Escherichia coli

J Virol, 1988 Jan, 62(1), 120 - 6
Localization of sequences responsible for trans-activation of the equine infectious anemia virus long terminal repeat; Sherman L et al.; We used the Escherichia coli chloramphenicol acetyltransferase gene (cat) to study sequences that influence expression of the equine infectious anemia virus (EIAV) genome . The EIAV long terminal repeat (LTR) directed CAT activity in a canine cell line, but at levels much lower than those achieved with other eucaryotic viral promoters . In the same cells infected with EIAV or cotransfected with molecularly cloned EIAV genomic DNA, LTR-directed activity was markedly enhanced . Comparison of cat mRNA and protein levels in these cells indicated that this trans-activating effect could be accounted for by a bimodal mechanism in which both transcriptional and posttranscriptional events are enhanced . trans-Activation but not promoter activity was abolished by deletion of the R-U5 region of the EIAV LTR . EIAV sequences responsible for the trans-activating function could be localized to a region encompassing the 3' and 5' termini of the pol and env genes, respectively (nucleotides 4474 to 5775) . Interestingly, this stretch harbors a short open reading frame with some amino acid sequence similarity to the human immunodeficiency virus type I tat gene product.

Arch Immunol Ther Exp (Warsz), 1988, 36(5), 537 - 45
Effects of interferons, interferon inducers and growth factors on phagocytosis measured by quantitative determination of synthetic compound ingested by mouse bone marrow-derived macrophages; Szulc B et al.; Sodium salt of 9-oxo-10-acridineacetohydroxamic acid (HCA), a new synthetic compound, forms small crystals in aqueous solution . These crystals were easily phagocytized by the mouse bone marrow-derived macrophages . The ingested HCA crystals were visible under light microscope as dark granules . The degree of phagocytosis was estimated by the spectroscopic measurements of absorption of ingested HCA . About 10 day-old cultures of mouse bone marrow-derived macrophages were found to be suitable for a study on the effect of murine or human interferons . It was observed that murine interferons alpha, beta and gamma at low concentrations (10-200 U/ml) stimulated and higher concentrations (400-1000 U/ml) had no effect on the phagocytosis . Previous treatment of interferons with anti-IFN sera abolished the effect of the interferons . CMA-induced interferon and growth factors were found to modify the phagocytic activity of macrophage cultures.

Arch Immunol Ther Exp (Warsz), 1988, 36(3), 273 - 85
Effect of copper-dextran complex (C-79) on the immunity indices in normothermic rabbits and in postpyrogenic fever; Obminska-Domoradzka B; The complex combination of bivalent copper with 3-mercapto-2-hydroxypropyl ether of dextran (preparation C-79) was intravenously injected to normothermic rabbits in the doses 0.04 mg/kg, 0.4 mg/kg and 0.8 mg/kg and additionally in the dose of 0.4 mg/kg to febrile rabbits with fever induced by i.v . administration of 0.32 micrograms/kg LPS from E . coli . The following indices were determined: killing and phagocytic capability of peripheral blood neutrophils, migration of lymphocytes, number of T and B cells and number of cells producing IgM after SRBC immunization . Normothermic rabbits responded to C-79 administration by: increase of killing capability of peripheral blood neutrophils, enhanced formation of rosettes against RRBC and EAC rosettes, increased amount of IgM producing cells and inhibition of migration activity of peripheral blood lymphocytes . As a result of the interaction with bacterial pyrogen, C-79 potentiates and prolongs the stimulatory effect of LPS on the killing ability and phagocytic activity of neutrophils, prolongs the mitogenic effect of LPS on B lymphocytes and potentiates the immunogenic effect evidenced by the increased number of IgM producing cells.

J Basic Microbiol, 1988, 28(8), 541 - 51
Characterization of a thermosensitive mutant of Streptomyces hygroscopicus defective in both DNA and RNA syntheses; Suss F et al.; A stable temperature sensitive mutant of Streptomyces hygroscopicus JA6599 defective in both DNA and RNA syntheses is described . The mutant ts35 is characterized by an immediate stop of DNA synthesis and continued protein synthesis after transfer to restrictive temperature . The reinitiation of DNA synthesis begins immediately after a return to the permissive temperature . This kinetics of macromolecular synthesis at restrictive temperature appears to share similarities with a defect in the DNA elongation process, as described for Escherichia coli (Carl 1970, Hanna and Carl 1975) . The simultaneous stop of both DNA and RNA syntheses may be caused by an additional mutational event affecting also the RNA synthesis . The data were discussed with respect to similar results in E . coli.

Nucleic Acids Symp Ser, 1988, (19), 135 - 8
Sequence-specific cleavage of RNA using chimeric DNA splints and RNase H; Inoue H et al.; To cleave RNA molecules using E . coli RNase H in a site-specific manner, a short oligodeoxyribonucleotide (3-5 mer) linked with oligo(2'-O-methyl)ribonucleotide(s) was designed to be used as a DNA splint . Our model experiments with ribooligomer the splint duplexes (9 mers) and RNase H demonstrated that a tetradeoxynucleotide cluster seems to be sufficient for the enzyme recognition and the short DNA-containing splint directs a unique cleavage of RNA by RNase H . The method could be applied to longer ribooligonucleotide substrates . For example, when 3'm (GA)d(AGAA)m(GGU)5' was used as a hybridization strand, 32pUCUUUCUUCUUCCAGGAU was cleaved specifically between U11 and C12 to yield 32pUCUUUCUUCUU . This method will have a variety of applications for the study of RNA.

Nucleic Acids Symp Ser, 1988, (19), 115 - 6
3'-end labeling of DNA fragments by AMV-reverse transcriptase; Oyama F et al.; The efficiency on 3'-end labeling of DNA fragments was compared between Klenow fragment of DNA polymerase I from E . coli and AMV-reverse transcriptase . In the case of Klenow fragment, the exonuclease activity of this enzyme rapidly removed the labeled dNMP incorporated in 3'-end of DNA fragment by the polymerase activity of it . AMV-reverse transcriptase caused no decrease of the labeled dNMP incorporated by the polymerase activity because it has no exonuclease activity . Therefore, AMV-reverse transcriptase is easier to use than Klenow fragment for labeling the 3'-end of DNA fragment.

Protein Seq Data Anal, 1988, 1(6), 479 - 85
Escherichia coli glutaminyl-tRNA synthetase: a single amino acid replacement relaxes rRNA specificity; Uemura H et al.; We described previously the isolation, by genetic selection, of a mutant of Escherichia coli glutaminyl-tRNA synthetase that misaminoacylates supFtRNATyr with glutamine . The single amino acid change responsible for the mischarging phenotype was identified at amino acid 235 in the translated glnS gene . The mutant, called glnS7, has an Asp----Asn change, and studies with the purified glnS7 gene product show it may mischarge a number of presumably different tRNAs . We have carried out extensive homology searches that show E . coli glutaminyl-tRNA synthetase shares regions of homology with other aminoacyl-tRNA synthetases, although little apparent similarity at the site of the glnS7 mutation . In addition to the conserved 'HIGH' motif implicated in aminoacyl adenylate formation, there are regions of homology of glutaminyl-tRNA synthetase with other synthetases which may be involved in tRNA binding . These include short stretches of homology in sequences acting as a tRNA 'anchor' as well as homology of some aminoacyl-tRNA synthetases to a recently identified motif in ribonucleoproteins . Therefore, our results show that E . coli glutaminyl-tRNA synthetase may share with other aminoacyl-tRNA synthetases regions responsible for tRNA binding, while other regions of the protein, of which the glnS7 mutation may be a component, are responsible for tRNA discrimination.

C R Acad Sci III, 1988, 307(3), 99 - 104
{Restoration, by glycine betaine, of growth and enzyme activities of Escherichia coli Lac-mutants}; Bernard T et al.; Lac- mutants of Escherichia coli which presented a growth triggered by adding glycine betaine to the medium were isolated and characterized . Glycine betaine restores beta-galactosidase (strain AM 12) and lactose permease (strain AT42) activities . It is suggested that the right and active conformation of these enzymes, lost during mutagenesis, is restored, in vivo, in presence of this betaine.

Life Sci, 1988, 43(8), 673 - 81
Expression of domains of mouse alpha-fetoprotein in Escherichia coli; Boismenu R et al.; A mouse alpha-fetoprotein complementary DNA containing the coding region for amino acids 256 to 548 was fused to the lac transcriptional and translational control elements contained on the expression vector pOP203-28 . The expression of a 35 kD hybrid lac Z'- alpha-fetoprotein polypeptide in Escherichia coli was demonstrated by the chloramphenicol release assay and by immunoprecipitation using rabbit anti-mouse alpha-fetoprotein antibodies as probe.

Gene, 1988, 63(1), 123 - 34
Sequence determinants for promoter strength in the leuV operon of Escherichia coli; Bauer BF et al.; The promoter for the leuV tRNA operon of Escherichia coli has been studied . Derivatives of this promoter were examined in vivo, fused to the cat gene or to the lacZ gene . When compared to other promoters, the leuV promoter was found to be at least three times stronger than the tyrT promoter (for the tyrT tRNA operon), or the lac promoter (trp::lac promoter fusion) and as strong as the P1,P2 promoter of the rrnB operon (a ribosomal RNA operon) . Deletion analysis revealed that, while removal of sequences downstream from +11 (relative to the transcription start point) did not affect activity, removal of sequences upstream from -39 resulted in a ten-fold reduction in expression . Unlike rRNA operons which also display upstream activation, sequences responsible for this effect in the leuV promoter are separated into two regions, one between -76 and -47, and the other between -45 and -39 . DNA fragments carrying the leuV promoter migrate aberrantly on polyacrylamide gels, a phenomenon usually associated with DNA bending . One sequence thought to be involved in bending is a TTTTT run centered around -71 . Point mutations engineered at this T5 region resulted in a loss of activation but had no apparent effect on migration rate . Transcription efficiency of promoter derivatives was examined in vitro using supercoiled, relaxed, or linearized plasmids as templates . Upstream activation was observed only when using relaxed templates, although maximum activity was obtained using supercoiled forms . Insertion of the very efficient 16S transcription terminator between the leuV promoter and the cat gene resulted in barely detectable activities, indicating that no antitermination mechanism was present.

Bioorg Khim, 1988 Jan, 14(1), 27 - 30
{Preparative isolation of individual tRNA Phe by high performance liquid reversed-phase chromatography}; Bulychev NV et al.; A method of rapid preparative isolation of Phe-tRNA(Phe) (E . coli) of almost 100% purity (1800 pmoles per 1 OE260) is developed . In includes three successive chromatographies of total tRNA on C18-modified Lichrosphere Si-1000 in acetonitrile gradients . Other individual tRNAs can be isolated by the same approach.

Chem Biol Interact, 1988, 65(3), 275 - 81
Investigation of the specificity of O6-alkylguanine-DNA-alkyltransferase; Pegg AE et al.; Comparison of the abilities of alkylated RNA and DNA to serve as substrates for the O6-alkylguanine-DNA-alkyltransferase have been carried out . It was found that the O6-methylguanine in tRNA was much less active as a substrate for the protein than O6-methylguanine in double stranded DNA . The difference in rates of repair was such that it is unlikely that the alkyltransferase would act on RNA in vivo and, therefore, the reaction with RNA should not contribute towards the exhaustion of its repair capacity.

Proteins, 1988, 3(1), 53 - 9
Ribosome-inhibiting proteins, retroviral reverse transcriptases, and RNase H share common structural elements; Ready MP et al.; Plant ribosome-inhibiting proteins are shown to be homologous at the domain level to RNase H from Escherichia coli and to two regions of the pol gene product of retroviral reverse transcriptases . One of these regions carries the viral integrase or int function, while the other has previously been suggested to contain the viral RNase H exo activity . Several residues conserved among the ribosome inhibitors, E . coli RNase H, and the integrase proteins are seen to occupy a prominent cleft in the tertiary structure of the ribosome inhibitor ricin, suggesting roles in binding or catalysis . It is likely that these homologous sequences represent modern derivatives of an ancient protein-folding unit capable of nucleic acid binding and modification which has been incorporated into a variety of enzyme functions.

Mol Biol (Mosk), 1988 Jan-Feb, 22(1), 267 - 9
{Eukaryotic tryptophanyl-tRNA synthetase acquires affinity to high molecular weight RNA after limited proteolysis}; Fedorov AN et al.; It is shown that the native tryptophanyl-tRNA synthetase (Mr 2X60 kDa) isolated from bovine pancreas does not interact with high-molecular weight RNAs (E . coli rRNAs) . The enzyme acquires the affinity for high-molecular weight RNAs after the cleavage of the 20 kDa fragment from each of the subunits upon digestion by protease . The possible functional significance of the discovered phenomenon is discussed.

Environ Mol Mutagen, 1988, 11(4), 461 - 72
Mutagenesis of bleomycin-damaged lambda phage in SOS-deficient and repair endonuclease-deficient Escherichia coli; Povirk LF et al.; Previous DNA sequence analysis of bleomycin-induced forward mutations in repackaged lambda phage has suggested SOS-dependent replicative bypass of oxidized apyrimidinic sites as a possible mechanism of mutagenesis . In order to evaluate this hypothesis further, frequencies of mutation to a clear-plaque phenotype were compared for bleomycin-damaged phage grown in various repair-deficient strains of Escherichia coli . Survival of bleomycin-damaged phage was virtually identical in all host strains . Studies in SOS-deficient strains indicated specific requirements for functional recA+ and umuC+ alleles in the generation of the majority of bleomycin-induced mutations, as well as a less stringent requirement for induction of the SOS response by ultraviolet irradiation of the host cells . These results are expected for mutagenesis resulting from apyrimidinic sites . However, the mutation frequency for bleomycin-damaged phage was the same whether the phage were grown in a wild-type strain or in strains deficient in apurinic/apyrimidinic repair endonucleases; this was true even for an nth-nfo-xth- strain lacking all three major apurinic/apyrimidinic endonucleases (endonuclease III, endonuclease IV, and exonuclease III) . Likewise, phage grown in an endonuclease IV-overproducing strain showed the same mutation frequency as those grown in wild-type cells . These data suggest that either i) bleomycin-induced mutagenesis results from SOS-dependent bypass of lesions other than apyrimidinic sites or ii) the number of apyrimidinic sites available for SOS processing is virtually independent of the level of apurinic/apyrimidinic endonuclease activity in the cell . It is possible that a fraction of the apyrimidinic sites induced by bleomycin either are intrinsically resistant to repair or undergo secondary reactions that render them resistant.

Anal Biochem, 1988 Jan, 168(1), 25 - 30
Purification of Escherichia coli alkaline phosphatase on an ion-exchange high-performance liquid chromatographic column using carboxymethyl dextrans; Dunn BE et al.; Carboxymethyl dextrans (CM-Ds) were used on an HPLC ion-exchange column to obtain significantly enriched alkaline phosphatase (EC 3.1.3.1) from a sample of Escherichia coli periplasmic space proteins without significant loss of enzymatic activity . The ability of CM-Ds to separate alkaline phosphatase even when the column was 80-85% saturated with protein demonstrates the potential for high column capacity using CM-Ds . In addition, the fractions containing alkaline phosphatase and CM-Ds were reapplied to the same ion-exchange column under different buffer conditions and purified to homogeneity by salt gradient elution chromatography, thus demonstrating the compatibility of CM-Ds with the latter chromatographic method . The two-step chromatographic procedure yielded enzyme of purity comparable to that of electrophoretically purified E . coli alkaline phosphatase obtained commercially . These studies demonstrate that HPLC displacement chromatography is a mild procedure which allows rapid, quantitative purification of an enzyme . Scaling up with larger columns should allow purification of enzymes of a commercial basis.

EMBO J, 1988 Jan, 7(1), 225 - 30
Major surface antigen p190 of Plasmodium falciparum: detection of common epitopes present in a variety of plasmodia isolates; Gentz R et al.; Plasmodium falciparum merozoites are covered with polymorphic proteins that are processed from a 190 kd (p190) precursor protein . These are candidates for an antimalarial vaccine . We cloned and expressed a number of DNA fragments, comprising almost the entire p190 gene of the K1 isolate, in Escherichia coli . Pooled human endemic-area sera and rabbit antibodies raised against p190 protein isolated from K1 parasites react with only a limited number of the recombinant proteins . From these studies we could select two antigenic polypeptides containing conserved amino acid stretches of the otherwise highly polymorphic protein . Rabbits and mice injected with the purified recombinant proteins produce antibodies reacting differentially with various isolates of P . falciparum . We obtained antibodies detecting all isolates tested and a monoclonal antibody specific for isolates containing a K1 type allele of the p190 gene.

Biochem Int, 1988 Jan, 16(1), 101 - 9
Enhanced production of hepatitis B virus surface antigen in mouse C127 cell on a bovine papillomavirus-metallothionein vector; Lee YH et al.; We have constructed a recombinant plasmid pCPS12 containing the hepatitis B viral surface antigen (HBsAg) gene linked to the mouse metallothionein promoter on a BPV-pML2 vector . Two stable clones S12-8 and S12-2, obtained by transfection of the mouse C127 cells with pCPS12 propagated in dam+ dcm+ and dam- dcm- Escherichia coli respectively, exhibited different types of response to 5-azacytidine (5-aza-CR) and cadmium (Cd) induction . In S12-8, the productivity of HBsAg was enhanced by 5-aza-CR or 5-aza-CR plus Cd, but not by Cd alone . In S12-2, the expression of HBsAg was not affected by 5-aza-CR but was induced by Cd in the presence or absence of 5-aza-CR . This suggests that methylation may be important in controlling the HBsAg expression and the inducibility of Cd in the transfectants.

J Steroid Biochem, 1988 Jan, 29(1), 27 - 31
Ribonucleic acid association with androgen receptor from rat submandibular gland; Ohara-Nemoto Y et al.; The transformed androgen receptor from rat submandibular gland converts to a faster sedimenting form (6-8S) on a glycerol gradient centrifugation after withdrawal of a transformation-inducing reagent (KCl or ATP) . In this report, the association of cytosolic RNA with the transformed androgen receptor was investigated as a possible mechanism of molecular conversion of the androgen receptor . When the transformed and converted androgen receptors were treated with RNase A, these receptors sedimented at 4.5S in a low-salt glycerol gradient . Addition of RNA from rat submandibular gland to the RNase-Sepharose-treated transformed receptor caused a shift of receptor peak from 4.5S to 5.8S . RNA from rat submandibular gland, yeast RNA and E . coli rRNA inhibited DNA-cellulose binding of a RNase-treated transformed receptor in the absence of molybdate . These observations suggest that conversion from the transformed 4S androgen receptor to a 6-8S form resulted from the association of RNA(s) with the transformed receptor.

Arch Biochem Biophys, 1988 Jan, 260(1), 452 - 60
Synthesis and assembly of H+-ATPase complex by isolated "rough" thylakoids; Shinohara K et al.; The synthesis and assembly of chloroplast H+-ATPase complex were studied by analyzing the incorporation of {35S}methionine into the constituent subunits with isolated intact chloroplasts and with thylakoid membranes that had been prepared from the chloroplasts so that they would retain ribosomes . The complex was isolated from thylakoids after labeling and identified by immunoprecipitation with an antiserum specific to CF1 . The mechanism for the assembly of the complex was demonstrated to be active in the isolated chloroplasts by the following observations: the plastid genome-regulated subunits (alpha, beta, epsilon, I, and III) were labeled by in organello translation and recovered with the complex, and three other subunits (gamma, delta, and II) were labeled when intact chloroplasts were incubated with translation products from polyadenylated RNA . The two largest subunits, alpha and beta, were translated on thylakoid-bound ribosomes when the thylakoid membranes were incubated with soluble factors from Escherichia coli . They were recovered with the H+-ATPase complex, suggesting that they are translated on the bound ribosomes in the chloroplast, and that the isolated membranes retain the ability to assemble a complete complex . Provided that these observations are the result of de novo assembly of the complex, the imported and processed nuclear-coded subunits are presumed to be pooled not in stroma but on the membrane.

J Bacteriol, 1988 Jan, 170(1), 431 - 5
Variation in precursor pool size during the division cycle of Escherichia coli: further evidence for linear cell growth; Kubitschek HE et al.; The magnitudes of several pools of radioactively labeled precursors for RNA and protein synthesis were determined as a function of cell age during the division cycle of Escherichia coli 15 THU . Uracil, histidine, and methionine pools increased from low initial values for cells at birth to maxima during midcycle and then subsided again . These pools were small or nonexistent at the beginning and the end of the cycle, and their average values during the cycle were less than 4% of the total cellular radioactivity . The results are consistent with a linear pattern of growth for cells during the division cycle and provide strong evidence against exponential or bilinear growth of E . coli cells.

Biochem Biophys Res Commun, 1987 Dec 31, 149(3), 859 - 65
Molecular cloning and in vivo expression of a precursor to rat mitochondrial aspartate aminotransferase; Mattingly JR Jr et al.; A 2.4 kilobase cDNA for rat mitochondrial aspartate aminotransferase (E.C . 2.6.1.1.) was isolated and sequenced . The predicted presequence is 93% homologous to the presequences of the enzyme from pig and mouse . The predicted amino acid sequence of the mature enzyme differs from that determined directly by amino acid sequencing (Huynh, Q.K., Sakakibara, R., Watanabe, T., and Wada, H . (1981) J . Biochem . (Tokyo) 90, 863-875) at 13 amino acids residues . The most important difference is at position 140 where the cDNA encodes a tryptophanyl residue rather than the previously reported glycine . This critical residue is now seen to be conserved in all aspartate aminotransferases . The coding region of this cDNA was inserted into the plasmid cloning vector pKK233-2 and used to stably express an unfused precursor in Escherichia coli JM105.

Biochem Biophys Res Commun, 1987 Dec 31, 149(3), 846 - 51
A method to isolate DNA sequences that are promoter-active in Escherichia coli and in yeast; Kwak JW et al.; A method convenient for isolation of DNA sequences capable of directing gene transcription in both organisms of E . coli and yeast is described . The method is composed of sequential steps of phenotypic selection for chloramphenicol resistance, first in E . coli and then in yeast . A series of promoter-probe, shuttle plasmid vectors between yeast and E . coli were constructed and utilized in the method.

Eur J Biochem, 1987 Dec 30, 170(1-2), 59 - 67
Translation of an mRNA in rat L6 muscle cells is regulated within the cell cycle; Pramanik SK et al.; In muscle cells two populations of mRNA are present in the cytoplasm . The majority of mRNA is associated with ribosomes and active in protein synthesis . A small population of cytoplasmic mRNA occur as free mRNA-protein complex and is not associated with ribosomes . This apparently repressed population of mRNA from rat L6 myoblast cells was used to construct a cDNA library . Radioactively labeled cDNA preparations of polysomal and free (or repressed) mRNA populations showed that at least ten recombinant clones preferentially annealed to the cDNA from repressed mRNA . One of these clones was extensively studied . The DNA from a recombinant plasmid D12 hybridized to a 1.3-kb poly(A)-rich mRNA . In proliferating myoblast cells, the 1.3-kb mRNA was more abundant in the polysomal fraction and mostly free in the non-dividing myotubes . In contrast to this mRNA, 90% of alpha and beta actin mRNAs were translated in both myoblasts and myotubes . Further analysis of distribution of the 1.3-kb RNA in the polysomal (active) and free (repressed) fractions in fusion-arrested postmitotic myotubes suggested that fusion of myoblasts was not necessary for the control of translation of this mRNA . Withdrawal of muscle cells from the cell cycle appeared to be involved in regulating translation of this mRNA . The presence of this mRNA was not, however, limited to muscle cells . This mRNA was also present in the repressed state in rat liver and kidney cells . These results, therefore, suggest that the 1.3-kb mRNA is probably translated during a particular phase of the cell cycle and is not translated in terminally differentiated non-dividing cells . Messenger RNA homologous to the 600-base-pair insert of the recombinant plasmid D12 was isolated by hybrid selection procedure from both polysomal mRNA of myoblasts and free mRNA of myotubes . Translation of the hybrid selected mRNAs from both myoblasts and myotubes in rabbit reticulocyte lysate cell-free system synthesized a 40-kDa polypeptide . These results suggest that the repressed population of 1.3-kb mRNA can be translated in vitro . The hybridization pattern of DNA from the recombinant plasmid D12 with rat genomic DNA suggested that the 1.3-kb mRNA is derived from moderately repetitive rat DNA with a repetition frequency of approximately 100 copies per haploid genome.

Eur J Biochem, 1987 Dec 30, 170(1-2), 335 - 42
The role of the N-terminus of the large subunit of ribulose-bisphosphate carboxylase investigated by construction and expression of chimaeric genes; Kettleborough CA et al.; The genes for the large and small subunits of ribulose bisphosphate carboxylase/oxygenase (Rubisco) from Anacystis nidulans have been expressed in Escherichia coli under the control of the lac promoter to produce active enzyme . The enzyme can be purified from the cells to yield up to 200 mg Rubisco/l cultured bacteria, and is indistinguishable from the enzyme extracted from A . nidulans . In order to investigate the role of the N-terminus of the large subunit in catalysis, chimaeric genes were constructed where the DNA coding for the 12 N-terminal amino acids in A . nidulans was replaced by DNA encoding the equivalent, but poorly conserved, region of either the wheat or maize large subunit . These genes, in constructs also containing the gene for the A . nidulans small subunit, were expressed in E . coli and produced enzymes with similar catalytic properties to the wild-type Rubisco of A . nidulans . In contrast, when the N-terminal region of the large subunit was replaced by unrelated amino acids encoded by the pUC8 polylinker, enzyme activity of the expressed protein was reduced by 90% under standard assay conditions, due to an approximately tenfold rise in the Km for ribulose 1,5-bisphosphate . This confirms that the N-terminus of the large subunit has a function in catalysis, either directly in substrate binding or in maintaining the integrity of the active site.

Biochemistry, 1987 Dec 29, 26(26), 8599 - 606
Catalytic mechanism and inhibition of tRNA (uracil-5-)methyltransferase: evidence for covalent catalysis; Santi DV et al.; tRNA (Ura-5-)methyltransferase catalyzes the transfer of a methyl group from S-adenosylmethionine (AdoMet) to the 5-carbon of a specific Urd residue in tRNA . This results in stoichiometric release of tritium from {5-3H}Urd-labeled substrate tRNA isolated from methyltransferase-deficient Escherichia coli . The enzyme also catalyzes an AdoMet-independent exchange reaction between {5-3H}-Urd-labeled substrate tRNA and protons of water at a rate that is about 1% that of the normal methylation reaction, but with identical stoichiometry . S-Adenosylhomocysteine inhibits the rate of the exchange reaction by 2-3-fold, whereas an analogue having the sulfur of AdoMet replaced by nitrogen accelerates the exchange reaction 9-fold . In the presence (but not absence) of AdoMet, 5-fluorouracil-substituted tRNA (FUra-tRNA) leads to the first-order inactivation of the enzyme . This is accompanied by the formation of a stable covalent complex containing the enzyme, FUra-tRNA, and the methyl group of AdoMet . A mechanism for catalysis is proposed that explains both the 5-H exchange reaction and the inhibition by FUra-tRNA: the enzyme forms a covalent Michael adduct with substrate or inhibitor tRNA by attack of a nucleophilic group of the enzyme at carbon 6 of the pyrimidine residue to be modified . As a result, an anion equivalent is generated at carbon 5 that is sufficiently reactive to be methylated by AdoMet . Preliminary experiments and precedents suggest that the nucleophilic catalyst of the enzyme is a thiol group of cysteine . The potent irreversible inhibition by FUra-tRNA suggests that a mechanism for the "RNA" effects of FUra may also involve irreversible inhibition of RNA-modifying enzymes.

Biochemistry, 1987 Dec 29, 26(26), 8591 - 8
Construction of an altered proton donation mechanism in Escherichia coli dihydrofolate reductase; Howell EE et al.; We have explored the substrate protonation mechanism of Escherichia coli dihydrofolate reductase by changing the location of the proton donor . A double mutant was constructed in which the proton donor of the wild-type enzyme, aspartic acid-27, has been changed to serine and simultaneously an alternative proton donor, glutamic acid, has replaced threonine at position 113 . The active site of the resulting variant enzyme molecule should therefore somewhat resemble that proposed for the R67 plasmid-encoded dihydrofolate reductase {Matthews, D . A., Smith, S . L., Baccanari, D . P., Burchall, J . J., Oatley, S . J., & Kraut, J . (1986) Biochemistry 25, 4194} . At pH 7, the double-mutant enzyme has a 3-fold greater kcat and an unchanged Km(dihydrofolate) as compared with the single-mutant Asp-27----Ser enzyme described previously {Howell, E . E., Villafranca, J . E., Warren, M . S., Oatley, S . J., & Kraut, J . (1986) Science (Washington, D.C.) 231, 1123} . Additionally, its activity vs pH profiles together with observed deuterium isotope effects, suggest that catalysis depends on an acidic group with a pKa of 8 . It is concluded that the dihydropteridine ring of a bound substrate molecule can indeed be protonated by a glutamic acid side chain at position 113 (instead of an aspartic acid side chain at position 27), but with greatly decreased efficiency: at pH 7, the double mutant still has a 25-fold lower kcat (1.2 s-1) and a 2900-fold lower kcat/km(dihydrofolate) (8.6 X 10(3) s-1 M-1) than the wild-type enzyme.

J Biol Chem, 1987 Dec 25, 262(36), 17450 - 4
The properties of hybrid F1-ATPase enzymes suggest that a cyclical catalytic mechanism involving three catalytic sites occurs; Rao R et al.; Maximal rates of ATP hydrolysis catalyzed by F1-ATPase enzymes are known to involve strong positive catalytic site cooperativity . There are three potential catalytic nucleotide-binding sites on F1 . Two important and unanswered questions are (i) whether all three potential catalytic sites must interact cooperatively to yield maximal rates of ATP hydrolysis and (ii) whether a cyclical three-site mechanism operates as suggested by several authors . We have studied these two questions here by measuring the ATPase activities of hybrid enzymes containing normal beta-, gamma-, delta-, and epsilon-subunits together with different combinations of mutant and normal alpha-subunits . The mutant alpha-subunits were derived from uncA401, uncA447, and uncA453 mutant E . coli F1-ATPase, in which positive cooperativity between catalytic sites is strongly attenuated by defined mis-sense mutations . Our data show that three normal catalytic sites are required to interact in order to achieve maximal ATPase rates and suggest that a cyclical mechanism does operate . Hybrid enzyme containing one-third mutant alpha-subunit and two-thirds normal alpha-subunits had substantial but submaximal activity, showing that cooperativity between three sites in a noncyclical fashion, or between pairs of sites, can achieve effective catalysis.

J Biol Chem, 1987 Dec 25, 262(36), 17750 - 6
Expression and accurate processing of yeast penta-ubiquitin in Escherichia coli; Jonnalagadda S et al.; An expression vector (pSJyub-5) was constructed which contained five repeats of the "yeast ubiquitin gene" regulated by a heat-inducible lambda PL promoter . The vector, when expressed in Escherichia coli, produced a penta-ubiquitin of approximately 42 kDa . Purified penta-ubiquitin was found to be as active as the human mono-ubiquitin in the in vitro ATP/ubiquitin-dependent proteolytic assay of the reticulocyte lysate, indicating that the expressed gene product was recognized by the enzymes involved in the ATP/ubiquitin-dependent proteolytic pathway . The inability of penta-ubiquitin to act as a substrate in the pyrophosphate exchange reaction with the ubiquitin-activating enzyme E1 suggested that it had a carboxyl-terminal Asn, in agreement with the nucleotide sequence . In E . coli, the expressed penta-ubiquitin was processed correctly to mono-ubiquitin . The fidelity of processing in E . coli was confirmed by the following observations . The amino acid compositions of the processed mono-ubiquitin and human ubiquitin were similar . The 1H NMR spectrum of peaks representing amide hydrogens of the carboxyl-terminal Arg-74, Gly-75, and Gly-76 of the processed mono-ubiquitin was identical with that of human ubiquitin . The immunoreactivity of processed mono-ubiquitin and human ubiquitin against polyclonal antibodies that recognized epitope(s) only on the carboxyl terminus of ubiquitin were similar . The human and processed mono-ubiquitin were equally active in degrading 125I-bovine serum albumin in the ATP/ubiquitin-dependent in vitro proteolytic assay with reticulocyte lysates . In the pyrophosphate exchange assay with isolated ubiquitin activating enzyme E1, they were also equally reactive, confirming that the expressed and processed ubiquitin contained an intact carboxyl-terminal Gly-76 . Purified penta-ubiquitin should prove to be a useful substrate for identifying and isolating processing enzyme(s) involved in the ATP/ubiquitin-dependent proteolytic pathway.

J Biol Chem, 1987 Dec 25, 262(36), 17556 - 62
Purification, properties, and genetic location of Escherichia coli cytidine 5'-monophosphate N-acetylneuraminic acid synthetase; Vann WF et al.; N-Acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-NeuAc synthetase) catalyzes the formation of cytidine monophosphate N-acetylneuraminic acid . We have purified CMP-NeuAc synthetase from an Escherichia coli O18:K1 cytoplasmic fraction to apparent homogeneity by ion exchange chromatography and affinity chromatography on CDP-ethanolamine linked to agarose . The enzyme has a specific activity of 2.1 mumol/mg/min and migrates as a single protein and activity band on nondenaturing polyacrylamide gel electrophoresis . The enzyme has a requirement for Mg2+ or Mn2+ and exhibits optimal activity between pH 9.0 and 10 . The apparent Michaelis constants for the CTP and NeuAc are 0.31 and 4 mM, respectively . The CTP analogues 5-mercuri-CTP and CTP-2',3'-dialdehyde are inhibitors . The purified CMP-N-acetylneuraminic acid synthetase has a molecular weight of approximately 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The gene encoding CMP-N-acetylneuraminic acid synthetase is located on a 3.3-kilobase HindIII fragment . The purified enzyme appears to be identical to the 50,000 Mr polypeptide encoded by this gene based on insertion mutations that result in the loss of detectable enzymatic activity . The amino-terminal sequence of the purified protein was used to locate the start codon for the CMP-NeuAc synthetase gene . Both the enzyme and the 50,000 Mr polypeptide have the same NH2-terminal amino acid sequence . Antibodies prepared to a peptide derived from the NH2-terminal amino acid sequence bind to purified CMP-NeuAc synthetase.

J Immunol Methods, 1987 Dec 24, 105(2), 275 - 80
A novel polycarbonate (Nuclepore) membrane demonstrates chemotaxis, unaffected by chemokinesis, of polymorphonuclear leukocytes in the Boyden chamber; Bignold LP; A novel polycarbonate (Nuclepore) membrane having capillary pores of 3 microns diameter and occupying 0.1% of surface area (average minimum spacing of 48 +/- 18.5 microns), a standard polycarbonate filtration membrane (pores 3 microns in diameter occupying 5% of surface area and having average minimum spacing of 3.9 +/- 2.8 microns) as well as a 3 microns pore cellulose acetate filtration membrane, were compared for their abilities to demonstrate chemotactic movement uninfluenced by chemokinetic movement of polymorphonuclear leukocytes in a Boyden-type chemotactic chamber . The chemoattractant used was dilute Escherichia coli culture filtrate employed in a gradient across the membranes to measure chemotaxis and in non-gradient conditions to measure chemokinesis . The new polycarbonate (Nuclepore) membrane provided the clearest demonstration of chemotaxis unaffected by chemokinesis of the polymorphonuclear leukocytes.

Cell, 1987 Dec 24, 51(6), 1079 - 90
Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain; Kadonaga JT et al.; Transcription factor Sp1 is a protein present in mammalian cells that binds to GC box promoter elements and selectively activates mRNA synthesis from genes that contain functional recognition sites . We have isolated a cDNA that encodes the 696 C-terminal amino acid residues of human Sp1 . By expression of truncated fragments of Sp1 in E . coli, we have localized the DNA binding activity to the C-terminal 168 amino acid residues . In this region, Sp1 has three contiguous Zn(II) finger motifs, which are believed to be metalloprotein structures that interact with DNA . We have found that purified Sp1 requires Zn(II) for sequence-specific binding to DNA . Thus, it is likely that Sp1 interacts with DNA by binding of the Zn(II) fingers . To facilitate the identification of mutant variants of Sp1 that are defective in DNA binding, we have also devised a bacterial colony assay for detection of Sp1 binding to DNA.

Cell, 1987 Dec 24, 51(6), 1131 - 43
Differential mRNA stability controls relative gene expression within a polycistronic operon; Newbury SF et al.; In this paper we demonstrate a role for mRNA stability in controlling relative gene expression within a polycistronic operon . The polycistronic malEFG operon of E . coli contains two REP sequences (highly conserved inverted repeats) within the malE-malF intercistronic region . Deletion of these REP sequences from the chromosomal operon not only destabilizes upstream malE mRNA, but also results in a 9-fold reduction in the synthesis of MalE protein . A single REP sequence seems to be as efficient as the two normally found in this intergenic region at stabilizing translationally active upstream mRNA . The widespread occurrence of REP sequences and other sequences that could potentially stabilize upstream mRNA suggests that this mechanism of control of gene expression may be rather common.

Cell, 1987 Dec 24, 51(6), 1123 - 30
Transcriptional activation of ColE1 DNA synthesis by displacement of the nontranscribed strand; Masukata H et al.; Plasmid ColE1 can replicate using RNAase H and DNA polymerase I . However, it can also replicate in the absence of these enzymes . In this case, formation of a persistent hybrid between a transcript (RNA II) and the DNA indirectly activates subsequent DNA synthesis, instead of providing a primer as it does in the presence of these enzymes . To activate DNA synthesis, a certain length is required for the hybridized region and the region of minimum length cannot include a palindrome . These results show that the single-stranded region of DNA displaced by the hybridization is responsible for the activation . A single-stranded region was identified on the nontranscribed strand by its enhanced reactivity to dimethyl sulfate . The necessary length for the single-stranded region is at least 40 nucleotides . The region probably provides a site for initial binding of a helicase that further unwinds the template DNA for initiation of DNA synthesis.

Cell, 1987 Dec 24, 51(6), 1113 - 22
Multiple mechanisms for initiation of ColE1 DNA replication: DNA synthesis in the presence and absence of ribonuclease H; Dasgupta S et al.; A transcript (RNA II) of plasmid ColE1 that hybridizes with the template DNA is cleaved by RNAase H and used as a primer by DNA polymerase I . However, the plasmid can replicate in bacteria lacking both enzymes, apparently using a different mechanism of initiation of replication . Here we report in vivo and in vitro studies on initiation of DNA replication in the presence or absence of either or both enzymes . Hybridization of RNA II with the template DNA is always required for initiation . Hybridized RNA II is cleaved by RNAase H to form a primer or used as a primer without cleavage by RNAase H . Hybridization also creates a single-stranded region on the nontranscribed strand that can serve as a template for synthesis of the lagging strand in a reaction that does not require DNA polymerase I . Lagging strand synthesis terminates 17 nucleotides upstream of the normal replication origin, forcing unidirectional replication.

Nucleic Acids Res, 1987 Dec 23, 15(24), 10331 - 43
A cyanogen bromide fragment of S4 that specifically rebinds 16S RNA; Conrad RC et al.; Escherichia coli ribosomal protein S4 was subjected to cyanogen bromide cleavage and was found to generate a complete cleavage product capable of rebinding 16S rRNA . This fragment, consisting of residues 1-103, was found to bind with an apparent association constant of 11 microM-1 . This fragment was used in place of S4 in an in vitro reconstitution experiment . Although the particles formed had a protein composition not significantly different from reconstituted 30S ribosomal subunits, their sedimentation behavior was more like that of particles reconstituted without S4 . These results indicate to us that, although residues 104-203 of S4 are involved in the assembly of the 30S ribosome, they are not necessary for the binding of S4 to 16S RNA . Taken with previous results, the domain of S4 involved in specific binding of 16S RNA can be confined to residues 47-103.

Nucleic Acids Res, 1987 Dec 23, 15(24), 10199 - 210
Molecular cloning and nucleotide sequence of the gene for Escherichia coli leucyl-tRNA synthetase; Hartlein M et al.; The gene for Escherichia coli leucyl-tRNA synthetase leuS has been cloned by complementation of a leuS temperature sensitive mutant KL231 with an E.coli gene bank DNA . The resulting clones overexpress leucyl-tRNA synthetase (LeuRS) by a factor greater than 50 . The DNA sequence of the complete coding regions was determined . The derived N-terminal protein sequence of LeuRS was confirmed by independent protein sequencing of the first 8 aminoacids . Sequence comparison of the LeuRS sequence with all aminoacyl-tRNA synthetase sequences available reveal a significant homology with the valyl-, isoleucyl- and methionyl-enzyme indicating that the genes of these enzymes could have derived from a common ancestor . Sequence comparison with the gene product of the yeast nuclear NAM2-1 suppressor allele curing mitochondrial RNA maturation deficiency reveals about 30% homology.

Nucleic Acids Res, 1987 Dec 23, 15(24), 10393 - 404
The mitochondrial S13 ribosomal protein gene is silent in wheat embryos and seedlings; Bonen L; The sequence of a wheat mitochondrial reading frame encoding a protein homologous to the E . coli S13 small subunit ribosomal protein has been determined . The gene is located immediately downstream of a 1.4 kb recombinationally-active repeat element that contains the ATPase subunit 6 gene . The coding regions of the two genes are separated by only 153 bp, the shortest distance yet observed between protein-coding genes in plant mitochondria . However, their transcript profiles differ markedly . The ATPase 6 gene displays a single, prominent mRNA of approximately 1.4 kb, whereas the S13 gene shows no stable transcript as judged by Northern blot analysis of wheat mitochondrial RNA isolated from different developmental stages . A short segment of the 26S rRNA gene is located downstream of the S13 gene and its presence illustrates the frequent DNA duplication/rearrangements found in wheat mitochondria.

EMBO J, 1987 Dec 20, 6(13), 4235 - 9
Mutants of elongation factor Tu promote ribosomal frameshifting and nonsense readthrough; Hughes D et al.; This is the first report of ribosomal frameshifting promoted by mutants of the elongation factor Tu (EF-Tu) . EF-Tu mutants can suppress both -1 and +1 frameshift mutations . The level of nonsense readthrough is also increased at some UGA (this paper) and UAG (Hughes, 1987) sites by these mutants . Suppression occurs when a mutant tuf allele is paired with a wild-type copy of the other tuf gene but is most efficient when both tuf genes are mutant . Frameshifting mediated by the tuf alleles studied, tufA8 and tufB103, is not general; indeed most frameshift mutations are not suppressed . Several possible mechanisms by which mutant EF-Tu may cause frameshifting are discussed.

EMBO J, 1987 Dec 20, 6(13), 3901 - 7
GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants; Jefferson RA et al.; We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants . Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made . We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants . Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue . Plants expressing GUS are normal, healthy and fertile . GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage . Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.

EMBO J, 1987 Dec 20, 6(13), 4227 - 34
A new target for CRP action at the malT promoter; Menendez M et al.; In Escherichia coli, the transcription of the malT gene is activated by the complex formed between cAMP and its receptor protein, CRP . Kinetics of formation of polyribonucleotide products from the corresponding promoter were studied in vitro by two sets of techniques, abortive initiation assays and run-off experiments . The first type of assay indicated that open complexes were formed at malT with an equivalent efficiency, and at comparable rates, whether CRP-cAMP was present or not . Secondary effects due to the activating complex were observed (increased stability of the open complex, elimination of a weaker binding site for the enzyme, improved Michaelis constants of RNA polymerase for the substrates of the assay, UTP in particular) . But, primarily, CRP-cAMP did not exert a significant role in the rate of formation of the initiation complex . In contrast, run-off assays showed that the yield of the full-length transcripts was markedly enhanced by prior incubation of the DNA fragment with CRP-cAMP . Both in the presence and in the absence of activator, the rate-limiting step for this process was markedly slower than the formation of the initial open complex . Short oligonucleotides (n less than 9), probably arising from a recycling process, were found when the initiation complex was formed in the absence of CRP-cAMP . They were abolished by prior incubation with the activator . Unexpectedly, CRP-cAMP appears to favour the escape of RNA polymerase from the initiation complex at this promoter.

EMBO J, 1987 Dec 20, 6(13), 4205 - 12
Antagonistic controls regulate copy number of the yeast 2 mu plasmid; Murray JA et al.; The endogenous 2 mu plasmid of yeast encodes a site-specific recombinase FLP that can cause an amplification of plasmid copy number . Using strains overexpressing 2 mu plasmid proteins from chromosomal constructs to disrupt the normal balance of gene products, we show here that copy number is controlled by regulating the transcript level of FLP . Expression of FLP is negatively regulated over a 100-fold range by the joint action of the plasmid-encoded REP1 and REP2 proteins, which also have a role in plasmid partition . We also show that the product of the fourth plasmid open reading frame D increases FLP expression by relieving the repression caused by REP1 and REP2 . This is the first demonstration of a function for this gene, which we call RAF . The transcription of RAF is also repressed by REP1 and REP2 acting together, but requires a higher level for complete inhibition than that required to repress FLP . Copy number is therefore negatively regulated by REP1-REP2 concentration both by direct repression of FLP and indirectly, by control of the positive element, the anti-repressor RAF . We propose that these antagonistic regulatory mechanisms amplify the signal produced by a small fall in copy number.

J Mol Biol, 1987 Dec 20, 198(4), 693 - 703
Role of SecA and SecY in protein export as revealed by studies of TonA assembly into the outer membrane of Escherichia coli; Baker K et al.; The growth of secAts or secYts mutants at the restrictive temperature has been shown to inhibit the export of many outer membrane proteins . We report here that in two secAts strains the rate of incorporation of newly synthesized protein into both inner and outer membrane fractions decreased by about 70% at the restrictive temperature . The export of the outer membrane protein TonA was used as a model system in which to study the effects of SecA or SecY inactivation . pre-TonA that accumulated at the restrictive temperature was found to co-sediment with the outer membrane fraction . However, the precursor was sensitive to protease and did not float up a sucrose gradient with the membrane fractions . It was therefore concluded that pre-TonA was not integrated into the outer membrane fraction but probably accumulated in the cytoplasm . Studies on the rate of processing of pre-TonA, pulse-labelled at the restrictive temperature then chased at the permissive temperature, revealed differences between secA and secY mutants . In the secAts mutant the great majority of cytoplasmic pre-TonA was not apparently processed to the mature form, whereas in the secYts mutant significant amounts of precursors were rapidly chased into mature TonA, which appeared in the outer membrane . These results suggest that SecA and SecY may act sequentially in the export of proteins to the outer membrane . In particular these data indicate that SecA is required to maintain pre-TonA in a translocationally competent form prior to interaction with the SecY export site.

J Mol Biol, 1987 Dec 20, 198(4), 633 - 41
Molecular analysis of the promoter region of the Escherichia coli K-12 phoE gene . Identification of an element, upstream from the promoter, required for efficient expression of phoE protein; Tommassen J et al.; The phoE gene of Escherichia coli codes for an outer membrane pore protein whose expression is induced under phosphate limitation . The promoter of this gene contains a 17 base-pair fragment, designated a pho box, which is present also in other phosphate-controlled promoters . The mRNA start site was determined and found to be located downstream from the pho box, such that this element is located in the -35 region of the phoE promoter . A set of promoter deletions was generated in vitro and analysis of these deletions revealed that sequences upstream from the pho box are required for the efficient expression of phoE . The required upstream region is located (in part) between positions -106 and -121 relative to the mRNA start site, and contains sequences homologous to a pho box and a correctly spaced Pribnow box, but in the reversed orientation relative to the regular -35 and -10 regions . A proper spacing between this upstream region and the -35 region appears to be important, since an oligonucleotide insertion in the intervening region interferes with phoE expression . By cloning the upstream region in a lacZ operon fusion vector, a weak phosphate limitation-inducible promoter activity could be detected.

Nature, 1987 Dec 17-23, 330(6149), 662 - 4
Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia; Tracey KJ et al.; Bacterial infection of the mammalian bloodstream can lead to overwhelming sepsis, a potentially fatal syndrome of irreversible cardiovascular collapse (shock) and critical organ failure . Cachectin, also known as tumour necrosis factor, is a macrophage-derived peptide hormone released in response to bacterial lipopolysaccharide, and it has been implicated as a principal mediator of endotoxic shock, although its function in bacterial sepsis is not known . Anaesthetized baboons were passively immunized against endogenous cachectin and subsequently infused with an LD100 dose of live Escherichia coli . Control animals (not immunized against cachectin) developed hypotension followed by lethal renal and pulmonary failure . Neutralizing monoclonal anti-cachectin antibody fragments (F(ab')2) administered to baboons only one hour before bacterial challenge protected against shock, but did not prevent critical organ failure . Complete protection against shock, vital organ dysfunction, persistent stress hormone release and death was conferred by administration of antibodies 2 h before bacterial infusion . These results indicate that cachectin is a mediator of fatal bacteraemic shock, and suggest that antibodies against cachectin offer a potential therapy of life-threatening infection.

Biochim Biophys Acta, 1987 Dec 17, 894(3), 499 - 506
Distinct phases of the fluorescence response of the lipophilic probe N-phenyl-1-naphthylamine in intact cells and membrane vesicles of Escherichia coli; Sedgwick EG et al.; The fluorescence of the lipophilic probe N-phenyl-1-naphthylamine (NPN) bound to intact cells of Escherichia coli is quenched by the addition of glucose, succinate, D-lactate, pyruvate, formate and glycerol . Partial recovery of fluorescence occurs on anaerobiosis . Use of mutants with defects in the ATP synthase or the respiratory chain show that quenching of fluorescence may be energized either by ATP hydrolysis or by substrate oxidation through the respiratory chain . Permeabilization of the outer membrane by treatment of intact cells with EDTA, or use of a mutant with an outer membrane permeable to lipophilic substances, results in a more rapid binding of NPN and in a decrease in quenching observed on substrate addition . NPN binds rapidly to everted membrane vesicles, but does not respond to membrane energization . It is proposed that inner membrane energization in intact cells alters the binding or environment of NPN in the outer membrane . The fluorescence recovery which occurs on anaerobiosis has two components . One component represents a reversal of the changes which occur on membrane energization . The other component of the fluorescence change is insensitive to the uncoupler CCCP and resembles the behaviour of NPN with everted membrane vesicles . It is suggested that a portion of the fluorescence events seen with NPN involves a response of the probe to changes in the inner membrane.

Biochim Biophys Acta, 1987 Dec 17, 894(3), 399 - 406
The proton pore in the Escherichia coli F0F1-ATPase: a requirement for arginine at position 210 of the a-subunit; Lightowlers RN et al.; Site-directed mutagenesis was used to generate three mutations in the uncB gene encoding the a-subunit of the F0 portion of the F0F1-ATPase of Escherichia coli . These mutations directed the substitution of Arg-210 by Gln, or of His-245 by Leu, or of both Lys-167 and Lys-169 by Gln . The mutations were incorporated into plasmids carrying all the structural genes encoding the F0F1-ATPase complex and these plasmids were used to transform strain AN727 (uncB402) . Strains carrying either the Arg-210 or His-245 substitutions were unable to grow on succinate as sole carbon source and had uncoupled growth yields . The substitution of Lys-167 and Lys-169 by Gln resulted in a strain with growth characteristics indistinguishable from a normal strain . The properties of the membranes from the Arg-210 or His-245 mutants were essentially identical, both being proton impermeable and both having ATPase activities resistant to the inhibitor DCCD . Furthermore, in both mutants, the F1-ATPase activities were inhibited by about 50% when bound to the membranes . The membrane activities of the mutant with the double lysine change were the same as for a normal strain . The results are discussed in relation to a previously proposed model for the F0 (Cox, G.B., Fimmel, A.L., Gibson, F . and Hatch, L . (1986) Biochim . Biophys . Acta 849, 62-69).

Biochem Biophys Res Commun, 1987 Dec 16, 149(2), 607 - 14
The design of a simple competitive ELISA using human proinsulin-alkaline phosphatase conjugates prepared by gene fusion; Lindbladh C et al.; The gene encoding human proinsulin has been fused in-frame with the E . coli alkaline phosphatase gene (pho A) (EC 3.1.3.1) . Two constructions are described . One construction consists of the entire proinsulin gene fused to the 5'-terminal end of pho A . In the other construction a 42 base pair DNA fragment has been deleted from the 3'-terminal end of the proinsulin gene . The two purified fusion proteins are enzymatically active showing a specific activity of 10-15 U/mg and 18-25 U/mg, respectively . The first construction exhibited insulin antigenicity and was used to design a simple competitive ELISA for insulin . The lower detection limit was found to be at least 2.5 ng/ml . Both fusion proteins were also shown to have potential for use in a competitive ELISA for proinsulin.

Biochem Biophys Res Commun, 1987 Dec 16, 149(2), 340 - 6
Observation of a possible pause mutant in the total synthesis of T4 lysozyme; Narang SA et al.; A DNA of 495 bp coding for T4-lysozyme was chemically synthesized and cloned in Escherichia coli . On DNA sequence analysis, clones pTLY.10 and pTLY.9 were identified to contain identical and complete T4-lysozyme coding sequences except that pTLY.9 had an additional 23 bp inverted repeat DNA at the 3'-end of the coding sequence . On expression and purification under similar conditions, T4-lysozymes from these two clones showed different degrees of retention time on HPLC as well as in the rate of enzymatic reaction . We speculate that this difference could be due to the generation of a pause mutant of T4-lysozyme in pTLY.9 under the influence of 3'-inverted repeat DNA that alters the rate of protein synthesis.

J Biol Chem, 1987 Dec 15, 262(35), 17212 - 20
Accumulation of lysophosphatidylinositol in RAW 264.7 macrophage tumor cells stimulated by lipid A precursors; Zoeller RA et al.; N2,O3-Diacylglucosamine 1-phosphate (lipid X), a monosaccharide precursor of Escherichia coli lipid A, was used to stimulate RAW 264.7 macrophage tumor cells, and the effects on macrophage phospholipid metabolism were examined . The addition of E . coli lipid X to the medium of cells that had been uniformly labeled with 32Pi resulted in a 4-8-fold increase in the level of lysophosphatidylinositol . This effect was maximal at 5 microM lipid X . Lysophosphatidylinositol levels reached a maximum 45 min after stimulation, followed by a gradual decline to near normal levels within 2 h . The formation of lysophosphatidylinositol was dependent upon extracellular calcium and was almost completely inhibited when cycloheximide was added at the time of stimulation . The addition of the disaccharide lipid A precursor IVA, commercial lipopolysaccharide (1 microgram/ml), phorbol 12-myristate 13-acetate (10(-7) M), or calcium ionophore A23187 (10(-6) M) to these cells resulted in a similar increase in lysophosphatidylinositol levels, but phosphatidic acid was inactive . The stimulation by IVA and phorbol myristate acetate was blocked by cycloheximide, but the stimulation by lipopolysaccharide was only partially blocked . The stimulation by A23187 was unaffected by cycloheximide . The increase in lysophosphatidylinositol levels might be related to the stimulation of arachidonate release and prostaglandin synthesis that is also observed in cells treated with lipid A precursors . The disaccharide precursor, IVA, was at least 100 times more effective than lipid X at stimulating lysophosphatidylinositol formation and prostaglandin release . The relative ability of lipid X and IVA to stimulate these cells correlated well with their effects on other lipopolysaccharide-responsive systems . Macrophage tumor cells also had the ability to inactivate lipid X by dephosphorylating it.

Biochemistry, 1987 Dec 15, 26(25), 8410 - 7
Kinetic mechanism of DNA polymerase I (Klenow); Kuchta RD et al.; The minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment of DNA polymerase I (KF) from Escherichia coli has been determined with short DNA oligomers of defined sequence . A key feature of this scheme is a minimal two-step sequence that interconverts the ternary KF.DNAn.dNTP and KF.DNAn+1.PPi complexes . The rate is not limited by the actual polymerization but by a separate step, possibly important in ensuring fidelity {Mizrahi, V., Henrie, R . N., Marlier, J . F., Johnson, K . A., & Benkovic, S . J . (1985) Biochemistry 24, 4010-4018} . Evidence for this sequence is supplied by the observation of biphasic kinetics in single-turnover pyrophosphorolysis experiments (the microscopic reverse of polymerization) . Data analysis then provides an estimate of the internal equilibrium constant . The dissociations of DNA, dNTP, and PPi from the various binary and ternary complexes were measured by partitioning (isotope-trapping) experiments . The rate constant for DNA dissociation from KF is sequence dependent and is rate limiting during nonprocessive DNA synthesis . The combination of single-turnover (both directions) and isotope-trapping experiments provides sufficient information to permit a quantitative evaluation of the kinetic scheme for specific DNA sequences.

Biochemistry, 1987 Dec 15, 26(25), 8242 - 6
Specificity of cotranslational amino-terminal processing of proteins in yeast; Huang S et al.; Polypeptides synthesized in the cytoplasm of eukaryotes are generally initiated with methionine, but N-terminal methionine is absent from most mature proteins . Many proteins are also N alpha-acetylated . The removal of N-terminal methionine and N alpha-acetylation are catalyzed by two enzymes during translation . The substrate preferences of the methionine aminopeptidase (EC 3.4.11.x) and N alpha-acetyltransferase (EC 2.3.1.x) have been partially inferred from the distribution of amino-terminal residues and/or mutations found for appropriate mature proteins, but with some contradictions . In this study, a synthetic gene corresponding to the mature amino acid sequence of the plant protein thaumatin, expressed in yeast as a nonexported protein, i.e., lacking a signal peptide, has been used to delineate the specificities of these enzymes with respect to the penultimate amino acid . Site-directed mutagenesis, employing synthetic oligonucleotides, was utilized to construct genes encoding each of the 20 amino acids following the initiation methionine codon, and each protein derivative was isolated and characterized with respect to its amino-terminal structure . All four possible N-terminal variants--those with and without methionine and those with and without N alpha-acetylation--were obtained . These results define the specificity of these enzymes in situ and suggest that the nature of the penultimate amino-terminal residue is the major determinant of their selectivity.

Biochemistry, 1987 Dec 15, 26(25), 8237 - 41
Protection of oligonucleotide primers against degradation by DNA polymerase I; Ott J et al.; By use of a mutational assay employing an octadecamer with a mismatch in the center, it is shown that the introduction of phosphorothioate groups near the 5'-end can protect the mismatch against degradation by the 5'-3'-exonuclease activity of Escherichia coli DNA polymerase I . An optimal level of protection is achieved when the phosphorothioate groups are incorporated in at least the second and third internucleotidic linkages from the 5'-end . However, gel electrophoretic analysis as well as the use of an octadecamer with a mismatch closer to the 5'-end in the mutational assay reveals that degradation of the oligonucleotide is not completely blocked but only slowed down.

Biochemistry, 1987 Dec 15, 26(25), 8221 - 7
Role of a bulged A residue in a specific RNA-protein interaction; Wu HN et al.; The translational operator of the R17 replicase gene contains a bulged A residue that is essential for the specific binding to R17 coat protein . A large number of operator variants have been synthesized to more precisely examine the role of the bulged A residue on this specific protein-RNA interaction . By use of RNA ligase and transcription of synthetic DNA templates by T7 RNA polymerase, 14 different nucleotides were introduced to the bulged A position of three different coat protein binding fragments . The affinity between coat protein and each fragment was determined by a nitrocellulose filter binding assay . The data indicate that while functional groups on N1, C2, C6, N7, and 2'OH of the bulged A can be substituted without greatly changing protein binding, bulky substituents cannot be tolerated at these positions . Data from additional fragments that have base-pair changes adjacent to the bulged A suggest that the propensity of the bulged A to intercalate into the helix can affect protein binding.

Biochemistry, 1987 Dec 15, 26(25), 8200 - 6
Mechanism of action of Escherichia coli endonuclease III; Kow YW et al.; Endonuclease III isolated from Escherichia coli has been shown to have both N-glycosylase and apurinic/apyrimidinic (AP) endonuclease activities . A nicking assay was used to show that the enzyme exhibited a preference for form I DNA when DNA containing thymine glycol was used as a substrate . This preference was reduced or eliminated either when the DNA was relaxed or when the type of damage was altered to urea residues or AP sites . The combined N-glycosylase/AP endonuclease activity was at least 10-fold higher than the AP endonuclease activity alone when urea-containing DNA was used as a substrate as compared to AP DNA . When DNA containing thymine glycol was used as a substrate, the combined N-glycosylase/AP endonuclease activity was about 2-fold higher than the AP endonuclease activity . Yet, when DNA containing thymine glycol or urea was used as substrate, no apurinic sites remained . Furthermore, magnesium selectively inhibited endonuclease III activity when AP DNA was used as a substrate but had no effect when DNA containing either urea or thymine glycol was used as substrate . These data suggest that both the N-glycosylase and AP endonuclease activities of endonuclease III reside on the same molecule or are in very tight association and that these activities act in concert, with the N-glycosylase reaction preceding the AP endonuclease reaction.

Biochem J, 1987 Dec 15, 248(3), 937 - 41
Expression of human glutathione S-transferase 2 in Escherichia coli . Immunological comparison with the basic glutathione S-transferases isoenzymes from human liver; Board PG et al.; A plasmid, termed pTacGST2, which contains the complete coding sequence of a GST2 (glutathione S-transferase 2) subunit and permits the expression of the protein in Escherichia coli was constructed . The expressed protein had the same subunit Mr as the enzyme from normal human liver and retained its catalytic function with both GST and glutathione peroxidase activity . Antiserum raised against the bacterially synthesized protein cross-reacted with all the basic GST isoenzymes in human liver . The electrophoretic mobility in agarose of the bacterially expressed isoenzyme suggested that its pI is identical with that of the cationic isoenzyme from human liver previously termed GST2 type 1 . The available evidence suggests that the three common cationic isoenzymes found in human liver are the products of two very similar gene loci.

J Immunol, 1987 Dec 15, 139(12), 4116 - 21
Molecular cloning and expression of hybridoma growth factor in Escherichia coli; Brakenhoff JP et al.; Human monocytes produce a factor that supports the growth of B lymphocyte hybridoma cells, termed hybridoma growth factor (HGF) . By using expression cloning in Escherichia coli of complementary DNA derived from human monocyte-poly(A+) RNA, we selected seven clones producing HGF activity as measured in a bioassay, based on the induction of proliferation of the HGF-dependent B cell hybridoma B9 . Sequence analysis of the cDNA revealed that HGF is identical with interferon-beta 2, 26,000 protein, and B cell stimulatory factor-2 . One of the active clones contained a cDNA that encoded a recombinant product lacking the 28-amino acid long signal peptide and the first 15 amino acids of the mature protein . Antibodies against the recombinant HGF inhibited the biologic activity of recombinant HGF as well as of monocyte-derived HGF.

Biochem Pharmacol, 1987 Dec 15, 36(24), 4287 - 91
A novel binding assay for phospholipase A2; Peers SH et al.; We have devised a rapid and simple assay for estimating the binding of pancreatic phospholipase A2 to a bilayer lipid membrane . The binding was observed to be extremely rapid at 37 degrees and was absolutely dependent upon Ca2+ . Amongst several drugs known to inhibit the catalytic activity of phospholipase only mepacrine at high concentrations (500 microM) and chlorpromazine (100 microM) were active . Treatment of the enzyme with p-bromophenacylbromide did not inhibit binding . Several alcohols potentiated binding whereas detergents tended to inhibit . Amongst several purified proteins tested, only the steroid-induced anti-phospholipase protein lipocortin prevented binding . The use of this assay in screening for antiphospholipase agents is discussed.

J Biol Chem, 1987 Dec 15, 262(35), 16865 - 70
Facilitated diffusion properties of melibiose permease in Escherichia coli membrane vesicles . Release of co-substrates is rate limiting for permease cycling; Bassilana M et al.; The mechanism of melibiose symport by the melibiose permease of Escherichia coli was studied by looking at the modifications of the facilitated diffusion properties of the permease which arise upon substitution of the coupled cations (H+, Na+, or Li+) . Kinetic analysis of melibiose influx and efflux down a concentration gradient, exchange at equilibrium, and counterflow were examined in de-energized membrane vesicles resuspended in media allowing melibiose to be co-transported with either H+, Na+, or Li+ . The data show that the maximal rates of melibiose efflux coupled to either H+, Na+, or Li+ are between 10 and 40 times faster than the corresponding influxes . This suggests that the permease functions asymmetrically . Cross-comparison between the rates of net {3H}melibiose entry during the influx reactions coupled to either cation and corresponding unidirectional sugar inflow during exchange and counterflow reactions leads to the conclusions that: 1) the step involving release of the co-substrates from the permease on the inner surface of the membrane is sequenced (sugar first and then coupled cation); 2) this step is rate determining for cycling of the permease . The Na+-melibiose passive flux data indicate in particular that release of Na+ ions rather than release of sugar into the intravesicular space is the slowest step during permease cycling . This property would hamper net passive Na+-melibiose influx but should allow exchange of sugar without concomitant exchange of the coupled cation . Finally, evidence is provided suggesting that the relative rates of release of the two co-substrates from the permease on the inner membrane surface varied considerably in relation to the identity of the coupled cation.

Carbohydr Res, 1987 Dec 15, 170(2), 151 - 65
Assignment of the 1H-n.m.r . spectra of heparin and heparan sulphate; Mulloy B et al.; Resonances from the main repeating unit of heparan, ----4)-beta-D-GlcA-(1----4)-alpha-D-GlcNAc-(1----, have been assigned by using a sample of the capsular polysaccharide of E . coli K5 . Comparison of the spectra of heparan sulphate samples before and after O- and/or N-desulphation, with re-N-acetylation or re-N-sulphation, allowed assignment of some of the H-1 doublets in terms of sequence effects . Chemical shifts for H-1 of unsulphated uronic acid residues are influenced by 6-sulphation of the nearest neighbour GlcN on the reducing side; those of GlcN residues vary according to whether they have IdoA or GlcA as the nearest neighbour on the reducing side . The H-1 doublets due to residues in the binding sequence for antithrombin have been assigned by comparison of the spectra of heparins having high and low affinities for immobilised antithrombin.

Philos Trans R Soc Lond B Biol Sci, 1987 Dec 15, 317(1187), 429 - 38
Replication of adenovirus and SV40 chromosomes in vitro; Kelly TJ et al.; As an approach to studying the mechanisms involved in the replication of eukaryotic chromosomes, we have developed and characterized cell-free replication systems for the animal viruses, adenovirus and SV40 . In this report we summarize recent work on the proteins required for the initiation of DNA synthesis in these two systems . The adenovirus origin of DNA replication was shown to consist of three functionally distinct sequence domains . Cellular proteins that specifically recognize each of these domains were purified and characterized . Initiation of adenovirus DNA replication was reconstituted from two virus-encoded and three cell-encoded factors . The SV40 origin of replication consists of a 65 base pair DNA segment that contains a high affinity binding site for the viral initiation protein T antigen . Evidence is presented that the first step in initiation of SV40 DNA replication involves the specific binding of T antigen to the origin, followed by the local unwinding of the two strands of the template . The unwinding reaction is specific for DNA templates containing the SV40 origin and requires ATP hydrolysis . In addition to T antigen, efficient unwinding requires a cellular factor(s) that can be replaced by the single-stranded DNA binding protein of Escherichia coli . These results indicate that the recently discovered helicase activity of T antigen plays a central role in initiation of viral DNA synthesis.

J Immunol, 1987 Dec 15, 139(12), 4061 - 6
In vitro generated human monoclonal trinitrophenyl-specific B cell lines . Evidence that human and murine anti-trinitrophenyl monoclonal antibodies cross-react with Escherichia coli beta-galactosidase; Golding B et al.; Stable human antigen-specific monoclonal B cell lines were established without prior in vivo immunization . This was accomplished by expanding the anti-trinitrophenyl (TNP) B cells in vitro with the antigen TNP-Brucella abortus and then immortalizing them with Epstein-Barr virus . Five anti-TNP clones were selected by sequential limiting dilution . All five anti-TNP clones secreted IgM kappa antibodies . When tested against a panel of self and environmental antigens, all five anti-TNP clones exhibited cross-reactivity with an Escherichia coli-derived beta-galactosidase . To determine whether this was a more general phenomenon, a panel of murine monoclonals were tested and found to bind to beta-galactosidase . It is therefore possible that human and murine anti-TNP beta cell responses reflect reactivity against an environmental antigen, namely an epitope present on E . coli-derived beta-galactosidase . This approach of expanding human antigen-specific B cells by antigen stimulation in vitro, with a T-independent hapten-carrier conjugate before Epstein-Barr virus transformation, may prove useful in the development of human monoclonals for therapeutic purposes.

J Biol Chem, 1987 Dec 15, 262(35), 17088 - 91
Roles of globotriosyl- and galabiosylceramide in verotoxin binding and high affinity interferon receptor; Cohen A et al.; The cellular specificity of the Escherichia coli-derived verotoxin is of particular interest because of its extreme toxicity and high selectivity toward certain primate cells . The human Burkitt lymphoma cell line (Daudi) is highly susceptible to the cytotoxicity of verotoxin and contains large amounts of the verotoxin-binding glycolipids on its surface . A mutant selected from Daudi cells for verotoxin resistance was found to be deficient in the verotoxin-binding glycolipids, globotriosylceramide and galabiosylceramide, and failed to bind verotoxin to its surface; interestingly, these mutant cells were found to be cross-resistant to inhibition of growth by alpha-interferon . Mutant cells also lack the high affinity component of alpha-interferon binding . These observations suggest that, in addition to providing the functional cell-surface receptor for verotoxin, these glycolipids may also play a role in the modulation of the affinity of alpha-interferon for its membrane protein receptors.

J Biol Chem, 1987 Dec 15, 262(35), 17072 - 82
Purified lac permease and cytochrome o oxidase are functional as monomers; Costello MJ et al.; Purified lac permease, the 46.5-kDa product of the lac Y gene that catalyzes lactose/H+ symport, or purified cytochrome o, a terminal oxidase of the Escherichia coli respiratory chain composed of four subunits with a composite molecular mass of 140 kDa, was reconstituted into proteoliposomes individually or in combination . The preparations were then examined by freeze-fracture electron microscopy employing conventional platinum/carbon replicas or by means of a new technique using thin tantalum replicas . In nonenergized proteoliposomes, both proteins appear to reconstitute as monomers based on (i) the variation of intramembrane particle density with protein concentration; (ii) the ratio of particles corresponding to each protein in proteoliposomes reconstituted with a known ratio of permease to oxidase; and (iii) the dimensions of the particles observed in tantalum replicas . The intramembrane particle diameters in tantalum replicas are about 20-25% smaller than those observed in conventional platinum/carbon replicas, indicating that the dimensions of the particles revealed with tantalum more accurately reflect the sizes of lac permease and cytochrome o . The diameters and heights of the permease and cytochrome o in tantalum replicas are 5.1 nm X 2.8 nm and 7.4 nm X 4.2 nm, respectively . Furthermore, a higher percentage of lac permease molecules exhibits a notch or cleft in tantalum replicas relative to platinum/carbon replicas . Importantly, the initial rate of lactose/H+ symport in proteoliposomes varies linearly with the ratio of lac permease to phospholipid, and no change is observed in either the size or distribution of lac permease molecules when the proteoliposomes are energized . The results taken as a whole provide a strong indication that both lac permease and cytochrome o reconstitute into proteoliposomes as monomers, that the permease does not dimerize in the presence of the H+ electrochemical gradient, and that both molecules are completely functional as monomers.

Philos Trans R Soc Lond B Biol Sci, 1987 Dec 15, 317(1187), 455 - 69
Structure and function of SV40 large-T antigen; Mole SE et al.; The small eukaryotic DNA tumour virus, SV40, has long provided a very useful model for the study of eukaryotic DNA replication and cellular transformation . The viral gene product, large-tumour (large-T) antigen, is essential for the initiation of viral DNA replication and the initiation and maintenance of SV40-virus-mediated cellular transformation . The large-T antigen is a complex multifunctional protein, and to delineate its activity more precisely in viral DNA replication and cellular transformation, small functional domains of the protein have been expressed in Escherichia coli and analysed by using a very extensive library of anti-T monoclonal antibodies.

J Biol Chem, 1987 Dec 15, 262(35), 16822 - 9
Characterization of the yeast HEM2 gene and transcriptional regulation of COX5 and COR1 by heme; Myers AM et al.; The respiratory deficiency of two noncomplementing mutants of Saccharomyces cerevisiae (C41 and N28) has been shown to be due to mutations in HEM2, the structural gene for delta-aminolevulinate dehydratase . The mutants are unable to convert delta-aminolevulinic acid to porphobilinogen and are not complemented by the hem2 mutant GL4 (Gollub, E . G., Liu, K.-P., Dagan, J., Adlersberg, M., and Sprinson, D . B . (1977) J . Biol . Chem . 252, 2846-2854) . A gene capable of complementing the respiratory deficiency of C41 and N28 has been cloned by transformation of a hem2 mutant with a recombinant plasmid library of wild type yeast nuclear DNA . The sequence of the protein encoded by the cloned gene exhibits extensive homology to the recently reported sequence of human delta-aminolevulinate dehydratase (Wetmur, J . G., Bishop, D . F., Cantelmo, C., and Desnick, R . J . (1986) Proc . Natl . Acad . Sci . U . S . A . 83, 7703-7707) . Several approaches were taken to study the effect of heme on transcription of PET genes known to code for subunit components of respiratory enzymes and of mitochondrial ATPase . The first involved measurements of the steady state levels of mRNAs for subunit 5 of cytochrome oxidase and the beta subunit of F1 ATPase in wild type and in a hem2 mutant . Secondly, transcription of the genes coding for the cytochrome oxidase and ATPase subunits as well as of the COR1 gene coding for the 44-kDa core 1 subunit of coenzyme QH2-cytochrome c reductase was quantitated by fusing the 5'-flanking and part of the coding region of each gene to the lacZ gene of Escherichia coli in vectors capable of integrating into yeast chromosomal DNA . The different lacZ fusions were integrated into nuclear DNA of a wild type strain and of hem2 mutants allowing expression of beta-galactosidase to be studied as a function of intracellular heme . These experiments indicate that the promoters of the genes for subunits of the respiratory complexes are regulated by heme . In contrast, the expression of the ATPase subunit appears to be heme-independent . Because neither subunit 5 of cytochrome oxidase nor the core 1 subunit of coenzyme QH2-cytochrome c reductase are hemoproteins, transcriptional regulation by heme may be a general mechanism for controlling the synthesis of mitochondrial proteins involved in respiration.

Nucleic Acids Res, 1987 Dec 10, 15(23), 9807 - 23
Molecular cloning and characterization of the gene encoding the DNA methyltransferase, M.CviBIII, from Chlorella virus NC-1A; Narva KE et al.; The gene encoding the DNA methyltransferase, M.CviBIII, from Chlorella virus NC-1A was cloned and expressed in E . coli plasmid pUC8 . Plasmid (pNC-1A.14.8) encoded M.CviBIII methylates adenine in TCGA sequences both in vivo in E . coli and in vitro . Transposon Tn5 mutagenesis localized the M.CviBIII functional domain to a 1.5 kbp region of pNC-1A.14.8 and also indicated that a virus promoter directs transcription of the gene in E . coli . The 2.1 kbp insert containing the M.CviBIII gene was sequenced and a single open reading frame of 1131 bp was identified within the domain determined by Tn5 mutagenesis . When the M.CviBIII gene was fused in-frame with the 19 amino-terminal codons of lacZ a 45 kD polypeptide was identified in maxicells as predicted by the DNA sequence . The M.CviBIII gene was not essential for virus replication since a virus M.CviBIII deletion mutant also replicated in Chlorella.

Nucleic Acids Res, 1987 Dec 10, 15(23), 9781 - 96
Cloning, sequencing and expression of the Taq I restriction-modification system; Slatko BE et al.; The Taq I modification and restriction genes (recognition sequence TCGA) have been cloned in E . coli and their DNA sequences have been determined . Both proteins were characterized and the N-terminal sequence of the endonuclease was determined . The genes have the same transcriptional orientation with the methylase gene 5' to the endonuclease gene . The methylase gene is 1089 bp in length (363 amino acids, 40,576 daltons); the endonuclease gene is 702 bp in length (234 amino acids, 27,523 daltons); they are separated by 132 bp . Both methylase and endonuclease activity can be detected in cell extracts . The clones fully modify the vector and chromosomal DNA but they fail to restrict infecting phage . Clones carrying only the restriction gene are viable even in the absence of modification . The restriction gene contains 7 Taq I sites; the modification gene contains none . This asymmetric distribution of sites could be important in the regulation of the expression of the endonuclease gene.

FEBS Lett, 1987 Dec 10, 225(1-2), 195 - 200
Hydroxyl radical footprinting of the sequence-selective binding of netropsin and distamycin to DNA; Portugal J et al.; Hydroxyl radicals, generated by allowing an iron (II).EDTA complex to react with hydrogen peroxide, have been employed to cleave the 160-base pair tyrT DNA fragment in the presence and absence of the minor groove-binding antibiotics netropsin and distamycin A . The control DNA cleavage pattern is practically independent of nucleotide sequence, which overcomes certain limitations of other footprinting techniques, so that additional information can be gained about the AT-rich sequence preference of the minor groove-binding ligands.

Biochim Biophys Acta, 1987 Dec 8, 910(3), 254 - 60
Escherichia coli RNA polymerase binding to a DNA terminus prevents formation of a closed promoter complex; Hofer B et al.; A 302 bp DNA fragment and a 113 bp subfragment of the former, both containing the fd gene VIII promoter (P VIII), were found to exhibit temperature-dependent differential behaviour in RNA chain initiation from P VIII . At 37 degrees C no significant differences were observed, while at 17 degrees C chain initiation was strongly suppressed only with the 113 bp fragment . This phenomenon depended on the presence of the (blunt) DNA terminus upstream from P VIII (position -70) . Footprinting revealed that at 17 degrees C RNA polymerase was bound to this DNA fragment in a different mode . Contacts were observed only upstream from position -25 . On the contrary, at 37 degrees C only the promoter complex footprint was visible . These results indicate that at 17 degrees C formation of the non-initiating complex is more favourable than formation of the promoter complex (which is closed at 17 degrees C; Hofer, B., Muller, D . and Koster, H . (1985) Nucleic Acids Res . 13, 5995-6013) and that formation of both complexes is mutually exclusive . No footprints of RNA polymerase were observed at other DNA termini . This indicates a sequence-specificity for the interaction at the terminus of the 113 bp fragment . The footprint pattern, together with features of the DNA sequence, suggests that the contacts involved in this interaction are similar to those promoter contacts formed upstream from position -20 and that DNA without a -10 region can be specifically recognized by RNA polymerase.

J Theor Biol, 1987 Dec 7, 129(3), 337 - 48
Elongation and surface extension of individual cells of Escherichia coli B/r: comparison of theoretical and experimental size distributions; Grover NB et al.; The way individual cells grow and divide uniquely determines the (time-invariant) cell size distribution of populations in steady-state exponential growth . In the preceding article, theoretical distributions were derived for two exponential and six linear models containing a small number of adjustable parameters but no assumptions other than that all cells obey the same growth law . The linear models differ from each other with respect to the timing of the presumptive doubling in their growth rate, the exponential models--according to whether there is or is not a part of the cell that does not contribute to the growth rate . Here we compared the size distributions predicted by each of these models with those of cell length and surface area measured by electron microscopy; the quality of the fit, as determined by the mean-square successive-differences test and the chi 2 goodness-of-fit test, was taken as a measure of the adequacy of the model . The actual data came from two slow-growing E . coli B/r cultures, an A strain (pi = 125 min) and a K strain (pi = 106 min), and a correction was introduced in each to account for the distortion caused by the finite size of the picture frame . The parameter estimates produced by the various models are quite reliable (cv less than 0.1%); we discuss them briefly and compare their values in the two strains . All the length extension models were rejected outright whereas most of the surface growth versions were not . When the same models were tested on A-strain data from a faster growing culture (tau = 21 min), those models that provided an adequate fit to the cell surface area data proved equally satisfactory in the case of cell length . These findings are evaluated and shown to be consistent with cell surface area rather than cell length being the dimension under active control . Three surface area models, all linear, are rejected--those in which doubling of the growth rate occurs with a constant probability from cell birth, at a particular cell age, and precisely at cell division . The evidence in the literature that appears to contradict this last result, rejection of the simple linear surface growth model, is shown to be faulty . The 16 original models are here reduced to five, two involving exponential surface growth and three linear, and possible reasons are presented for our inability to discriminate further at this stage.

J Biol Chem, 1987 Dec 5, 262(34), 16612 - 7
Biological equivalence of natural bovine and recombinant human alpha-endothelial cell growth factors; Jaye M et al.; The cDNA encoding human alpha-endothelial cell growth factor (alpha-ECGF) has been engineered for high-level expression in Escherichia coli . Induction of bacterial cultures harboring the recombinant plasmid pMJ26 results in the appearance of a prominent 16-kDa polypeptide . This protein has been purified from bacterial lysates using a rapid, 2-step procedure employing heparin-Sepharose affinity based chromatography and reversed-phase high pressure liquid chromatography . Recombinant human alpha-ECGF was compared to bovine brain-derived alpha-ECGF in three biological assays: receptor binding on murine lung capillary endothelial cells (LE-II cells), stimulation of {3H}thymidine incorporation in LE-II cells, and stimulation of human umbilical vein endothelial cell proliferation . The results demonstrate that the recombinant human mitogen has the same biological potency as the bovine brain-derived material . Fluorescence spectroscopy was used to study the interaction between recombinant ECGF and heparin . Heparin-binding resulted in a 40% reduction in the intrinsic fluorescence of ECGF, consistent with a heparin-induced conformational change . The intrinsic fluorescence of ECGF also varied as a function of pH.

J Mol Biol, 1987 Dec 5, 198(3), 361 - 9
A 38 base-pair segment of DNA is required in cis for conjugative mobilization of broad host-range plasmid R1162; Brasch MA et al.; oriT, the region required in cis for conjugative mobilization of broad host-range plasmid R1162, has been localized to a 38 base-pair segment of DNA . The oriT DNA is also required for conjugation-dependent recombination . Point mutations at the HinPI cleavage site within oriT affect both mobilization and recombination, and the crossover location has been mapped to this site . An inverted repeat ten base-pairs from the recombination site is also involved in mobilization and recombination, and may be a recognition site for proteins involved in cleavage of the oriT DNA . The properties of conjugation-dependent recombination suggest that mobilization entails the formation of a linear intermediate that is transferred with both a unique origin and polarity.

J Biol Chem, 1987 Dec 5, 262(34), 16644 - 54
The Escherichia coli preprimosome and DNA B helicase can form replication forks that move at the same rate; Mok M et al.; A DNA replication system was developed that could generate rolling-circle DNA molecules in vitro in amounts that permitted kinetic analyses of the movement of the replication forks . Two artificial primer-template DNA substrates were used to study DNA synthesis catalyzed by the DNA polymerase III holoenzyme in the presence of either the preprimosomal proteins (the primosomal proteins minus the DNA G primase) and the Escherichia coli single-stranded DNA binding protein or the DNA B helicase alone . Helicase activities have recently been demonstrated to be associated with the primosome, a mobile multiprotein priming apparatus that requires seven E . coli proteins (replication factor Y (protein n'), proteins n and n'', and the products of the dnaB, dnaC, dnaG, and dnaT genes) for assembly, and with the DNA B protein . Consistent with a rolling-circle mechanism in which a helicase activity permitted extensive (-) strand DNA synthesis on a (+) single-stranded, circular DNA template, the major DNA products formed were multigenome-length, single-stranded, linear molecules . The replication forks assembled with either the preprimosome or the DNA B helicase moved at the same rate (approximately 730 nucleotides/s) at 30 degrees C and possessed apparent processivities in the range of 50,000-150,000 nucleotides . The single-stranded DNA binding protein was not required to maintain this high rate of movement in the case of leading strand DNA synthesis catalyzed by the DNA polymerase III holoenzyme and the DNA B helicase.

J Biol Chem, 1987 Dec 5, 262(34), 16267 - 70
Escherichia coli mutT mutator effect during in vitro DNA synthesis . Enhanced A.G replicational errors; Schaaper RM et al.; The mechanism of the Escherichia coli mutT mutator effect was investigated using single-stranded phage as a mutational target . In vivo experiments showed that two M13mp2 lacZ alpha nonsense mutants reverted at a higher rate on a mutT1 host than on the wild-type host . The specificity of this mutator effect was identical to that observed for E . coli genes: A.T----C.G transversions . The mutT effect was subsequently demonstrated in vitro during DNA replication of M13mp2 DNA in cell-free extracts of E . coli . Replication (the single-stranded----replicative form conversion) in mutT1 extracts proceeded with a higher error rate than in wild-type extracts, and DNA sequence analysis of the in vitro revertants revealed the specific induction of A.T----C.G transversions . On the basis of the template specificity of the mutT effect in vitro, we conclude that the mutT effect involves the aberrant processing of A.G rather than T.C mispairs.

J Mol Biol, 1987 Dec 5, 198(3), 547 - 50
Feedback regulation of rRNA synthesis . A mutational alteration in the anti-Shine-Dalgarno region of the 16 S rRNA gene abolishes regulation; Yamagishi M et al.; It was shown that induction of rRNA (and ribosome) synthesis from the lambda PL promoter/operator by temperature shift-up causes a repression of rRNA and tRNA synthesis from chromosomal genes . We have carried out experiments using a similar conditional rRNA gene expression system in which a mutational alteration was introduced in the anti-Shine-Dalgarno region at the 3'-end of the 16 S rRNA gene . It was found that the repression observed with the wild-type gene was largely abolished by the mutation . It appears that ribosomes inefficient in translational initiation are unable to cause feedback regulation of rRNA synthesis . It is suggested that the cell regulates rRNA (and tRNA) synthesis by monitoring the production of ribosomes, and that this monitoring is apparently carried out through their activity in the initiation (and perhaps subsequent steps) of translation.

J Mol Biol, 1987 Dec 5, 198(3), 383 - 92
Feedback regulation of rRNA synthesis in Escherichia coli . Requirement for initiation factor IF2; Cole JR et al.; It has been shown that the transcription of rRNA in Escherichia coli is feedback-regulated by its own transcription products through a negative feedback loop which appears to require the assembly of rRNA into complete ribosomes . In order to examine whether the feedback loop involves the ribosomes' main function, translation, we have constructed a strain in which the chromosomal copy of infB, encoding IF2, was placed under lac promoter/operator control, and the effects of limitation of translation initiation factor IF2 on the regulation were examined . By varying the concentration of a lac operon inducer, isopropyl thiogalactoside (IPTG), it was possible to vary the cellular concentration of IF2 . Under the growth conditions used, decreasing the concentration of IF2 about twofold affected the growth rate only slightly, but further deprivation of IF2 resulted in a significant decrease in growth rate, an increase in RNA content and a large accumulation of non-translating ribosomes . These accumulated ribosomes were apparently unable to cause feedback regulation of rRNA synthesis in the absence of sufficient IF2 . When a higher concentration of IPTG was added to these IF2-deficient cells, a rapid increase in the IF2 level and a significant decrease in the rate of RNA accumulation were observed before the new steady-state growth was attained . These results indicate that IF2 apparently is necessary for feedback regulation of stable RNA and imply that ribosomes must enter translation for feedback regulation to occur.

J Mol Biol, 1987 Dec 5, 198(3), 371 - 81
Feedback regulation of the rplJL-rpoBC ribosomal protein operon of Escherichia coli requires a region of mRNA secondary structure; Climie SC et al.; The rplJ-rpoBC (L10) operon of Escherichia coli is regulated in part through translational repression (feedback regulation) by ribosomal protein L10 or a complex of ribosomal proteins L10 and L7/L12 (L10-L7/L12) . We have constructed mutants in the untranslated leader region of a rplJ-lacZ fusion by oligonucleotide-directed mutagenesis . The mutations include several deletions and a number of single base changes, all of which fail to exhibit normal feedback regulation . Chemical probing of part of the rplJ mRNA leader in the mutagenized region confirms that all of the mutations lie in a stem structure located 140 nucleotides upstream from the translation start-site . The structure includes a 12 base-pair stem, a four base stem-loop, and a six base bulge-loop . Point mutations that abolish feedback regulation are presumed to disrupt this stem structure . Pseudorevertants of selected point mutations were constructed by combining pairs of single base mutations . In these cases, both the secondary structure of the RNA and feedback regulation were restored . The results allow us to define a region of secondary structure in the rplJ mRNA leader that is necessary for feedback regulation.

Science, 1987 Dec 4, 238(4832), 1403 - 6
A complete mapping of the proteins in the small ribosomal subunit of Escherichia coli; Capel MS et al.; The relative positions of the centers of mass of the 21 proteins of the 30S ribosomal subunit from Escherichia coli have been determined by triangulation using neutron scattering data . The resulting map of the quaternary structure of the small ribosomal subunit is presented, and comparisons are made with structural data from other sources.

Cell, 1987 Dec 4, 51(5), 699 - 707
Lac repressor is a transient gene-activating protein; Straney SB et al.; We show, using a combination of methods, that contrary to the usual view, lac repressor increases, by more than 100-fold, the initial binding of RNA polymerase to E . coli lac UV5 promoter DNA . Kinetic studies revealed that the repressor acts to block the isomerization step in transcription initiation . When IPTG, a gratuitous inducer, is added, formation of open complex and productive transcription proceed . Because of the large increases in the binding constant, at low polymerase concentrations the presence of lac repressor (and then inducer) actually increases the rate of the first round of productive transcription, thus allowing the system to respond rapidly to the release of repression . This dual role of stabilization of a pretranscriptional complex coupled with blockage of transcription initiation may be a more general model for genetic regulation than that provided by the concept of simple repression.

Inflammation, 1987 Dec, 11(4), 389 - 400
Modulation of phospholipase A2 activity in human synovial fluid by cations; Fawzy AA et al.; Cell-free, Ca2+-dependent phospholipase A2 activity (PLA2) was measured in human synovial fluid of patients with various kinds of arthritis using {1-14C} oleate-labelled autoclaved Escherichia coli as substrate . PLA2 activity at pH 7.0 and with 5 mM added Ca2+ was stimulated and then inhibited in a dose-dependent fashion by NaCl; maximal stimulation of 8.8 fold was found at 150 mM Na+ . Similar effects were obtained with K+, Li+ and Ru+ . In the absence of added Na+, PLA2 activity was maximal with 25 mM Ca2+ (145 nmols/hr/mg), but in the presence of 150 mM Na+, activity was maximal with 4 mM Ca2+ (415 nmols/hr/mg) . PLA2 activity was optimal between pH 6.5-8.0 in presence of 150 mM Na+1 and 4 mM Ca2+ . There was no significant difference between PLA2 activity in synovial fluids from rheumatoid and other types of arthritis . Neutral active, Ca2+-dependent PLA2 activity in acid extracts of human platelets, plasma, polymorphonuclear leukocytes and synovial fluid varied in response to added Na+ . In presence of 150 mM added Na+ and 5 mM Ca2+, PLA2 activity in human synovial fluid was inhibited by all multivalent cations tested . In the absence of Na+, Cu2+ and Mg2+ stimulated PLA2 activity in a dose dependent fashion; whereas, Fe2+, Fe3+ and Al3+ were inhibitory . The extent of stimulation by Mg2+ was inversely related to the concentration of added Ca2+.

Eur J Biochem, 1987 Dec 1, 169(2), 245 - 52
The domain structure of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii; Hanemaaijer R et al.; Limited proteolysis with trypsin has been used to study the domain structure of the dihydrolipoyltransacetylase (E2) component of the pyruvate dehydrogenase complex of Azotobacter vinelandii . Two stable end products were obtained and identified as the N-terminal lipoyl domain and the C-terminal catalytic domain . By performing proteolysis of E2, which was covalently attached via its lipoyl groups to an activated thiol-Sepharose matrix, a separation was obtained between the catalytic domain and the covalently attached lipoyl domain . The latter was removed from the column after reduction of the S-S bond and purified by ultrafiltration . The lipoyl domain is monomeric with a mass of 32.6 kDa . It is an elongated structure with f/fo = 1.62 . Circulair dichroic studies indicates little secondary structure . The catalytic domain is polymeric with S20.w = 17 S and mass = 530 kDa . It is a compact structure with f/fo = 1.24 and shows 40% of the secondary structure of E2 . The cubic structure of the native E2 is retained by this fragment as observed by electron microscopy . Ultracentrifugation in 6 M guanidine hydrochloride in the presence of 2 mM dithiothreitol yields a mass of 15.8 kDa . An N-terminal sequence of 36 amino acids is homologous with residues 370-406 of Escherichia coli E2 . The catalytic domain possesses the catalytic site, but in contrast to the E . coli subunit binding domain the pyruvate dehydrogenase (E1) and lipoamide dehydrogenase (E3) binding sites are lost during proteolysis . From comparison with the E . coli E2 sequence a model is presented in which the several functions, such as lipoyl domain, the E3 binding site, the catalytic site, the E2/E2 interaction sites, and the E1 binding site, are indicated.

J Biochem (Tokyo), 1987 Dec, 102(6), 1511 - 8
Structural gene of cytochrome b-562 from the cytochrome b-c1 complex of Rhodobacter sphaeroides; Iba K et al.; The structural gene coding for cytochrome b-562 isolated from the cytochrome b-c1 complex of Rhodobacter (Rhodopseudomonas) sphaeroides has been cloned . Its nucleotide sequence has been determined and the amino acid sequence was deduced therefrom . It consists of 157 amino acids (Mr 17,237) and contains four hydrophobic segments . The first 30 residues in the predicted amino acid sequence are the same as those determined for the NH2-terminal portion of purified cytochrome b-562 . The amino acid composition is in accord with that determined for the pure protein . From the hydropathy profile and molar ratio of protoheme to cytochrome b-562, it is suggested that the structural and functional unit of the cytochrome is a two-heme cross-linked homodimer.

J Leukoc Biol, 1987 Dec, 42(6), 621 - 7
Potentiation of neutrophil function by recombinant DNA-produced interleukin 1a; Ozaki Y et al.; The effect of interleukin 1a (IL-1a) produced by E . coli-derived recombinant DNA was evaluated on various parameters of human neutrophil function . IL-1a alone stimulated neutrophil hydrogen peroxide production in a dose-dependent manner, but the rate was much lower than that of opsonized zymosan . IL-1a induced release of specific granule contents, but not azurophilic granule contents . Cytochalasin B did not augment the rate of release . IL-1a was chemotactic for neutrophils at the optimal concentrations of 0.1-10 ng/ml . Pretreatment of the neutrophils with IL-1a augmented neutrophil oxygen radical production induced by opsonized zymosan, and this synergistic effect was evident as early as 10 min after IL-1a was added to the neutrophil culture . Phagocytosis of opsonized particles by neutrophils, and degranulation induced by opsonized zymosan were also enhanced by IL-1a in a dose-dependent manner . The present results suggest that IL-1a is weak as a direct activator of neutrophil function and that IL-1a in vivo may augment the response of neutrophils to other stimulators such as foreign bodies.

Mol Gen Genet, 1987 Dec, 210(2), 314 - 22
The ribosomal protein gene cluster of Mycoplasma capricolum; Ohkubo S et al.; The DNA sequence of the part of the Mycoplasma capricolum genome that contains the genes for 20 ribosomal proteins and two other proteins has been determined . The organization of the gene cluster is essentially the same as that in the S10 and spc operons of Escherichia coli . The deduced amino acid sequence of each protein is also well conserved in the two bacteria . The G + C content of the M . capricolum genes is 29%, which is much lower than that of E . coli (51%) . The codon usage pattern of M . capricolum is different from that of E . coli and extremely biased to use of A and U(T): about 91% of codons have A or U in the third position . UGA, which is a stop codon in the "universal" code, is used more abundantly than UGG to dictate tryptophan.

Proc Natl Acad Sci U S A, 1987 Dec, 84(23), 8235 - 8
The function of acyl carrier protein in the synthesis of membrane-derived oligosaccharides does not require its phosphopantetheine prosthetic group; Therisod H et al.; An enzyme system catalyzing the synthesis of the beta 1,2-linked glucan backbone of the membrane-derived oligosaccharides of Escherichia coli from UDP-glucose has an essential requirement for the E . coli acyl carrier protein (ACP) . This finding was surprising, because all other characterized functions of ACP involve acyl thioester residues linked to the phosphopantetheine moiety covalently bound to ACP . We now report that the activity of ACP in the synthesis of membrane-derived oligosaccharides is not altered by treatment with the sulfhydryl reagent N-ethylmaleimide nor by complete removal of the phosphopantetheine residue by treatment with a specific phosphodiesterase . The function of ACP in the synthesis of membrane-derived oligosaccharides is thus clearly different from that involved in lipid biosynthesis . We have nevertheless found that the same molecular species of ACP that undergo enzymic acylation with long-chain fatty acid residues also function in the synthesis of membrane-derived oligosaccharides.

Mol Cell Probes, 1987 Dec, 1(4), 327 - 35
A DNA probe for detecting Mycoplasma genitalium in clinical specimens; Risi GF Jr et al.; Despite decades of careful study, the etiologies of all cases of pelvic inflammatory disease (PID) and non-gonococcal urethritis (NGU) have yet to be described . Mycoplasma genitalium is a newly described organism which has been implicated as a cause of both PID and NGU . Because of fastidious growth requirements, prolonged incubation time and frequent overgrowth in clinical specimens by Mycoplasma hominis, non-culture methods need to be developed for its detection . We have cloned M . genitalium DNA by transfection into Escherichia coli using M13 as the vector . Using these segments as templates, we synthesized radiolabelled cDNAs that were tested for specific hybridization with M . genitalium, and clinically isolated genital mycoplasmas presumptively identified as M . hominis, and Ureaplasma urealyticum . A 256 base-pair segment was found to hybridize with M . genitalium with a sensitivity of 10(2) colour-changing units (CCUs) . No cross-hybridization was observed with M . hominis, and cross-hybridization was observed only with large concentrations (greater than 10(6) CCUs) of U . urealyticum . Because of our choice of M13 as the vector, which contains the Lac Operon of E . coli, slight hybridization occurred with E . coli as well . This cDNA can be used against clinical specimens to determine the ecologic niche and spectrum of disease caused by M . genitalium.

Anticancer Drug Des, 1987 Dec, 2(3), 289 - 96
Structural studies on bio-active compounds . Part 9 . 2,4-Diamino-6-azidoquinazoline hydrochloride; Schwalbe CH et al.; C8H7N7.HCl has a formula weight Mr237.7, and crystallizes in space group P21/n with a = 6.498(1), b = 10.333(8), c = 15.406(5) A, beta = 91.72(2) degrees . The final R factor was 0.049 for 1010 unique observed reflections . The azido substituent is almost linear, coplanar with the heterocycle, and parallel to the C(7)-C(6) ring bond . The heterocycle is protonated at N(1) and extensively hydrogen bonded . The 6-azido compound is at least a 100-fold better inhibitor of Escherichia coli dihydrofolate reductase than the analogue lacking the azido group, according to I50 values . Superimposing the quinazoline moiety upon the pteridine ring of the methotrexate-dihydrofolate reductase (E . coli) complex reveals two orientations of the N3 group in which it can fit the enzyme, making van der Waals contacts . Ab initio molecular orbital calculations suggest that one of these conformations, with torsion angle phi = C(5)-C(6)-N(61)-N(62) = -163.8 degrees, is low in energy.

Biochem J, 1987 Dec 1, 248(2), 403 - 8
Active-site-directed inactivation of wheat-germ aspartate transcarbamoylase by pyridoxal 5'-phosphate; Cole SC et al.; Treatment of 1 microM wheat-germ aspartate transcarbamoylase with 1 mM-pyridoxal 5'-phosphate caused a rapid loss of activity, concomitant with the formation of a Schiff base . Complete loss of activity occurred within 10 min when the Schiff base was reduced with a 100-fold excess of NaBH4 . Concomitantly, one amino group per chain was modified . No further residues were modified in the ensuing 30 min . The kinetics of inactivation were examined under conditions where the Schiff base was reduced before assay . Inactivation was apparently first-order . The pseudo-first-order rate constant, kapp., showed a hyperbolic dependence upon the concentration of pyridoxal 5'-phosphate, suggesting that the enzyme first formed a non-covalent complex with the reagent, modification of a lysine then proceeding within this complex . Inactivation of the enzyme by pyridoxal was 20 times slower than that by pyridoxal 5'-phosphate, indicating that the phosphate group was important in forming the initial complex . Partial protection against pyridoxal phosphate was provided by the leading substrate, carbamoyl phosphate, and nearly complete protection was provided by the bisubstrate analogue, N-phosphonoacetyl-L-aspartate, and the ligand-pair carbamoyl phosphate plus succinate . Steady-state kinetic studies, under conditions that minimized inactivation, showed that pyridoxal 5'-phosphate was also a competitive inhibitor with respect to the leading substrate, carbamoyl phosphate . Pyridoxal 5'-phosphate therefore appears to be an active-site-directed reagent . A sample of the enzyme containing one reduced pyridoxyl group per chain was digested with trypsin, and the labelled peptide was isolated and shown to contain a single pyridoxyl-lysine residue . Partial sequencing around the labelled lysine showed little homology with the sequence surrounding lysine-84, an active-centre residue of the catalytic subunit of aspartate transcarbamoylase from Escherichia coli, whose reaction with pyridoxal 5'-phosphate shows many similarities to the results described in the present paper . Arguably the reactive lysine is conserved between the two enzymes whereas the residues immediately surrounding the lysine are not . The same conclusion has been drawn in a comparison of reactive histidine residues in the two enzymes {Cole & Yon (1986) Biochemistry 25, 7168-7174}.

Protein Eng, 1987 Dec, 1(6), 493 - 8
Diphtheria toxin receptor binding domain substitution with interleukin-2: genetic construction and properties of a diphtheria toxin-related interleukin-2 fusion protein; Williams DP et al.; We have genetically replaced the diphtheria toxin receptor binding domain with a synthetic gene encoding interleukin-2 (IL-2) and a translational stop signal . The diphtheria toxin-related T-cell growth factor fusion gene encodes a 70 586-d polypeptide, pro-IL-2-toxin . The mature form of IL-2-toxin has a deduced mol . wt of 68,086 and is shown to be exported to the periplasmic compartment of Escherichia coli (pABI508), and contain immunologic determinants intrinsic to both its diphtheria toxin and IL-2 components . IL-2-toxin has been purified from periplasmic extracts of recombinant strains of E . coli (pABI508) by immunoaffinity chromatography using immobilized anti-IL-2 . The purified chimeric toxin is shown to selectively inhibit protein synthesis in IL-2 receptor bearing targeted cells, whereas cell lines which do not express the IL-2 receptor are resistant to IL-2-toxin action.

Protein Eng, 1987 Dec, 1(6), 487 - 92
Efficient cleavage by alpha-thrombin of a recombinant fused protein which contains insulin-like growth factor I; Nishikawa S et al.; The gene for insulin-like growth factor I (IGF-I) was constructed from chemically synthesized deoxyoligonucleotides and expressed in Escherichia coli, under the control of a trp promoter, as a set of fusion proteins which were connected with a portion of human growth hormone through the recognition sequence for a sequence-specific protease, either blood coagulation factor Xa or alpha-thrombin . Upon induction with 3-indoleacrylic acid, fusion proteins accumulated with a yield of 10-30% of the total protein . A fusion protein connected through a tetradecapeptide (Asp-Asp-Pro-Pro-Thr-Val-Glu-Leu-Gln-Gly-Leu-Val-Pro-Arg) was efficiently and correctly cleaved by alpha-thrombin, and the purified IGF-I possessed somatomedin-like activity, as determined by the enhancement of sulfation of glycosaminoglycans in cultured costal chondrocytes from rabbits.

Protein Eng, 1987 Dec, 1(6), 481 - 5
Hierarchical strategy for protein folding and design: synthesis and expression of T4 lysozyme gene and two putative folding mutants; Narang SA et al.; A T4 lysozyme-coding DNA sequence of 495 bp was chemically synthesized and cloned by ligation of 26 deoxyribooligonucleotide fragments in two steps with a linearized plasmid followed by transformation . On selection by colony hybridization and DNA sequence analysis, clone pTLY.10 was identified to contain a complete T4 lysozyme synthetic DNA . On expression under lac-promoter, unfused T4 lysozyme was obtained in approximately 4-6% yield . The design and synthesis of two putative folding mutants, flexible (Gly-Gly-Gly) and rigid (Asn-Asp-Gly) at position 73-74-75, were based on hierarchical principles . Both mutants lost enzymatic activity of the wildtype . These results are readily understandable if the hierarchical organization of the structure is taken into account . A possible explanation is that the catalytic sites are blocked in both mutants.

J Gen Microbiol, 1987 Dec, 133 ( Pt 12), 3289 - 98
Isolation of genes required for hydrogenase synthesis in Escherichia coli; Chaudhuri A et al.; A mutant strain of Escherichia coli, strain AK23, is devoid of hydrogenase activity when grown anaerobically on glucose and cannot grow on H2 plus fumarate . From E . coli chromosomal DNA library, a plasmid, pAK23, was isolated which restored hydrogenase activity in this strain . Two smaller plasmids, pAK23C and pAK23S, containing different parts of the insert DNA fragment of plasmid pAK23, were isolated . The former plasmid restored activity in strain AK23 while the latter did not . The smallest active DNA fragment in plasmid pAK23C was 0.9 kb . This gene is designated hydE . Plasmids pAK23 and pAK23S restored activity in another hydrogenase-negative strain, SE-3-1 (hydB), while plasmid pAK23C did not, suggesting that plasmid pAK23 contains two genes required for hydrogenase expression . Strain AK23 was also devoid of formate hydrogenlyase and formate dehydrogenase activities and these activities were restored by some of the plasmids . Hydrogenase and formate-related activities in strain AK23 were restored by growth of cells in a high concentration of nickel . Plasmid pAK23C led to synthesis of a polypeptide of subunit molecular mass 36 kDa and plasmid pAK23S led to synthesis of polypeptides of subunit molecular masses 30 and 41 kDa.

Biochem Cell Biol, 1987 Dec, 65(12), 1022 - 30
Reassembly of active 30S ribosomal subunits with an unmethylated in vitro transcribed 16S rRNA; Melancon P et al.; A plasmid has been constructed, which contains the 16S ribosomal RNA gene of Escherichia coli immediately downstream from a phage T7 promoter . In vitro transcription of this gene by RNA polymerase of the T7 phage yielded an unmethylated 16S rRNA that could be used for the assembly of functional 30S subunits . These subunits when assayed in a poly(U)-directed translation assay, were as active as 30S subunits reconstructed with native 16S rRNA . This system opens the possibility of investigating the role of the methylations of the rRNA and the functional consequences of site-directed mutagenesis in a rRNA gene . An application of this system was provided by generating a 16S rRNA transcript shortened by about 30 nucleotides at the 3' end . This truncated rRNA could be reassembled into 30S subunits, about 70% as active as 30S subunits reconstructed with the full-length 16S rRNA transcript.

Biosci Rep, 1987 Dec, 7(12), 969 - 74
Expression in Escherichia coli of active human alcohol dehydrogenase lacking N-terminal acetylation; Hoog JO et al.; Human alcohol dehydrogenase (ADH, beta beta isozyme of class I) was expressed in Escherichia coli, purified to homogeneity, and characterized regarding N-terminal processing . The expression system was obtained by ligation of a cDNA fragment corresponding to the beta-subunit of human liver alcohol dehydrogenase into the vector pKK 223-3 containing the tac promoter . The enzyme, detected by Western-blot analysis and ethanol oxidizing activity, constituted up to 3% of the total amount of protein . Recombinant ADH was separated from E . coli ADH by ion-exchange chromatography and the isolated enzyme was essentially pure as judged by SDS-polyacrylamide gel electrophoresis and sequence analysis . The N-terminal sequence was identical to that of the authentic beta-subunit except that the N-terminus was non-acetylated, indicating a correct removal of the initiator methionine, but lack of further processing.

Environ Health Perspect, 1987 Dec, 76, 29 - 32
Nearest neighbor affects G:C to A:T transitions induced by alkylating agents; Glickman BW et al.; The influence of local DNA sequence on the distribution of G:C to A:T transitions induced in the lacI gene of E . coli by a series of alkylating agents has been analyzed . In the case of nitrosoguanidine, two nitrosoureas and a nitrosamine, a strong preference for mutation at sites proceeded 5' by a purine base was noted . This preference was observed with both methyl and ethyl donors where the predicted common ultimate alkylating species is the alkyl diazonium ion . In contrast, this preference was not seen following treatment with ethylmethanesulfonate . The observed preference for 5'PuG-3' site over 5'-PyG-3' sites corresponds well with alterations observed in the Ha-ras oncogene recovered after treatment with NMU . This indicates that the mutations recovered in the oncogenes are likely the direct consequence of the alkylation treatment and that the local sequence effects seen in E . coli also appear to occur in mammalian cells.

Biochem Int, 1987 Dec, 15(6), 1079 - 88
An ultraviolet light sensitive target in nitrate reductase of Escherichia coli K-12; Francisco R et al.; Ultraviolet light was shown to inactivate purified nitrate reductase in the presence of reduced benzyl viologen . Loss of activity was not complete, reaching 60 to 70% . Photolysis was maximum at 345 nm . The differential spectrum between native and irradiated enzyme exhibited absorption bands at 216, 275, 314 and 365 nm . The photosensitive electron carrier could be extracted by organic solvents . It had the following absorption bands: 225, 275 and 285 nm . It was reduced by Nile blue A but not by methylene blue . The precise nature of this light sensitive molecule could not be determined although the results support the idea that this chromophore might be a naphthoquinone.

Mol Cell Biol, 1987 Dec, 7(12), 4564 - 7
Plasminogen activator inhibitor 2: regulation of gene transcription during phorbol ester-mediated differentiation of U-937 human histiocytic lymphoma cells; Schleuning WD et al.; We have isolated and sequenced two cDNA clones coding for plasminogen activator inhibitor 2 (PAI-2) . The cDNA was used to study the regulation of PAI-2 gene transcription by the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate in the human histiocytic lymphoma cell line U-937 . The tumor promoter caused a transient, 50-fold increase of PAI-2 gene transcription.

Mol Gen Genet, 1987 Dec, 210(2), 364 - 8
Nucleotide sequence of putC, the regulatory region for the put regulon of Escherichia coli K12; Nakao T et al.; The nucleotide sequence of the putC region, from which divergent transcription of the putP and putA genes starts, was determined . The promoter region for the putA gene was restricted to the location between the HindIII site and the NcoI site or at the NcoI site by using putA-lacZ fusion plasmids and the transcriptional start for the putA gene was identified in the region between the HindIII site and the NcoI site by Sl mapping . This region also contains a potential CAP binding site, a ribosome binding site, and a sequence that is highly homologous to argTr . Five potential promoters (putPp1-putPp5) for the putP gene, which were separate from the promoter region for the putA gene, were indicated by S1 mapping analysis of the putP gene transcripts . We concluded that the putC region is 419 bp long and contains two independent sets of promoters, regulating the expression of putP and putA genes in opposite directions . In addition, this region was found to contain an open reading frame (orf) capable of encoding a polypeptide of 111 amino acids in overlapping fashion . But studies using an orf-lacZ fusion gene showed that this open reading frame was not expressed.

Mol Gen Genet, 1987 Dec, 210(2), 331 - 7
The organization of the fuc regulon specifying L-fucose dissimilation in Escherichia coli K12 as determined by gene cloning; Chen YM et al.; In Escherichia coli the six known genes specifying the utilization of L-fucose as carbon and energy source cluster at 60.2 min and constitute a regulon . These genes include fucP (encoding L-fucose permease), fucI (encoding L-fucose isomerase), fucK (encoding L-fuculose kinase), fucA (encoding L-fuculose 1-phosphate aldolase), fucO (encoding L-1,2-propanediol oxidoreductase), and fucR (encoding the regulatory protein) . In this study the fuc genes were cloned and their positions on the chromosome were established by restriction endonuclease and complementation analyses . Clockwise, the gene order is: fucO-fucA-fucP-fucI-fucK-fucR . The operons comprising the structural genes and the direction of transcription were determined by complementation analysis and Southern blot hybridization . The fucPIK and fucA operons are transcribed clockwise . The fucO operon is transcribed counterclockwise . The fucR gene product activates the three structural operons in trans.

Mol Gen Genet, 1987 Dec, 210(2), 288 - 93
Transductional analysis of chromosome replication time; Hanks MC et al.; Following transduction of exponentially growing cultures of Escherichia coli with phage P1, cells with recombinant phenotype begin to increase in number after an initial lag of about one generation time . We show that transductants for markers located at different positions on the chromosome begin to increase at different times, in reverse order to that in which they are replicated . The period over which this happens is equal in duration to the time taken to replicate the chromosome and we have used this relationship to calculate the C-period of E . coli K12 growing at 30 degrees C . We exclude transduction-induced filamentation as the cause of the initial lag and suggest that the lag may result from the way in which donor DNA is inherited.

Mol Gen Genet, 1987 Dec, 210(2), 234 - 40
Isolation and characterization of Escherichia coli birA intragenic suppressors; Uchida KM et al.; The biotin (bio) operon in Escherichia coli is negatively regulated by BirA, a bifunctional protein with both repressor and biotin-activating functions . Twenty-five heat-resistant revertants of three temperature-sensitive birA alleles (birA85, birA104 and birA879) were isolated and categorized into five growth and six repression classes . The revertants appear to increase biotin activation by raising the specific activity of BirA and/or increasing the number of enzyme molecules . The 19 birA85 revertants displayed a broad range of activity for both enzyme and repressor functions, and may represent intragenic second-site suppressor mutations . The birA85 revertants included a novel class of bio superrepressor mutations . Repressor titration experiments suggested that many of the birA85 revertants increase BirA concentrations above wild-type levels because the repressors were not competed from the chromosomal bio operator by multicopy bio operator plasmids . The majority of the birA104 revertants resulted in both wild-type repressor and enzyme activity; they are possibly true revertants in which the amino acid residue altered by the birA104 mutation has been substituted by the wild-type or a chemically similar amino acid.

Vet Immunol Immunopathol, 1987 Dec, 17(1-4), 193 - 209
Bovine interleukin 2: cloning, high level expression, and purification; Baker PE; We utilized a human IL2 probe to isolate bovine IL2 sequences from a lymph node cDNA library . Bovine IL2 was subsequently expressed in both bacteria and yeast . Using a rapid, two-step purification scheme, we have been able to isolate over 20 mg/l of homogenous bovine rIL2 secreted from the yeast . The availability of sizable quantities of bovine rIL2 should make it possible to ascertain potential therapeutic or prophylactic utility of this lymphokine in cattle.

Neth J Surg, 1987 Dec, 39(6), 194 - 6
Intrabiliary rupture of a hydatid cyst of the liver with concomitant intraperitoneal rupture into the lesser sac; van Gulik TM et al.; A case is reported of a young man of Moroccan origin, admitted with fever, aerobilia and an epigastric mass . Laparotomy revealed a suppurated hydatid cyst extending from the left lobe of the liver, which had ruptured into the left hepatic duct and intraperitoneally, into the lesser sac . A left hepatic lobectomy was performed . Gas producing micro-organisms were cultured from the cyst contents whereas no viable scolices were demonstrated . Medical therapy with mebendazole was instituted postoperatively . Six months later, the patient underwent subtotal pancreaticoduodenectomy because of an obstructive, lymphoblastic non-Hodgkin lymphoma in the ampullary region . One year after the initial operation, there were no signs of recurrence of the hydatid disease.

Mol Gen Genet, 1987 Dec, 210(3), 535 - 42
Involvement of the ntrA gene product in the anaerobic metabolism of Escherichia coli; Birkmann A et al.; The ntr A gene product, required for expression of genes involved in nitrogen fixation (nif) and regulation (ntr), was shown to be necessary for the expression of the two enzymes of the anaerobically inducible formate hydrogenlyase (FHL) pathway, formate dehydrogenase (FDHH) and hydrogenase isoenzyme 3 . Consistent with this finding, the gene encoding the selenopolypeptide (fdhF) of FDHH was shown to have a nif consensus promoter . The levels of six other anaerobically inducible enzymes were examined and found to be ntrA independent . Significantly, these latter six enzymes are dependent upon the fnr gene product for their expression while FDHH and hydrogenase 3 are fnr independent . These findings indicate that there are at least two classes of anaerobically regulated promoters: one class which is ntrA dependent and fnr independent and a second class which is fnr dependent and ntr A independent.

Mol Gen Genet, 1987 Dec, 210(3), 504 - 8
The Escherichia coli cell division proteins FtsY, FtsE and FtsX are inner membrane-associated; Gill DR et al.; The cell division genes ftsY, ftsE and ftsX form an operon mapping at 76 min on the Escherichia coli chromosome . The protein products of these genes have been identified previously . We have studied the cellular location of the radiolabelled Fts proteins using maxicells and standard fractionation procedures . Previous protein sequence homologies suggested an inner membrane location for FtsE . We have confirmed this predicted location and have shown that FtsY and FtsX are also inner membrane-associated . These results are in agreement with the hypothesis that FtsE may act at the inner membrane, in a "septalsome" complex, by coupling ATP hydrolysis to the process of bacterial cell division.

Mol Gen Genet, 1987 Dec, 210(3), 449 - 59
Transcriptional regulation of sporulation genes in yeast; Holaway BL et al.; The relative transcription rates of three sporulation-regulated genes of yeast (SPR1, SPR2 and SPR3) were determined at intervals during sporulation, using a filter binding assay . The binding of in vivo labeled RNA to the corresponding DNAs increased 3- to 12-fold at the time of meiosis I, in parallel with the accumulation of the SPR transcripts . SPR1 and SPR3 mRNA abundance increased from less than 0.7 to 130 and 90 copies per cell, respectively, between the time of shift to sporulation medium and the initiation of spore formation . This represented a 150-to 200-fold increase in the steady-state levels of these RNAs . Similarly, the levels of beta-galactosidase present in sporulating cells harboring fusions between SPR3 and Escherichia coli lacZ increased at least 700-fold . We conclude that SPR1, SPR2 and SPR3 transcription is modulated during sporulation, possibly in response to earlier events in the process.

Mol Gen Genet, 1987 Dec, 210(3), 394 - 8
Replication intermediate of a hybrid plasmid carrying the replication terminus (ter) site of R 6K as revealed by agarose gel electrophoresis; Horiuchi T et al.; A 4.32 kb DNA fragment, on which the DNA replication terminus (terR) site of plasmid R 6K was located, was inserted into the unique EcoRI site of plasmid pUC9 . To detect replication intermediate molecules with a replication fork halted at the terR site, a cell DNA extract was digested with EcoRI, electrophoresed through an agarose gel and stained with ethidium bromide . In addition to two major bands, one derived from vector DNA and the other from the ter insert fragment, two extra minor bands were detected . Following DNA-DNA hybridization and electron microscopic observation we concluded that the two minor bands corresponded to the two Y-shaped molecules, produced from the theta-shaped intermediate molecules by EcoRI digestion.

Mol Gen Genet, 1987 Dec, 210(3), 381 - 4
Direct selection for the exchange of alleles between a plasmid and the Escherichia coli chromosome; Merryweather A et al.; Recombination is extensively used in order to move alleles between replicons . The exchange of wild-type chromosomal and mutant plasmid-borne alleles is a two-step process entailing the formation of a cointegrate between the entire plasmid and the chromosome, followed by resolution of such cointegrates to give a mutant chromosome and a plasmid carrying the wild-type chromosomal sequence . Often the cointegrate and the resolved forms cannot be distinguished phenotypically . To enable the direct isolation of the resolved products we have developed a positive selection technique . Cells containing a cointegrated plasmid R1 were constructed by transduction using a P1 lysate prepared from cells harbouring a plasmid comprising a mutant chromosomal allele and the so-called omega fragment which carries an aad (aminoglycoside adenylyltransferase) gene . P1 transduction from the cointegrate strain into an SmD recipient allowed direct selection for the resolved complex, since transduction of the aad gene is lethal to an SmD strain.

Mech Ageing Dev, 1987 Dec, 41(3), 241 - 50
Endotoxin-induced liver injury in aged and subacutely hypervitaminotic A rats; Hendriks HF et al.; The plasma disappearance of endotoxin and endotoxin-induced hepatic injury were studied in two rat models: the aging rat and the subacutely hypervitaminotic A rat . The choice of these models was based on their respective association with a decreased or increased Kupffer cell endocytic activity . The half-life of endotoxin (E . coli O26: B6, phenol extracted) in plasma was significantly prolonged in aged rats as measured by both the Limulus assay (t1/2 = 2.1 +/- 0.1 h in 3-6-month-old, and 3.3 +/- 0.3 h in 24-36-month-old rats) and 51Cr-labeled endotoxin radioactivity assay (t1/2 = 5.3 +/- 0.3 h in 3-6-month old and 7.7 +/- 0.6 h in 24 36-month-old rats) . In subacute hypervitaminosis A, the half-life of endotoxin was significantly decreased in the Limulus assay (t1/2 = 2.1 +/- 0.1 h in 3-6-month old and 1.4 +/- 0.2 h in subacutely hypervitaminotic A rats), but not in the radioactivity assay (t1/2 = 5.3 +/- 0.3 h in 3-6-month-old and 5.0 +/- 0.4 h in subacutely hypervitaminotic A rats) . Hundred percent mortality was observed at a dose of 2 mg endotoxin/100 g body wt . in old rats, but not in young rats . Only 1 of 7 young subacutely hypervitaminotic A rats died following injection of this dose of endotoxin . The dose of endotoxin which caused only minimal parenchymal liver cell injury in young rats induced substantial parenchymal cell injury in old rats and subacutely hypervitaminotic A rats as determined by both histological and biochemical parameters . It is concluded that some basic characteristics of experimental animals, such as age and nutritional status, can dramatically influence the sensitivity to endotoxin and this is not necessarily correlated with the rate of endotoxin clearance.

J Clin Pathol, 1987 Dec, 40(12), 1402 - 4
HeLa cell and buccal epithelial cell adhesion assays for detecting intestinal Escherichia coli with adhesive properties in ulcerative colitis; Burke DA et al.; The two methods used to detect intestinal Escherichia coli in patients with ulcerative colitis, HeLa cell and buccal epithelial cell (BEC) adhesion assays, were compared . Both methods showed a significant difference between the adhesive property of colitic and control E coli: p less than 0.0002 and p less than 0.01 for the buccal epithelial cell and HeLa cell methods, respectively . HeLa cells did not detect all BEC positive isolates which were quantitatively more adhesive than any control isolate . All BEC negative isolates were HeLa cell negative . The BEC technique seems to be more sensitive for detecting adhesive E coli in ulcerative colitis because the true incidence of adhesive E coli in ulcerative colitis was higher than that determined by the HeLa cell method.

DNA, 1987 Dec, 6(6), 583 - 91
A standardized vector system for manipulation and enhanced expression of genes in Escherichia coli; Botterman J et al.; Different families of cloning and expression vectors were engineered on a standard plasmid . They contain several regulatory signals for transcription and/or translation initiation and termination . The plasmids in each series differ only in the number, type, and order of unique restriction cleavage sites clustered in front of a transcription terminator . The pLK30 plasmids are general cloning vectors and the corresponding pLK50 plasmids carry the lambda pL promoter . The pLK60 vectors carry the lambda pR promoter and translation initiation signals of the cro gene containing the Shine-Dalgarno sequence and initiation codon . The pLK70 series is similar to pLK60 except that additional 5'-translated cro sequences are included . The pLK80 plasmids have a lacZ gene fragment suitable for the construction of hybrid genes . The presence of translational stop signals in the pLK90 series facilitates the manipulation of genes truncated at the 3' end . This standardized pLK vector system offers great versatility in gene manipulation and in optimization of gene expression under the control of strong regulatable promoters . Measurement of expression levels under repressed conditions permits the identification of optimal promoter-gene configurations in constructions directing high-level expression.

DNA, 1987 Dec, 6(6), 539 - 51
Cloning, characterization, and sequencing of the genes encoding the large and small subunits of the periplasmic {NiFe}hydrogenase of Desulfovibrio gigas; Li C et al.; The structural genes for the large and small subunits of Desulfovibrio gigas periplasmic {NiFe}hydrogenase were identified and isolated by immunological and oligonucleotide screening . The gene for the small subunit codes for a 266-amino-acid, 28,724-dalton polypeptide which is separated by 63 nucleotides from the large subunit gene that codes for a 560-amino-acid, 61,707-dalton polypeptide . A putative signal peptide precedes the small subunit coding region, which may direct transport of the enzyme into the periplasmic compartment . Comparison of the amino acid sequence of this enzyme with those of two other classes of hydrogenase found in Desulfovibrio revealed that the D . gigas periplasmic hydrogenase has some homologies to the periplasmic {NiFeSe}hydrogenase of D . baculatus but none to the periplasmic {Fe}hydrogenase of D . vulgaris . The genes for the large and small subunits of the D . gigas hydrogenase hybridize strongly to genomic DNAs from several species of Desulfovibrio, indicating molecular similarity of the {NiFe}hydrogenase among sulfate reducers.

Biochemistry, 1987 Dec 1, 26(24), 7702 - 7
Location and activity of ubiquinone 10 and ubiquinone analogues in model and biological membranes; Cornell BA et al.; Deuteriated analogues of ubiquinone 10 (Q10) have been dispersed with plasma membranes of Escherichia coli and with the inner membranes of beetroot mitochondria . Orientational order at various deuteriated sites was measured by solid-state deuterium nuclear magnetic resonance (2H NMR) . Similar measurements were made, using the compounds dispersed in dimyristoylphosphatidylcholine (DMPC) and egg yolk lecithin and dispersions prepared from the lipid extracts of beetroot mitochondria . In all cases only a single unresolved 2H NMR spectrum (typically 1000-Hz full width at half-height) was observed at concentrations down to 0.02 mol % Q10 per membrane lipid . This result shows that most Q10 is in a mobile environment which is physically separate from the orientational constraints of the bilayer lipid chains . In contrast, a short-chain analogue of Q10, in which the 10 isoprene groups have been replaced by a perdeuteriated tridecyl chain, showed 2H NMR spectra with quadrupolar splittings typical of an ordered lipid that is intercalated into the bilayer . The NADH oxidase activity and O2 uptake in Escherichia coli and in mitochondria were independent of which analogue was incorporated into the membrane . Thus, despite the major difference in their physical association with membranes, or their lipid extracts, the electron transport function of the long- and short-chain ubiquinones is similar, suggesting that the bulk of the long-chain ubiquinone does not have a direct function in electron transporting activity . The physiologically active Q10 may only be a small fraction of the total ubiquinone, a fraction that is below the level of detection of the present NMR equipment.(ABSTRACT TRUNCATED AT 250 WORDS)

Aviat Space Environ Med, 1987 Dec, 58(12), 1211 - 4
The influence of high and low pressure on phagocytosis of Escherichia coli by human neutrophils in vitro; Lingaas E et al.; Human polymorphonuclear neutrophils were exposed to high (4 atm) and low (0.4 atm) pressures in vitro before and during phagocytosis of radiolabelled Escherichia coli . Preexposure of the neutrophils to high pressure before phagocytosis did not influence uptake . However, when phagocytosis took place at 4 atm the uptake of bacteria opsonized by active serum and IgG was significantly depressed . On the other hand, uptake in the presence of heat-inactivated serum or albumin and uptake of non-opsonized E . coli was not changed at high pressure . The results suggest an influence on uptake mechanisms related to receptors in the cell membrane . When the neutrophils were exposed to hypobaric pressure both before and during phagocytosis, the uptake of nonopsonized E . coli was slightly depressed . However, neither preexposure nor exposure during phagocytosis was sufficient alone to elicit this inhibiting effect . The uptake of E . coli opsonized by active serum or IgG was not influenced by low pressure.

Arch Biochem Biophys, 1987 Dec, 259(2), 316 - 30
Kinetic mechanism of native Escherichia coli aspartate transcarbamylase; Hsuanyu Y et al.; Equilibrium isotope exchange kinetics have been used to reinvestigate the kinetic mechanism of Escherichia coli aspartate transcarbamylase (aspartate carbamoyl-transferase) at pH 7.0, 30 degrees C . Keq = 5.9 (+/- 0.6) X 10(3), allowing variation of substrate concentrations above and below their Km values in all experiments, a condition not possible at pH 7.8 {F . C . Wedler and F . J . Gasser (1974) Arch . Biochem . Biophys . 163, 57-68} . The rate of the {14C}Asp in equilibrium N-carbamoyl L-aspartate (C-Asp) exchange reaction was five times faster than that of {32P}carbamyl phosphate (C-P) in equilibrium Pi, which argues strongly against the rapid equilibrium random mechanism previously proposed by E . Heyde, A . Nagabhushanam, and J . F . Morrison {Biochemistry 12, 4718-4726 (1973} . Substrate concentrations were varied either as reactant-product pairs (holding the other pair constant) or together simultaneously in constant ratio at equilibrium . The resulting kinetic saturation patterns were most consistent with a preferred order random kinetic mechanism, with C-P binding prior to Asp and with C-Asp being released before Pi . Weak inhibition effects at high substrate levels could be accounted for by multiple weak dead-end complexes or ionic strength effects . Computer-based simulations have led to a set of rate constants that fit the experimental data, are in agreement with rate constants measured previously by pre-steady-state methods, and predict the correct initial velocities in the forward and reverse directions . Simulations also show that rate constants consistent with any of the various alternative mechanisms do not provide good fit to the experimental data . A model for the kinetic mechanism is considered, in which the binding of Asp prior to C-P may restrict access of C-P to the active site, but C-P binding prior to Asp potentiates the enzyme for the allosteric (T-R) transition, centered entirely upon the Asp binding process.

Am J Physiol, 1987 Dec, 253(6 Pt 2), R868 - 76
Thermoregulatory responses of febrile sheep to spinal and hypothalamic heating; Blatteis CM et al.; Fever was induced by the intravenous injection of 0.25 microgram/kg of lipopolysaccharide (LPS) from Escherichia coli in eight conscious sheep exposed to ambient temperatures adjusted to the lower range of thermoneutrality . Chronic spinal or hypothalamic thermodes were perfused with water of 44 degrees C for 20 min or for most of the rising phase of fever (100 min of the mean 166 min total rise time) . The effects of spinal and hypothalamic heating were identical . Thus, before LPS, spinal or hypothalamic heating did not affect the rate of O2 consumption (VO2) but increased skin blood flow (as indicated by skin temperatures) and elicited panting; therefore rectal temperature (Tre) fell . During fever rise, the already reduced skin blood flow and respiratory rate were not affected by spinal or hypothalamic heating, but the increased VO2 was reduced; consequently, the rise in Tre was attenuated . During the plateau phase of fever, all responses were similar to those seen before LPS . In febrilysis, heating strongly enhanced the operating heat loss mechanisms and, hence, augmented the fall in Tre . Thus, although the thermoeffectors activated by spinal or hypothalamic heating were modified during the different stages of fever, the effect on body temperature was nearly the same . Therefore there seems to be no change in spinal or hypothalamic thermosensitivity during fever in sheep.

Scand J Immunol, 1987 Dec, 26(6), 673 - 81
Analysis of the antigenic profile of Mycobacterium leprae: cross-reactive and unique specificities of human and rabbit antibodies; Ehrenberg JP et al.; Thirty-two mycobacterial components were detected by antibodies contained in leprosy patients' sera across the clinical spectrum and rabbit anti-M . leprae hyperimmune sera by western blot analysis of armadillo-derived M . leprae antigen preparations . Sera of borderline tuberculoid patients were found to contain antibodies recognizing 18 M . leprae components . While the reactivity of the sera on the lepromatous pole seemed to be distributed over the entire molecular weight range, most of the reactivity in the borderline tuberculoid patients was directed at higher molecular weight components (greater than 70,000) . Identification of a series of previously unrecognized M . leprae components offers new possibilities in regard to the potential use of these antigens as targets for immunodiagnosis . Antibodies contained in the rabbit anti-M . leprae sera reacted with 19 M . leprae components . Antigens migrating at 64,000, 38,000, and 22,000 were detected by the rabbit sera only . Evidence of extensive cross-reactivity between M . leprae and BCG organisms emphasizes the need to use well-characterized antibody probes to determine the specificity of select mycobacterial antigens . The potential usefulness of rabbit monospecific hyperimmune sera to select M . leprae fractions in immunodiagnosis, in immune regulation studies, or as a tool to screen for mycobacterial products in lambda gt11 phage lysates of E . coli is discussed . Select M . leprae components were partially purified and their recovery assessed through SDS-PAGE analysis of Coomassie blue-stained gels.

Proc Natl Acad Sci U S A, 1987 Dec, 84(24), 8961 - 5
High-level expression of sperm whale myoglobin in Escherichia coli; Springer BA et al.; Sperm whale myoglobin was expressed in Escherichia coli from a totally synthetic gene inserted in the expression vector pUC19 . The gene was constructed as 23 overlapping oligonucleotides encoding both strands of the DNA . Gene synthesis provides several advantages over traditional eukaryotic gene-cloning techniques, allowing the incorporation of an efficient ribosome binding site, appropriate initiation and termination sequences, restriction enzyme sites for convenient subcloning and future mutagenesis, and frequently used codons for highly expressed E . coli genes . The sperm whale myoglobin expressed from the synthetic gene constituted approximately 10% of the total soluble protein as holo-protein, indicating that iron-protoporphyrin IX biosynthesis and prosthetic-group incorporation are not limiting in the high-level expression of this heme protein in E . coli . We credit the use of frequently used E . coli codons for the observed high-level expression . The sperm whale myoglobin produced is stable, easily purified to homogeneity, and indistinguishable from commercially available sperm whale myoglobin by optical and magnetic spectroscopic methods.

Proc Natl Acad Sci U S A, 1987 Dec, 84(24), 8903 - 6
Human immunodeficiency virus protease expressed in Escherichia coli exhibits autoprocessing and specific maturation of the gag precursor; Debouck C et al.; The mature gag and pol proteins of human immunodeficiency virus (HIV) and all retroviruses derive from large gag and gag-pol polyprotein precursors by posttranslational cleavage . A highly specific, virally encoded protease is required for this essential proteolytic processing . In this study, the HIV protease gene product was expressed in Escherichia coli and shown to autocatalyze its maturation from a larger precursor . In addition, this bacterially produced HIV protease specifically processed an HIV p55 gag polyprotein precursor when coexpressed in E . coli . This system will allow detailed structure-function analysis of the HIV protease and provides a simple assay for the development of potential therapeutic agents directed against this critical viral enzyme.

J Med Microbiol, 1987 Dec, 24(4), 359 - 62
Verotoxin production among porcine strains of Escherichia coli and its association with oedema disease; Linggood MA et al.; Strains of Escherichia coli isolated from documented cases of disease in pigs and belonging to a wide range of pathogenic serotypes were tested for their ability to produce a heat labile verotoxin (VT) . The strains isolated from oedema disease all produced VT, indicating that the cytotoxin detected by the vero-cell assay was identical to "oedema disease principle" . Strains belonging to the serotypes associated with enterotoxic diarrhoea were VT- . Not all the strains belonging to the recognised oedema disease serotypes (O141:K85, O139:K82 and O138:K81) produced VT, but the VT- strains were not associated with outbreaks of clinical disease.

J Am Dent Assoc, 1987 Dec, 115(6), 861 - 3
Brain abscess of odontogenic origin: report of case; Aldous JA et al.; Advanced dental infection rarely causes brain abscess resulting in death . Good dental hygiene and removing abscessed teeth are advised for prevention of any such occurrence . An intercranial infection is described in a 29-year-old male who also had a dental phobia.

J Cell Biol, 1987 Dec, 105(6 Pt 2), 2999 - 3005
Expression in Escherichia coli of a functional Dictyostelium myosin tail fragment; De Lozanne A et al.; The amino acid sequence of the myosin tail determines the specific manner in which myosin molecules are packed into the myosin filament, but the details of the molecular interactions are not known . Expression of genetically engineered myosin tail fragments would enable a study of the sequences important for myosin filament formation and its regulation . We report here the expression in Escherichia coli of a 1.5-kb fragment of the Dictyostelium myosin heavy chain gene coding for a 58-kD fragment of the myosin tail . The expressed protein (DdLMM-58) was purified to homogeneity from the soluble fraction of E . coli extracts . The expressed protein was found to be functional by the following criteria: (a) it appears in the electron microscope as a 74-nm-long rod, the predicted length for an alpha-helical coiled coil of 500 amino acids; (b) it assembles into filamentous structures that show the typical axial periodicity of 14 nm found in muscle myosin native filaments; (c) its assembly into filaments shows the same ionic strength dependence as Dictyostelium myosin; (d) it serves as a substrate for the Dictyostelium myosin heavy chain kinase which phosphorylates myosin in response to chemotactic signaling; (e) in its phosphorylated form it has the same phosphoamino acids and similar phosphopeptide maps to those of phosphorylated Dictyostelium myosin heavy chain; (f) it competes with myosin for the heavy chain kinase . Thus, all the information required for filament formation and phosphorylation is contained within this expressed protein.

Cell Tissue Res, 1987 Dec, 250(3), 695 - 9
The early postnatal development of the primary immune response in rat popliteal lymph node, stimulated with thymus-independent type-1 and type-2 antigens; van Rees EP et al.; To examine the development of the postnatal immune response to thymus-independent type-1 (TI-type 1) and TI type-2 antigens, respectively, trinitrophenyl-lipopolysaccharide (TNP-LPS) or TNP-Ficoll was injected subcutaneously into the hind footpads of young rats of various ages . After 5 days the popliteal lymph nodes (PLNs) were removed and the localization pattern of specific anti-TNP antibody-containing cells was studied . The first specific antibody-containing cells elicited in rats by TNP-LPS appeared in animals at day 19 after birth . The results suggest that the development of these cells from lymphocyte to plasma cell occurs while they migrate from cortex to medulla . An unexpected finding was the low response to TNP-Ficoll in PLN; from 6 weeks after birth only very few specific antibody-containing cells were found . However, in the spleen numerous anti-TNP antibody-containing cells were found in the periarteriolar lymphocyte sheaths . To test the exclusive role of the spleen in the appearance of anti-TNP antibody-containing cells in lymph node after subcutaneous administration of TNP-Ficoll, the experiment was repeated in rats that had been splenectomized . Evidence from these experiments suggests that the spleen plays a major role in the appearance of the above-mentioned cells in lymph nodes.

Proc Natl Acad Sci U S A, 1987 Dec, 84(23), 8544 - 7
Partition site of the P1 plasmid; Martin KA et al.; We have defined a minimal partition site, parS, from the plasmid P1 . It contains sufficient cis-acting information to direct accurate segregation of low-copy-number plasmids that contain it as long as the two essential P1 Par proteins are supplied in trans . The site is, at most, 34 base pairs and contains a perfect 13-base-pair inverted repeat . Site-directed mutations were made within the repeat sequence that abolished activity whether or not the symmetry of the palindrome was maintained . Partition appears to be a competitive process, as differentially marked plasmids carrying the same type of partition site are not independently segregated but are randomly distributed with respect to each other . We have studied competition between plasmids carrying various fragments encompassing the parS site . As expected, two plasmids carrying the minimal parS site compete with each other . However, a sequence that lies to the left of the minimal parS site acts as a major modulator of this competition, changing the specificity of the competitive effect completely . Thus, this adjacent sequence appears to be an important determinant of the specificity of the wild-type P1 partition system without being necessary for its efficient function.

Proc Natl Acad Sci U S A, 1987 Dec, 84(23), 8525 - 9
Determinants of membrane protein topology; Boyd D et al.; The topology of the integral membrane protein MalF, which is required for maltose transport in Escherichia coli, has been analyzed using fusions of alkaline phosphatase (EC 3.1.3.1) . The properties of such fusion strains support a MalF structure previously proposed on theoretical grounds . Several transmembrane segments within MalF can act as signal sequences in exporting alkaline phosphatase . Other transmembrane sequences, in conjunction with cytoplasmic domains, can stably anchor alkaline phosphatase in the cytoplasm . Our results suggest that features of the amino acid sequence (possibly the positively charged amino acids) of the cytoplasmic domains of membrane proteins are important in anchoring these domains in the cytoplasm . These studies in conjunction with our earlier results show that alkaline phosphatase fusions to membrane proteins can be an important aid in analyzing membrane topology and its determinants.

Proc Natl Acad Sci U S A, 1987 Dec, 84(23), 8365 - 9
Correlation between the 32-kDa sigma factor levels and in vitro expression of Escherichia coli heat shock genes; Skelly S et al.; S-30 extracts from Escherichia coli cells were used to express heat shock (HS) and non-HS genes in vitro in a DNA-directed protein synthesis system . The S-30 extracts prepared from cells that have been shifted to 45 degrees C express HS genes in vitro approximately 8 times better than extracts from cells at 33 degrees C . In contrast, the expression of non-HS genes in extracts from heat-induced cells is only 40% of that seen in extracts from cells at 33 degrees C . These results correlate well with the levels of HS sigma factor and normal sigma factor bound to RNA polymerase . Thus, there was an 8-fold increase in the HS sigma factor and a 60% decrease in the normal sigma factor associated with RNA polymerase at the higher temperature . Part of the increase in the level of the HS sigma factor could be accounted for by a 3-fold increase in the level of HS sigma factor mRNA during heat induction.

J Virol, 1987 Dec, 61(12), 3946 - 9
Interference with viral infection by defective RNA replicase; Inokuchi Y et al.; RNA-dependent RNA and DNA polymerases have a conserved segment, Tyr-X-Asp-Asp (G . Karmer and P . Argos, Nucleic Acids Res . 12:7269-7282, 1984) . To investigate the function of this segment, we changed the Gly residue at position 357 in the conserved sequence Tyr-356-Gly-357-Asp-358-Asp-359 of the replicase of RNA coliphage Q beta to Ala, Ser, Pro, Met, or Val and examined the replicase activity in vivo . Cells carrying the variant plasmids lost the replicase activity and severely inhibited the proliferation of phage Q beta (group III) and related phage SP (group IV) by suppressing phage RNA synthesis . In contrast, substitution of the Gly residue at 390 showed only a slight inhibitory effect, although replicase activity was also lost . These results suggest that the cells harboring an altered replicase at the conserved segment can interfere specifically with the wild-type phage and different but related phage infections.

J Bacteriol, 1987 Dec, 169(12), 5856 - 8
Requirement of a heat-labile factor(s) for in vitro expression of the amp gene of pBR322; Kuriki Y; The amp gene of pBR328 and pBR322 was expressed less efficiently at 45 than at 30 degrees C in an in vitro coupled transcription-translation system of Escherichia coli . Preincubation of S30 extract at 45 degrees C reduced specifically its ability to express the amp gene at 30 degrees C, indicating inactivation of a factor(s) required for efficient expression of the amp gene.

J Bacteriol, 1987 Dec, 169(12), 5776 - 81
In vivo cell division gene product interactions in Escherichia coli K-12; Ferreira LC et al.; Overexpression of plasmid-coded PBP 3 was analyzed in strains harboring ftsA, ftsH, pbpB (ftsI), ftsQ, ftsZ, or recA441 (Tif) mutations . Higher cellular levels of PBP 3, the pbpB gene product, could not restore septum formation of ftsA, ftsQ, ftsZ, and recA (Tif) mutants at 42 degrees C . However, filamentation in strains harboring pbpB and ftsH mutations was fully suppressed by PBP 3 overexpression . Additional observations indicated that the Y16 (ftsH) strain, not transformed with the PBP 3-overproducing plasmid, had no detectable PBP 3 in envelopes after incubation at the restrictive temperature . These results suggest that suppression of filamentation of fts strains overexpressing wild-type cell division proteins after the shift to the restrictive temperature can be a useful strategy to demonstrate in vivo interactions of cell division gene products.

J Bacteriol, 1987 Dec, 169(12), 5735 - 44
Sequence analysis of the Escherichia coli dnaE gene; Tomasiewicz HG et al.; We have determined the sequence of a 4,350-nucleotide region of the Escherichia coli chromosome that contains dnaE, the structural gene for the alpha subunit of DNA polymerase III holoenzyme . The dnaE gene appeared to be part of an operon containing at least three other genes: 5'-lpxB-ORF23-dnaE-ORF37-3' (ORF, open reading frame) . The lpxB gene encodes lipid A disaccharide synthase, an enzyme essential for cell growth and division (M . Nishijima, C.E . Bulawa, and C.R.H . Raetz, J . Bacteriol . 145:113-121, 1981) . The termination codons of lpxB and ORF23 overlapped the initiation codons of ORF23 and dnaE, respectively, suggesting translational coupling . No rho-independent transcription termination sequences were observed . A potential internal transcriptional promoter was found preceding dnaE . Deletion of the -35 region of this promoter abolished dnaE expression in plasmids lacking additional upstream sequences . From the deduced amino acid sequence, alpha had a molecular weight of 129,920 and an isoelectric point of 4.93 for the denatured protein . ORF23 encoded a more basic protein (pI 7.11) with a molecular weight of 23,228 . In the accompanying paper (D.N . Crowell, W.S . Reznikoff, and C.R.H . Raetz, J . Bacteriol . 169:5727-5734, 1987), the sequence of the upstream region that contains lpxA and lpxB is reported.

J Bacteriol, 1987 Dec, 169(12), 5692 - 9
Genes encoding two lipoproteins in the leuS-dacA region of the Escherichia coli chromosome; Takase I et al.; The coding of two rare lipoproteins by two genes, rlpA and rlpB, located in the leuS-dacA region (15 min) on the Escherichia coli chromosome was demonstrated by expression of subcloned genes in a maxicell system . The formation of these two proteins was inhibited by globomycin, which is an inhibitor of the signal peptidase for the known lipoproteins of E . coli . In each case, this inhibition was accompanied by formation of a new protein, which showed a slightly lower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and which we suppose to be a prolipoprotein with an N-terminal signal peptide sequence similar to those of the bacterial major lipoproteins and lysis proteins of some bacteriocins . The incorporation of 3H-labeled palmitate and glycerol into the two lipoproteins was also observed . Sequencing of DNA showed that the two lipoprotein genes contained sequences that could code for signal peptide sequences of 17 amino acids (rlpA lipoprotein) and 18 amino acids (rlpB lipoprotein) . The deduced sequences of the mature peptides consisted of 345 amino acids (Mr 35,614, rlpA lipoprotein) and 175 amino acids (Mr 19,445, rlpB lipoprotein), with an N-terminal cysteine to which thioglyceride and N-fatty acyl residues may be attached . These two lipoproteins may be important in duplication of the cells.

J Bacteriol, 1987 Dec, 169(12), 5641 - 7
Method for localization of cloned DNA fragments on the Escherichia coli chromosome; Fayet O et al.; In exponentially growing cultures of Escherichia coli strains carrying the dnaC28 mutation, DNA replication can be synchronized by temperature changes (R . L . Rodriguez, M . S . Dalbey, and C . I . Davern, J . Mol . Biol . 74:599-604, 1973) . We used this synchronization procedure and DNA-DNA hybridization to develop a technique for the localization of cloned chromosomal fragments on the genetic map . Because of the bidirectional nature of replication in E . coli, our method gave two possible positions (one on each replication arm) . However because of the precision obtained for each position (+/- 1 map unit), the final mapping with various genetic techniques was greatly facilitated . Using this technique and a simple chromosomal mobilization test, we located at 93.2 +/- 1 min a cloned DNA fragment carrying an extragenic suppressor of dnaA46, a thermosensitive mutation in the dnaA initiation gene . Further analysis showed that the groES (mopA) and groEL (mopB) genes, both located at 94.2 min on the standard map, were indeed carried by the cloned suppressor fragment.

J Bacteriol, 1987 Dec, 169(12), 5434 - 44
Distribution of newly synthesized lipoprotein over the outer membrane and the peptidoglycan sacculus of an Escherichia coli lac-lpp strain; Hiemstra H et al.; The insertion of newly synthesized lipoprotein molecules into the cell wall of Escherichia coli was studied topographically by immunoelectron microscopy . Lipoprotein was briefly induced with isopropyl-beta-D-thiogalactopyranoside in cells carrying lac-lpp on a low-copy-number plasmid in an E . coli lpp host . Specific antibodies bound to the newly inserted lipoprotein molecules, which were exposed at the cell surface after treatment of the cells with Tris-EDTA, were detected with a protein A-gold probe . The average distribution of the gold particles over the cell surface of noninduced cells was determined for cells induced for 5 and 10 min . Analysis of 250 to 350 cells showed that the distribution of newly synthesized lipoprotein over the cell surface was homogeneous in both cases . The binding of lipoprotein to the peptidoglycan layer was studied by the same technique, and visual inspection again revealed a homogeneous distribution of bound lipoprotein over the entire sacculus surface . It is therefore concluded that free lipoprotein is inserted equally over the entire cell wall of E . coli, while binding to peptidoglycan also occurs over the entire cell surface . The rate of lipoprotein synthesis increased with cell length in nondividing cells, whereas it was constant in cells which had initiated constriction . Analysis of cells having different amounts of lipoprotein in their cell wall revealed that the cell shape depended on the total lipoprotein content of the cell . Cells having no or only a small amount of lipoprotein grew as spheres, whereas cells with increasing numbers of lipoprotein molecules gradually changed their shape to short rods.

J Bacteriol, 1987 Dec, 169(12), 5429 - 33
Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product; Ishino Y et al.; The iap gene in Escherichia coli is responsible for the isozyme conversion of alkaline phosphatase . We analyzed the 1,664-nucleotide sequence of a chromosomal DNA segment that contained the iap gene and its flanking regions . The predicted iap product contained 345 amino acids with an estimated molecular weight of 37,919 . The 24-amino-acid sequence at the amino terminus showed features characteristic of a signal peptide . Two proteins of different sizes were identified by the maxicell method, one corresponding to the Iap protein and the other corresponding to the processed product without the signal peptide . Neither the isozyme-converting activity nor labeled Iap proteins were detected in the osmotic-shock fluid of cells carrying a multicopy iap plasmid . The Iap protein seems to be associated with the membrane.

J Bacteriol, 1987 Dec, 169(12), 5343 - 52
Cloning and promoter identification of the iron-regulated cir gene of Escherichia coli; Griggs DW et al.; The cir gene, which encodes the colicin I receptor protein and is regulated by both cellular iron content and growth temperature, was cloned into a multicopy-number plasmid . Physical mapping and complementation analysis established the position of cir between mgl and nfo on the Escherichia coli chromosome . A gene encoding a 32,000-dalton polypeptide was located downstream of and adjacent to cir, but did not appear to be part of the same transcriptional unit . A 525-base-pair fragment from the 5' end of the 1.8-kilobase-pair receptor-coding region directed iron-regulated transcription and translation of a hybrid cir-lacZ gene . Two overlapping promoters were identified by determination of the transcriptional start sites and by sequence analysis . A small open reading frame (120 nucleotides) of unknown significance preceded the receptor-coding sequence . Examination of the amino acid sequence of the receptor purified from the outer membrane revealed that the gene product was processed by removal of a signal peptide and that the mature form had an amino acid sequence near its amino terminus which closely resembled that of several other TonB-dependent proteins.

Clin Immunol Immunopathol, 1987 Dec, 45(3), 348 - 55
Production and clearance of tumor necrosis factor in rats exposed to endotoxin and dexamethasone; Waage A; The production and clearance of tumor necrosis factor (TNF) in relation to endotoxinemia was studied by the injection of lipopolysaccharide (LPS) in rats . TNF was released into the circulation as a burst when the serum concentration of LPS was rapidly or gradually increased . The maximum concentration of TNF in serum was attained 60 to 90 min after the injection of LPS . TNF was eliminated from the serum according to a first-order kinetics; the half-life was calculated to be 27 +/- 7 min . No additional release of TNF could be evoked by a persistent high level of LPS . When two LPS injections were given within 3 days, the peak concentration of TNF detected after the second injection was 15% of the concentration detected after the first injection . The results indicate that if TNF is a mediator of septic shock, its role is restricted to the initial phase after the appearance of endotoxin in the circulation . Treatment of the rats with dexamethasone (0.5-2.0 micrograms/g) reduced the LPS-induced peak concentrations of TNF in serum by 70-90% . Maximal suppression of the TNF release was observed when dexamethasone was given 5 hr or more prior to LPS, but was gradually lost at shorter intervals.

Blood, 1987 Dec, 70(6), 1836 - 41
Kinetic evaluation of the pool sizes and proliferative response of neutrophils in bacterially challenged aging mice; Rothstein G et al.; Clinical observations during infection suggest that in aged patients, the kinetic or proliferative responses of neutrophils to infection may be deranged . To test this hypothesis, the neutrophil responses of 6-month-old and 30-month-old mice were compared . After intrapulmonary injection of Escherichia coli, young mice exhibited neutrophilia and diminution of the neutrophil storage pool (NSP) by a mean of 6.4 x 10(6) neutrophils/two femurs . This was accompanied by an increase in the pool of CFU-GM from a control value of 1.1 x 10(5) cells/two femurs (range 0.7 to 1.4) to 1.5 x 10(5) (1.1 to 1.9) (P less than .05) and the thymidine suicide (relative proliferative rate) of CFU-GM rose from 27% (19 to 42) to 51% (31 to 61) (P less than .05) . Furthermore, the CFU-GM of infected young mice displayed enhanced differentiation to the neutrophil series . In contrast, old mice exhibited a greater mean diminution of the NSP: 12.8 x 10(6) neutrophils . Also, old mice experienced a reduction in CFU-GM from 2.3 x 10(5) (1.0 to 3.9) (controls) to 1.3 x 10(5) (1.2 to 1.5)/two femurs (P less than .05), a reduction in the proliferation of CFU-GM and reduced differentiation of CFU-GM to neutrophils . These experiments establish that the neutrophil response of infected old mice is disordered, with exaggerated depletion of the NSP and lack of stimulus-driven granulocytopoiesis as reflected by a paradoxical reduction in the number and proliferative rate of precursors . This defect may be compounded by decreased differentiation of precursors to neutrophils.

J Biomol Struct Dyn, 1987 Dec, 5(3), 601 - 14
Conformational peculiarities of polynucleotides with a nonrandom base sequence according to the 1H----3H exchange rate in C8H groups of purinic residues; Lesnik EA et al.; We have determined the 1H----3H exchange rate constants between water and C8H groups of purinic residues of alternating polynucleotides poly(dA-dT).poly(dA-dT), poly(dG-dC).poly(dG-dC) and poly(dA-dC).poly(dG-dT) as well as homopolynucleotides poly(dA).poly(dT) and poly(dG).poly(dC) in aqueous solutions with high-salt concentrations (3 M NaCl and 4-6 M CsF), in water-ethanol (60%) solution and in 0.15 M NaCl at 25 degrees C . The rate constants for adenine (kA) and guanine (kG) of polynucleotides were compared with corresponding constants for E . coli DNA . dGMP nd dAMP at the same conditions . The relation between exchange rates and conformations of polynucleotides permits the study of their conformational peculiarities in solution . Of three alternating polynucleotides examined in 0.15 M NaCl the exchange retardation was observed only for poly(dA-dT).poly(dA-dT) as compared with that in B-DNA, which is in good agreement with the B-alternating "wrinkled" DNA model . The conformations of poly(dG-dC).poly(dG-dC) and poly(dA-dC).poly(dG-dT), according to the exchange data obtained are within the B form . For homopolynucleotides in 0.15 M NaCl, the KA value for poly(dA).poly(dT) is nearly the same as kA for B-DNA, which indicates the similarity of their conformations, whereas the kG value for poly(dG).poly(dC) is 1.7-fold lower in comparison with the kG value in B-DNA . This seems to be connected with the existence of B = A conformation equilibrium for poly(dG).poly(dC) in solution . The increase of NaCl concentration to 3 M results in a B----Z transition in the case of poly(dG-dC).poly(dG-dC) and in the shift of B = A equilibrium towards the A-form in the case of poly(dG).poly(dC) as is evidenced by alterations of their KG values . Poly(dA-dT).poly(dA-dT) in 6 M CsF and poly(dA-dC).poly(dG-dT) in 4.3 M CsF maintain their inherent conformations in 0.15 M NaCl in spite of the fact that they are characterised by the "X-type" CD-spectrum at these conditions . According to the exchange data the conformation of poly(dA).poly(dT) in 6 M CsF corresponds to the "heteronomous" DNA model or some other structure with lower accessibility of C8H groups of adenylic residues.

Biochemistry, 1987 Dec 1, 26(24), 7732 - 7
NADH-ubiquinone oxidoreductases of the Escherichia coli aerobic respiratory chain; Matsushita K et al.; Deamino-NADH/ubiquinone 1 oxidoreductase activity in membrane preparations from Escherichia coli GR19N is 20-50% of NADH/ubiquinone 1 oxidoreductase activity . In comparison, membranes from E . coli IY91, which contain amplified levels of NADH dehydrogenase, exhibit about 100-fold higher NADH/ubiquinone 1 reductase activity but about 20-fold less deamino-NADH/ubiquinone 1 reductase activity . Deamino-NADH/ubiquinone 1 reductase is more sensitive than NADH/ubiquinone 1 reductase activity to inhibition by 3-undecyl-2-hydroxyl-1,4-naphthoquinone, piericidin A, or myxothiazol . Furthermore, GR19N membranes exhibit two apparent Kms for NADH but only one for deamino-NADH . Inside-out membrane vesicles from E . coli GR19N generate a H+ electrochemical gradient (interior positive and acid) during electron transfer from deamino-NADH to ubiquinone 1 that is large and stable relative to that observed with NADH as substrate . Generation of the H+ electrochemical gradient in the presence of deamino-NADH is inhibited by 3-undecyl-2-hydroxy-1,4-naphthoquinone and is not observed in IY91 membrane vesicles or in vesicles from GR19N that are deficient in deamino-NADH/ubiquinone 1 reductase activity . The data provide a strong indication that the E . coli aerobic respiratory chain contains two species of NADH dehydrogenases: (i) an enzyme (NADH dh I) that reacts with deamino-NADH or NADH whose turnover leads to generation of a H+ electrochemical gradient at a site between the primary dehydrogenase and ubiquinone and (ii) an enzyme (NADH dh II) that reacts with NADH exclusively whose turnover does not lead to generation of a H+ electrochemical gradient between the primary dehydrogenase and ubiquinone 1.

Infect Immun, 1987 Dec, 55(12), 3149 - 54
Immunological activity of a 14-kilodalton recombinant protein of Mycobacterium tuberculosis H37Rv; Kingston AE et al.; A 14-kilodalton peptide antigen from Mycobacterium tuberculosis was isolated from an Escherichia coli lambda gt 11 recombinant DNA clone and was identified by Western blotting (immunoblotting) with monoclonal antibody TB68 . Immunization of mice and guinea pigs with the recombinant peptide (rTB68) induced in vitro lymphoproliferative responses in draining lymph node lymphocyte cultures as well as in vivo delayed-type hypersensitivity reactions . Moreover, rTB68 was found both to induce and to cross-react with Mycobacterium leprae immune lymphocytes, but did not generate protective effects against live M . leprae challenge in mice . These findings showed that a 14-kilodalton peptide which has been characterized as specific for M . tuberculosis on the basis of B-cell recognition was capable of generating cell-mediated immune responses and moreover contained T-cell epitopes which were cross-reactive with M . leprae antigens.

Zh Mikrobiol Epidemiol Immunobiol, 1987 Dec, (12), 33 - 7
{The structure, morbidity level and the frequency of acute diarrheal diseases in children in the preschool period (longitudinal cohort research)}; Artemov VG et al.; Some new aspects of acute intestinal infections incidence rate have been revealed in longitudinal cohort study, whose results are proposed for use in the system of prophylactic measures . The group of susceptible children frequently suffering from acute intestinal infections is considered to be of particular scientific and practical importance, for it is this group which determines the level of morbidity in these infections.

Mol Gen Genet, 1987 Dec, 210(2), 248 - 55
Efficient RecABC-dependent, homologous recombination between coliphage lambda and plasmids requires a phage ninR region gene; Hollifield WC et al.; A phage lambda gene that gives a 100-fold increase in recombinant frequencies for RecABC pathway-mediated, phage-plasmid homologous recombination (Shen and Huang 1986) maps to ninG (orf 204) of lambda . We call this gene rap, for recombination adept with plasmid . A similar determinant exists in Charon 4A and maps in phi 80-derived sequences, between nin5 and the Rz homology with lambda . The absence of the Rap+ phenotype from certain lambda vectors explains the inefficiency of screening the resulting phage libraries using phage-plasmid homologous recombination . The mapping of rap permits the construction of lambda vectors more suitable for this screening technique.

Proc Natl Acad Sci U S A, 1987 Dec, 84(24), 8814 - 8
Arabinose-induced binding of AraC protein to araI2 activates the araBAD operon promoter; Lee N et al.; The state of Escherichia coli araI DNA occupancy by AraC protein has been found to change from a two-turn to a four-turn occupancy upon the addition of the inducer arabinose . The araI site is separable into two contiguous regions, araI1 and araI2 . araI1 binds both ligand-bound and ligand-free AraC protein, whereas araI2 binds AraC protein in the presence of arabinose only . A mutation in araI and a known mutation in araC led to the loss of araI2 binding, while binding to araI1 was unaffected . Both mutants failed to activate the promoter of the araBAD operon . We propose that araI2 occupancy by AraC protein leads to RNA polymerase recognition of the araBAD promoter and that araI1 acts as a switch mechanism allowing both the repressor and the activator forms of AraC protein to regulate the araBAD promoter.

Eur J Biochem, 1987 Dec 1, 169(2), 359 - 64
Activation with plasmin of tow-chain urokinase-type plasminogen activator derived from single-chain urokinase-type plasminogen activator by treatment with thrombin; Lijnen HR et al.; Thrombin converts single-chain urokinase-type plasminogen activator (scu-PA) to an inactive two-chain derivative (thrombin-derived tcu-PA) by hydrolysis of the Arg-156--Phe-157 peptide bond . In the present study, we show that inactive thrombin-derived tcu-PA (specific activity 1000 IU/mg) can be converted with plasmin to active two-chain urokinase-type plasminogen activator (specific activity 43,000 IU/mg) by hydrolysis of the Lys-158--Ile-159 peptide bond . This conversion follows Michaelis-Menten kinetics with a Michaelis constant Km of 37 microM and a catalytic rate constant k2 of 0.013 s-1 . The catalytic efficiency (k2/Km) for the activation of thrombin-derived tcu-PA by plasmin is about 500-fold lower than that for the conversion of intact scu-PA to tcu-PA . tcu-PA, generated by plasmin treatment of thrombin-derived tcu-PA, has similar properties to tcu-PA obtained by digestion of intact scu-PA with plasmin (plasmin-derived tcu-PA); its plasminogen activating potential and fibrinolytic activity in an in vitro plasma clot lysis system appear to be unaltered . These observations confirm that the structure of the NH2-terminal region of the B chain of u-PA is an important determinant for its enzymatic activity, whereas that of the COOH-terminal region of the A chain is not.

J Bacteriol, 1987 Dec, 169(12), 5880 - 3
Mutational analysis of the lambda int gene: DNA sequence of dominant mutations; Bear SE et al.; We have combined techniques of genetic and physical mapping with rapid DNA sequence analysis to identify the nucleotide change in lambda int mutations . These mutations define two dominant phenotypic classes: (i) recombination that is partially independent of accessory factors, and (ii) inhibition of wild-type Int by missense or nonsense proteins, i.e., negative complementation.

Mol Gen Mikrobiol Virusol, 1987 Dec, (12), 20 - 6
{Identification of protein products of the operon for leukocytosis (lymphocytosis)-stimulating factor from Bordetella pertussis cloned in Escherichia coli}; Rozinov MN et al.; The hybrid plasmid pRH119 was constructed on the basis of vector plasmid pUC19 and shown to carry Bordetella pertussis PT operon in the same transcriptional orientation with the lac-promoter of the vector plasmid . Expression of PT genes in E . coli cells harbouring pRH119 was not registered . Weak expression of PT genes was found by immunoscreening of recombinant clones in situ with antiserum against PT when PT genes were put closer to lac-promoter . 0.95 kb SalGI fragment was deleted from pRH119 . The derivative plasmid pRH122 was digested by SalGI and the ends were polymerized to "blunt" by polIK and ligated . The obtained plasmid pRH122K was deleted for 40 bp in XbaI site by Bal31 deletion . The lysate of E . coli cells harbouring the resulting plasmid pRH134 passed through Sepharose 4B with covalently bound immunoglobulins from antiserum against PT . The eluted protein contains S2 multimers identified by immunoblotting . The experiments with CHO-cells and active mice protection have shown the absence of S2 multimers protectiveness.

Mol Gen Genet, 1987 Dec, 210(3), 523 - 7
Deletion analysis of the promoter region of the Escherichia coli pepN gene, a gene subject in vivo to multiple global controls; Foglino M et al.; The pepN gene codes for aminopeptidase N whose expression is regulated at the transcriptional level by anaerobiosis and phosphate starvation . To define and characterize the functional region of the pepN promoter (pepNp), various promoter fragments were fused to the malPQ operon of Escherichia coli and transferred to the chromosome . The expression of the single copy operon fusion was measured by assaying the amylomaltase activity . Sequences upstream from the canonical promoter elements, located 110 to 60 bp preceding the transcription start site, are important for promoter functioning . This region plays a role in the expression of the two divergent promoters pepNp and pncBp . The regulation of pepNp under phosphate starvation was conserved in the various constructs in which pepNp is functional . However, no particular sequence specific for phosphate regulation was detected . In addition, the regulation of pncBp by Pi starvation was demonstrated.

EMBO J, 1987 Dec 1, 6(12), 3855 - 61
DCCD inhibits protein translocation into plasma membrane vesicles from Escherichia coli at two different steps; Muller M et al.; In vitro translocation of periplasmic and outer membrane proteins into inverted plasma membrane vesicles from Escherichia coli was completely prevented by the H+-ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) . DCCD was inhibitory to both co- and post-translational translocations, suggesting an involvement of the H+-translocating F1F0-ATPase in either mode of transport . This was verified by (i) the dependence of efficient co-translational translocation upon a low salt, i.e . F1-containing extract from membrane vesicles; (ii) the co-purification of the translocation activity present in this extract and F1-ATPase; (iii) the inability of either vesicles or their low-salt extract, derived from F1F0-ATPase-lacking mutant strains, to support translocation; and (iv) the greatly diminished extent of ATP-dependent, post-translational translocation into F1-deprived vesicles . Membranes devoid of F1 did show, however, residual translocation activity that was also found to be inhibitable by DCCD . These results suggest a dual target for DCCD in bacterial protein export, one being the H+-ATPase and the other an as yet unidentified translocation factor.

Arch Biochem Biophys, 1987 Dec, 259(2), 423 - 30
Enhanced degradation of oxidized glutamine synthetase in vitro and after microinjection into hepatoma cells; Rivett AJ et al.; Mixed-function oxidation of Escherichia coli glutamine synthetase has previously been suggested to mark the enzyme for intracellular degradation, and in vitro studies have demonstrated that oxidation renders the enzyme susceptible to proteolytic attack . In this study, the susceptibility of glutamine synthetase to degradation by purified proteases has been compared with the rate of degradation after microinjection into hepatoma cells . Upon exposure to an ascorbate mixed-function oxidation system the enzyme rapidly loses most of its activity, but further oxidation is required to cause susceptibility to extensive proteolytic attack either by a high-molecular-weight liver cysteine proteinase or by trypsin . The rate of degradation of biosynthetically 14C-labeled native and oxidized glutamine synthetase preparations after injection into hepatoma cells parallels their susceptibility to proteolysis in vitro . Native enzyme preparations and enzyme oxidatively inactivated, but not susceptible to extensive degradation by purified proteases, had similar intracellular half-lives; however, oxidized enzyme preparations that were susceptible to proteolytic breakdown in vitro were degraded almost ten times faster than the native enzyme within the growing hepatoma cells . These results suggest that the same features of the oxidized enzyme that render it susceptible to proteolysis in vitro are also recognized by the intracellular degradation system . In addition, they show that loss of enzyme activity does not necessarily imply decreased metabolic stability.

Am J Physiol, 1987 Dec, 253(6 Pt 1), G775 - 80
T84 cell receptor binding and guanyl cyclase activation by Escherichia coli heat-stable toxin; Guarino A et al.; Escherichia coli heat-stable enterotoxin (STa) induces intestinal secretion by binding to enterocyte receptors and activating the guanylate cyclase-guanosine 3',5'-cyclic monophosphate (cGMP) system . The intermediate steps between binding of STa and secretion are poorly understood, due in part to the lack of a convenient system to study the effects of STa at the cellular level . To establish such a model, we investigated the binding of 125I-STa, STa activation of guanylate cyclase, and STa-induced increase in cGMP production in a well-characterized human colonic cell line, T84 . Binding was specific, linear with cell number, and time, temperature and pH dependent, and reversible . ST may also be internalized by these cells . Addition of unlabeled STa competitively inhibited binding of 125I-STa . These parameters closely resemble those described in intact rat enterocytes and cell-free membrane preparations . STa stimulated guanylate cyclase and cGMP production in a dose-related manner . The similar dose-response relationships for binding, guanylate cyclase stimulation by STa, and cGMP production suggest that the guanylate cyclase-cGMP system is coupled to ST occupancy of specific receptors . These data, together with the fact that STa induces chloride secretion from T84 cells suggest that T84 cells are a suitable and convenient system to study the cellular mechanism of action of STa.

J Bacteriol, 1987 Dec, 169(12), 5610 - 4
Cloning of genes that suppress an Escherichia coli K-12 alanine auxotroph when present in multicopy plasmids; Wang MD et al.; To facilitate molecular analyses of a previously uncharacterized gene involved in alanine synthesis, attempts were made to clone the wild-type allele of this gene, alaA, with a mini-Mu plasmid element used for in vivo cloning . Seventy-six independent Ala+ plasmids were isolated and characterized . Physiological, enzymological, and restriction endonuclease analyses indicated that three different genes, none of them alaA, were cloned . These genes were avtA+, which encodes the alanine-valine transaminase (transaminase C); tyrB+, which encodes the tyrosine-repressible transaminase (transaminase D); and a previously undescribed gene, called alaB, which encodes an alanine-glutamate transaminase.

J Bacteriol, 1987 Dec, 169(12), 5530 - 6
Identification of two ancillary subunits of Escherichia coli type 1 fimbriae by using antibodies against synthetic oligopeptides of fim gene products; Abraham SN et al.; We have chemically synthesized oligopeptides corresponding to the NH2-terminal stretch of two gene products, designated FimG and FimH, of the fim gene cluster of Escherichia coli . These synthetic peptides, designated S-T1FimG(1-16) and S-T1FimH(1-25)C, evoked antibodies in rabbits that reacted with 14- and 29-kilodalton subunits, respectively, of dissociated fimbriae encoded by the recombinant plasmid pSH2 carrying the genetic information for the synthesis and expression of functional type 1 fimbriae . Neither of these fimbrial proteins was detected in dissociated fimbrial preparations from nonadhesive E . coli cells carrying the mutant plasmid pUT2002, containing a restriction site-specific deletion of fimG and fimH . Anti-S-T1FimH(1-25)C inhibited the adherence of type 1 fimbriated E . coli to epithelial cells . Immunoelectron microscopy revealed that anti-S-T1FimH(1-25)C, but not anti-S-T1FimG(1-16), bound to intact type 1 fimbriae of E . coli at the fimbrial tips and at long intervals along the fimbrial filaments . Anti-S-T1FimG(1-16) appeared to be directed at epitopes not accessible on the intact fimbriae and consequently failed to bind to intact fimbriae or to block fimbrial attachment . Our results suggest that the fimG and fimH gene products are components of type 1 fimbriae and that FimH may be the tip adhesin mediating the binding of type 1 fimbriated E . coli to D-mannose residues on mucosal surfaces.

Infect Immun, 1987 Dec, 55(12), 3168 - 73
Molecular cloning of the Escherichia coli O75X adhesin; Nowicki B et al.; The uropathogenic strain Escherichia coli IH11128 (O75:K5:H-) exhibits a mannose-resistant O75X adhesin . The genes encoding the O75X adhesin were cloned from a clinical strain and transferred to E . coli K-12 derivatives . The recombinant plasmids were found to express a 15-kilodalton fimbrial subunit protein, a fimbrialike extracellular structure, and mannose-resistant hemagglutination . An indirect immunofluorescence assay was used to study attachment of the clone and purified adhesin to frozen sections of human kidney . The clone bound selectively to the interstitial areas and notably to Bowman's capsule . The purified adhesin bound to the basement membrane of the tubules and to Bowman's capsule.

Infect Immun, 1987 Dec, 55(12), 3111 - 6
In vivo emergence of enterotoxigenic Escherichia coli variants lacking genes for K99 fimbriae and heat-stable enterotoxin; Mainil JG et al.; Neonatal pigs were inoculated with porcine enterotoxigenic Escherichia coli 431, which carries genes for K99 fimbriae and STaP enterotoxin . Colonies of strain 431 were recovered from feces of pigs for up to 17 days after inoculation and tested for hybridization with gene probes for K99 and STaP . Variants of strain 431 that did not hybridize with the probes were considered to have lost the genes . Variants were recovered from 10 of 13 suckling pigs that survived the infection . Only 0.4% of the isolates recovered during the first 2 days after inoculation were variants . Of the isolates recovered 3 to 5 days after inoculation, 20 to 36% were variants . Variant colonies were detected more frequently among pigs in some litters than in others . The litter with the highest number of variant-shedding pigs had the dam with the highest titer of K99 antibody in her colostrum . Variants also occurred in colostrum-deprived, artificially reared pigs . However, the number of variants detected was lower and they occurred later in the course of the infection in colostrum-deprived pigs than in suckling pigs . More variants were detected and they were detected earlier in colostrum-deprived pigs fed anti-K99 monoclonal antibody than in controls fed anti-K88 monoclonal antibody . Loss of STaP appeared to be secondary to loss of K99 in that some variants lacked only K99 (K99- STaP+) and some lacked both genes (K99- STaP-), but none was of the K99+ STaP- type . Our results confirmed reports of gene loss from enterotoxigenic E . coli during infection . They are consistent with the hypothesis that variants emerge under in vivo selection pressure of K99 antibody and with the speculation that gene loss may be an important component of protection in vaccinated populations . However, the emergence of variants did not appear to play a major role in the recovery of individual pigs from clinical disease.

Microbiol Sci, 1987 Dec, 4(12), 371 - 5
Positive regulation of gene expression by cyclic AMP and its receptor protein in Escherichia coli; Busby S et al.; The role of cyclic AMP in the modulation of gene expression in Escherichia coli is briefly reviewed . Recent results concerning the molecular mechanism by which cyclic AMP and its receptor protein activate transcription initiation are discussed.

J Gen Microbiol, 1987 Dec, 133 ( Pt 12), 3279 - 88
Regulation and over-expression of the fnr gene of Escherichia coli; Spiro S et al.; The fnr gene of Escherichia coli encodes a transcriptional activator (FNR) which is required for the expression of a number of genes involved in anaerobic respiratory pathways . From the study of a translational fusion of fnr to the gene for beta-galactosidase (lacZ) it has been concluded that the fnr gene is expressed under both aerobic and anaerobic conditions and is subject to autoregulation and repression by glucose, particularly during anaerobic growth . These findings imply that during anaerobiosis the FNR protein adopts an active conformation, in which it functions both as a repressor of the fnr gene and as an activator of fnr-dependent genes . Sequences in the 5' non-coding region of fnr which could be involved in autoregulation are discussed . The fnr coding region was cloned into an expression vector which has allowed an amplification of FNR synthesis such that it accounts for about 2% of total cell protein . The ability to over-produce FNR in this way should be very useful for future biochemical studies.

Bioorg Khim, 1987 Dec, 13(12), 1681 - 2
{Inorganic pyrophosphatase--a new enzyme label for biochemical analysis}; Baikov AA et al.; Inorganic pyrophosphatase isolated from Escherichia coli has been proposed as a label in heterogeneous enzyme immunoassays . The enzyme is remarkably stable and insensitive to sodium azide . Enzyme-antibody conjugates were prepared with glutaraldehyde and purified by gel filtration . Enzyme activity was measured by means of a sensitive colour reaction between phosphomolybdate and malachite green . A 5-10-fold increase is sensitivity in terms of absorbance readings was observed compared to peroxidase-based assays . The colour change (yellow/greenish blue) inherent in the use of pyrophosphatase as the labelling agent is highly suitable for visual analysis.

J Biochem (Tokyo), 1987 Dec, 102(6), 1555 - 63
Molecular cloning and expression in Escherichia coli of a cDNA encoding human pancreatic elastase 2; Shirasu Y et al.; We have cloned a DNA that is complementary to the messenger RNA that encodes human pancreatic elastase 2 from a human pancreatic cDNA library using a cloned cDNA for rat pancreatic elastase 2 messenger RNA . This complementary DNA contains the entire protein coding region of 807 nucleotides which encodes preproelastase of 269 amino acids, and 4 and 82 nucleotides of the 5'- and 3'-untranslated sequences, respectively . When this deduced amino acid sequence was compared with known amino acid sequences it showed 82% homology with rat pancreatic elastase 2 . This deduced sequence also contains a 16-amino-acid peptide identical with the N-terminal sequence determined for native human pancreatic proelastase 2 . Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 241 amino acids including 16 and 12 amino acids for a signal peptide and an activation peptide, respectively . Moreover, the predicted key amino acid residues involved in determining the substrate specificity of mammalian pancreatic elastase 2 are retained in the human enzyme . Cloned human pancreatic elastase 2 cDNA was expressed in E . coli as a mature and pro-form protein . Both resulting proteins showed immunoreactivity toward anti-elastase serum and enzymatic activity . We have also cloned and sequenced a porcine pancreatic elastase 2 cDNA.

Mol Gen Mikrobiol Virusol, 1987 Dec, (12), 11 - 6
{Construction of derivatives of the diphtheria toxin gene and their expression in Escherichia coli cells}; Iakubovich NV et al.; A fragment of diphtheria toxin (tox) gene from beta 45 phage DNA was cloned on pUC19 plasmid in E . coli cells . The fragment is coding for toxA fragment of the toxin and contains the control region of the tox gene . The tox gene promoter is active in E . coli . The toxA protein is found mainly in periplasm of E . coli cells . The protein is enzymatically active in ADP-ribosilation of elongation factor 2 from eucaryotic cells . An in frame toxA-lacZ' fusion was constructed on pUC8 plasmid . The hybrid protein expresses both toxA and lacZ' activities . Two or seven base pairs were deleted from the central part of toxA gene by means of S1 nuclease digestion . Translation of hybrid toxA-lacZ' mRNA should be terminated downward the delections due to the frameshifts caused by them . Nevertheless, a functionally active alpha-peptide of beta-galactosidase is expressed by both the deletion fusions . The existence of another translational start site functioning in E . coli and located inside 3'-end region of toxA mRNA is suggested.

Genetika, 1987 Dec, 23(12), 2120 - 7
{Genetic mapping of transposon Tn5-induced mutation blocking threonine dehydrogenase in Escherichia coli K-12}; Shakalis IO et al.; The paper deals with a mutant of Escherichia coli K-12 obtained by transposon Tn5 mutagenesis . Insertion of this transposon inactivated the gene for L-threonine dehydrogenase catalysing the first step of L-threonine degradation . The insertion of Tn5 was mapped by using conjugation as well as transduction by T4GT7 and P1 . It is located at 81 min of the E . coli genetic map between mtl and pyrE genes.

Mol Gen Genet, 1987 Dec, 210(2), 262 - 9
Integrative suppression of dnaA(Ts) mutations mediated by plasmid F in Escherichia coli is a DnaA-dependent process; Kogoma T et al.; The thermosensitivity of dnaA(Ts) mutations can be suppressed by integration of plasmid F (integrative suppression) . In the light of the recent finding that F requires DnaA protein for both establishment and maintenance, integrative suppression of 11 dnaA(Ts) mutations by a mini-F, pML31, integrated near oriC was examined . The plating efficiency of integratively suppressed strains was dnaA(Ts) allele-dependent and medium-dependent . The initiation capability of suppressed dnaA(Ts) strains lacking the oriC site and their F- counterparts was determined at various temperatures between 30 degrees C and 42 degrees C . The degree of integrative suppression measured by the initiation capability varied in a dnaA(Ts) allele-dependent manner . F-directed DNA replication was most affected by the dnaA(Ts) mutations mapping in the middle of the gene whereas oriC-dependent replication was most thermosensitive in strains carrying mutations mapping in the carboxy-terminal half of the gene . The results indicated that the integrative suppression by F plasmid is a DnaA-dependent process and suggested that the requirements for DnaA protein in the oriC-dependent replication and F replication processes are qualitatively different.

J Appl Physiol, 1987 Dec, 63(6), 2426 - 32
Dimethylthiourea attenuates endotoxin-induced acute respiratory failure in pigs; Olson NC et al.; We hypothesized that toxic O2 radicals might be important mediators of endotoxin-induced acute respiratory failure in pigs . As a relatively specific scavenger of .OH, we infused dimethylthiourea (DMTU, 1 g/kg) before endotoxemia . Escherichia coli endotoxin (055-B5) was infused intravenously into anesthetized 10- to 14-wk-old pigs at 5 micrograms/kg the 1st h, followed by 2 micrograms.kg-1.h-1 for 3.5 h . During phase 1 (i.e., 0-2 h) and phase 2 (i.e., 2-4.5 h), endotoxin decreased cardiac index (CI) and increased mean pulmonary arterial pressure (Ppa), pulmonary vascular resistance (PVR), alveolar-arterial O2 gradient (AaDo2), and hematocrit (Hct) . Endotoxemia also caused leukopenia and increased the postmortem bronchoalveolar lavage fluid (BALF) albumin concentration and wet weight-to-dry weight ratio of bloodless lung . Dimethylthiourea did not significantly modify the phase 1 response . However, during phase 2, DMTU attenuated the endotoxin-induced decrease in CI and increases in Ppa, PVR, Hct, AaDo2, lung water, and BALF albumin concentration . In separate groups of endotoxin- and DMTU + endotoxin-treated pigs, lung microvascular hydrostatic pressure was increased to approximately 16 Torr (by fluid overload) to assess alveolar-capillary membrane permeability . Under these conditions, DMTU markedly attenuated the endotoxin-induced increase in alveolar-capillary membrane permeability . Under these conditions, DMTU markedly attenuated the endotoxin-induced induced increase in alveolar-capillary membrane permeability . We conclude that .OH (and possibly H2O2) significantly contributes to endotoxin-induced lung injury in anesthetized pigs.

Biochem J, 1987 Dec 1, 248(2), 495 - 500
Proton-linked L-fucose transport in Escherichia coli; Bradley SA et al.; 1 . Addition of L-fucose to energy-depleted anaerobic suspensions of Escherichia coli elicited an uncoupler-sensitive alkaline pH change diagnostic of L-fucose/H+ symport activity . 2 . L-Galactose or D-arabinose were also substrates, but not inducers, for the L-fucose/H+ symporter . 3 . L-Fucose transport into subcellular vesicles was dependent upon respiration, displayed a pH optimum of about 5.5, and was inhibited by protonophores and ionophores . 4 . These results showed that L-fucose transport into E . coli was energized by the transmembrane electrochemical gradient of protons . 5 . Neither steady state kinetic measurements nor assays of L-fucose binding to periplasmic proteins revealed the existence of a second L-fucose transport system.

Vaccine, 1987 Dec, 5(4), 270 - 4
Stimulation of non-specific host resistance against Sendai virus and Escherichia coli infections by chitin derivatives in mice; Iida J et al.; The efficacy of chitin derivatives on non-specific host resistance to Sendai virus and Escherichia coli infections was studied in mice . Seventy percent deacetylated chitin (DAC-70) and N-trimethylated DAC-70 {DAC-70(Me)3} showed protective activity against Sendai virus infection; however, carboxymethyl-chitin (CM-chitin) did not . DAC-70 also showed protective activity against E . coli infection.

Proc Natl Acad Sci U S A, 1987 Dec, 84(24), 9123 - 7
Detection in situ of genomic regulatory elements in Drosophila; O'Kane CJ et al.; We have developed an approach for the in situ detection of genomic elements that regulate transcription zin Drosophila melanogaster . The approach is analogous to a powerful method of bacterial genetics, the random generation of operon fusions, that enables the isolation and characterization of genes simply by knowing or postulating their pattern of expression; it is not necessary initially to screen for mutant phenotypes . To apply this approach to Drosophila, we have used the expression of the lacZ gene of Escherichia coli from the P-element promoter in germ-line transformant flies to screen for chromosomal elements that can act at a distance to stimulate expression from this apparently weak promoter . Of 49 transformed fly lines obtained, approximately 70% show some type of spatially regulated expression of the lacZ gene in embryos; many of these express lacZ specifically in the nervous system . The P-lacZ fusion gene is, therefore, an efficient tool for the recovery of elements that may regulate gene expression in Drosophila and for the generation of a wide variety of cell-type-specific markers.

Proc Natl Acad Sci U S A, 1987 Dec, 84(24), 9118 - 22
Identification of base pairs in the outside end of insertion sequence IS50 that are needed for IS50 and Tn5 transposition; Phadnis SH et al.; Short DNA sequences at ends of transposable elements are needed as sites for transposition . Previous deletion mapping showed that, in Tn5 and its component IS50 elements, these essential sites are about 19 base pairs long . To determine which positions are important in transposition, we made one or more sequence changes at each position in the IS50 outside (O) end and assayed the effects of these changes on transposition . Our results indicate that the specific base pairs at 18 of the 19 positions are important in transposition . A 9-base-pair segment in the O end corresponds to a binding site for the Escherichia coli DnaA protein . Comparisons of effects of mutations at different positions in this site, and also measurements of Tn5 transposition in dnaA- cells, indicate that DnaA protein participates in O-end-mediated transposition.

Proc Natl Acad Sci U S A, 1987 Dec, 84(24), 9108 - 12
Site-specific insertion of DNA into a pseudorabies virus vector; Sauer B et al.; A simple, efficient method for introducing recombinant DNA into a herpesvirus vector and retrieving it at a later time has been developed . By using the Cre-lox site-specific recombination system of coliphage P1, DNA can be readily inserted in vitro into a pseudorabies virus (PRV) vector containing the lox recombination site . The vector PRV42 contains a lox site within the nonessential gIII gene, which encodes a virion envelope glycoprotein . Incubation in vitro of PRV42 DNA with Cre protein and a circular plasmid containing a lox site generates approximately 5% recombinant molecules having the plasmid integrated into the PRV genome at the lox site . Transfection of the reaction mixture into cultured cells allows recovery of the infectious recombinant virus, which is readily identified by a nondestructive "black-plaque assay" using a gIII-specific monoclonal antibody . PRV42 plaques stain black when treated with the gIII monoclonal antibody and a peroxidase-linked second anti-antibody because the lox site placed within the gIII gene of PRV42 does not destroy the gIII epitope . However, Cre-mediated integration of heterologous DNA at the lox site disrupts the gIII epitope so that the resulting recombinant virus produces white plaques . The recombinant virus is infectious, stable, and grows as well as the parental PRV42 vector . The inserted plasmid can be efficiently excised (greater than 50%) from viral DNA by Cre and recovered by transformation of Escherichia coli.

Proc Natl Acad Sci U S A, 1987 Dec, 84(24), 8971 - 5
Expression of yeast DNA topoisomerase I can complement a conditional-lethal DNA topoisomerase I mutation in Escherichia coli; Bjornsti MA et al.; We show that, despite differences in primary structure, substrate preference, and mechanism of catalysis, yeast DNA topoisomerase I can functionally substitute for Escherichia coli DNA topoisomerase I . A family of plasmids expressing the yeast TOP1 gene or 5'-deletion mutations of it were used to complement the temperature-sensitive phenotype of an E . coli topA mutant . These plasmids were then isolated from the cells by a rapid lysis procedure and examined for their degrees of supercoiling . Functional complementation of a conditional-lethal mutation in topA, which encodes E . coli DNA topoisomerase I, correlates with the expression of a catalytically active yeast enzyme that reduces the degree of negative supercoiling of intracellular DNA . We also show that approximately 130 amino acids of the amino-terminal portion of the yeast enzyme can be deleted without affecting its activity in vitro; activity of the enzyme inside E . coli, however, is more sensitive to such deletions.

Proc Natl Acad Sci U S A, 1987 Dec, 84(24), 8907 - 11
Importance of disulfide bridges in the structure and activity of Escherichia coli enterotoxin ST1b; Gariepy J et al.; A 13-amino acid sequence of the Escherichia coli heat-stable enterotoxin ST1b encodes its receptor-binding and diarrheal functions . This sequence includes six cysteines involved in three intramolecular disulfide bridges . To determine the importance of disulfide bridges to the biological activity of ST1b, we synthesized 15 analogues of the tridecapeptide representing all possible replacements of two of the six cysteines by alanines . Only 2 analogues--namely, A6,11ST1b-(6-18) and A10,18ST1b(6-18)--could inhibit the binding of a radiolabeled analogue of ST1b to rat intestinal cells . The purified peptides were, respectively, 4200 and 130 times less effective as inhibitors than ST1b(6-18), the sequence that includes all six cysteines . In addition, both peptides produce diarrhea when given orally to suckling mice . These analogues share in common only two cysteines (Cys-7 and Cys-15), suggesting that four cysteines, two of which are Cys-7 and Cys-15, are necessary for activity . A pattern of disulfide linkages is proposed where Cys-7 is paired to Cys-15, Cys-6 to Cys-11, and Cys-10 to Cys-18, the preceding disulfide bridges being ranked in descending order of importance in terms of their respective contribution to the activity of the enterotoxin . Using this disulfide bridge arrangement and constraints derived from NMR spectroscopy, we propose a folding pattern for the toxic domain of ST1b.

Virology, 1987 Dec, 161(2), 555 - 60
Synthesis in Escherichia coli of the HTLV-I trans-acting protein p40x; Sanchez A et al.; The pX gene of human T-cell leukemia virus type I (HTLV-I) encodes a protein that activates the expression of viral genes in trans . Plasmid constructs designed to express the pX gene under the control of either the temperature-inducible lambda PL promoter or the trp promoter were used to transform several Escherichia coli strains, including murein-lipoprotein and Ion mutant strains . Upon induction it was possible to detect the synthesis of a new polypeptide of approximately 40 kDa which reacted specifically with serum from an ATL patient.

Virology, 1987 Dec, 161(2), 348 - 56
Formation of poliovirus RNA polymerase 3D in Escherichia coli by cleavage of fusion proteins expressed from cloned viral cDNA; Richards OC et al.; The poliovirus polymerase 3D was synthesized in Escherichia coli by cleavage of fusion proteins expressed from cloned viral cDNA inserted into several plasmid expression vectors . Cleavage was accomplished by the action of viral protease 3C sequences expressed in the same bacteria, either from a second plasmid or from the same plasmid, cloned so as to produce contiguous sequences in the same protein . In the case of two plasmids, protease 3C functioned in trans to cleave the fusion protein at or very near the normal Gln/Gly cleavage site . When protease and polymerase sequences were produced in the same protein, the protease sequences acted in the precursor form to release the polymerase from itself . Thus, cleavage can occur to generate polymerase 3D both as an intermolecular reaction and, very likely, also as an intramolecular event.

Proc Natl Acad Sci U S A, 1987 Dec, 84(23), 8623 - 7
Human aldolase A deficiency associated with a hemolytic anemia: thermolabile aldolase due to a single base mutation; Kishi H et al.; Fructose-1,6-bisphosphate aldolase A (fructose-bisphosphate aldolase; EC 4.1.2.13) deficiency is an autosomal recessive disorder associated with hereditary hemolytic anemia . To clarify the molecular mechanism of the deficiency at the nucleotide level, we have cloned aldolase A cDNA from a patient's poly(A)+ RNA that was expressed in cultured lymphoblastoid cells . Nucleotide analysis of the patient's aldolase A cDNA showed a substitution of a single nucleotide (adenine to guanine) at position 386 in a coding region . As a result, the 128th amino acid, aspartic acid, was replaced with glycine (GAT to GGT) . Furthermore, change of the second letter of the aspartic acid codon extinguished a F ok I restriction site (GGATG to GGGTG) . Southern blot analysis of the genomic DNA showed the patient carried a homozygous mutation inherited from his parents . When compared with normal human aldolase A, the patient's enzyme from erythrocytes and from cultured lymphoblastoid cells was found to be highly thermolabile, suggesting that this mutation causes a functional defect of the enzyme . To further examine this possibility, the thermal stability of aldolase A of the patient and of a normal control, expressed in Escherichia coli using expression plasmids, was determined . The results of E . coli expression of the mutated aldolase A enzyme confirmed the thermolabile nature of the abnormal enzyme . The Asp-128 is conserved in aldolase A, B, and C of eukaryotes, including an insect, Drosophila, suggesting that the Asp-128 of the aldolase A protein is likely to be an amino acid residue with a crucial role in maintaining the correct spatial structure or in performing the catalytic function of the enzyme.

Proc Natl Acad Sci U S A, 1987 Dec, 84(23), 8345 - 9
Escherichia coli replication factor Y, a component of the primosome, can act as a DNA helicase; Lee MS et al.; The primosome is a mobile multienzyme DNA replication-priming complex that requires seven Escherichia coli proteins for assembly (the products of the dnaB, dnaC, dnaG, and dnaT genes as well as proteins n and n" and replication factor Y) . It has been shown previously that the primosome, in combination with the E . coli DNA polymerase III holoenzyme, can form replication forks in vitro that move at rates similar to those measured in vivo and that the primosome and one of the components of the primosome, the DNA B protein, have DNA helicase activity . Evidence is presented here that another component of the primosome, replication factor Y, possesses DNA helicase activity as well . Factor Y helicase activity requires the presence of E . coli single-stranded DNA binding protein, Mg2+, and hydrolyzable ATP or dATP . Helicase activity is stimulated 15-fold when the enzyme is actively loaded onto single-stranded DNA through a primosome assembly site, and duplex DNA is unwound unidirectionally, 3'----5', along the DNA strand to which the protein is bound.

Proc Natl Acad Sci U S A, 1987 Dec, 84(23), 8315 - 9
Mutants of the catabolite activator protein of Escherichia coli that are specifically deficient in the gene-activation function; Irwin N et al.; In the presence of cyclic AMP, the catabolite activator protein (CAP) of Escherichia coli binds DNA and stimulates transcription at a number of promoters . We have examined a model of CAP bound at the gal promoter and, using directed mutagenesis, have isolated CAP mutants that are analogous to the lambda repressor positive control (pc) mutants . These CAP mutants bind DNA but are defective in stimulating transcription at the gal P1 promoter . These mutants are also altered in positive control at the lac and malT promoters, where CAP binds to sites further upstream from the transcription start site.

Proc Natl Acad Sci U S A, 1987 Dec, 84(23), 8311 - 4
Elevated glucose 6-phosphate levels are associated with plasmid mutations in vivo; Lee AT et al.; The incubation in vitro of plasmid pBR322 DNA with glucose 6-phosphate (Glc-6-P) has been shown to have a mutagenic effect when the plasmid was transformed into wild-type Escherichia coli . To further investigate the modifications of DNA by the reducing sugar Glc-6-P, we have developed an in vivo model to monitor plasmid DNA mutations . E . coli strains that are defective for phosphoglucose isomerase (strain DF40) alone or phosphoglucose isomerase and glucose-6-phosphate dehydrogenase (strain DF2000) accumulate Glc-6-P when grown in gluconate minimal medium in the presence of glucose . These strains and the control strain K10 were transformed with pAM006, a plasmid that carries the genes for ampicillin resistance and beta-galactosidase production, and grown for 24 hr under conditions that prompted the accumulation of Glc-6-P . An increase in plasmid mutations was observed (7- and 13-fold) that was associated with the increased intracellular levels of Glc-6-P (20- and 30-fold) present in the DF40 and DF2000 E . coli strains, respectively . Growth of the mutant bacteria in gluconate minimal medium does not increase the intracellular levels of Glc-6-P or the rate of plasmid mutations over background . Further characterization of the mutated plasmid DNA showed that insertions, deletions, and point mutations were responsible for the loss of beta-galactosidase production . The increase in plasmid mutations as a function of increased intracellular Glc-6-P levels suggests that the accumulation of adducts formed by Glc-6-P and other reducing sugars may contribute to DNA damage.

Proc Natl Acad Sci U S A, 1987 Dec, 84(23), 8220 - 4
Specific binding of transposase to terminal inverted repeats of transposable element Tn3; Ichikawa H et al.; Tn3 transposase, which is required for transposition of Tn3, has been purified by a low-ionic-strength-precipitation method . Using a nitrocellulose filter binding assay, we have shown that transposase binds to any restriction fragment . However, binding of the transposase to specific fragments containing the terminal inverted repeat sequences of Tn3 can be demonstrated by treatment of transposase-DNA complexes with heparin, which effectively removes the transposase bound to the other nonspecific fragments at pH 5-6 . DNase I "footprinting" analysis showed that the transposase protects an inner 25-base-pair region of the 38-base-pair terminal inverted repeat sequence of Tn3 . This protection is not dependent on pH . Interestingly, binding of the transposase to the inverted repeat sequences facilitates DNase I to nick at the end of the Tn3 sequence . It was also observed that the transposase protects the end regions of restriction fragments with a cohesive sequence at their 5' end or with a flush end from DNase I cleavage . The specific and nonspecific binding of transposase to DNA is ATP-independent.

J Bacteriol, 1987 Dec, 169(12), 5795 - 800
Isolation and characterization of temperature-sensitive pantothenate kinase (coaA) mutants of Escherichia coli; Vallari DS et al.; Escherichia coli mutants conditionally defective in the conversion of pantothenate to coenzyme A were isolated and characterized . The gene was designated coaA and localized between argEH and rpoB near min 90 of the chromosome . The coaA15(Ts) mutation caused a temperature-sensitive growth phenotype and temperature-dependent inactivation of pantothenate kinase activity assayed both in vivo and in vitro . At 30 degrees C, coaA15(Ts) extracts contained less than 20% of the wild-type pantothenate kinase activity; the kinase had near normal kinetic constants for the substrates ATP and pantothenate and was inhibited by coenzyme A to the same degree as the wild-type enzyme . These data define the coaA gene as the structural gene for pantothenate kinase.

J Bacteriol, 1987 Dec, 169(12), 5727 - 34
Nucleotide sequence of the Escherichia coli gene for lipid A disaccharide synthase; Crowell DN et al.; The lpxB gene of Escherichia coli, believed to be the structural gene for lipid A disaccharide synthase, is located in the min 4 region of the chromosome . It is adjacent to and clockwise of the lpxA gene, which is thought to encode UDP-N-acetylglucosamine acyltransferase . Preliminary evidence suggests that lpxA and lpxB are cotranscribed in the clockwise direction and thus constitute part of a previously unknown operon (D . N . Crowell, M . S . Anderson, and C . R . H . Raetz, J . Bacteriol . 168:152-159, 1986) . We now report the complete nucleotide sequence of a 1,522-base-pair PvuII-HincII fragment known to carry the lpxB gene . This sequence contained an open reading frame of 1,149 base pairs, in agreement with the predicted size, location, and orientation of lpxB . There was a second open reading frame 5' to, and in the same orientation as, lpxB that corresponded to lpxA . The ochre codon terminating lpxA was shown to overlap the methionine codon identified as the initiation codon for lpxB, suggesting that these genes are cotranscribed and translationally coupled . A third open reading frame was also shown to begin at the 3' end of lpxB with analogous overlap between the opal codon terminating lpxB and the methionine codon that putatively initiates translation downstream of lpxB in the clockwise direction . These results argue that at least three genes constitute a translationally coupled operon in the min 4 region of the E . coli chromosome . The accompanying paper by Tomasiewicz and McHenry (J . Bacteriol . 169:5735-5744, 1987) presents 4.35 kilobases of DNA sequence, beginning at the 3' end of lpxB, and argues that dnaE and several other open reading frames may be members of this operon.

J Bacteriol, 1987 Dec, 169(12), 5569 - 74
The phoBR operon in Escherichia coli K-12; Wanner BL et al.; The phoB and phoR genes encode a transcription activator and a sensory protein of the phosphate regulon, respectively . It is shown here that they were transcribed as an operon in which the phoB gene was promoter proximal . Although an operon structure was suggested previously (K . Makino, H . Shinagawa, M . Amemura, and A . Nakata, J . Mol . Biol . 190:37-44 and 192:549-556, 1986), previous results showed only that phoR gene expression during phosphate limitation is dependent on the upstream phoB promoter . The phoR gene could still have had its own promoter for expression in the presence of phosphate . Two polar transposon-induced mutations are described which simultaneously abolished phoB and phoR gene function in cis; one mutation mapped in the phoB gene, and the other mapped upstream of the phoB gene . These results demonstrate an operon structure, in which phoR gene function required expression from the phoB promoter . Unexpectedly, an antisense pho omega Mu d1(lacZ) insertion within the promoter-proximal end of the phoB gene expressed the lacZ reporter gene, thus allowing for the possibility that the phoBR operon is regulated by an antisense RNA.

J Bacteriol, 1987 Dec, 169(12), 5416 - 22
Regulation of gluconeogenesis by the glucitol enzyme III of the phosphotransferase system in Escherichia coli; Yamada M et al.; The gut operon was subcloned into various plasmid vectors (M . Yamada and M . H . Saier, Jr., J . Bacteriol . 169:2990-2994, 1987) . Constitutive expression of the plasmid-encoded operon prevented utilization of alanine and Krebs cycle intermediates when they were provided as sole sources of carbon for growth . Expression of the gutB gene alone (encoding the glucitol enzyme III), subcloned downstream from either the lactose promoter or the tetracycline resistance promoter, inhibited utilization of the same compounds . On the other hand, overexpression of the gutA gene (encoding the glucitol enzyme II) inhibited the utilization of a variety of sugars as well as alanine and Krebs cycle intermediates by an apparently distinct mechanism . Phosphoenolpyruvate carboxykinase activity was greatly reduced in cells expressing high levels of the cloned gutB gene but was nearly normal in cells expressing high levels of the gutA gene . A chromosomal mutation in the gutR gene, which gave rise to constitutive expression of the chromosomal gut operon, also gave rise to growth inhibition on gluconeogenic substrates as well as reduced phosphoenolpyruvate carboxykinase activity . Phosphoenolpyruvate synthase activity in general varied in parallel with that of phosphoenolpyruvate carboxykinase . These results suggest that high-level expression of the glucitol enzyme III of the phosphotransferase system can negatively regulate gluconeogenesis by repression or inhibition of the two key gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and phosphoenolpyruvate synthase.

J Bacteriol, 1987 Dec, 169(12), 5408 - 15
Sequence analysis, transcriptional organization, and insertional mutagenesis of the envA gene of Escherichia coli; Beall B et al.; The Escherichia coli cell permeability-cell separation gene envA and the region immediately downstream were sequenced . The envA gene consisted of 305 codons which encoded a 34-kilodalton polypeptide that lacked a signal sequence and hydrophobic membrane-spanning regions . The envA1 mutation was determined to be a missense mutation in codon 19 resulting in a change in the amino acid sequence from histidine to tyrosine . Located 299 base pairs downstream of the envA gene was an unidentified open reading frame consisting of 147 codons . This open reading frame was followed by an additional open reading frame starting 59 base pairs further downstream and corresponded to the secA gene . A transcription terminator was located just downstream of envA on a fragment that contained a sequence corresponding to a typical rho-independent terminator . Transcription of envA and the upstream fts genes terminated at this terminator and was probably uncoupled from the downstream genes, including secA . Gene disruption experiments indicated that the envA gene was an essential gene.

J Biochem (Tokyo), 1987 Dec, 102(6), 1565 - 70
Characterization of a novel acidic protein of 38 kDa, A0, in yeast ribosomes which immunologically cross-reacts with the 13 kDa acidic ribosomal proteins, A1/A2; Mitsui K et al.; A new ribosomal protein of 38 kDa, named A0, was detected in yeast ribosomes on immunoblotting . The antibody used here was that against A1/A2, 13 kDa acidic ribosomal proteins which cross-reacted with A0 . Although A0 and A1/A2 share common antigenic determinants, they differ in the following biochemical properties . While A1/A2 could be extracted from ribosomes with ethanol and ammonium sulfate, A0 could not . A0 gave two protein spots in a less acidic region than for A1/A2 on two-dimensional gel electrophoresis . The heterogeneity observed for A0 was ascribable to phosphorylation because one spot disappeared after treatment of the ribosomes with phosphatase . The syntheses of A0 and A1/A2 are directed by different mRNA species, as judged with a cell-free translation system, ruling out the possibility that A0 is a precursor of A1/A2 . Although a mammalian ribosomal protein equivalent to A0 has been shown to be associated with 13 kDa acidic proteins in the cytoplasm, essentially no A0 was detected on immunoblotting in the yeast cytosol, while a small but detectable amount of A1/A2 was present . The possibility that A0 is a eukaryotic equivalent of L10 of Escherichia coli is discussed.

Immunol Cell Biol, 1987 Dec, 65 ( Pt 6), 473 - 82
Responses in mice to Sj26, a glutathione S-transferase of Schistosoma japonicum worms; Davern KM et al.; The genetic variation in antibody responses of mice to glutathione S-transferase (GST) enzymes of Schistosoma japonicum worms, and in particular to a Mr 26,000 species termed Sj26, was analysed . Sera from infected mice, or mice immunized with adjuvant and an Sj26 beta-galactosidase fusion protein produced in Escherichia coli (Sj26FP), or purified near-native recombinant Sj26 produced in E . coli (rSj26), were assayed by enzyme-linked immunosorbent assay (ELISA) for antibody titres to GST purified from adult worms . Anti-GST antibody levels are high in a mouse strain, WEHI 129/J, that is genetically resistant to infection with S . japonicum . Antibody responses to GST are low in BALB/c mice and intermediate in most other mouse strains analysed such as CBA/H and C57B1/6 . Responsiveness to Sj26 in adjuvant is dominant in (BALB/c x WEHI 129/J)F1 hybrids . BALB/c.H-2b and BALB/c.H-2k mice are higher responders than BALB/c . One feature of antibody responses to Sj26 is the variability within a group of mice . When rSj26 conjugated to the hapten azobenzenearsonate was used as immunogen, BALB/c mice produced substantial amounts of anti-Sj26 antibodies . In an attempt to correlate antibody levels with resistance in infected mice, a new functional assay was devised to measure the ability of sera to inhibit the binding of rSj26 to glutathione . However, there was no correlation between inhibitory titre in this assay and the numbers of worms recovered . In regard to the candidacy of GST as a vaccinating antigen in schistosomiasis japonica, the data raise the problem of variable responsiveness to the antigen that will need to be overcome by antigen modification and/or powerful adjuvants.

Gen Comp Endocrinol, 1987 Dec, 68(3), 387 - 99
The primary structure of coho salmon growth hormone and its cDNA; Nicoll CS et al.; Total RNA was extracted from coho salmon growth hormone (sGH) cell regions and used to synthesize double-stranded cDNA, which was inserted into a plasmid vector and used to transform Escherichia coli HB101 . The total RNA was also separated according to size by electrophoresis on agarose gels and the fraction that directed the cell-free synthesis of protein in the size range of GHs of other species was isolated and used to screen the transformed colonies of E . coli . A clone containing the putative sGH cDNA was identified and its nucleotide sequence was determined . To verify that the cDNA was that of sGH, the GH cell region of coho pituitary glands was incubated in organ culture . The secreted GH was purified by HPLC and the sequence of its 42 amino-terminal amino acids was determined . Comparison of this sequence with the amino acid sequence derived from the cDNA showed that it encoded sGH . Medium containing the presumptive sGH as the only prominent protein was active in a GH radioreceptor assay that involved labeled bovine GH and pregnant mouse liver membranes: the sGH was approximately 10% as active as the bGH standard . RNA blotting analysis showed that sGH was the major species of RNA produced by the GH cell region of the salmon pituitary . The mRNA of sGH differed from those of human, rat, and bovine GH in that its 3'-untranslated region was unusually large (about 500 nucleotides) but the coding region showed significant homology with mammalian GHs and resembled them in having a strong (78%) preference for G and C in the third positions of the codons . The amino acid sequence of sGH showed 32-34% and 19-22% identical homology with mammalian GHs and prolactins, respectively . Several conserved regions between sGH and mammalian GH and PRL molecules were also revealed that could indicate conservation of structurally and/or functionally important domains . Hydropathy analysis disclosed that although sGH and the GH of a representative mammal (pig) had similar profiles in some regions, the sGH was overall more hydrophobic than the pig (p) GH . Similarities and differences, were also noted in the predicted secondary structure of sGH and pGH.

Mol Biochem Parasitol, 1987 Dec, 26(3), 267 - 76
Giardia intestinalis antigens expressed in Escherichia coli; Upcroft JA et al.; cDNA and genomic DNA of Giardia intestinalis have been cloned in pUC vectors and used to express Giardia antigens in Escherichia coli . Several expression libraries have been produced and positive clones identified by immuno-colony assays with antisera raised against whole parasites and partially purified antigen(s) . Those clones which express G . intestinalis antigens have been used to raise antisera in mice and the antisera used in immunofluorescence assays . The proteins expressed by the clones have been shown to represent a 32 kDa protein of the flagellae and axonemes, a protein associated with the spiral part of the ventral disc, proteins covering the surface of the trophozoite or associated with the coat, and other proteins associated with axonemes of posterolateral flagellae, kinetosomes and funis, and the anterolateral axonemes . mRNA was purified from G . intestinalis and translated in a cell free lysate . A rabbit antiserum raised against trophozoites immunoprecipitated several translation products while an antiserum raised against a purified 32 kDa protein only immunoprecipitated this protein . G . intestinalis rRNA subunits also were examined in the course of mRNA purification . Two rRNA species were evident, the small rRNA and the post-transcriptionally processed large rRNA.

Mol Immunol, 1987 Dec, 24(12), 1373 - 82
Antigenic regions of ribosomal protein S1 as defined by monoclonal antibodies; Hahn V et al.; Analysis of the antigenic structure of the E . coli ribosomal protein S1 was undertaken using a set of 13 monoclonal antibodies (MAbs) directed against the isolated S1 . The location of the epitopes was mapped using a series of large fragments and truncated forms of S1 . Most of the epitopes were localized in the C-terminal half of the molecule, while only one antibody bound to the N-terminal region . Two MAbs were able to bind to more than one region of S1, suggesting the presence of repeated epitopes related to internal sequence homologies . Six distinct antigenic domains were identified by competitive binding assays . Competition between some antibodies suggested that the C-terminal region of S1 might be in spatial proximity with the N-terminal domain in the tertiary structure of the protein . The binding of a few MAbs induced conformational changes in the protein which resulted in the complete inhibition of antibody binding at non-adjacent sites . All the MAbs reacted with the isolated form of S1 or with the protein bound to the small ribosomal subunit . This indicated that the same epitopes were expressed in the two forms of the antigen and that they were accessible to antibody binding when S1 was part of the ribosomal subunit.

Scand J Immunol, 1987 Dec, 26(6), 639 - 43
The development of IgA-specific antibodies to Escherichia coli O antigen in children; Gleeson M et al.; One hundred and sixty-five infants were studied longitudinally from birth to 5 years of age . One hundred and twenty-three school-age children and 27 adults were examined cross-sectionally . Total salivary IgA levels and IgA antibodies against Escherichia coli O antigens were measured . Total IgA levels were low (less than 20 mg/l) from birth to 4 years of age . At 5 years of age there was a dramatic increase in the total IgA level (geometric mean = 100.7 mg/l), after which the levels fell to values similar to those observed in adults (adult geometric mean = 53.2 mg/l) . Low levels of IgA-specific E . coli antibodies were observed for the first 4 years of life (less than 1.0 ELISA units) . There was a gradual increase in specific antibodies between 5 and 9 years of age (geometric men at 9 years = 4.66 ELISA units) to levels similar to those observed in adults (adult geometric mean = 8.20 ELISA units) . It is suggested that the patterns of development for these variables reflect a balance between antigenic exposure and immune control mechanisms.

Proc Natl Acad Sci U S A, 1987 Dec, 84(24), 8849 - 53
Host control of plasmid replication: requirement for the sigma factor sigma 32 in transcription of mini-F replication initiator gene; Wada C et al.; Replication of F factor or mini-F plasmid is strongly inhibited in the rpoH (htpR) mutants of Escherichia coli deficient in the sigma factor (sigma 32) known to be required for heat shock gene expression . Transcription of the mini-F repE gene encoding a replication initiator protein (E protein) was examined by operon fusion and by direct determination of repE mRNA . The synthesis rate and the level of repE mRNA were found to increase transiently upon temperature upshift (30 degrees C to 42 degrees C) in wild-type cells but to decrease rapidly in the rpoH mutants . Thus sigma 32 appeared to be directly involved in transcription of repE whose product, E protein, in turn activates DNA replication from the mini-F ori2 region . This scheme of host-controlled plasmid replication is further supported by the analysis of transcription in vitro: RNA synthesis can be initiated from the repE promoter by a minor form of RNA polymerase containing sigma 32 but not by the major polymerase containing the normal sigma factor sigma 70 . The sigma 32-mediated transcription from the repE promoter is strongly inhibited by the E protein . We conclude that transcription of the mini-F repE gene is mediated by the host transcription factor sigma 32 and is negatively controlled by its own product.

J Gen Virol, 1987 Dec, 68 ( Pt 12), 3137 - 43
Fusion proteins with multiple copies of the major antigenic determinant of foot-and-mouth disease virus protect both the natural host and laboratory animals; Broekhuijsen MP et al.; Proteins consisting of one, two or four copies of the amino acid sequence 137 to 162, which contains the major immunogenic site of VP1 of foot-and-mouth disease virus, attached to the N-terminus of beta-galactosidase have been expressed in Escherichia coli cells . In guinea-pigs the protein containing one copy (P71) of the viral determinant elicited only low levels of neutralizing antibody whereas protective levels were elicited by the proteins containing two (P72) or four (P74) copies of the determinant . Single inoculations of the P72 and P74 proteins containing as little as 2 micrograms or 0.8 micrograms of peptide respectively were sufficient to protect all the animals against challenge infection . Moreover, the equivalent of 40 micrograms of peptide in P74 protected pigs against challenge infection after one inoculation.

Mutat Res, 1987 Dec, 192(4), 247 - 52
Role of rec genes in SOS-induced inhibition of cell division in Escherichia coli; Knezevic-Vukcevic J et al.; The inhibition of cell division induced by bleomycin (BM) and UV irradiation in the set of rec mutants of E . coli K12 was studied . Data presented in this work indicate that BM treatment requires mainly the RecBC pathway for the induction of cell filamentation . In the recB21 mutant cell filamentation is delayed and reduced compared to the wild type . Cell filamentation is BM-induced with similar kinetics in strains with a proficient RecBC recombination pathway (rec+, recF143 and recN262), as well as in the strain with a fully expressed RecF pathway (recB21recC22sbcB15) . Induction is completely abolished in the recB21recF143 double mutant . On the other hand cell filamentation was induced similarly by UV irradiation in all strains with a functional recF gene and in the strain with a fully operative RecF pathway, but it was delayed in the recF143 and recB21recF143 mutants.

J Exp Med, 1987 Dec 1, 166(6), 1798 - 813
Opsonin-independent ligation of Fc gamma receptors . The 3G8-bearing receptors on neutrophils mediate the phagocytosis of concanavalin A-treated erythrocytes and nonopsonized Escherichia coli; Salmon JE et al.; We report that phagocytosis by human neutrophils of Con A-treated erythrocytes (E-Con A) and nonopsonized Escherichia coli with mannose-binding adhesions is mediated by the Fc gamma receptor bearing the 3G8 epitope . Modulation of Fc receptors by pretreating with aggregated-IgG or with 3G8 anti-Fc gamma receptor mAb markedly inhibited internalization of E-Con A and E . coli without altering their cell surface attachment . Phagocytosis of these probes was specifically blocked by alpha-methylmannoside and D-mannose and not by other monosaccharides . Thus, recognition of E-Con A and E . coli by the Fc receptor is dependent upon the mannose-specific interaction with lectin or lectin-like adhesions . These data demonstrate that ligands other than the classical IgG opsonins can bind to classical immune receptors for IgG through lectin-carbohydrate interactions.

J Immunol, 1987 Dec 1, 139(11), 3828 - 33
Molecular cloning of the liver-specific rat F antigen; Gershwin ME et al.; F antigen is a 43-kDa widely conserved liver protein that has been intensively used in studies of immunogenicity and tolerance; two murine allotypes have been identified . Immunization of specific responder inbred strains with liver homogenates from the opposite allotype leads to precipitating antibody and cell-mediated immunity against F . The antibodies produced are autoantibodies as they react equally well with self . We have identified a cDNA clone from rat liver that reacts with alloantisera to F . The fused polypeptide produced by the clone was shown to correspond to F by several experiments . First, alloantisera to F antigen reacted with the cloned fused polypeptide, but not control recombinant clones . Second, mice immunized with the fused polypeptide generate an antibody response that reacts specifically with the 43-kDa protein of mouse liver homogenates and with highly purified F antigen . Finally, both anti-F allosera and sera from mice immunized with the fused polypeptide react with the same 43-kDa liver protein on two-dimensional immunoblots . The nucleotide and deduced amino acid sequence of the clone are presented and the sequence was found to have a significant homology with L28, an Escherichia coli ribosomal protein . The availability of recombinant F antigen will allow definitive questions to be addressed with respect to epitopes and specifically the identification of the T cell epitope which allows for autoimmune responses.

J Bacteriol, 1987 Dec, 169(12), 5870 - 2
Two domains at the origin are required for replication and maintenance of broad-host-range plasmid R1162; Kim YJ et al.; Two domains at the replicative origin of broad-host-range plasmid R1162 are required in cis for plasmid maintenance in Escherichia coli and for plasmid DNA replication in cell extracts . Increasing the distance between the domains reduces replication in vitro, without substantially changing plasmid DNA content or stability in vivo.

J Bacteriol, 1987 Dec, 169(12), 5678 - 85
Chlamydial rRNA operons: gene organization and identification of putative tandem promoters; Engel JN et al.; We isolated and characterized the rRNA operons of murine Chlamydia trachomatis . By exhaustively screening a library of chlamydial DNA and by blot hybridization of genomic DNA, we showed that there are only two rRNA operons in C . trachomatis . S1 nuclease protection and primer extension analysis were used to map the 5' and 3' ends of the mature 16S and 23S transcripts in both rRNA cistrons and, additionally, to demonstrate the lack of intervening sequences in these genes . The 5' ends of the presumed primary rRNA transcript were located and found to originate at two tandem sites separated by 100 base pairs . The two tandem chlamydial rDNA transcripts were not differentially regulated . Their products were coordinately expressed and were detectable as early as 9 h postinfection . However, the upstream transcript was only 10% as abundant as the downstream transcript . The sequences surrounding the transcription initiation sites bore little homology with each other or with the classic Escherichia coli -10 and -35 promoter sequences . This finding suggests that chlamydial transcription signals may differ from those of previously studied procaryotes.

J Bacteriol, 1987 Dec, 169(12), 5353 - 63
Transcriptional pausing in a region important for plasmid NR1 replication control; Dong XN et al.; The results of in vitro single-round transcription experiments indicated that RNA polymerase pauses during transcription of the leader region that precedes the repA1 gene of IncFII plasmid NR1 . Transcription initiated at either of the two transcription promoter sites of the repA1 gene, which encodes the essential replication initiation protein of NR1, was observed to pause in this region . Pausing was specifically enhanced by addition of NusA protein, an Escherichia coli transcription accessory factor . Northern blot RNA-DNA hybridization analysis of repA1 mRNA synthesized in vivo revealed RNA species that had lengths equivalent to those of the in vitro-paused intermediates . The steady-state rate of in vivo repA1 mRNA transcription downstream from the pause sites (measured by quantitative hybridization of pulse-labeled RNA to DNA probes complementary to different segments of repA1 mRNA) was not appreciably affected, which suggests that the pause sites do not promote premature termination of transcription . The pause sites were located between the target sequence within the leader region of the mRNA that interacts with a 91-base countertranscript and the beginning of the repA1 coding sequence . Because the countertranscript is an inhibitor of translation of repA1 mRNA, transcriptional pausing in this region may be an important feature of the regulation of RepA1 synthesis, which is the mechanism by which plasmid NR1 controls its replication.

Biochem Biophys Res Commun, 1987 Nov 30, 149(1), 13 - 20
Interaction between the gene 5 protein, gene 5 protein/single stranded fd DNA complex and gene 8 protein of the filamentous phage fd; Boehler-Kohler BA et al.; An affinity column consisting of gene 8 protein, the major coat protein of fd phage, bound to Sepharose was prepared . Isolated gene 5 protein/single stranded fd DNA complex was found to bind to this column and was eluted with fd phage single stranded fd DNA . pH changes, and 1 M CaCl2 were not effective in eluting the protein from the affinity column . Gene 5 protein/single stranded fd DNA complex from the crude extracts of fd-infected E . coli also bound to the column, as did isolated gene 5 protein; whereas fd single stranded DNA alone did not . These results may be relevant for the illucidation of the molecular events occurring in the early stages of fd phage assembly.

FEBS Lett, 1987 Nov 30, 224(2), 306 - 10
Purification and characterization of a recombinant human IgE Fc epsilon fragment lacking the C4 domain; Ikeyama S; Complementary DNA of human IgE Fc epsilon fragment (residues 226-480) lacking the C epsilon 4 domain was expressed in Escherichia coli and the product was purified by immunoaffinity chromatography on a monoclonal antibody (E12 0.02)-Affi-Gel 10 column . About 1.8 mg of an apparent dimer and 5.9 mg of a monomer were obtained from 65 g E . coli cells with 9.3% recovery . The purified products were found to lack more than half of the COOH-terminal portion of the C epsilon 3 domain . The apparent dimer showed high immunological specific activity (3.6 x 10(6) U/mg protein) comparable to that of natural human IgE when measured by commercial human IgE determination kits.

Biochem Biophys Res Commun, 1987 Nov 30, 149(1), 93 - 101
Thymine glycol-DNA glycosylase/AP endonuclease of CEM-C1 lymphoblasts: a human analog of Escherichia coli endonuclease III; Lee K et al.; A thymine glycol-DNA glycosylase/AP endonuclease has been identified in human CEM-C1 lymphoblasts . The enzyme is active in the absence of divalent cations and has an apparent molecular size of approximately 60,000 daltons . The enzyme releases thymine glycol from osmium tetroxide-damaged DNA via an N-glycosylase activity and is associated with an endonuclease activity that mediates phosphodiester bond cleavage at sites of thymine glycol and apurinic sites . We propose that this enzyme, which we call redoxyendonuclease, is the human analog of a bacterial enzyme, E . coli endonuclease III, that recognizes oxidative DNA damage.

Biochem Biophys Res Commun, 1987 Nov 30, 149(1), 34 - 9
Identification of functional regions in the C-terminal domain of Escherichia coli ribosomal protein S1 using monoclonal antibodies; Hahn V et al.; Monoclonal antibodies specific for defined regions of E . coli ribosomal protein S1 were used in a R17 mRNA-directed protein synthesis assay to reveal functionally important sites of the protein . Two distinct sites for mRNA binding were identified in the regions 349-437 and 438-547 located in the C-terminal domain of protein S1.

Biochim Biophys Acta, 1987 Nov 27, 905(1), 227 - 30
An evaluation of the contribution of membrane lipids to protection against ultraviolet radiation; Anderson R et al.; Using dioleoylphosphatidylcholine liposomes incorporating various fatty acids and neutral lipids, we have examined the ability of such lipids to provide protection of Escherichia coli and vesicular stomatitis virus (VSV) against the lethal effect of ultraviolet (254 nm) radiation . While the presence of varying amounts of saturated (palmitic) or polyunsaturated (arachidonic) fatty acids or the lipid antioxidant, alpha-tocopherol, had little effect on killing by ultraviolet radiation, considerable radioprotection was observed with beta-carotene, retinal and vitamin K-1 at final concentrations of 1 mg/ml . In another approach, vesicular stomatitis virus grown under conditions in which its envelope fatty acid composition was substantially modified, showed little change in its sensitivity to inactivation by ultraviolet radiation . The results provide strong evidence for a radioprotective role of certain, relatively rare natural lipid components with conjugated polyene systems, but not of the more ubiquitous and abundant membrane fatty acids.

Biochim Biophys Acta, 1987 Nov 27, 905(1), 109 - 17
Escherichia coli haemolysin forms voltage-dependent ion channels in lipid membranes; Menestrina G et al.; The action of the 107 kDa hemolysin from Escherichia coli on planar lipid membranes was investigated . We report that a single toxin molecule can form a cation-selective, ion-permeable channel of large conductance in a planar phospholipid bilayer membrane . The conductance of the pore is proportional to that of the bulk solution, indicating that the channel is filled with water . A pore diameter of about 2 nm can be evaluated . The pore formation mechanism is voltage-dependent and essentially resembles that of pore-forming colicins; this implies that opening of the channel is dependent on transfer of an electrical charge through the membrane . We propose that the physiological effects of E . coli hemolysin result from its ability to form ion channels in the membrane of attacked cells, and show that there is quantitative agreement between the effects of this toxin on model membranes and its hemolytic properties.

Nucleic Acids Res, 1987 Nov 25, 15(22), 9515 - 30
Transcription of the tRNA-tufB operon of Escherichia coli: activation, termination and antitermination; van Delft JH et al.; Signals setting the level of transcription of the tRNA-tufB operon have been studied by deletion mapping . TufB transcription was measured in vivo with plasmid-borne tRNA-tufB:galk operon fusions . Removal of the sequences from -133 to -58 with respect to the transcription start point, results in a 90% decrease of tufB transcription . This demonstrates the presence of a region, upstream of the tRNA-tufB promoter, that enhances the expression of the operon . DNA fragments bearing this upstream activator region do not display an abnormal electrophoretic mobility, as has been observed for the rrnB P1 upstream activator . Deletions starting in the first tRNA gene and directing towards tufB reveal at least two sites that influence tufB transcription . One signals transcription termination in the intergenic region between thrT and tufB . The other may be involved in antitermination . Possible mechanisms underlying antitermination and termination are considered in the light of the nucleotide sequence.

J Biol Chem, 1987 Nov 25, 262(33), 16037 - 40
The speEspeD operon of Escherichia coli . Formation and processing of a proenzyme form of S-adenosylmethionine decarboxylase; Tabor CW et al.; We have previously shown that the gene (speD) for S-adenosylmethionine decarboxylase is part of an operon that also contains the gene (speE) for spermidine synthase (Tabor, C . W., Tabor, H., and Xie, Q.-W . (1986) Proc . Natl . Acad . Sci . U . S . A . 83, 6040-6044) . We have now determined the nucleotide sequence of this operon and have found that speD codes for a polypeptide of Mr = 30,400, which is considerably greater than the subunit size of the purified enzyme . Our studies show that S-adenosylmethionine decarboxylase is first formed as a Mr = 30,400 polypeptide and that this proenzyme is then cleaved at the Lys111-Ser112 peptide bond to form a Mr = 12,400 subunit and a Mr = 18,000 subunit . The latter subunit contains the pyruvoyl moiety that we previously showed is required for enzymatic activity . Both subunits are present in the purified enzyme . These conclusions are based on (i) pulse-chase experiments with a strain containing a speD+ plasmid which showed a precursor-product relationship between the proenzyme and the enzyme subunits, (ii) the amino acid sequence of the proenzyme form of S-adenosylmethionine decarboxylase (derived from the nucleotide sequence of the speD gene), and (iii) comparison of this sequence of the proenzyme with the N-terminal amino acid sequences of the two subunits of the purified enzyme reported by Anton and Kutny (Anton, D . L., and Kutny, R . (1987) J . Biol . Chem . 262, 2817-2822).

J Biol Chem, 1987 Nov 25, 262(33), 15908 - 14
Kinetic analysis of the mechanism of Escherichia coli dihydrofolate reductase; Penner MH et al.; A kinetic mechanism is presented for Escherichia coli dihydrofolate reductase which describes the full time course of the enzymatic reaction over a wide range of substrate and enzyme concentrations at pH 7.2 and 20 degrees C . Specific rate constants were estimated by computer simulation of the full time course of single turnover, burst, and steady-state experiments using both nondeuterated and deuterated NADPH . The mechanism involves the random addition of substrates, but the substrates and enzyme are not at equilibrium prior to the chemical transformation step . The rate-limiting step follows the chemical transformation, and the maximum velocity of the reaction is limited by the release of the product tetrahydrofolate . The full time course of the reaction is markedly affected by the formation of the enzyme-NADPH-tetrahydrofolate abortive complex, but not by the enzyme-NADP-dihydrofolate abortive complex.

J Biol Chem, 1987 Nov 25, 262(33), 16100 - 4
DNA polymerase-primase from embryos of Drosophila melanogaster . The DNA polymerase subunit; Cotterill S et al.; The DNA polymerase-primase from Drosophila melanogaster has been separated into its constituent polymerase and primase subunits by sedimentation in glycerol gradients containing 50% ethylene glycol . Both activities have been obtained in good yield . The properties of the 182-kDa polymerase subunit are similar to those of the intact four-subunit enzyme . However, there are three significant differences . (i) The polymerase activity of the 182-kDa subunit shows an increased thermolability; (ii) the pause sites during replication of singly primed, single-stranded circular DNA by the 182-kDa subunit are altered; and (iii) unlike the intact enzyme, the 182-kDa subunit is highly processive in the presence of the single-stranded DNA-binding protein of Escherichia coli.

Nucleic Acids Res, 1987 Nov 25, 15(22), 9461 - 9
Rapid sequencing of cloned DNA using a transposon for bidirectional priming: sequence of the Escherichia coli K-12 avtA gene; Liu L et al.; A new approach to determining the sequence of cloned DNA is described . Unique regions near each end of the transposable element gamma-delta provide a pair of "portable" primer-specific sites for bidirectional sequencing by the dideoxy chain termination method . A set of gamma-delta insertions positioned about 200 bp apart over the entire cloned DNA allowed us to determine the sequence of both strands in a single parental plasmid without subcloning . The avtA (alanine-valine transaminase) gene of E . coli K-12 was sequenced by this approach . Surprisingly, gamma-delta insertions downstream of the coding region were found to significantly reduce avtA expression . We suggest that these nondisruptive insertions probably change the DNA topology and thereby alter gene expression.

J Biol Chem, 1987 Nov 25, 262(33), 16095 - 9
Isocitrate dehydrogenase kinase/phosphatase exhibits an intrinsic adenosine triphosphatase activity; Stueland CS et al.; In Escherichia coli, isocitrate dehydrogenase (IDH) is regulated by reversible phosphorylation . The bifunctional enzyme which catalyzes this phosphorylation cycle, IDH kinase/phosphatase, also exhibits a specific ATPase activity . Mutant derivatives of this protein which are nearly devoid of IDH phosphatase activity retain both IDH kinase and ATPase activity, indicating that ATP hydrolysis does not result from the cyclic phosphorylation of IDH . However, the IDH kinase and ATPase activities of these mutant proteins differ significantly from those of the wild-type IDH kinase/phosphatase expressed from the parental allele . This observation suggest that IDH kinase and IDH phosphatase do not reside on structurally independent domains . In contrast to many enzymes which catalyze kinetically unfavorable side reactions, the maximum velocity of the ATPase substantially exceeded those of IDH kinase and IDH phosphatase . ATP hydrolysis was only partially inhibited by phospho- and dephospho-IDH, with saturating levels of phospho-IDH decreasing the rate of ATP hydrolysis by a factor of approximately 5 . Even in the presence of near-saturating concentrations of phospho-IDH, the rate of ATP hydrolysis was 4-fold greater than the rate of the cyclic phosphorylation of IDH.

J Immunol Methods, 1987 Nov 23, 104(1-2), 31 - 42
A two-site sandwich immunoradiometric assay of human lymphotoxin with monoclonal antibodies and its applications; Meager A et al.; Three monoclonal antibodies (MoAbs L49-15, L81-11 and L238-14) were raised against recombinant human lymphotoxin (rLT) derived from E . coli containing the cDNA sequence specifying LT . MoAb L81-11 strongly neutralised the cytotoxicity of LT derived either from E . coli or the RPMI 1788 lymphoblastoid cell line, whilst the other two MoAbs were only weakly neutralising in this respect . L81-11 and L238-14 MoAbs bound to different antigenic determinants on the rLT molecule, but neither bound to other lymphokines such as the structurally related tumour necrosis factor (TNF) . As such, these MoAbs were ideal reagents for immunoassay of LT and a very sensitive, highly specific immunoradiometric assay (IRMA) was developed . This assay was rapid to perform and was capable of detecting as little as 10 pg/ml of LT . Application of the LT IRMA in combination with previously developed human gamma-interferon (IFN-gamma) and human TNF-specific IRMA (Crane et al., 1985; Meager et al., 1987) permitted independent estimations of these three substances to be carried out in parallel . By these means, it was found that RPMI 1788 produced both LT and TNF, but not IFN-gamma . Extensive analyses on cytokine (monokine and lymphokine) preparations derived from a variety of activated lymphocytes are also reported . Co-production of LT, TNF and IFN-gamma was a common finding, even occurring in alloantigen-specific T helper cell clones.

J Mol Biol, 1987 Nov 20, 198(2), 159 - 70
Organization and sequence of the hsd genes of Escherichia coli K-12; Loenen WA et al.; The nucleotide sequence of the hsdR and M genes, together with that for hsdS comprises an 8400 base segment spanning the entire hsd region of Escherichia coli K-12 . The three hsd genes are transcribed in the same direction, but from two promoters . hsdR and hsdM are separated by 492 base-pairs, whereas the termination codon of hsdM overlaps the initiation codon of hsdS . pres precedes hsdR, and our data indicate a transcription termination signal in the interval between hsdR and pmod, as expected if transcription of hsdM and S is dependent on pmod . Transcription from pres is not influenced by the products of the hsdM and S genes, and the mechanism whereby restriction is prevented when the hsd region is transferred to a modification-deficient cell remains to be elucidated . A segment of the predicted amino acid sequence of the M polypeptide shares homology with a variety of adenine methylases and may identify part of the active site for methylation of specific adenine residues . The R polypeptide shows homology with a variety of ATPases, and pronounced regions of alpha-helical structure are predicted, one of which is amphipathic.

Biochim Biophys Acta, 1987 Nov 20, 910(2), 130 - 41
Transcription initiation by Escherichia coli RNA polymerase at the gene II promoter of M13 phage: stability of ternary complex, direct photocrosslinking to nascent RNA, and retention of sigma subunit; Osumi-Davis PA et al.; The initial stages of transcription have been characterized using a template containing the gene II promoter region of M13 phage . Initiation of transcription in the presence of all four nucleotides gives rise to the 140-residue run-off transcript, with a minor pause at the RNA hexamer stage . Cycling, leading to the accumulation of significant amounts of short oligonucleotides {1}, was not observed . An RNA hexamer GUUUUU was the sole product when GpU and UTP were used and the ternary complex with the hexamer was stable and resistant to high salt (0.4 M) and S1 nuclease attack . After direct ultraviolet photocrosslinking of the RNA hexamer to RNA polymerase in the ternary complex, the radioactive label incorporation into various subunits was determined by autoradiography after sodium tetradecyl sulfate gel electrophoresis to be as follows: sigma, 86%; beta, 14%; beta' and alpha, negligible . Both electrophoresis and sucrose gradient centrifugation experiments indicate that the sigma subunit is not released from the ternary complex when either the RNA hexamer or the 140-residue RNA is synthesized on this template, even though the complexes are stable.

J Mol Biol, 1987 Nov 20, 198(2), 311 - 26
Structure of a complex of catabolite gene activator protein and cyclic AMP refined at 2.5 A resolution; Weber IT et al.; The structure of a dimer of the Escherichia coli catabolite gene activator protein has been refined at 2.5 A resolution to a crystallographic R-factor of 20.7% starting with coordinates fitted to the map at 2.9 A resolution . The two subunits are in different conformations and each contains one bound molecule of the allosteric activator, cyclic AMP . The amino-terminal domain is linked to the smaller carboxy-terminal domain by a nine-residue hinge region that exists in different conformations in the two subunits, giving rise to approximately a 30 degree rotation between the positions of the small domains relative to the larger domains . The amino-terminal domain contains an antiparallel beta-roll structure in which the interstrand hydrogen bonding is well-determined . The beta-roll can be described as a long antiparallel beta-ribbon that folds into a right-handed supercoil and forms part of the cyclic AMP binding site . Each cyclic AMP molecule is in an anti conformation and has ionic and hydrogen bond interactions with both subunits.

J Mol Biol, 1987 Nov 20, 198(2), 223 - 34
Nucleotide sequences required for a ColE1-type plasmid to replicate in Escherichia coli cells with or without RNase H; Ohmori H et al.; To elucidate the replication mechanism of a ColE1-type plasmid in RNase H-deficient (rnh-) strains of Escherichia coli, we constructed plasmid derivatives that deleted the whole, or a part, of the 5'-AAAAA-3' sequence (positions -3 to +2) that acts as the origin of replication in vivo and in vitro in the presence of RNase H . The activity of plasmid replication in rnh+ cells was found to be reduced by alterations of the AAAAA sequence . The activity could be restored when the derivatives, retaining the upstream sequence down to -8, regained a sequence containing at least two A residues in the region from -3 to +2 . By contrast, replication in rnh- cells was maintained at high levels even when the deletion included the AAAAA sequence and extended up to position -7 . The activity in rnh- cells decreased as deletions proceeded to -8 and further up to -17, and was abolished completely by further upward deletions . We concluded that in rnh- cells the plasmid replicates by a mechanism that operates only when RNase H is inactive . This RNase H-sensitive replication in rnh- cells seems to require the RNA-DNA hybrid formation that is also required for RNase H-dependent replication in rnh+ cells . The hybrid formation probably contributes by unwinding a portion of DNA from which replication can be initiated.

J Mol Biol, 1987 Nov 20, 198(2), 203 - 10
Escherichia coli dnaA initiation function is required for replication of plasmids derived from coliphage lambda; Kur J et al.; The dnaA gene function, indispensable for the initiation of Escherichia coli replication from oriC is not essential for the growth of phage lambda . The in-vitro replication of plasmids derived from phage lambda does not seem to require DnaA protein either . However, we present evidence that in vivo the normal replication of lambda plasmids is dnaA-dependent . After inactivating the dnaA gene function, half of the plasmid molecules may enter a single round of replication . Rifampicin sensitivity of this abortive, as well as normal, replication indicates involvement of RNA polymerase . The rifampicin resistance of the normal replication of lambda plasmids in E . coli carrying the dnaAts46 or dnaAts5, but not the dnaAts204 allele at 30 degrees C implies the interaction of DnaA protein and RNA polymerase in this process . We propose that DnaA protein co-operates with RNA polymerase in the initiation of replication at ori lambda . The dispensability of DnaA in the growth of phage lambda and in lambda plasmid replication in vitro is discussed.

J Mol Biol, 1987 Nov 20, 198(2), 187 - 202
Mechanisms of ultraviolet-induced mutation . Mutational spectra in the Escherichia coli lacI gene for a wild-type and an excision-repair-deficient strain; Schaaper RM et al.; We have analyzed the DNA sequence changes in a total of 409 ultraviolet light-induced mutations in the lacI gene of Escherichia coli: 227 in a Uvr+ and 182 in a UvrB- strain . Both differences and similarities were observed . In both strains the mutations were predominantly (60 to 75%) base substitutions, followed by smaller contributions of single-base frameshifts, deletions and frameshift hotspot mutations . The base substitutions proved largely similar in the two strains but differences were observed among the single-base frameshifts, the deletions and the hotspot mutations . Among the base substitutions, both transitions (72.5%) and transversions (27.5%) were observed . The largest single group was G.C----A.T (60% of all base substitutions) . The sites where G.C----A.T changes occurred were strongly correlated (97.5%) with sequences of adjacent pyrimidines, indicating mutation targeted ultraviolet photoproducts . Comparable amounts of mutation occurred at cytosine/cytosine and (mixed) cytosine/thymine sites . From an analysis of the prevalence of mutation at either the 5' or 3' side of a dipyrimidine, we conclude that both cyclobutane dimers and (6-4) lesions may contribute to mutation . Despite the general similarity of the base-substitution spectra between the wild-type and excision-defective strains, a number of sites were uniquely mutable in the UvrB- strain . Analysis of their surrounding DNA sequences suggested that, in addition to damage directly at the site of mutation, the potential for nearby opposite-strand damage may be important in determining the mutability of a site . The ultraviolet light-induced frameshift mutations were largely single-base losses . Inspection of the DNA sequences at which the frameshifts occurred suggested that they resulted from targeted mutagenesis, probably at cyclobutane pyrimidine dimers . The prevalence of frameshift mutations at homodimers (TT or CC) suggests that their formation involves local misalignment (slippage) and that base-pairing properties are partially retained in cyclobutane dimers . While the frameshift mutations in the Uvr+ strain were distributed over many different sites, more than half in the UvrB- strain were concentrated at a single site . Ultraviolet light-induced deletions as well as frameshift hotspot mutations (+/- TGGC at positions 620 to 632) are considered to be examples of untargeted or semitargeted mutagenesis . Hotspot mutations in the Uvr+ strain showed an increased contribution by (-)TGGC relative to (+)TGGC, indicating that ultraviolet light may specifically promote the loss of the four bases.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell, 1987 Nov 20, 51(4), 623 - 30
Factor-independent termination of transcription in a stretch of deoxyadenosine residues in the template DNA; Tomizawa J et al.; The primer transcript of plasmid ColE1 extends beyond the replication origin in either of two different modes . It does or does not form a hybrid with the template DNA . When a stretch of 20 deoxyadenosine residues is inserted into the template strand downstream of the origin, more than 90% of hybridized transcripts and about 10% of unhybridized transcripts terminate at the insert . When the number of inserted residues is reduced to ten, the corresponding values are decreased considerably, while the sites of termination are almost unchanged . A palindrome immediately before the stretch increased the efficiency of termination of unhybridized transcripts . Upon termination of hybridized transcripts, RNA polymerase detaches from the template . A structure made by a hybrid or by folded RNA may affect a property of transcription, and a stretch of deoxyadenosine residues including those beyond the actual site of termination may facilitate detachment of RNA polymerase from the template DNA.

Nature, 1987 Nov 19-25, 330(6145), 266 - 9
HIV F/3' orf encodes a phosphorylated GTP-binding protein resembling an oncogene product; Guy B et al.; Apart from the retroviral gag, pol and env the HIV genome contains the F (3' orf) gene which encodes a polypeptide of 206 amino acids which is myristylated at the N-terminal and whose function is unknown . We have expressed the F gene in Escherichia coli and from a recombinant vaccinia virus, VVTGfHIV . The F-protein produced in VVTGfHIV-infected mammalian cells is myristilated, and is phosphorylated by protein kinase C at a residue close to the N-terminus like pp60-src (ref . 5) . Purified bacterial F-protein also shows the GTPase, autophosphorylation and GTP-binding activities reported for the ras gene product . Furthermore, we show that expression of F in a CD4+ cell line down-regulates the CD4(T4) antigen . These results suggest that F is important in the pathophysiology of AIDS (acquired immune deficiency syndrome).

Biochim Biophys Acta, 1987 Nov 19, 894(2), 127 - 37
Ligand-induced conformational changes in the Escherichia coli F1 adenosine triphosphatase probed by trypsin digestion; Bragg PD et al.; Digestion of the F1-ATPase of Escherichia coli with trypsin stimulated ATP hydrolytic activity and removed the delta and epsilon subunits of the enzyme . A species represented by the formula alpha 1(3) beta 1(3) gamma 1, where alpha 1, beta 1 and gamma 1 are forms of the native alpha, beta and gamma subunits which have been attacked by trypsin, was formed by trypsin digestion in the presence of ATP . In the presence of ATP and MgCl2, conversion of gamma to gamma 1 was retarded and the enzyme retained the epsilon subunit . These results imply that binding of ATP to the beta subunits alters the conformation of ECF1 to increase the accessibility of the gamma subunit to trypsin . The likely trypsin cleavage sites in the alpha, beta and gamma subunits are discussed . ECF1 from the alpha subunit-defective mutant uncA401, or after treatment with N,N'-dicyclohexylcarbodiimide or 4-chloro-7-nitrobenzofurazan, was present in a conformation in which the gamma subunit was readily accessible to trypsin and could not be protected by the presence of ATP and MgCl2 . In a similar manner to native E . coli F1-ATPase, the hydrolytic activity of the trypsin-digested enzyme was stimulated by the detergent lauryldimethylamine N-oxide . Since the digested enzyme lacked the epsilon subunit, a putative inhibitor of hydrolytic activity, a mechanism for the stimulation which involves loss or movement of this subunit is untenable.

Biochemistry, 1987 Nov 17, 26(23), 7234 - 8
Footprinting DNA-protein complexes in situ following gel retardation assays using 1,10-phenanthroline-copper ion: Escherichia coli RNA polymerase-lac promoter complexes; Kuwabara MD et al.; Protein-DNA complexes isolated in gel retardation assays can be digested within the acrylamide matrix by the nuclease activity of 1,10-phenanthroline-copper ion (OP-Cu) . When the oligonucleotide products are eluted and analyzed on a sequencing gel, a footprint of the DNA-protein complex is obtained . Therefore, any protein-DNA complex isolated by the widely used gel retardation technique can be defined in terms of sequence-specific interactions by this simple methodology . The binding of the lac repressor and Escherichia coli RNA polymerase to an EcoRI fragment containing the lac control region has been studied by the combined gel retardation-1,10-phenanthroline-copper ion footprinting procedure . Footprints of lac repressor binding correspond to those obtained in solution with OP-Cu and DNase I and verify the experimental procedures . In studying E . coli RNA polymerase-promoter complexes, we have found that magnesium ion is required to form single-stranded DNA structures characteristic of kinetically competent open transcription complexes.

Biochemistry, 1987 Nov 17, 26(23), 7262 - 71
Lambda cro repressor complex with OR3 DNA: 15N NMR observations; Leighton P et al.; 15N NMR studies of the coliphage lambda cro repressor are presented . The protein has been uniformally labeled with 15N, and individual amino acids have been incorporated . Although the four C-terminal residues (63-66) were not located in the original crystallographic studies of the protein {Anderson, W.F., Ohlendorf, D.H., Takeda, Y., & Matthews, B.W . (1981) Nature (London) 290, 754}, it has been proposed that the C-terminus is involved in DNA binding {Ohlendorf, D.H., Anderson, W.F., Fisher, R.G., Takeda, Y., & Matthews, B.W . (1982) Nature (London) 298, 718} . These experiments give direct verification of that proposal . {15N}Amide resonances are assigned for residues 56, 62, 63, and 66 in the C-terminus by enzymatic digestion and by 13C-15N double-labeling experiments . 15N{1H} nuclear Overhauser effects show that the C-terminus is mobile on a nanosecond time scale . Exchange experiments using distortionless enhancement via polarization transfer, which is sensitive to proton exchange on the 1/JNH (10 ms) time scale, indicate that the amide protons in the C-terminus are freely accessible to solvent . It is thus a flexible arm in solution . The binding of both specific operator and nonspecific DNA is shown to reduce both the mobility and the degree of solvent exposure of this arm . Two-dimensional 15N-1H correlation experiments using 15N-labeled cro reveal inconsistencies with previously reported 1H NMR assignments for the lysine amides {Weber, P.L., Wemmer, D.E., & Reid, B.R . (1985) Biochemistry 24, 4553} . This result suggests that those assignments require reexamination, illustrating the utility of 15N labeling for obtaining 1H resonance assignments of biomolecules . Furthermore, isomerization of the peptide bond of Pro-59, which has been previously suggested (Weber et al., 1985) and which would significantly affect the properties of the C-terminal arm, is shown to not occur.

Biochemistry, 1987 Nov 17, 26(23), 7304 - 10
Base dynamics of nitroxide-labeled thymidine analogues incorporated into (dA-dT)n by DNA polymerase I from Escherichia coli; Pauly GT et al.; Nitroxide-labeled thymidine substrates (dL) for Escherichia coli DNA polymerase I (pol I) were used to synthesize spin-labeled alternating double-stranded copolymers with (dA-dT)n as a template . All dL substrates use an alkane or alkene tether substituted into the 5-position of the pyrimidine ring to link a five- or six-membered ring nitroxide to the pyrimidine base . The kinetics of dL incorporation show some tether dependence with respect to tether length and tether geometry . The electron spin resonance (ESR) spectra of (dA-dT,dL)n duplexes directly formed by polymerization with pol I are compared with the ESR spectra of (dA)n(dT,dL)n duplexes, which are obtained after annealing of nitroxide-labeled single strands with complementary unlabeled single strands . The ESR spectra indicate that nitroxide-labeled analogues with tethers short enough to let the nitroxide ring reside in the major groove are excellent reporter groups for monitoring hybridization . A small difference between the ESR line shapes of the alternating duplexes (dA-dT,dL)n and the homopolymer duplexes (dA)n(dT,dL)n containing the same dL is detectable, suggesting the presence of subtle differences in the base dynamics between both systems . Computer simulation of the ESR spectra of the (dA-dT,dL)n duplexes was successful with the same motional model reported earlier {Kao, S.-C., & Bobst, A.M . (1985) Biochemistry 24, 5465-5469} . The thymidine motion arising from tilting and torsion of base pairs and base twisting in (dA-dT)n is similar to that in (dA)n(dT)n and is of the order of 4 ns.

FEBS Lett, 1987 Nov 16, 224(1), 125 - 7
Expression of soybean glycinin subunit precursor cDNAs in Escherichia coli; Fukazawa C et al.; As the cDNAs encoding A1aB1b and A2B1a subunit precursors of the glycinin A2 subfamily contain a unique NcoI site sequence, (A)CCATGG, occurring at their translation initiation sites, plasmids were constructed to direct the synthesis of those precursor proteins by inserting NcoI/PstI fragments derived from those cDNA clones into the NcoI/PstI-pKK233-2 expression vector in Escherichia coli MV1190, respectively . The resultant plasmids directed the expression of 57-kDa protein components that have molecular masses in agreement with those of the in vitro translation products directed by glycinin A2 subfamily mRNAs, by the addition of isopropyl beta-D-thiogalactoside . These proteins, which comprised as much as 1% of the total bacterial protein, are immunoprecipitable with rabbit antibodies specific for glycinin subunits . This procedure makes glycinin subunits available as a model for studying structure-function relationships in seed proteins using site-directed mutagenesis . This is the first expression of glycinin-like storage protein in E . coli.

FEBS Lett, 1987 Nov 16, 224(1), 121 - 4
Covalent flavinylation of 6-hydroxy-D-nicotine oxidase involves an energy-requiring process; Brandsch R et al.; E . coli cells harbouring the recombinant plasmid pDB222 with the 6-HDNO gene under the control of the tac-promotor were induced with IPTG to synthesize a high amount of 6-HDNO protein . Part of this protein was present as 6-HDNO apoenzyme . The proportion of 6-HDNO apoenzyme formed could be increased when the induction of 6-HDNO synthesis by IPTG was performed in the presence of the inhibitor diphenyleneiodonium . The 6-HDNO apoenzyme thus formed could be transformed into enzymatically active holoenzyme in the presence of FAD by a process requiring an energy-generating system consisting of ATP, phosphoenolpyruvate and pyruvate kinase . This finding suggests that an enzymatic step(s) is (are) involved in the covalent flavinylation of 6-HDNO.

Eur J Biochem, 1987 Nov 16, 169(1), 59 - 64
Mistranslation in twelve Escherichia coli ribosomal proteins . Cysteine misincorporation at neutral amino acid residues other than tryptophan; Laughrea M et al.; The misincorporation of cysteine (codon: UGU/C) into twelve ribosomal proteins devoid of cysteine has been studied . Although it is generally assumed that cysteine is misincorporated at arginine and tryptophan residues (codons: CGU/U and UGG respectively), our results are consistent with the idea that cysteine is also misincorporated at phenylalanine residues (codon: UUU/C) through a second-position C:U mismatch . Cysteine was found in ribosomal proteins L29, L32/L33 and S10, under conditions where only its misincorporation at neutral residues was measured . Since these proteins contain no tryptophan, the date imply that cysteine has replaced a neutral amino acid other than tryptophan . Because there was a statistically significant correlation between the total level of cysteine in the twelve proteins under study and their content of phenylalanine and arginine residues, we conclude that there is a likelihood of cysteine misincorporation at phenylalanine residues, in addition to its misincorporation at arginine and tryptophan residues . Our measurements are consistent with the existence of a cluster of ribosomal proteins having an average mistranslation frequency of 2.5 X 10(-4)/residue and another having an average mistranslation frequency of 10(-3)/residue . There was three times less cysteine misincorporated into ribosomal protein L1 than into L7/L12, although the L1 mRNA contains eleven CGU/C codons and four UUU/C codons while the L7/L12 mRNA contains only one arginine and two phenylalanine codons (both proteins are free of tryptophan) . Furthermore, the mRNAs for both L1 and L7/L12 contain a CGU codon located in the context GUA-codon-GG and there was as much cysteine incorporated at this codon in L7/L12 {Bouadloun, F., Donner, D . and Kurland, C.G . (1983) EMBO J . 2, 1351-1356} than in the whole of L1 . This suggests that, relatively speaking, little cysteine is to be found at the phenylalanine and the other ten arginine positions of L1 and that the phenylalanine residues of L7/L12 are particularly error-prone.

Eur J Biochem, 1987 Nov 16, 169(1), 27 - 31
Endotoxic properties of synthetic pentaacyl lipid A precursor Ib and a structural isomer; Rietschel ET et al.; A pentaacyl precursor of lipid A biosynthesis, termed precursor Ib, and a structural isomer have been chemically synthesized . These compounds were, in comparison to synthetic Escherichia-coli type lipid A or lipopolysaccharide, analyzed for their activity in typical endotoxin test systems . It was found that both precursor Ib and the isomer exhibited similar or only slightly lower pyrogenic, lethal and Shwartzman-phenomenon-inducing activity than lipid A . All preparations were comparable in their B-lymphocyte mitogenicity, macrophage-activating capacity and immunoreactivity towards lipid A antisera . The proton nuclear magnetic resonance spectra of the 1-dephospho derivative of synthetic and bacterial precursor Ib were indistinguishable proving that the previously proposed structure for precursor Ib is correct.

Eur J Biochem, 1987 Nov 16, 169(1), 65 - 71
Insertion mutagenesis on a cell-surface-exposed region of outer membrane protein PhoE of Escherichia coli K-12; Agterberg M et al.; Amino acid residue arginine-158 of the outer membrane protein PhoE of Escherichia coli K-12 has been shown to be cell-surface-exposed {Korteland et al . (1985) Eur . J . Biochem . 152, 691-697} . To study the effects of small insertions in this region of the protein on its biogenesis and characteristics, a unique restriction site was created by site-directed mutagenesis in a plasmid carrying the phoE gene and oligonucleotides of 12-74 bp were inserted . The insertions did not interfere with incorporation into the outer membrane since (a) several monoclonal antibodies, directed against the cell-surface-exposed part of PhoE protein, bound to whole cells producing the altered proteins and (b) the proteins formed functional pores for the uptake of beta-lactam antibiotics . The binding of one monoclonal antibody and of the PhoE-specific phages TC45 and TC45hrN3 was disturbed by the insertions, showing that this region of the protein is immunogenic and is involved in the binding of both of these phages . The functioning of the mutant pores was characterized both in vivo by studying the uptake of beta-lactam antibiotics and in vitro after the reconstitution of the proteins in black lipid films . The pore characteristics changed depending on the nature of the inserted amino acids . Addition of a negatively charged amino acid resulted in decreased anion-selectivity, whereas insertion of a positive charge and deletion of a negative charge had only a small influence.

Biochem J, 1987 Nov 15, 248(1), 43 - 51
Lysyl-tRNA synthetase from Escherichia coli K12 . Chromatographic heterogeneity and the lysU-gene product; Charlier J et al.; In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene . During its purification from E . coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5')tetraphospho(5')adenosine (Ap4A) synthesis activities . Ap4A synthesis was measured by a new assay using DEAE-cellulose filters . The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage . Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected . LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not . Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock . Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1 . LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+ . These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

J Biol Chem, 1987 Nov 15, 262(32), 15457 - 65
Purification of the yeast PHR1 photolyase from an Escherichia coli overproducing strain and characterization of the intrinsic chromophores of the enzyme; Sancar GB et al.; We have placed the PHR1 gene of Saccharomyces cerevisiae under the transcriptional and translational control of the tac expression cartridge . Under inducing conditions Escherichia coli cells harboring plasmids carrying this construct accumulate approximately 8% of total cellular protein as the Phr1 photolyase . Using a strain devoid of E . coli photolyase activity, we have obtained milligram quantities of the yeast enzyme at greater than 95% purity and have characterized the enzyme . Phr1 photolyase is a monomer in solution with an Mr of 60,000, has a turnover number of 0.7 dimers min-1 molecule-1 in vitro, exhibits absorbance maxima at lambda = 277 and 377 nm, and has a fluorescence excitation maximum at 390 nm and an emission maximum at 475 nm . The near UV absorbance peak is shown to reflect the contributions of two intrinsic chromophores which are noncovalently bound to the enzyme . Spectroscopic, fluorescence, and thin layer chromatographic studies indicate that one of these chromophores is 1,5-reduced FAD rather than 4a,5-reduced FAD as previously proposed (Iwatsuki, N., Joe, C . O., and Werbin, H . (1980) Biochemistry 19, 1172-1176), while the other chromophore has properties similar to the second chromophore of E . coli photolyase . The fact that yeast and E . coli photolyases are similar both with respect to amino acid sequence and chromophore composition provides strong evidence that the enzymes share a common action mechanism which may also be utilized by photolyases from other organisms throughout the phylogenetic tree.

J Biol Chem, 1987 Nov 15, 262(32), 15327 - 9
Intersubunit location of the active site of ribulose-bisphosphate carboxylase/oxygenase as determined by in vivo hybridization of site-directed mutants; Larimer FW et al.; Ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum is a homodimer of 50.5-kDa subunits with two substrate binding sites per molecule of dimer . To determine whether each subunit contains an independent active site or whether the active sites are created by intersubunit interactions, we have used a novel in vivo approach for producing heterodimers from catalytically inactive, site-directed mutants of the carboxylase . When the alleles encoding these mutant proteins are placed separately into compatible plasmids and coexpressed in the same Escherichia coli host, activity is observed at about 20% of the wild-type level . Analysis of the carboxylase purified from these cells reveals the presence of heterodimers of the two mutant proteins . This interallelic complementation demonstrates that domains from each of the subunits interact to form a shared active site.

Cancer Res, 1987 Nov 15, 47(22), 5971 - 4
Inhibition of mammalian DNA polymerases by recombinant alpha-interferon and gamma-interferon; Tanaka M et al.; Interferons (IFNs) have been shown to suppress the growth of both normal and malignant cells . We examined the effect of gene-cloned IFN-alpha and IFN-gamma on the in vitro activities of human, calf, or rat DNA polymerases . IFN-alpha strongly inhibited the reactions of DNA polymerase alpha and beta at apparent Ki values of 1.25 and 0.35 x 10(5) antiviral units/ml, respectively, but inhibited DNA polymerase gamma only slightly . IFN-gamma inhibited the reaction of DNA polymerase alpha more strongly (Ki, 1.2 x 10(4) units/ml) than IFN-alpha, but not that of DNA polymerase beta . On the other hand, neither IFN-alpha nor IFN-gamma inhibited the reactions of DNA polymerase I from Escherichia coli, Klenow fragment, T-4 DNA polymerase, and RNA polymerase . The fact that Ki values for IFN-alpha of DNA polymerase from calf thymus, human leukemic cells, and rat liver were similar suggests the absence of species specificity among animals with regard to the inhibition of DNA polymerases by IFNs . These results indicate that DNA polymerase may be one of the targets of the action of IFNs.

J Biol Chem, 1987 Nov 15, 262(32), 15624 - 9
Isolation and characterization of the Escherichia coli mutH gene product; Welsh KM et al.; The Escherichia coli mutH gene product has been isolated in near homogeneous form using an in vitro complementation assay for DNA mismatch correction (Lu, A.-L., Clark, S., and Modrich, P . (1983) Proc . Natl . Acad . Sci . U.S.A . 80, 4639-4643) which is dependent on mutH function . The protein has a subunit Mr of 25,000, and purified preparations contain a Mg2+-dependent endonuclease activity which cleaves 5' to the dG of d(GATC) sequences to generate 5'-phosphoryl and 3'-hydroxyl termini . Symmetrically methylated d(GATC) sites are resistant to the endonuclease, hemimethylated sequences are cleaved on the unmethylated strand, and unmethylated d(GATC) sites are usually subject to scission on only one DNA strand . Although this endonuclease activity is extremely weak (less than 1 scission/h/mutH monomer equivalent) and cleavage at a d(GATC) site does not depend on the presence of a mismatched base pair within the DNA substrate, the activity does not appear to be a contaminant of mutH preparations . d(GATC) endonuclease activity and mutH complementing activity co-purify through multiple column steps without change in relative specific activities, and both activities co-electrophorese under native conditions . These findings suggest that the mutH product functions at the strand discrimination stage of mismatch correction and that this stage of the reaction involves scission of the unmethylated DNA strand.

J Biol Chem, 1987 Nov 15, 262(32), 15428 - 35
Yeast phosphatidylethanolamine methylation pathway . Cloning and characterization of two distinct methyltransferase genes; Kodaki T et al.; The structural genes (PEM1 and PEM2) encoding the enzymes involved in the yeast phosphatidylethanolamine (PE) methylation pathway were cloned by means of genetic complementation using yeast mutants . The cloned genes were expressed in a yeast mutant that was completely deficient in the PE methylation pathway . The membrane fraction obtained from the transformants carrying PEM1 only catalyzed the conversion of PE to phosphatidyl-N-monomethylethanolamine (PMME), the first step of the methylation pathway . Therefore, the enzyme encoded by PEM1 was designated as PE methyltransferase . In contrast, the membrane fraction from the transformants carrying PEM2 catalyzed the synthesis of phosphatidylcholine (PC) from PE, indicating that it contains all of the three methylation activities . PMME and phosphatidyl-N,N-dimethylethanolamine were found to be utilized more preferentially than PE . Because of its rather broad substrate specificity, the enzyme encoded by PEM2 is designated as phospholipid methyltransferase . The results of phospholipid composition analysis showed that the PEM1 transformant accumulated PMME whereas the PEM2 transformant contained a decreased amount of PC . Both genes were required for maintenance of the PC content of the yeast at a normal level . The results of nucleotide sequence analysis demonstrated that the coding frames of the PEM1 and PEM2 genes were capable of encoding 869- and 206-amino acid residues with calculated molecular weights of 101,202 and 23,150, respectively . The sizes of the PEM1 and PEM2 transcripts detected in the exponentially growing wild-type yeast were consistent with those of the deduced translation products . PE methyltransferase exhibits internal sequence homology as well as homology with phospholipid methyltransferase, suggesting that these two methyltransferase genes evolved through gene duplication . Furthermore, there was significant sequence homology between PE methyltransferase and bovine phenylethanolamine N-methyltransferase, and between phospholipid methyltransferase and Escherichia coli S-adenosylmethionine-6-N',N'-adenosyl (rRNA) dimethyltransferase.

Biochem Biophys Res Commun, 1987 Nov 13, 148(3), 954 - 61
Aspects of model building applied to the C-terminal domain of the L12 protein from chloroplast ribosomes: a molecular dynamics study; Horjales E et al.; A 170 picosecond molecular dynamics trajectory has been calculated starting from a model-built structure of chloroplast CTF . Local conformational changes occur during the equilibration period . Thereafter, a dynamically stable structure is attained . The conformational changes involve a turn connecting two structural subdomains which has an amino acid insertion and several substitutions with respect to the E . coli sequence . Potential energy minimisation alone fails to detect such a change . The overall folding and atomic positional fluctuations are very similar to those found in MD simulations of the E . coli molecule . The combined use of computer graphics based model building and MD calculations has lead to a thermally stable putative structure for the chloroplast CTF.

Biochim Biophys Acta, 1987 Nov 13, 904(2), 191 - 200
Cation specificity for sugar substrates of the melibiose carrier in Escherichia coli; Wilson DM et al.; A study has been made of the sugar substrate specificities and the cation specificities of the melibiose transport system of Escherichia coli . The following beta-galactosides were found to be transported: lactose, L-arabinose-beta-D-galactoside, D-fructose-beta-D-galactoside, o- and p-nitrophenyl-beta-D-galactosides . These beta-galactosides were cotransported with Na+ but not with H+ . The alpha-galactosides raffinose, melibiose and p-nitrophenyl-alpha-galactoside were transported with either H+ or Na+ . Of the monosaccharides tested D-galactose could use either Na+ or H+ for cotransport whereas D-fucose, L-arabinose and D-galactosamine could use only Na+ . The sugar specificity requirements for H+ cotransport are therefore more exacting than those for Na+ cotransport.

Nucleic Acids Res, 1987 Nov 11, 15(21), 8845 - 60
Alteration of a mitochondrial tRNA precursor 5' leader abolishes its cleavage by yeast mitochondrial RNase P; Hollingsworth MJ et al.; A mitochondrial specific RNase P is required to process 5' leaders from mitochondrial tRNA precursors in Saccharomyces cerevisiae . Experiments with a pair of mitochondrial pretRNAs(Asp) having leaders of different base composition suggest that this enzyme is unexpectedly sensitive to leader sequence or structure . Asp-AU (75% AU leader) is cleaved by the mitochondrial RNase P while Asp-GC (39% AU) is not . Both are substrates for E . coli RNase P . Partial nuclease digestions show that the tRNA portions of the two precursors differ in tertiary structure, while their 5' leaders differ in secondary structure . It is unusual for an RNaseP to have substrate specificity requirements which preclude processing of a pretRNA known to be a suitable substrate for an RNaseP from another species.

Nucleic Acids Res, 1987 Nov 11, 15(21), 8919 - 34
Cloning and characterization of a human ribosomal protein gene with enhanced expression in fetal and neoplastic cells; Ou JH et al.; Hepatocellular carcinoma is strongly associated with hepatitis B virus carrier patients who usually have HBV sequences integrated in the chromosomal DNA of liver cells . To assess the possible effects of HBV regulatory sequences (e.g., the enhancer) on expression of neighboring host genes we have screened for cellular genes that are both overexpressed and adjacent to integrated HBV sequences in hepatocellular carcinoma cells . The cloned cDNA for one such gene encodes a protein similar to the E . coli L-3 ribosomal protein which is thought to play a role in mRNA binding to the ribosome . The protein encoded by the cDNA localizes to the nucleolus and is also found in ribosomes; possibly it is the mammalian homologue of L-3 (MRL3) . The expression of MRL3 is higher in colon carcinoma and lymphoma cell lines than in normal liver, placenta and diploid fibroblasts, and is also higher in fetal than in adult liver . Therefore, MRL3 overexpression seems to be a property of rapidly dividing cells and is not directly linked to oncogenesis.

Nucleic Acids Res, 1987 Nov 11, 15(21), 8861 - 75
Sequence effect on alkali-sensitive sites in UV-irradiated SV40 DNA; Bourre F et al.; Ultraviolet light at 254 nm induces various kinds of DNA damage . We have located and quantified the pyrimidine (6-4) pyrimidone photoproducts along three hundred and forty two nucleotides of SV40 DNA . The level of photoproduct induction varies greatly according to the position on the DNA, but unlike what happens with pyrimidine dimers, the very adjacent nucleotides do not play a major role in the frequency of formation . A new alkali-sensitive site has been found on the ACA sequence after UV irradiation . This complex lesion is insensitive to the T4 endonuclease V and the E . coli photolyase, and may be involved with mutagenesis.

Nucleic Acids Res, 1987 Nov 11, 15(21), 8799 - 813
Genomic organization of low copy number sequences that are associated with deca-satellite DNA in the monkey genome; Maresca A et al.; A previously described segment of African green monkey DNA (cloned in phage lambda MkA) contains deca-satellite linked to DNA sequences that are estimated to occur once per genome . Sequences homologous to the low copy number sequences in lambda MkA are also associated with species-specific satellite DNAs in the human and mouse genomes . A second clone, lambda Mk8, contains a monkey DNA region that is colinear and homologous to a portion of the low copy number sequences in lambda MkA, but no satellite sequences . The two cloned segments are markedly different starting at a point proximal to the satellite DNA region in lambda MkA . DNA-blotting experiments indicate that lambda Mk8 but not lambda MkA represents the typical genomic organization and that the low copy number segments occur only once per haploid genome . The data suggest that rearrangements such as deletions or inversions occurring in monkey cells account in part for the structure of lambda MkA . Additional rearrangements may have occurred during cloning in E . coli . This unique chromosomal region may be particularly susceptible to recombination.

Science, 1987 Nov 6, 238(4828), 773 - 7
Left-handed DNA in vivo; Jaworski A et al.; Left-handed DNA is shown to exist and elicit a biological response in Escherichia coli . A plasmid encoding the gene for a temperature-sensitive Eco RI methylase (MEco RI) was cotransformed with different plasmids containing inserts that had varying capacities to form left-handed helices or cruciforms with a target Eco RI site in the center or at the ends of the inserts . Inhibition of methylation in vivo was found for the stable inserts with the longest left-handed (presumably Z) helices . In vitro methylation with the purified MEco RI agreed with the results in vivo . Supercoil-induced changes in the structure of the primary helix in vitro provided confirmation that left-handed helices were responsible for this behavior . The presence in vivo of left-handed inserts elicits specific deletions and plasmid incompatibilities in certain instances.

Cell, 1987 Nov 6, 51(3), 493 - 501
Transposition of Mu DNA: joining of Mu to target DNA can be uncoupled from cleavage at the ends of Mu; Craigie R et al.; Transposition of Mu involves transfer of the 3' ends of Mu DNA to the 5' ends of a staggered cut in the target DNA . We find that cleavage at the 3' ends of Mu DNA precedes cutting of the target DNA . The resulting nicked species exists as a noncovalent nucleoprotein complex in which the two Mu ends are held together . This cleaved donor complex completes strand transfer when a target DNA, Mu B protein, and ATP are provided . Mu end DNA sequences that have been precisely cut at their 3' ends by a restriction endonuclease, instead of by Mu A protein and HU, are efficiently transferred to a target DNA upon subsequent incubation with Mu A protein, Mu B protein, and ATP . Cleavage of the Mu ends therefore cannot be energetically coupled with joining these ends to a target DNA . We discuss the DNA strand transfer mechanism in view of these results, and propose a model involving direct transfer of the 5' ends of the cut target DNA, from their original partners, to the 3' ends of Mu.

Cell, 1987 Nov 6, 51(3), 483 - 92
The remarkable specificity of a new transcription termination factor suggests that the mechanisms of termination and antitermination are similar; Robert J et al.; E . coli lysogenic for the temperate, lambda-related phage HK022 do not support lambda growth . The exclusion of lambda is caused by the HK022 nun gene product, which blocks the expression of genes located downstream of and in the same transcription unit as the lambda nutL and nutR sequences . Transcripts terminating prematurely at or near nutR have been detected after inactivation of lambda repressor in lambda, HK022 dilysogens . Nun therefore appears to be a transcription termination factor with a remarkable specificity; it converts the lambda nut sequences, which normally interact with lambda N protein to suppress transcription termination, into terminators . These and other similarities between Nun-promoted termination and N-promoted antitermination argue strongly that the mechanisms of the two reactions have steps in common.

Cell, 1987 Nov 6, 51(3), 455 - 62
A replication initiator protein enhances the rate of hybrid formation between a silencer RNA and an activator RNA; Patel I et al.; The replication origin gamma of plasmid R6K in certain miniplasmids is kept silent by a silencer RNA . We have identified a major and three minor transcripts that are synthesized in a direction antiparallel and complementary to the silencer RNA . The major RNA, called the activator, is essential for replication from ori gamma . The complementary nature of the activator and silencer RNAs strongly suggests that the former is a target of the latter . We have also discovered that the initiator protein is a sequence-specific double-stranded RNA-binding protein that accelerates the rate of activator-silencer hybrid formation . Thus the efficient silencing of ori gamma can be explained by silencer RNA-activator RNA hybrid formation that is driven to completion by the initiator protein.

J Mol Biol, 1987 Nov 5, 198(1), 91 - 107
Binding of Escherichia coli ribosomal protein S8 to 16 S rRNA . A model for the interaction and the tertiary structure of the RNA binding site; Mougel M et al.; We have investigated in detail the secondary and tertiary structures of the 16 S rRNA binding site of protein S8 using a variety of chemical and enzymatic probes . Bases were probed with dimethylsulfate (at A(N-1), C(N-3) and G(N-7)), with N-cyclohexyl-N'-(2-(N-methylmorpholino)-ethyl)-carbodiimide-p- toluenesulfonate (at G(N-1) and U(N-3)) and with diethylpyrocarbonate (at A(N-7)) . The involvement of phosphates in hydrogen bonds or ion co-ordination was monitored with ethylnitrosourea . RNases T1, U2 and nuclease S1 were used to probe unpaired nucleotides and RNase V1 to monitor base-paired or stacked nucleotides . The RNA region, encompassing nucleotides 582 to 656 was probed within: (1) the complete 16 S rRNA molecule; (2) a 16 S rRNA fragment corresponding to nucleotides 578 to 756 obtained by transcription in vitro; (3) the S8-16 S rRNA complex; (4) the S8-RNA fragment complex; (5) the 30 S subunit . Cleavage or modification sites were detected by primer extension with reverse transcriptase . We present a three-dimensional model derived from mapping experiments and graphic modeling . Nucleotides in area 594-599/639-645 display unusual features: a non-canonical base-pair is formed between U598 and U641; and A595, A640 and A642 are bulging out of the major groove . The resulting helix is slightly unwound . Comparative analysis of probing experiments leads to several conclusions . (1) The synthesized fragment adopts the same conformation as the corresponding region in the complete RNA molecule, thus confirming the existence of independent folding domains in RNAs . (2) A long-range interaction involving cytosine 618 and its 5' phosphate occurs in 16 S rRNA but not in the fragment . (3) The fragment contains the complete information required for S8 binding . (4) The RNA binding site of S8 is centered in the major groove of the slightly unwound helix (594-599/639-645), with the three bulged adenines appearing as specific recognition sites . (5) This same region of the 16 S RNA is not exposed at the surface of the 30 S subunit.

J Mol Biol, 1987 Nov 5, 198(1), 137 - 8
Preliminary X-ray crystallographic study of cyanase from Escherichia coli; Kim KH et al.; Cyanase, an oligomeric enzyme of Escherichia coli that catalyzes the decomposition of cyanate to ammonia and bicarbonate, crystallizes in the space group P1 with unit cell parameters a = 85.96 A, b = 83.17 A, c = 83.28 A, alpha = 110.29 degrees, beta = 118.29 degrees and gamma = 72.40 degrees . Crystals diffract to a resolution of at least 2.5 A . The crystal data, in conjunction with a subunit molecular weight of 17,008, suggest that two oligomers are in the asymmetric unit of the crystal and that eight subunits comprise a single oligomer.

J Mol Biol, 1987 Nov 5, 198(1), 123 - 7
Novel blunt-end addition reactions catalyzed by DNA polymerase I of Escherichia coli; Clark JM et al.; DNA polymerase I (Klenow fragment) of Escherichia coli catalyzes the addition of deoxynucleotides to 3' hydroxyl termini of blunt-ended DNA fragments . The product of the reaction, which we call +1 addition, is found only in very low yield under conditions that permit editing by the 3'----5' exonuclease activity of the wild-type polymerase . A mutant form of the Klenow fragment that lacks detectable 3'----5' exonuclease activity shows an elevated accumulation of the +1 addition product . The mutant enzyme can use any one of the four dNTPs to carry out the reaction when each precursor is provided individually . However, in the presence of all four dNTPs the addition of dATP is strongly preferred . Suppression of the editing function of the wild-type polymerase through the use of high concentrations of exogenous deoxynucleoside monophosphates also results in a significant increase in the amount of +1 addition product formed . The presence of a high dNMP concentration also alters the specificity of the nucleotide addition reaction carried out by the wild-type enzyme . Thus, in addition to dATP, the dNTP which is complementary to the exogenous deoxynucleoside monophosphate, is also used in the +1 addition reaction . A similar effect of dNMPs on the specificity of nucleotide addition was obtained with the mutant Klenow fragment . These results define two pathways for the +1 addition reaction: one that does not require coding information from the DNA template and a second in which coding information is provided by the exogenous dNMP.

J Mol Biol, 1987 Nov 5, 198(1), 1 - 11
Surface exclusion genes traS and traT of the F sex factor of Escherichia coli K-12 . Determination of the nucleotide sequence and promoter and terminator activities; Jalajakumari MB et al.; The DNA encoding the surface exclusion genes traS and traT of the F sex factor of Escherichia coli K-12 has been sequenced and the biological activity of the various terminators and promoters determined . The data show that traS encodes a 16,861 Mr protein with no apparent signal sequence, as expected for its cytoplasmic membrane location . The protein is extremely hydrophobic . traS has its own promoter and a weak terminator region follows the gene . After the traS termination loop there is a small intergenic region before the traT promoter . The traT gene encodes a 25,932 Mr precursor for the 23,709 Mr mature protein . The amino-terminal signal peptide is 21 amino acid residues, consistent with it being an outer membrane lipoprotein . A very strong termination loop follows the gene and adjacent to this a further loop can be predicted from the sequence . These secondary structures would be expected to enhance the stability of the mRNA in the presence of 3' specific ribonucleases accounting for the apparent long half-life of the messenger . The amino acid sequence of the mature product of traT of F differs from that of R100 by only a single amino acid substitution (Gly for Ala at position 119), whereas that of pED208 (Folac) differs at 40 positions . traT lies in a region of heteroduplex homology between F and R100, and the nucleotide sequence confirms this and demonstrates that this homology breaks down immediately preceding and following the coding region . Sequence analysis shows that this is also so for pED208 . Thus the entire traS of F, R100 and pED208 are very different at the DNA level . An open reading frame, preceded by a typical promoter sequence and a weak and poorly located Shine-Dalgarno sequence, follows traT and corresponds to the start of traD . Alone, this promoter appears to be inactive.

Nature, 1987 Nov 5-11, 330(6143), 80 - 2
Two calcium-binding proteins in infiltrate macrophages of rheumatoid arthritis; Odink K et al.; The aetiology and cellular mechanism of chronic inflammatory processes are poorly understood . Macrophages act prominently in the inflammatory response and we report here that they express two calcium-binding proteins . The expression of these proteins, referred to as MRP-8 and MRP-14, is specific for cells of myeloid origin, namely granulocytes, monocytes and macrophages, and is observed in blood granulocytes and monocytes but not in normal tissue macrophages . In acutely inflamed tissues, macrophages can express MRP-14 but not MRP-8, and in chronic inflammations, such as primary chronic polyarthritis, infiltrate macrophages express both MRP-8 and MRP-14 . Characterization of MRP-8 and MRP-14 could therefore be useful to the understanding of cellular processes induced in chronic inflammation.

J Biol Chem, 1987 Nov 5, 262(31), 15225 - 31
Identification, purification, and characterization of Escherichia coli virus T1 DNA methyltransferase; Scherzer E et al.; An Escherichia coli virus T1-induced DNA methyltransferase was identified by activity gel analysis in homogenates of infected E . coli DNA-adenine-methylation-deficient strains . Although the Mr of this protein (31,000) is in the same range as that of the E . coli DNA adenine methyltransferase, the two proteins are not closely related; the E . coli dam gene does not hybridize with T1 DNA . Selective conditions for measurement of the T1 activity were developed, and the enzyme was purified to functional homogeneity, as shown by activity analysis in polyacrylamide gels . Requirements for optimal activity of the viral enzyme were determined to be pH 6.9, ionic strengths below 0.1 M KCl, and a temperature between 40 and 43 degrees C . The Km for S-adenosyl-L-methionine is 4.9 microM . The purified T1 DNA methyltransferase is capable of methylating adenine in 5'-GATC-3' sites in vitro.

J Biol Chem, 1987 Nov 5, 262(31), 14929 - 34
Protein substrates activate the ATP-dependent protease La by promoting nucleotide binding and release of bound ADP; Menon AS et al.; The interaction of protein substrates with protease La from Escherichia coli enhances its ability to hydrolyze ATP and peptide bonds . These studies were undertaken to clarify how unfolded proteins allosterically stimulate this ATPase activity . The tetrameric protease can bind four molecules of ATP, which activates proteolysis, or four molecules of ADP, which inhibits enzymatic activity . Protein substrates stimulate binding of the nonhydrolyzable ATP analog {3H} adenyl-5'yl imidodiphosphate, although they do not increase the net binding of {3H}ATP or {3H}ADP . Once bound, ATP is quickly hydrolyzed to ADP, which remains noncovalently associated with protease La even through repeated gel filtrations . Exposure to protein substrates (e.g . denatured bovine serum albumin at 37 degrees C) induces the release of all the bound ADP from the enzyme . Nonhydrolyzable ATP analogs bound to the enzyme were not released by these substrates . Proteins that are not degraded (e.g . native bovine serum albumin) and oligopeptides that only bind to the catalytic site do not induce ADP release . Thus, polypeptide substrates have to interact with an allosteric site to induce this effect . The protein-induced ADP release is inhibited by high concentrations of Mg2+ and is highly temperature-dependent . Protein substrates promoted {3H}ATP binding in the presence of ADP and Mg2+ (i.e . ATP-ADP exchange) and reduced the ability of ADP to inhibit the enzyme's peptidase and ATPase activities . These results indicate that: 1) ADP release is a rate-limiting step in protease La function; 2) bound ADP molecules inhibit protein and ATP hydrolysis in vivo; 3) denatured proteins interact with the enzyme's regulatory site and promote ADP release, ATP binding, and their own hydrolysis.

J Biol Chem, 1987 Nov 5, 262(31), 14921 - 8
Binding of nucleotides to the ATP-dependent protease La from Escherichia coli; Menon AS et al.; A critical enzyme in protein breakdown in Escherichia coli is the ATP-hydrolyzing protease La, the lon gene product . In order to clarify the role of ATP in proteolysis, we studied ATP and ADP binding to this enzyme using rapid gel filtration to separate free from bound ligands . In the presence of Mg2+ or Mn2+ and 10 microM ATP, two molecules of ATP were bound to the tetrameric enzyme, while at 100 microM ATP (or higher), four ATP molecules were bound, both at 0 and 37 degrees C . Protease La thus has two high affinity sites (S0.5 less than 10(-7) M) for ATP and two lower affinity sites (S0.5 = 12-15 microM) . Binding was reversible . In the absence of a divalent ion, ATP bound to only two sites . However, much lower Mg2+ concentrations (50 microM) were required for maximal ATPase binding than for maximal proteolytic and ATPase activity (2 mM) . Decavanadate, which is a potent inhibitor of proteolysis, also blocked ATP binding, but orthovanadate had neither effect . Different ATP analogs bind to these sites in distinct ways . Adenyl-5'-yl imidodiphosphate binds to only one high affinity site, while adenyl-5'-yl methylene monophosphonate binds to two . Nevertheless, both non-metabolizable analogs can activate oligopeptide hydrolysis as well as ATP . Although binding of a single nucleotide can activate peptide hydrolysis, occupancy of all four sites appears necessary for maximal protein breakdown . The ATP molecules on all four sites are hydrolyzed rapidly . The Pi is released, but ADP remains on the enzyme . ADP binds to the same four sites, but this process does not require divalent ions . Protease La shows higher affinity for ADP than for ATP . Therefore, in vivo, ADP should inhibit ATP binding and protease La function.

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