntheses (DNA-polymerase I, RNA-polymerase, polynucleotide phosphorylase) as well as those of the biosynthesis of nucleic acids precursors (nucleotide kinases, acetokinase and nucleoside deoxyribosyltransferases) during fractionation of Escherichia coli MRE-600 cell extract was studied . On the basis of the results obtained a technological scheme was developed that enabled to combine routine procedures of purification of the above mentioned enzymes. Proc Natl Acad Sci U S A, 1987 Mar, 84(5), 1399 - 403 Synthetic gene construct expressing a repeated and highly immunogenic epitope of the Plasmodium falciparum antigen Pf155; Aslund L et al.; The Plasmodium falciparum-derived antigen Pf155 contains two blocks of tandemly repeated amino acid sequences . A pair of complementary oligonucleotides, encoding the C-terminally located repeat Val-Glu-His-Asp-Ala-Glu-Glu-Asn, were synthesized . The oligonucleotides were polymerized by ligation, and the resulting multimers were cloned into an expression vector . One construct that contained four copies of the repeat was expressed in Escherichia coli . The product, a fusion protein, was soluble and produced in high amounts . It reacted in immunoblotting with a monoclonal antibody to a synthetic octapeptide (Glu-Glu-Asn-Val-Glu-His-Asp-Ala) . Rabbits immunized with partially purified fusion protein, either with or without adjuvant, formed antibodies against this octapeptide . These antibodies reacted with Pf155 both in parasite extracts and when deposited in the membrane of infected erythrocytes . Furthermore, these antibodies inhibited merozoite reinvasion in vitro as efficiently as human antibodies to the octapeptide sequence in Pf155, induced by natural infection . The results suggest that products of synthetic gene constructs may be a suitable basis for an anti-merozoite vaccine. Proc Natl Acad Sci U S A, 1987 Mar, 84(5), 1345 - 9 Isolation of temperature-sensitive tyrosine kinase mutants of v-abl oncogene by screening with antibodies for phosphotyrosine; Kipreos ET et al.; Temperature-sensitive protein-tyrosine kinase (EC 2.7.1.112) mutants of the oncogene v-abl have been obtained by a direct screening of kinase mutants in bacteria . The v-abl oncogene was expressed in Escherichia coli as a trpE/v-abl fusion protein from the trp promoter . The expression plasmid was mutagenized in vitro and then transfected into E . coli . Bacteria that produced defective tyrosine kinases were distinguished from those producing wild-type v-abl kinases by hybridization with antibodies specific for phosphotyrosine . Two independent mutations that generated temperature-sensitive tyrosine kinases were found to be located in a 12-amino acid region in the tyrosine kinase domain of the v-abl-encoded protein . These mutant v-abl oncogenes displayed temperature-sensitive transforming activity when expressed in NIH 3T3 cells . Cells transformed by these temperature-sensitive tyrosine kinase mutants could be shifted between the transformed and untransformed states by changing their growth temperature . These results confirmed the crucial role of tyrosine kinase activity in the v-abl-mediated oncogenesis. Proc Natl Acad Sci U S A, 1987 Mar, 84(5), 1152 - 6 Site-directed mutagenesis of the COOH-terminal region of colicin A: effect on secretion and voltage-dependent channel activity; Baty D et al.; A large number of mutants introducing point mutations and deletions into the COOH-terminal domain of colicin A have been constructed by using site-directed mutagenesis . The COOH-terminal domain carries the channel activity . The effects of the alterations in the polypeptide chain on the secretion of colicin A by colicinogenic cells have been investigated . All deletions and some mutations were found to lead to protein aggregation in the cytoplasm, thereby preventing release into the medium . The mutated colicin A proteins have been purified, and their activity in vivo (on sensitive cells) and in vitro (in planar lipid bilayers) has been assayed . Deletions in the region containing putative helices 4, 5, and 6 (predicted to be involved in pore formation) and the transitions (Ala----Asp-492, Phe----Pro-493) in helix 4 abolished the activity . No correlation was observed between mutations leading to protein aggregation and those leading to loss of channel activity . Some mutations were found to alter characteristic properties of the single channels, such as stability, current-relaxation kinetics, voltage dependence, and pore conductance . Site-directed mutagenesis provides a powerful tool for studying structure-function relationships of voltage-sensitive ionic channels. Proc Natl Acad Sci U S A, 1987 Mar, 84(5), 1147 - 51 Partly native epitopes are already present on early intermediates in the folding of tryptophan synthase; Blond S et al.; Two monoclonal antibodies directed against the native beta 2 subunit of Escherichia coli tryptophan synthase {L-serine hydro-lyase (adding indoleglycerol-phosphate), EC 4.2.1.20}, one recognizing the C-terminal F1 domain and the other the N-terminal F2 domain, were used to detect immunoreactive intermediates in the folding of the protein . For that purpose, the association of the monoclonal antibodies with either the beta 2 subunit or its isolated domains was studied by using fluorescence energy transfer between tryptophan residues of the antibodies and a dansyl group covalently linked to the antigen . It is shown that the association of both monoclonal antibodies with the antigen occurs within a few seconds after initiation of the renaturation, whereas complete refolding of the beta 2 subunit requires several minutes under the same experimental conditions . Thus, immunoreactive intermediates appear to be formed at an early stage of the folding process . While the isolated F1 domain alone is able to rapidly refold into a conformational intermediate already well recognized by the anti-native-beta 2 antibody, it cannot, in the absence of the F2 domain, reach its native conformation . However, its association with the anti-native-beta 2 antibody induces a structural change of F1 that brings it closer to the conformation it normally adopts when interacting with F2 inside the native beta 2 subunit. J Bacteriol, 1987 Mar, 169(3), 1217 - 22 Activities of the RNAI and RNAII promoters of plasmid pBR322; Lin-Chao S et al.; The synthesis rates of the replication control RNAs of plasmid pBR322, RNAI, an inhibitor of replication, and RNAII, the preprimer, have been determined by hybridizing in vivo pulse-labeled RNA to specific, single-stranded DNA probes for RNAI and RNAII . In Escherichia coli growing in glycerol minimal medium, RNAI transcripts were made at a rate of one molecule per 30 s per plasmid; RNAII was transcribed fivefold less, at a rate of one molecule per 3 min per plasmid . It is estimated that only 1 in 20 prepriming events leads to replication. Biochim Biophys Acta, 1987 Feb 27, 908(2), 131 - 42 Absolute values of ras p21 defined by direct binding liquid competition radioimmunoassays; Horan Hand P et al.; Several distinct and high-conserved genes comprise the ras gene family of rodents and humans, i.e., rodent Harvey and Kirsten, and human Harvey, Kirsten and neuroblastoma . Transformation, either by a point-mutation resulting in a change in one amino acid of the 21 kDa ras gene product (p21), or by increased expression of ras p21, has been demonstrated to be mediated by members of this gene family . We report here the development of direct binding liquid competition radioimmunoassays for the detection and quantitation of the ras oncogene and proto-oncogene products . Using these radioimmunoassays and ras p21 purified from Escherichia coli containing the full-length T24 mutant human Harvey ras gene protein product as a standard, we have defined the actual amount of ras p21 per micrograms of total cellular protein, or per cell, in various ras transformed and 'normal' mammalian cell lines . One of the radioimmunoassays developed is group-specific, since the antigenic determinant recognized is shared by both the point-mutated and proto-forms of Harvey, Kirsten and neuroblastoma members of the ras gene family, while the second may be termed type-selective, since it recognizes an antigenic determinant localized primarily on the Harvey ras p21 . Both radioimmunoassays are interspecies, since they detect a ras p21 antigenic determinant common to cells of human and rodent origin . These studies thus describe the first means for defining absolute values of any oncogene or proto-oncogene protein product . The assays described, when used in combination with specific c-DNA probes to define specific ras proto-oncogenes or point-mutated oncogenes being expressed, will now permit truly quantitative analyses of ras p21 expression in experimental cell culture systems, animal models and human biopsy material. Biochem Biophys Res Commun, 1987 Feb 27, 143(1), 323 - 8 A sensitive colorimetric detection of virus DNA and oncogene; Inoue H et al.; Advantage of cloning probe DNA fragment in phage M13 DNA was taken to provide a larger single stranded DNA as a hybridization probe . High level of direct enzyme labels was introduced via the M13 DNA moiety as well as probe DNA . A highly sensitive colorimetric detection of virus DNA and oncogene was developed. Biochem Biophys Res Commun, 1987 Feb 27, 143(1), 104 - 9 Proofreading of a mutagenic nucleotide, N4-aminodeoxycytidylic acid, by Escherichia coli DNA polymerase I; Takahashi M et al.; N4-Aminodeoxycytidine triphosphate, a putative metabolite of N4-aminocytidine which is a potent mutagen, is incorporated, in vitro, into polynucleotides in place of dCTP and at a much lesser extent, but significantly, in place of dTTP by E . coli DNA polymerase I large fragment . The activity of the polymerase to proofread this unnatural nucleotide has now been investigated . The results indicate that the 3'-5' exonuclease in the polymerase recognizes N4-aminocytosine as an incorrect base when N4-aminocytosine is incorporated opposite adenine but the enzyme cannot distinguish N4-aminocytosine from cytosine when it is incorporated opposite guanine. Biochim Biophys Acta, 1987 Feb 27, 908(2), 113 - 22 Organization of genes for ribosomal proteins S7 and S12, elongation factors EF-Tu and EF-G in the cyanobacterium Spirulina platensis; Tiboni O et al.; The gene encoding ribosomal proteins S12 and probably S7 as well as protein synthesis elongation factors Tu (EF-Tu) and G (EF-G) of Spirulina platensis have been identified and cloned . Gene expression was determined for ribosomal protein S12 by genetic complementation of the appropriate Escherichia coli mutant, whereas for the EF-Tu gene it was determined by production of the protein in E . coli minicells . On the basis of these experiments we suggest the following gene order in the S . platensis chromosome: S12, S7, EF-G, EF-Tu. Biochem Biophys Res Commun, 1987 Feb 27, 143(1), 300 - 8 Repetitive region of calpastatin is a functional unit of the proteinase inhibitor; Maki M et al.; A cDNA portion coding for one of the repetitive regions of pig heart calpastatin (107 kDa) was subcloned into E . coli plasmid pUC119 to express the portion of the proteinase inhibitor gene in bacteria . The expressed protein was a chimaeric protein whose calpastatin segment (130 amino acid residues) was fused with an amino-terminus portion (7 amino acid residues) of beta-galactosidase . The chimaeric protein could inhibit proteolytic activity of calpain (Ca2+-dependent cysteine proteinase), and maintained properties of the authentic calpastatin concerning inhibition specificity and heat stability . These findings led us to conclude that the repetitive region is a functional unit of the proteinase inhibitor. Cell, 1987 Feb 27, 48(4), 555 - 66 The inducible lac operator-repressor system is functional in mammalian cells; Hu MC et al.; We have investigated the use of the Escherichia coli lac operator-repressor system to regulate expression of transfected genes in mammalian cells . We show that lac repressor produced in mouse L cells by transfection of a lacl expression vector blocks transcription of an MSV-CAT fusion gene when the lac operator is inserted at any one of the following sites within the promoter region: between the initiation codon (ATG) and the transcription start site; between the transcription start and TATA box regions; or upstream of the TATA box region . This last result suggests that the repressor may prevent protein-protein interactions involved in transcription activation . The inducer IPTG causes a marked derepression of CAT expression . The lac repressor-operator complex may be useful as an on/off "switch" in the regulation of gene expression for gene transfer experiments. J Immunol Methods, 1987 Feb 26, 97(1), 19 - 27 A sensitive micro-immunoassay using beta-galactosidase/anti-beta-galactosidase complexes; Durbin H et al.; This paper describes a new sensitive microELISA based on enzyme/anti-enzyme complexes following an unlabelled antibody bridging step . beta-Galactosidase/anti-beta-galactosidase complexes were made using a monoclonal antibody raised against bacterial (E . coli) beta-galactosidase and enzyme activity was quantified with a fluorogenic substrate . Because of its high sensitivity the assay is particularly suitable for the detection of limited amounts of antigen . One application illustrated is the analysis of Class I and Class II histocompatibility antigens on peripheral blood lymphocytes using 5000 cells/well in 60-well Terasaki or 96-well microtitre plates. Nucleic Acids Res, 1987 Feb 25, 15(4), 1763 - 77 Substrate specificity of DNA polymerases . I . Enzyme-catalysed incorporation of 5-'1-alkenyl)-2'-deoxyuridines into DNA; Otvos L et al.; A series of (E)-5-(1-alkenyl)-dUTPs as well as 5-vinyl-and (Z)-5-(1-propenyl)-dUTP have been synthesized to study steric requirements in DNA polymerase reactions . Experiments were carried out in E . coli DNA polymerase I Klenow fragment enzyme system . Substrates were characterized by KM and Vmax-values, initial incorporation rates as well as by total extent of incorporation of the analogues into poly(dA-dT) as a template-primer . Incorporation of the analogues could be best correlated with Vmax-values as well as the very similar initial incorporation rate values . Reactivity (Vmax/KM) showed no correlation with the extent of incorporation . 5-Vinyl-dUTP proved to be as good a substrate of the enzyme as dTTP, whereas (E)-5-(1-heptenyl)-and (E)-5-(1-octenyl)-dUTPs were very poor substrates, their incorporation was strongly limited and they also proved to be very efficient inhibitors of DNA replication, as shown by Ki-values . Substrate specificity of the Klenow enzyme can be explained by the steric hindrance of C-5 substituent, by the "orientational steric substituent effect" concept. J Biol Chem, 1987 Feb 25, 262(6), 2590 - 6 The kinetics of bovine growth hormone folding are consistent with a framework model; Brems DN et al.; The framework model of protein folding requires the hydrogen-bonded secondary structure to be formed early in folding (i.e . the formation of secondary structure precedes the tertiary structure) (Kim, P . S., and Baldwin, R . L . (1982) Annu . Rev . Biochem . 51, 459-489) . To test the framework model directly the kinetics of bovine growth hormone (bGH) folding were compared utilizing two methods of detection, one that measures the secondary structure (far ultraviolet circular dichroism) and another that measures the tertiary structure (near ultraviolet absorbance) . The results demonstrate that, under identical folding conditions, the kinetics observed by far ultraviolet circular dichroism are faster than those observed by ultraviolet absorption . The faster kinetics observed by circular dichroism indicate the existence of a helix-containing intermediate which is consistent with the framework model . The effect of protein concentration and denaturant concentration on the kinetics of refolding were studied . The rate of refolding measured by absorbance and circular dichroism was dependent on protein concentration . The protein concentration dependence on refolding is due to the transient formation of an associated intermediate . The concentration dependence of folding is taken as evidence that folding is a sequential process with partially folded monomers responsible for the observed association effect . At dilute protein concentrations the refolding can be studied independent of the association phenomena . The growth hormones utilized in this study were derived from Escherichia coli through recombinant DNA technology and from bovine pituitaries . The pituitary-derived bGH has been shown to be heterogeneous at the NH2 terminus (Lorenson, M . F., and Ellis, S . (1975) Endocrinology 96, 833-838), whereas the recombinant DNA-derived bGH contains a single NH2 terminus . No differences in the folding kinetics between the recombinant DNA and pituitary derived-bGH were observed . It is concluded that the heterogeneity of the NH2 terminus of growth hormone obtained from bovine pituitaries does not affect the observed in vitro folding kinetics. Nucleic Acids Res, 1987 Feb 25, 15(4), 1521 - 42 Effect of the deletion of upstream DNA sequences on expression from the ilvGp2 promoter of the ilvGMEDA operon of Escherichia coli K-12; Ortuno MJ et al.; Transcription in vitro of the regulatory region of the ilvGMEDA operon yields two attenuated RNAs initiated from the tandem promoters ilvGp1 and ilvGp2 . Both S1 nuclease analysis and the fusion of ilvGp1 to galK indicate that transcription is not initiated in vivo from ilvGp1 . However deletion of DNA sequences 150 to 100 bp upstream of ilvGp2 drastically reduces expression in vivo from ilvGp2 . Both the distance separating ilvGp2 from the upstream DNA sequences and their relative orientation to each other on the DNA helix affect expression from ilvGp2 . Deletion of DNA sequences approximately 400 bp upstream of ilvGp2 increases expression in vivo from this promoter . Analysis of products of transcription in vitro indicates that the effects observed in vivo are probably not due to DNA conformation or interactions of RNA polymerase. J Biol Chem, 1987 Feb 25, 262(6), 2852 - 3 Crystallization and preliminary X-ray investigation of uridine phosphorylase from Escherichia coli; Cook WJ et al.; Crystals of uridine phosphorylase from Escherichia coli K12 have been grown from solutions of polyethylene glycol 4000 . The crystals are trigonal, space group R3; the hexagonal axes are a = 154.4 A and c = 49.4 A . The crystals are quite stable to x-rays and diffract beyond 2.6 A resolution . It appears that the molecule is a hexamer with a subunit molecular weight of 27,500 and utilizes the 3-fold symmetry of the space group, resulting in two subunits/asymmetric unit. J Biol Chem, 1987 Feb 25, 262(6), 2478 - 84 Physical characteristics of ribosomal protein S4 from Escherichia coli; Dodd J et al.; A hydrodynamic study of protein S4 from Escherichia coli 30 S ribosomal subunits indicates that this protein is moderately asymmetric . A sedimentation coefficient of 1.69 S and a diffusion coefficient of 7.58 X 10(-7) cm2/s suggest that S4 has an axial ratio of about 5:1 using a prolate ellipsoidal model . This structure should give a radius of gyration of about 29-30 A from small-angle neutron or small-angle x-ray scattering studies . This study has utilized quasi-elastic light scattering as an analytical tool to obtain a diffusion coefficient as well as a method to monitor sample quality . Using quasi-elastic light scattering in this manner allows an assessment of problems associated with protein purity which may be responsible for the many disparate results reported for ribosomal proteins and especially protein S4. Nucleic Acids Res, 1987 Feb 25, 15(4), 1615 - 25 Characterization of the 5'-flanking region for the human fibrinogen beta gene; Huber P et al.; To identify the possible regulatory sequences in the genetic expression of fibrinogen, a human genomic DNA library raised in lambda EMBL 4 phage was screened using cDNA probes coding for the A alpha, B beta and gamma chains of human fibrinogen . The entire fibrinogen locus was characterized and its organization analysed by means of hybridization and restriction mapping . Among the clones identified, a single recombinant lambda phage contained the beta gene and its 5'- and 3'-flanking regions . A 1.5 kb fragment of the immediate 5'-flanking region was sequenced and S1 mapping experiments revealed three transcription start points . Comparison of this sequence with that previously reported for the same region upstream from the human gamma gene revealed no significant homology which suggests that the potential promoting sequences of these genes are different . In contrast, comparison of the 5'-flanking regions of human and rat beta genes revealed a 142 bp sequence of 80% homology situated 16 bp upstream from the human beta gene . This highly conserved region may well represents a potential candidate for a regulatory sequence of the human beta gene. J Biol Chem, 1987 Feb 25, 262(6), 2502 - 6 Circular dichroism investigation of Escherichia coli adenylate kinase; Monnot M et al.; We examined by circular dichroism (CD) spectroscopy in far- and near-ultraviolet three different molecular forms of Escherichia coli adenylate kinase: the wild type protein, the enzyme carboxymethylated at a single cysteine residue (Cys-77), and the thermosensitive adenylate kinase . The thermosensitive enzyme differs from the wild type protein in that a serine is substituted for a proline residue at position 87 (Gilles, A.-M., Saint Girons, I., Monnot, M., Fermandjian, S., Michelson, S., and Barzu, O . (1986) Proc . Natl . Acad . Sci . U . S . A., 83, 5798-5802) . We also examined the CD spectra of isolated peptides resulting from chemical cleavage of adenylate kinase at Cys-77 (C1, residues 1-76; C2, residues 77-214) . The secondary structure composition of wild type bacterial adenylate kinase (50% alpha-helix and 15% beta-sheet) was close to that derived from x-ray analysis of pig muscle enzyme (Schulz, G.E., Elzinga, M., Marx, F., and Schirmer, R . H . (1974) Nature 250, 120-123) . Carboxymethylation of wild type protein did not greatly affect the CD spectrum . The secondary structure of the thermosensitive adenylate kinase was observed to be significantly different from that of the wild type enzyme (reduction in alpha-helix content to 39%) . Changes in ellipticities at 222 nm as a function of temperature indicated that the melting temperature for thermosensitive adenylate kinase was 38 degrees C and that for the wild type enzyme was 54 degrees C . Isolated C1 and C2 peptides had a large proportion of unordered structures . When mixed, C1 and C2 fragments reassociated into structures resembling native, uncleaved adenylate kinase . The recovery of ordered structures, indicated by CD spectroscopy, paralleled the recovery of catalytic activity. J Biol Chem, 1987 Feb 25, 262(6), 2468 - 71 Regulation of pantothenate kinase by coenzyme A and its thioesters; Vallari DS et al.; Pantothenate kinase catalyzes the rate-controlling step in the coenzyme A (CoA) biosynthetic pathway, and its activity is modulated by the size of the CoA pool . The effect of nonesterified CoA (CoASH) and CoA thioesters on the activity of pantothenate kinase was examined to determine which component of the CoA pool is the most effective regulator of the enzyme from Escherichia coli . CoASH was five times more potent than acetyl-CoA or other CoA thioesters as an inhibitor of pantothenate kinase activity in vitro . Inhibition by CoA thioesters was not due to their hydrolysis to CoASH . CoASH inhibition was competitive with respect to ATP, thus providing a mechanism to coordinate CoA production with the energy state of the cell . There were considerable differences in the size and composition of the CoA pool in cells grown on different carbon sources, and a carbon source shift experiment was used to test the inhibitory effect of the different CoA species in vivo . A shift from glucose to acetate as the carbon source resulted in an increase in the CoASH:acetyl-CoA ratio from 0.7 to 4.3 . The alteration in the CoA pool composition was associated with the selective inhibition of pantothenate phosphorylation, consistent with CoASH being a more potent regulator of pantothenate kinase activity in vivo . These results demonstrate that CoA biosynthesis is regulated through feedback inhibition of pantothenate kinase primarily by the concentration of CoASH and secondarily by the size of the CoA thioester pool. Biochemistry, 1987 Feb 24, 26(4), 1132 - 6 Role of cysteine residues in the lac permease of Escherichia coli; Menick DR et al.; Oligonucleotide-directed, site-specific mutagenesis has been utilized to replace cysteine residues 117, 333, or 353 and 355 with serine in the lac permease of Escherichia coli . Replacement of Cys-117 or Cys-333 has no significant effect on permease activity, while permease with serine residues in place of Cys-353 and Cys-355 has about 50% of wild-type permease activity . The results provide a clear demonstration that cysteine residues at positions 117, 333, 353, and 355 are not obligatory for lactose/H+ symport . When considered in conjunction with previous findings, the results indicate that, of the eight cysteine residues in the lac permease, only Cys-154 is important for lactose transport . As discussed, the conclusion has important implications for the hypothesis that sulfhydryl-disulfide interconversion plays an important role in the symport mechanism. FEBS Lett, 1987 Feb 23, 212(2), 271 - 5 Simplified in vitro synthesis of mutated RNA molecules . An oligonucleotide promoter determines the initiation site of T7RNA polymerase on ss M13 phage DNA; Krupp G et al.; We describe a simplified method for the in vitro synthesis of mutated RNA molecules . The method makes use of an oligodeoxyribonucleotide (T7-oligo) which contains the T7RNA polymerase promoter sequence . In combination with a second oligonucleotide, a series of transcripts initiating and terminating at any chosen position on a cloned ss DNA (e.g . M13 phage DNA) can be generated . The phage DNA represents the non-coding DNA strand for the desired transcript; the T7-oligo determines the transcription start site, whereas the second oligonucleotide permits the choice of the transcription termination site . The synthesis of the required template DNA is achieved by hybridizing the two oligonucleotides to the phage DNA and subsequently synthesizing the coding DNA strand by a fill-in reaction with Klenow enzyme . The reaction product is used directly as a template for T7RNA polymerase; cloning of mutants is not required. J Mol Biol, 1987 Feb 20, 193(4), 723 - 50 Selection of DNA binding sites by regulatory proteins . Statistical-mechanical theory and application to operators and promoters; Berg OG et al.; We present a statistical-mechanical selection theory for the sequence analysis of a set of specific DNA regulatory sites that makes it possible to predict the relationship between individual base-pair choices in the site and specific activity (affinity) . The theory is based on the assumption that specific DNA sequences have been selected to conform to some requirement for protein binding (or activity), and that all sequences that can fulfil this requirement are equally likely to occur . In most cases, the number of specific DNA sequences that are known for a certain DNA-binding protein is very small, and we discuss in detail the small-sample uncertainties that this leads to . When applied to the binding sites for cro repressor in phage lambda, the theory can predict, from the sequence statistics alone, their rank order binding affinities in reasonable agreement with measured values . However, the statistical uncertainty generated by such a small sample (only 6 sites known) limits the result to order-of-magnitude comparisons . When applied to the much larger sample of Escherichia coli promoter sequences, the theory predicts the correlation between in vitro activity (k2KB values) and homology score (closeness to the consensus sequence) observed by Mulligan et al . (1984) . The analysis of base-pair frequencies in the promoter sample is consistent with the assumption that base-pairs at different positions in the sites contribute independently to the specific activity, except in a few marginal cases that are discussed . When the promoter sites are ordered according to predicted activities, they seem to conform to the Gaussian distribution that results from a requirement for maximal sequence variability within the constraint of providing a certain average activity . The theory allows us to compare the number of specific sites with a certain activity to the number that would be expected from random occurrence in the genome . While strong promoters are "overspecified", in the sense that their probability of random occurrence is very low, random sequences with weak promoter-like properties are expected to occur in very large numbers . This leads to the conclusion that functional specificity is based on other properties in addition to primary sequence recognition; some possibilities are discussed . Finally, we show that the sequence information, as defined by Schneider et al . (1986), can be used directly (at least in the case of equilibrium binding sites) to estimate the number of protein molecules that are specifically bound at random "pseudosites" in the genome.(ABSTRACT TRUNCATED AT 400 WORDS) Biochim Biophys Acta, 1987 Feb 20, 923(2), 275 - 85 Studies on the catalytic rate constant of ribosomal peptidyltransferase; Synetos D et al.; A detailed kinetic analysis of a model reaction for the ribosomal peptidyltransferase is described, using fMet-tRNA or Ac-Phe-tRNA as the peptidyl donor and puromycin as the acceptor . The initiation complex (fMet-tRNA X AUG X 70 S ribosome) or (Ac-Phe-tRNA X poly(U) X 70 S ribosome) (complex C) is isolated and then reacted with excess puromycin (S) to give fMet-puromycin or Ac-Phe-puromycin . This reaction (puromycin reaction) is first order at all concentrations of S tested . An important asset of this kinetic analysis is the fact that the relationship between the first order rate constant kobs and {S} shows hyperbolic saturation and that the value of kobs at saturating {S} is a measure of the catalytic rate constant (k cat) of peptidyltransferase in the puromycin reaction . With fMet-tRNA as the donor, this kcat of peptidyltransferase is 8.3 min-1 when the 0.5 M NH4Cl ribosomal wash is present, compared to 3.8 min-1 in its absence . The kcat of peptidyltransferase is 2.0 min-1 when Ac-Phe-tRNA replaces fMet-tRNA in the presence of the ribosomal wash and decreases to 0.8 min-1 in its absence . This kinetic procedure is the best method available for evaluating changes in the activity of peptidyltransferase in vitro . The results suggest that peptidyltransferase is subjected to activation by the binding of fMet-tRNA to the 70 S initiation complex. J Mol Biol, 1987 Feb 20, 193(4), 651 - 9 Specific strand loss in N-2-acetylaminofluorene-modified DNA; Koffel-Schwartz N et al.; N-2-Acetylaminofluorene (AAF), a well-known chemical carcinogen, when covalently linked to guanine residues constitutes a premutagenic lesion that is converted in vivo into frameshift mutations . In Escherichia coli, it is thought that -AAF adducts block the replication fork and that the mutagenic processing of the -AAF adducts is mediated by the SOS response . The construction in vitro of plasmids containing -AAF adducts in one strand only of a double-stranded DNA molecule enabled us to investigate the segregation of the strands and the mutagenicity of the lesions in vivo . The two DNA strands were "genetically labelled" by means of a single base-pair mismatch in the tetracycline-resistance gene, one strand carrying the wild-type allele and the other strand a mutant tetracycline-sensitive allele . The two strands contained either no -AAF adducts, -AAF adducts in one strand or -AAF adducts in both strands . When such constructions are used to transform bacterial cells the following are found . When no -AAF adducts are present on either strand of the DNA, a mixture of plasmids having information from both parent strands is found in 80% of the transformed bacterial clones . With -AAF adducts present in one strand only, in 90% of the transformants there is a consistent loss of the parent strand information that contained the -AAF adducts . In the constructions having -AAF adducts in both strands, the transformed bacteria carry either one or the other allele in a pure form . Our results suggest that when blocking lesions (-AAF adducts) are present in one strand only, they trigger the specific loss of that strand . The forward mutation frequency (i.e . the tetracycline-resistance gene inactivation frequency) was found to be more than ten times lower when the -AAF adducts are bound to one strand only compared with the situation where both strands carry the premutagenic lesions . Moreover, when the isolated mutants were sequenced, the mutations were found to consist of a mixture of true -AAF-induced mutations (i.e . -1 or -2 frameshift mutation at previously determined mutation hot spots) and of mutations that are not targeted at -AAF adducts . We suggest that these "background" mutants arose from the mutagenic processing of cryptic lesions present in our DNA . The low mutagenic efficiency of -AAF adducts, when present in one strand only of a duplex DNA, most probably results from the above-described loss of the damaged strand.(ABSTRACT TRUNCATED AT 400 WORDS) J Mol Biol, 1987 Feb 20, 193(4), 637 - 41 Ultraviolet light-induced mutagenesis in the Escherichia coli chromosome . Sequences of mutants in the cI gene of a lambda lysogen; Wood RD et al.; DNA sequences were determined for 56 mutations induced by ultraviolet light in the lambda cI gene of an Escherichia coli uvr+ lysogen, which should reflect those occurring in the E . coli chromosome . The spectrum of mutagenesis was similar to that found in the cI gene of irradiated phase assayed in uvr- host cells, except that the fraction of transversions is about 35% in prophage and about 15% in phage . The cause of this difference is not known . Of 17 frameshifts in phage and prophage, six have an accompanying base substitution . These double mutational events are consistent with a model in which a photoproduct in a template can cause a DNA polymerase to insert a wrong base and destabilize the next few bases added, thus leading to a frameshift by a slippage mechanism. J Mol Biol, 1987 Feb 20, 193(4), 609 - 36 Mechanisms of frameshift mutagenesis by aflatoxin B1-2,3-dichloride; Refolo LM et al.; In order to characterize frameshift mutagenesis by aflatoxin B1-2,3-dichloride (AFB1Cl2), we have introduced a +1 (BK8) or a -1 (HS8) frameshift within the lacZ alpha gene segment contained in the phage M13mp8 to obtain lacZ alpha- derivatives . BK8 or HS8 replicative form DNA was modified with AFB1Cl2 in vitro, transfected into appropriate Escherichia coli hosts and lacZ alpha+ revertants scored and defined by DNA sequencing . The -1 frameshift (BK8) results suggest the following . (1) The E . coli recA gene is not absolutely required for AFB1Cl2-induced frameshift mutagenesis; however, in recA+ cells, ultraviolet light (SOS) induction enhances AFB1Cl2 mutagenesis, but such ultraviolet induction is not required . The plasmid pGW270 (mucAB+) significantly enhances the AFB1Cl2-induced frameshift mutagenesis . The uvrABC+ excision system plays a major role in the repair of AFB1Cl2-induced damage . (2) Sequence analysis reveals that AFB1Cl2 induces two classes of -1 frameshift mutations: the simple class in which the frameshift is due to the loss of one base-pair, and the complex class in which the loss of a base-pair is coupled to a vicinal base substitution . Both types of mutations occur predominantly at G.C runs, which are hotspots for AFB1Cl2 damage . The complex mutations appear to be concerted events targeted by a single AFB1Cl2 adduct . The frequency of these complex mutations is significantly enhanced by mucAB activity . In this system, recA activity is required for generation of significant levels of complex mutations . An analysis of the +1 frameshifts (HS8) reveals that AFB1Cl2 induces +1 frameshifts with an efficiency comparable to that for -1 frameshifts . Most +1 frameshifts occur by the addition of a base, and a third of the additions are complex mutations because they are accompanied by at least one base substitution . All simple additions occur at G.C runs; however, in a striking contrast to spontaneous insertions, a majority of the induced events introduce an A.T pair at these sites . Our data suggest a model for the generation of base substitution as well as simple and complex frameshift mutations induced by AFB1Cl2 . To the extent determined, the frameshift specificity of aflatoxin B1 activated by metabolic enzymes is similar to that of AFB1Cl2. J Mol Biol, 1987 Feb 20, 193(4), 661 - 71 Sites of action of two ribosomal RNA methylases responsible for resistance to aminoglycosides; Beauclerk AA et al.; Methylation of either of two residues (G-1405 or A-1408) within bacterial 16 S ribosomal RNA results in high level resistance to specific combinations of aminoglycoside antibiotics . The product of a gene that originated in Micromonospora purpurea (an actinomycete that produces gentamicin) gives resistance to kanamycin plus gentamicin by converting residue G-1405 to 7-methylguanosine . Resistance to kanamycin plus apramycin results from conversion of residue A-1408 to 1-methyladenosine catalysed by the product of a gene from Streptomyces tenjimariensis. Nature, 1987 Feb 19-25, 325(6106), 720 - 3 T gamma protein is expressed on murine fetal thymocytes as a disulphide-linked heterodimer; Nakanishi N et al.; During the search for genes coding for the mouse alpha and beta subunits of the antigen-specific receptor of mouse T cells we encountered a third gene, subsequently designated gamma . This gene has many properties in common with the alpha and beta genes, somatic assembly from gene segments that resemble the gene segments for immunoglobulin variable (V), joining (J) and constant (C) regions; rearrangement and expression in T cells and not in B cells; low but distinct sequence homology to immunoglobulin V, J and C regions; other sequences that are reminiscent of the transmembrane and intracytoplasmic regions of integral membrane proteins; and a cysteine residue at the position expected for a disulphide bond linking two subunits of a dimeric membrane protein . Despite these similarities the gamma gene also shows some interesting unique features . These include a relatively limited repertoire of the germ-line gene segments, more pronounced expression at the RNA level in immature T cells such as fetal thymocytes and an apparent absence of in-frame RNA in some functional, alpha beta heterodimer-bearing T cells or cultured T clones and hybridomas . To understand the function of the putative gamma protein it is essential to define the cell population that expresses this protein . To this end we produced a fusion protein composed of Escherichia coli beta-galactosidase and the gamma-chain (hereafter referred to a beta-gal-gamma) using the phage expression vector lambda gt11 and raised rabbit antisera against the gamma determinants . Using the purified anti-gamma antibody we detected a polypeptide chain of relative molecular mass 35,000 (Mr 35K) on the surface of 16-day old fetal thymocytes . The gamma-chain is linked by a disulphide bridge to another component of 45K . No such heterodimer was detected on the surface of a cytotoxic T lymphocyte (CTL) clone 2C from which an in-phase gamma cDNA clone was originally isolated. Eur J Biochem, 1987 Feb 16, 163(1), 67 - 71 Expression of bovine pancreatic ribonuclease A in Escherichia coli; Nambiar KP et al.; A synthetic gene for bovine pancreatic ribonuclease A (RNase A) has been expressed in Escherichia coli as a fusion protein with beta-galactosidase linked by the tetrapeptide Ile-Glu-Gly-Arg . RNase A was cleaved from the fusion using factor Xa, and the resulting product purified and reconstituted . The isolated RNase A was chromatographically, catalytically, and immunologically identical with authentic RNase A . This work argues that the method suggested by Nagai and Thogersen {Nagai, K . & Thogersen, H . C . (1984) Nature (Lond.) 309, 810-812} for releasing fusion proteins is quite general, even when applied to particularly complicated expression problem . The procedure here makes RNase A available for the first time as a model for studying structure-function relationships in proteins using site-directed mutagenesis. Eur J Biochem, 1987 Feb 16, 163(1), 113 - 21 Studies on the functional topography of Escherichia coli RNA polymerase . Highly selective affinity labelling by analogues of initiating substrates; Grachev MA et al.; RNA polymerase was treated in the presence of promoter-containing templates with 16 affinity reagents, derivatives on NMPs, NDPs and NTPs with reactive substituents at the terminal phosphate . This treatment was followed by addition of a pyrimidine {alpha-32P}NTP . Due to 'catalytic competence' of some of the residues of the affinity reagents bound covalently near the active center at the first stage, active-center-catalyzed synthesis of a phosphodiester bond occurred, and radioactive residues with the general formula -pNpN (where p = radioactive phosphate) appeared covalently attached to the enzyme . Such affinity labelling was super-selective because affinity reagent residues bound outside the active center were not elongated and thus remained non-radioactive . Labelling took place only when the combination of the reagent and {alpha-32P}NTP corresponded to the sequence of nucleotides of the promoter . With reagents having short 'arms', only the beta subunit was labelled; the targets were His and/or Lys residues . With reagents having longer 'arms', the sigma subunit was also labelled. Eur J Biochem, 1987 Feb 16, 163(1), 141 - 6 Modulation of human neutrophil function by C-reactive protein; Buchta R et al.; Investigation of the effect of C-reactive protein (CRP), an acute-phase reactant, on the functional capacities of human neutrophils was carried out as the basis for elucidating the biological function of C-reactive protein . An initial stimulation at low concentrations, followed by inhibition of superoxide production, and secretion of vitamin-B12-binding protein in the presence of two stimulants, phorbol myristate acetate and concanavalin A, and of neutrophil chemotaxis with increasing concentration of CRP was observed . Correlation between modulation of cell function, at least at relatively high CRP concentrations (greater than 50 micrograms/ml) and an increase in the intracellular level of cAMP is suggested . CRP was also found to enhance neutrophil phagocytosis of particles not containing phosphorylcholine, the native CRP ligand . The proposed role of CRP in neutrophil function is one of regulation and as a negative feedback for potential cytotoxic neutrophil functions. Biochem J, 1987 Feb 15, 242(1), 261 - 6 Characterization of argininosuccinate lyase (EC 4.3.2.1) from Chlamydomonas reinhardtii; Farrell K et al.; We have isolated and characterized argininosuccinate lyase (ASL; EC 4.3.2.1) from the photosynthetic green alga, Chlamydomonas reinhardtii . The general properties of Chlamydomonas ASL are very similar to those described previously for ASLs from phylogenetically diverse organisms . The algal ASL has a native Mr, determined by gel-filtration chromatography, of 218,000 +/- 25,000, and a pI of 5.4-5.6 . The Km for argininosuccinate at 37 degrees C and pH 7.5 is 0.26 mM . The subunit Mr of Chlamydomonas ASL is approx . 50,000, determined by SDS/polyacrylamide-gel electrophoresis, in contrast with a previously reported value of 39,000 . Rabbit antisera prepared against the Mr-50,000 protein completely abolished ASL activity in vitro . In contrast, serum prepared against the Mr-39,000 protein was ineffective in inhibiting ASL activity . Despite the general similarity of the physical properties of Chlamydomonas ASL and those of other ASLs, antiserum raised against the algal ASL did not cross-react with ASL preparations from Escherichia coli, Saccharomyces cerevisiae or bovine liver. J Biol Chem, 1987 Feb 15, 262(5), 2121 - 30 Properties of initiation complexes formed between Escherichia coli DNA polymerase III holoenzyme and primed DNA in the absence of ATP; Kwon-Shin O et al.; In the presence of ATP, the beta subunit of the Escherichia coli DNA polymerase III holoenzyme can induce a stable initiation complex with the other holoenzyme subunits and primed DNA that is capable of highly processive synthesis . We have recently demonstrated that the ATP requirement for processive synthesis can be bypassed by an excess of the beta subunit (Crute, J., LaDuca, R., Johanson, K., McHenry, C., and Bambara, R . (1983) J . Biol . Chem . 258, 11344-11349) . To examine the complex formed with excess beta subunit, and the lengths of the products of processive synthesis, we have designed a uniquely primed DNA template . Poly(dA)4000 was tailed with dCTP by terminal deoxynucleotidyl transferase and the resulting template annealed to oligo(dG)12-18 . In the presence of excess beta, the lengths of processively extended primers nearly equaled the full-length of the DNA template . Similar length synthesis occurred in the presence or absence of spermidine or single-stranded DNA-binding protein . When the beta subunit was present at normal holoenzyme stoichiometry it could induce highly processive synthesis without ATP, although inefficiently . Both ATP and excess beta increased the amount of initiation complex formation, but complexes produced with excess beta did so without the time delay observed with ATP, suggesting different mechanisms for formation . Almost 50% of initiation complexes formed without ATP survived a 30-min incubation with anti-beta IgG, reflecting a stability similar to those formed with ATP . The ability to form initiation complexes in the absence of ATP permitted the demonstration that cycling of the holoenzyme to a new primer, after chain termination with a dideoxynucleotide, is not affected by the presence of ATP. Experientia, 1987 Feb 15, 43(2), 174 - 6 Ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase and arginine decarboxylase from Mycobacterium bovis (BCG); Paulin L et al.; Ornithine decarboxylase (ODC), S-adenosyl-L-methionine decarboxylase (AMDC) and arginine decarboxylase (ADC) activities were detected for the first time in extracts of Mycobacterium bovis (BCG) . All the decarboxylases differed from corresponding known bacterial decarboxylases in that: a) ODC did not require GTP for activity; b) ODC was not inhibited by any known inhibitor of bacterial ODCs; c) AMDC and ADC did not require Mg2+-ion for activity and were not markedly inhibited by any known inhibitor of the decarboxylases of other bacteria. Arch Biochem Biophys, 1987 Feb 15, 253(1), 241 - 8 Purification and properties of the manganese superoxide dismutase from the liver of bullfrog, Rana catesbeiana; Abe Y et al.; A manganese superoxide dismutase (Mn-SOD) from the liver of bullfrog, Rana catesbeiana, was purified to electrophoretic homogeneity . The enzyme has a molecular weight of about 84,000 and is composed of four identical subunits, each containing one manganese atom . The amino acid composition of the enzyme is similar to that of Mn-SODs isolated from human and chicken livers, but differs considerably from that of the Escherichia coli enzyme (D . Barra et al . (1984) J . Biol . Chem . 259, 12595-12601; R . A . Weisiger and I . Fridovich (1973) J . Biol . Chem . 248, 3582-3592; H . M . Steinman (1978) J . Biol . Chem . 253, 8708-8720) . The N-terminal amino acid is lysine . The sequence of 23 amino acid residues in the N-terminal region was determined . It shows excellent homologies with those of the human and chicken enzymes (H . M . Steinmam and R . L . Hill (1973) Proc . Natl . Acad . Sci . USA 70, 3725-3729; C . Ditlow et al . (1982) Carlsberg Res . Commun . 47, 81-91) . The frog liver enzyme is also located exclusively in the mitochondrial matrix . Immunologically the same enzyme is also found in the tadpole liver, in an amount of about one-half of that in the adult bullfrog. J Biol Chem, 1987 Feb 15, 262(5), 2358 - 62 In vitro translocation of protein across Escherichia coli membrane vesicles requires both the proton motive force and ATP; Yamane K et al.; The energy requirement for protein translocation across membrane was studied with inverted membrane vesicles from an Escherichia coli strain that lacks all components of F1F0-ATPase . An ompF-lpp chimeric protein was used as a model secretory protein . Translocation of the chimeric protein into membrane vesicles was totally inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or valinomycin and nigericin and partially inhibited when either valinomycin or nigericin alone was added . Depletion of ATP with glucose and hexokinase resulted in the complete inhibition of the translocation process, and the inhibition was suppressed by the addition of ATP-generating systems such as phosphoenolpyruvate-pyruvate kinase or creatine phosphate-creatine kinase . These results indicate that both the proton motive force and ATP are required for the translocation process . The results further suggest that both the membrane potential and the chemical gradient of protons (delta pH), of which the proton motive force is composed, participate in the translocation process. J Biol Chem, 1987 Feb 15, 262(5), 2304 - 9 Formation of rolling-circle molecules during phi X174 complementary strand DNA replication; Mok M et al.; The primosome is a mobile multiprotein priming apparatus that requires seven Escherichia coli proteins for assembly (the products of the dnaB, dnaC and dnaG genes; replication factor Y (protein n'); and proteins i, n, and n") . While the primosome is analagous to the phage T7 gene 4 protein and phage T4 gene 41/61 proteins in its DNA G-catalyzed priming function, its ability to act similarly also as a DNA helicase has remained equivocal . The role of the primosome in unwinding duplex DNA strands was investigated in the coliphage phi X174 SS(c)----replicative form DNA replication reaction in vitro, which requires the E . coli single-stranded DNA binding protein, the primosomal proteins, and the DNA polymerase III holoenzyme . Multigenome-length, linear, double-stranded DNA molecules were generated in this reaction, presumably via a rolling circle-type mechanism . Synthesis of these products required the presence of a helicase-catalyzed strand-displacement activity to permit multiple cycles of continuous complementary (-) strand synthesis . The participation of the primosome in this helicase activity was supported by demonstrating that other SS(c) DNA templates (G4 and alpha-3), which lack primosome assembly sites, failed to support significant linear multimer production and that replication of phi X174 with the general priming system (the DNA B and DNA G proteins and DNA polymerase III holoenzyme) resulted in a 13-fold lower rate of linear multimer synthesis. J Immunol, 1987 Feb 15, 138(4), 1109 - 14 Expression in Escherichia coli of fully active recombinant human IL 1 beta: comparison with native human IL 1 beta; Tocci MJ et al.; Although complementary DNA (cDNA) encoding human interleukin 1 beta (IL 1 beta) have been cloned in several laboratories, there are as yet no data demonstrating that recombinant IL 1 beta (rIL 1 beta) molecules expressed from such cDNA are faithful, fully active replicas of the native protein secreted by human monocytes . To this purpose, cDNA sequences corresponding to the exact NH2-terminus and amino acid sequence of mature, monocyte-derived human IL 1 beta were placed under control of the inducible trp-lac (TAC) fusion promoter and were expressed in E . coli strain JM105 . rIL 1 beta was purified to homogeneity by high pressure liquid chromatography (HPLC) . Yields of 10 to 20 mg of rIL 1 beta/liter/10(11) cells were obtained . Purified rIL 1 beta was then compared in a number of biochemical and biologic assays to purified native IL 1 beta . Native and rIL 1 beta co-migrated on SDS-polyacrylamide gels as 17.5 kd polypeptides and reacted with specific polyclonal antisera raised to three synthetic peptides of human IL 1 beta in immunoblot experiments . Amino acid sequence analysis of rIL 1 beta showed that the native amino terminus, an ALA residue, was faithfully maintained . Purified native and rIL 1 beta co-chromatographed on reverse-phase HPLC . Specific biologic activities of rIL 1 beta were indistinguishable from those of the native protein in murine thymocyte and human dermal fibroblast proliferation assays, with half-maximal stimulation occurring at concentrations of 25 pM in the murine thymocyte assay and 2 pM in the human dermal fibroblast assay . Similarly, native and rIL 1 beta competed equally well for high affinity IL 1 receptor binding sites, each exhibiting a Ki of 20 pM on MRC-5 human embryonic lung fibroblasts . These observations indicate that E . coli efficiently expresses large quantities of rIL 1 beta, which emulates exactly the properties of the native protein. J Biol Chem, 1987 Feb 15, 262(5), 2180 - 6 Catalytic properties of the F1-adenosine triphosphatase from Escherichia coli K-12 and its genetic variants as revealed by 18O exchanges; Wood JM et al.; We have examined intermediate Pi-water oxygen exchange during {gamma-18O}ATP hydrolysis by the F1 adenosine triphosphatase from Escherichia coli K-12 . Water oxygen incorporation into each Pi released was increased as ATP concentration was lowered as observed previously for the same reaction catalyzed by the enzyme from eukaryotic sources . Heterogeneous distributions of 18O in product Pi were produced by coexisting epsilon subunit-replete and epsilon subunit-depleted enzyme molecules . The epsilon-replete enzyme showed a much higher probability for oxygen exchange . These data imply that the epsilon subunit inhibits net ATP hydrolysis by imposing conformational constraints which reduce the cooperative conformational interactions that promote ADP and Pi release . Four enzyme variants altered in