Escherichia coli

J Virol, 1985 Oct, 56(1), 135 - 43
Regulation of cytomegalovirus gene expression: alpha and beta promoters are trans activated by viral functions in permissive human fibroblasts; Spaete RR et al.; We have fused immediate (alpha) and delayed (beta) early promoter-regulatory sequences taken from the cytomegalovirus (CMV) genome to Escherichia coli lacZ (beta-galactosidase) as an indicator gene to study regulated expression of these promoters . After transfection of human fibroblast cells with plasmid constructs carrying beta-galactosidase fusions, and subsequent infection with CMV, we have demonstrated that viral trans-acting functions up-regulate the expression of these genes in a temporally authentic manner . The alpha promoter is activated even when de novo protein synthesis is blocked and when UV-inactivated virus is used, suggesting that, as for herpes simplex virus type 1 (HSV-1), a virion structural protein is responsible for its up-regulation . We have found that HSV-1, as well as CMV, is capable of trans activating the CMV alpha promoter . The beta promoter is activated by CMV but is completely unresponsive to HSV-1 infection . The temporal synthesis of the alpha and beta promoters in the transient expression system conforms with their natural regulation during viral replication . The beta-galactosidase fusions we describe provide a most exquisitely sensitive indicator system for the study of cis- and trans-acting viral regulatory functions.

J Immunol, 1985 Oct, 135(4), 2865 - 75
In vivo administration of purified human interleukin 2 . II . Half life, immunologic effects, and expansion of peripheral lymphoid cells in vivo with recombinant IL 2; Lotze MT et al.; Purified recombinant human interleukin 2 (RIL 2) derived from E . coli containing the inserted gene encoding for IL 2 was administered to 20 patients with a variety of malignancies . Toxicity was dose related and included fever, chills, malaise, arthralgias, myalgias, and unexpectedly, weight gain related to marked fluid retention . All patients receiving more than 10(5) U/kg total cumulative dose developed evidence of fluid retention, and all patients requiring discontinuance of RIL 2 (11/20) received total doses of between 2.54 X 10(5) U/kg to 15.4 X 10(5) U/kg . The limiting dose with this preparation was 3000 U/kg/hr by continuous administration or 10(6) U/kg by bolus administration . IL 2 was rapidly cleared from the plasma, with a half life of 6.9 min, and a later delayed clearance was consistent with a two-compartment model, with slower release from the extravascular space back into the plasma compartment . A marked change in lymphoid cells in the periphery was noted with an early depletion of all lymphoid cells, followed by an expansion of such cells with continuous IL 2 administration . A twofold to 16-fold expansion of total lymphoid cells in the peripheral blood could be demonstrated . TAC+ cells representing up to 25% of the circulating peripheral blood mononuclear cells could be demonstrated with 3 wk of continuous RIL 2 administration . Interferon-gamma levels increased in patients treated with IL 2 . Precursors of lymphokine-activated killer cells generated under standard conditions were depleted within 2 to 3 min after IL 2 administration, but repopulated the peripheral blood after 7 to 10 days of continuous IL 2 administration . No tumor regression was seen in any of the cancer patients treated with IL 2 alone.

Mol Cell Biol, 1985 Oct, 5(10), 2653 - 61
E1A 13S and 12S mRNA products made in Escherichia coli both function as nucleus-localized transcription activators but do not directly bind DNA; Ferguson B et al.; We previously purified and characterized functionally the Escherichia coli-expressed product of the human subgroup C adenovirus E1A 13S mRNA (B . Ferguson, N . Jones, J . Richter, and M . Rosenberg, Science 224:1343-1346, 1984; B . Krippl, B . Ferguson, M . Rosenberg, and H . Westphal, Proc . Natl . Acad . Sci . USA 81:6988-6992, 1984) . We have now expressed in E . coli and purified the protein product encoded by the human subgroup C adenovirus E1A 12S mRNA and have compared the functional properties of this protein with those of the E1A 13S mRNA product . Using microinjection techniques to introduce these proteins into mammalian cells, we found that the E1A 12S mRNA product, like the 13S mRNA product, localized rapidly to the cell nucleus and induced adenovirus gene expression . Although both E1A gene products localized to the nucleus and stimulated adenovirus gene transcription, these proteins did not directly bind to DNA under conditions in which a known DNA-binding protein, the human c-myc gene product, bound DNA efficiently . Thus, the E1A and myc gene products, which have been related both structurally and functionally, exhibit distinctly different biochemical properties.

J Biochem (Tokyo), 1985 Oct, 98(4), 1007 - 16
Gene organization of pldA and pldB, the structural genes for detergent-resistant phospholipase A and lysophospholipase L2 of Escherichia coli; Kobayashi T et al.; The genes coding for the phospholipid degradation enzymes in E . coli, detergent-resistant (DR-) phospholipase A (pldA) and lysophospholipase L2 (pldB), were cloned together on the plasmid pKO1 (Homma, H., Kobayashi, T., Ito, Y., Kudo, I., Inoue, K., Ikeda, H., Sekiguchi, M., & Nojima, S . (1983) J . Biochem . 94, 2079-2081) . To study their gene organization, a transducing lambda phage, lambda pldApldB, carrying both the pldA and pldB genes was constructed in vitro from plasmid pKO1 . Viable deletion mutants of lambda pldApldB were isolated by EDTA killing, and their deleted DNA regions were determined by electron microscopic analysis of appropriate heteroduplexes . The activities of DR-phospholipase A and lysophospholipase L2 were also measured in lysates of cells infected with the deletion phages . The DNA region essential for the expression of each lipolytic activity was determined . In addition, proteins coded by the bacterial DNA on the plasmids containing the pldApldB region to various extents were detected by the maxicell system . The results showed that the product of the pldB gene is a protein with molecular weight of 40,000 . It was also shown that the pldB gene is located at a region about 3 kilobase from the pldA gene.

Cell, 1985 Oct, 42(3), 967 - 77
Evidence that a phage T4 DNA packaging enzyme is a processed form of the major capsid gene product; Rao VB et al.; A phage T4 DNA packaging enzyme appears to arise as a processed form of the major T4 capsid structural protein gp23 . The enzyme activity and antigen are missing from all head gene mutants that block the morphogenetic proteolytic processing reactions of the head proteins in vivo . The enzyme antigen can be formed in vitro by T4 (gp21) specific processing of gp23 containing extracts . Enzyme antigen is found in active processed proheads but not in full heads . The enzyme and the major capsid protein show immunological cross-reactivity, produce common peptides upon proteolysis, and share an assembly-conformation-dependent ATP binding site . The packaging enzyme and the mature capsid protein (gp23*) both appear to arise from processing of gp23, the former as a minor product of a specific gp23 structure in the prohead, acting in DNA packaging as a DNA-dependent ATPase, and a headful-dependent terminase.

Proc Natl Acad Sci U S A, 1985 Oct, 82(20), 6774 - 8
Effect of DNA polymerase I and DNA helicase II on the turnover rate of UvrABC excision nuclease; Husain I et al.; UvrABC excision nuclease (UvrA, UvrB, and UvrC proteins) of Escherichia coli removes nucleotide mono- and diadducts from DNA in the form of oligonucleotides 12 or 13 bases long . We find that the purified enzyme dissociates from DNA very slowly, if at all, in the absence of other proteins implicated in excision repair . Addition of DNA polymerase I and helicase II (UvrD protein) to the reaction mixture stimulates the turnover rate of the excision nuclease to a level comparable to that observed in vivo.

Carcinogenesis, 1985 Oct, 6(10), 1501 - 6
By-pass of the major aminofluorene-DNA adduct during in vivo replication of single- and double-stranded phi X174 DNA treated with N-hydroxy-2-aminofluorene; Lutgerink JT et al.; To examine the effects of aminofluorene-DNA adduct formation on the biological activity of DNA, single-stranded (ss) phi X174 DNA and phi X174 replicative form (RF) DNA were modified to different extents with 3H-labeled N-hydroxy-2-aminofluorene and subsequently transfected to Escherichia coli spheroplasts with different repair capabilities . When the fraction of active ss phi X174 DNA molecules was measured as a function of the mean number of adducts per molecule, exponential survival curves were obtained from which it could be deduced that in wild-type, uvrA- and recA- cells at least 86%, and in uvrC- cells at least 82% of the introduced adducts do not cause inactivation . In the case of RF DNA the survival curves are non-exponential, but they nevertheless show that an exceptionally high number of adducts per RF molecule must be introduced to destroy its biological activity . On average 52 adducts per RF molecule were needed to reduce the survival to 37%, irrespective of whether wild-type, uvrA- or recA- cells were used . On the other hand, the survival of the uvrC- cells was considerably lower, but even in these cells a majority of the adducts is not lethal . By h.p.l.c . analysis of the modified DNA after hydrolysis with trifluoroacetic acid, 81 and 84% of the adducts in ss- and RF DNA, respectively, could be identified as N-(guanin-8-yl)-2-aminofluorene . The results strongly indicate that this type of major modification product is very frequently by-passed during replication of both single- and double-stranded DNA . The results together with the data obtained by sucrose gradient analysis both before and after an alkali treatment and those obtained by h.p.l.c . analysis suggest that inactivation of ssDNA is mainly due to minor modifications such as secondary lesions consisting of chain breaks and alkali-labile sites together with unidentified interaction products.

Avian Dis, 1985 Oct-Dec, 29(4), 1078 - 83
Immunogenicity of an Escherichia coli (serotype O1) pili vaccine in chickens; Gyimah JE et al.; Immunogenicity of an oil-emulsified Escherichia coli (serotype O1) pili vaccine was evaluated in chickens . Chickens were vaccinated with 116 micrograms or 29 micrograms of pili protein and challenged with the homologous E . coli via the posterior thoracic air sac . Unvaccinated chickens were challenged to serve as positive controls or left unchallenged to serve as negative controls . Vaccinated chickens were protected against challenge because they suffered low or no mortality; had mild gross lesions in air sacs, pericardial sacs, and livers, and the scored values were significantly (P less than or equal to 0.05) lower than those in the positive controls; and eliminated E . coli from tissues more efficiently than the positive controls.

Anal Biochem, 1985 Oct, 150(1), 235 - 7
Glutathione specifically labeled with isotopes; Murata K et al.; A procedure for synthesis of glutathione selectivity labeled with isotopes is described . A strain of Escherichia coli enriched in its content of gamma-glutamylcysteine synthetase and glutathione synthetase by recombinant DNA techniques is immobilized in a carrageenan matrix and treated with toluene to render the cells more permeable to the substrates . The immobilized cell matrix is incubated with a mixture containing the appropriately labeled amino acid, the other amino acid constituents of glutathione, ATP, and acetylphosphate . The radiolabeled product is isolated by column chromatography.

J Gen Microbiol, 1985 Oct, 131 ( Pt 10), 2665 - 71
Conjugation systems of IncT plasmids; Bradley DE et al.; Four IncT plasmids were compared for various characters, in particular pilus synthesis and function at different temperatures . The prototype Rts1 differed in some respects from the others (R402, R394, pIN25) . At 37 degrees C, the supposedly temperature-sensitive conjugation systems of the plasmids could still function efficiently on a surface, but not in a liquid . Long conjugative pili were synthesized at 30 degrees C, but only short ones (approx . 200 nm) were produced at 37 degrees C . The long pili converted two surface-obligatory conjugation systems to surface + liquid ones at 30 degrees C.

J Bacteriol, 1985 Oct, 164(1), 310 - 5
AsnC: an autogenously regulated activator of asparagine synthetase A transcription in Escherichia coli; Kolling R et al.; The regulation of the asparagine synthetase A gene of Escherichia coli was studied in vitro with a coupled transcription-translation system . It was shown that the 17-kilodalton gene, which is transcribed divergently from the adjacent asnA gene, codes for an activator of asnA transcription . The synthesis of the 17-kilodalton protein, which we now call AsnC, is autogenously regulated . The stimulating effect of AsnC on asnA transcription is abolished by asparagine, while the autoregulation of asnC is not affected by asparagine . The N-terminal part of the asnC protein, inferred from the DNA sequence, is homologous to the DNA-binding domain of regulatory proteins like catabolite gene activator, cro, and cI . This homology and direct repeats found in the region of the two asn promoters suggest that the asnC protein regulates transcription by binding to DNA . The asn promoters were defined by mapping of the mRNA start sites of in vitro-generated transcripts.

Clin Immunol Immunopathol, 1985 Oct, 37(1), 124 - 34
Primary and secondary in vitro immune response of the rabbit Peyer's patch and spleen to RDEC-1 pili; Axelrod DA; The culture conditions and cellular requirements for antigen-induced B-cell activation of the rabbit Peyer's patch and spleen using the RDEC-1 pilus antigen were delineated . It was found that optimal conditions for both primary and secondary in vitro immunization are similar except for culture duration . Optimal cell density was 3 X 10(6) cells/ml and optimal antigen dose was 30 to 100 ng/ml . Antibody production was best carried out in flat-bottom culture plates . Both T cells and glass-plate-adherent cells (macrophages) were required for antigen induced antibody production for both the primary and secondary responses . Of note was the inability to induce a primary antibody response in the spleen cell cultures . This study demonstrates the effectiveness of the system in elucidating some of the complex in vitro events associated with primary and secondary, antigen-specific B-cell activation of the rabbit Peyer's patch to RDEC-1 pili and the ability of rabbit spleen cell cultures to respond to RDEC-1 pilus antigen only after in vivo challenge.

Bioorg Khim, 1985 Oct, 11(10), 1353 - 5
{General method of isolation and analysis of polynucleotide fragments cross-linked with proteins}; Abdurashidova GG et al.; A fragment of 16S RNA, cross-linked to S7 protein by UV irradiation of the 30S subunit of E . coli ribosome, was obtained by the action of T1 ribonuclease on the irradiated nucleoprotein . The digest was treated with polynucleotide kinase in the presence of {gamma-32P}ATP and the S7-cross-linked oligonucleotides were isolated . An individual oligonucleotide attached to S7 protein was obtained after proteinase treatment of the respective spot followed by electrophoresis . Sequencing of this oligonucleotide established its structure as 1233-1240 fragment of 16S RNA, the U1239 residue being the site of the S7 cross-linking . The developed general approach can be used for localizing protein - cross-linked residues in polynucleotides, whatever is the procedure employed for cross-linking.

Mol Biochem Parasitol, 1985 Oct, 17(1), 45 - 60
Expression in Escherichia coli of two Schistosoma mansoni genes that encode major antigens recognized by immune mice; Lanar DE et al.; Two clones which contain genes encoding Schistosoma mansoni proteins recognized by immune mouse sera were chosen from cDNA lambda gt11 expression vector library by preselecting clones from the library with rabbit antisera against adult worm phosphate-buffered saline (PBS)-soluble antigens . One clone, MAC 182, codes for part of a Mr 70 000 protein; the other clone, MAC 184, codes for a Mr 27 000 protein . The insert sizes of MAC 182 and MAC 184 are 400 bp and 800 bp, respectively . Both clones express S . mansoni beta-galactosidase fusion proteins as products of the construct . Antibodies from either chronically infected mice or mice vaccinated with irradiated cercariae recognize the MAC 182 fusion protein (MAC 182fp) but not the MAC 184 fusion protein (MAC 184fp) . Rabbit antibodies prepared against MAC 182fp immunoprecipitate a Mr 70 000 in vitro translation product from adult mRNA and react in Western blot with a corresponding Mr 70 000 protein present in eggs, cercariae and adult worms but absent in schistosomula . Although the MAC 184fp is not recognized directly by chronic infection or vaccinated mouse antibodies, antisera prepared against the purified fusion protein immunoprecipitate a Mr 27 000 in vitro translation product which also reacts with mouse chronic infection sera . The same Mr 27 000 protein appears to be present in eggs, cercariae, schistosomula and adults as determined by Western blots with rabbit antisera against the MAC 184fp . These results suggest that the S . mansoni polypeptide encoded by the MAC 184 gene, when expressed within a fusion protein, fails to present epitopes normally recognized during natural infection . We propose that these epitopes are conformationally determined and are destroyed when the MAC 184 protein is expressed within beta-galactosidase . This abrogation of conformational epitopes may explain the failure of antibodies from chronically infected or vaccinated mice and rabbits to effectively recognize gene products of certain lambda gt11-fusion protein clones.

Genetics, 1985 Oct, 111(2), 233 - 41
DNA sequences of frameshift and other mutations induced by ICR-170 in yeast; Ernst JF et al.; ICR-170-induced mutations in the CYC1 gene of the yeast Saccharomyces cerevisiae were investigated by genetic and DNA sequence analyses . Genetic analysis of 33 cyc1 mutations induced by ICR-170 and sequence analysis of eight representatives demonstrated that over one-third were frameshift mutations that occurred at one site corresponding to amino acid positions 29-30, whereas the remaining mutations were distributed more-or-less randomly, and a few of these were not frameshift mutations . The sequence results indicate that ICR-170 primarily induces G.C additions at sites containing monotonous runs of three G.C base pairs . However, some (Formula: see text) sites within the CYC1 gene were not mutated by ICR-170 . Thus, ICR-170 is a relatively specific mutagen that preferentially acts on certain sites with monotonous runs of G.C base pairs.

Cell, 1985 Oct, 42(3), 959 - 66
Alternative conformations of the ColE1 replication primer modulate its interaction with RNA I; Wong EM et al.; Replication of the ColE1 plasmid is regulated by the interaction of its primer RNA with a small countertranscript (RNA I) that acts as a repressor of functional primer formation . The interaction is dependent on the specific conformations of the complementary RNA molecules . Early in its synthesis, primer adopts an "anti-RNA I" configuration . As transcription proceeds, it is preempted by formation of an alternative domain designated stem-loop IV . This conformational transition has a significant effect on the rate of association of RNA I with the primer in vitro . Nascent primer in the "anti-RNA I" conformation (135 nucleotides) interacts with RNA I 6-fold faster than primer in the stem-loop IV conformation (241 nucleotides), and 35-fold faster than a 567 nucleotide primer precursor . We propose that a conformation-dependent "window of susceptibility" of primer to RNA I exists during primer transcription, and that altered conformations play a role in modulating the rate of functional primer formation.

Br J Pharmacol, 1985 Oct, 86(2), 399 - 403
The effects of the protease inhibitor, aprotinin, on the course of shock induced by endotoxin in cats; Hughes B et al.; The administration of endotoxin derived from Escherichia coli to anaesthetized cats resulted, within the first 5 min, in an initial increase in right atrial pressure and a reduction in systemic arterial blood pressure . Over the next 2 h shock was characterized by a reduced cardiac output, tachycardia, reduced arterial pH and an increased level of lactate . The survival rate at the end of the 8 h experimental period was only 10% . The protease inhibitor aprotinin (Trasylol), given as a continuous intravenous infusion 1000 kallikrein inhibitor units (k.i.u.) kg-1h-1 together with a bolus of 40,000 k.i.u . kg-1, significantly inhibited the severity and incidence of the initial endotoxin response (increase in right atrial pressure and systemic hypotension), perhaps suggesting the direct or indirect involvement of kinins . Aprotinin did not reduce the delayed effects of endotoxin (sustained reduction in cardiac output, lacticacidosis), nor did it improve survival at 8 h . Indeed, there was some evidence that aprotinin exaggerated the delayed effects of endotoxin in this model.

Arch Biochem Biophys, 1985 Oct, 242(1), 263 - 8
Methylglyoxal bis(guanylhydrazone) elimination of polyamine effects on protein synthesis; Ohnishi R et al.; The effect of methylglyoxal bis(guanylhydrazone) (MGBG), a structural analog of polyamines, on protein synthesis has been studied in the presence and absence of spermidine . The spermidine stimulation of polyphenylalanine- and MS2 RNA-directed RNA replicase synthesis in an Escherichia coli cell-free system and of globin synthesis in a rabbit reticulocyte cell-free system disappeared with the addition of MGBG . The spermidine reduction of misincorporation of leucine during polyphenylalanine synthesis in both E . coli and wheat germ cell-free systems was also disturbed by MGBG . MGBG noncompetitively interfered with polyamine stimulation of polyphenylalanine and globin synthesis, suggesting that MGBG could bind to both RNA and the complex of RNA and polyamine . MGBG was preferentially bound to ribosomal RNA among ribosomal RNA, poly(U), and calf thymus DNA, and strongly inhibited the amount of polyamine bound to ribosomal RNA . These results suggest that MGBG elimination of polyamine effects on protein synthesis may occur through the disturbance of polyamine binding to ribosomal RNA.

Proc Natl Acad Sci U S A, 1985 Oct, 82(19), 6427 - 30
RNase T is responsible for the end-turnover of tRNA in Escherichia coli; Deutscher MP et al.; A mutant strain deficient in RNase T was isolated and used to study the role of this enzyme in Escherichia coli . Strains lacking as much as 70% of RNase T activity, alone or in combination with the absence of other RNases, display normal growth properties . However, in cca strains, which lack tRNA nucleotidyltransferase, RNase T-deficient derivatives accumulate lower levels of defective tRNA and grow at increased rates compared to their RNase T+ parents . Slow-growing cca strains revert to a faster-growing form that contains less defective tRNA but which is still cca . All of these strains have decreased levels of RNase T . These data indicate that RNase T is responsible for nucleotide removal during the tRNA end-turnover process and that the amount of defective tRNA in cells is determined by the relative levels of RNase T and tRNA nucleotidyltransferase.

Infect Immun, 1985 Oct, 50(1), 333 - 5
Shwartzman reaction in germfree rabbits; Ito M; The local Shwartzman reaction occurred in germfree rabbits which had no natural antibody to endotoxin and none or only a very small amount of immunoglobulin G . From the results it was concluded that the presence of natural antibody to endotoxin is not a prerequisite of the production of the local Shwartzman reaction by bacterial endotoxin.

Infect Immun, 1985 Oct, 50(1), 279 - 83
Nucleotide sequences of four variants of the K88 gene of porcine enterotoxigenic Escherichia coli; Dykes CW et al.; The nucleotide sequences of four variants of the Escherichia coli K88 antigen gene, K88ab1, K88ab2, K88ac, and K88ad, have been determined . The K88ab2 and K88ac sequences have not been reported previously . The K88ab1 sequence is very similar to that determined by other workers, but the K88ad sequence differs considerably from that described in a previous report . Comparison of the amino acid sequences inferred from the gene sequences revealed certain clusters of amino acid substitutions which have been correlated with areas of potential antigenicity in the mature proteins.

Biochem Biophys Res Commun, 1985 Sep 30, 131(3), 1277 - 83
Induction of mutation in vitro in phage phi X174 am3 by N4-aminodeoxycytidine triphosphate; Takahashi M et al.; When phi X174 am3-phage-infected E . coli is treated with N4-aminocytidine, reversion of the phage to the wild type is efficiently induced . The mechanism of this reversion is considered to consist of metabolic conversion of N4-aminocytidine into its deoxynucleoside 5'-triphosphate followed by incorporation of the nucleotide into the replicating phage DNA, thereby causing AT-to-GC transition at the am3 locus . The second half of this mechanism has now been experimentally proved, using an in vitro mutagenesis system . Thus, by nick-translation, N4-aminodeoxycytidine 5'-triphosphate was incorporated into the replicative form of phi X174 am3 DNA, and the DNA was used to transfect CA++-treated E . coli HF4714 (sup+) . The reversion frequency of the phage produced was up to one-order of magnitude greater than that of the control in which the nick-translation had been done without the addition of N4-aminodeoxycytidine triphosphate . This nucleotide analog may be useful as a reagent for in vitro site-directed mutagenesis.

J Biol Chem, 1985 Sep 25, 260(21), 11659 - 62
19F nuclear magnetic resonance studies of communication between catalytic and regulatory subunits in aspartate transcarbamoylase; Wacks DB et al.; 19F nuclear magnetic resonance (NMR) spectroscopy was used to study "communication" between the catalytic and regulatory subunits in aspartate transcarbamoylase of Escherichia coli . Hybrid enzymes composed of fluorotyrosine-labeled regulatory subunits and native catalytic subunits or of native regulatory subunits and fluorotyrosine-labeled catalytic subunits were constructed and shown to have the allosteric kinetic properties of native enzyme . These hybrids exhibited the ligand-promoted "global" conformational changes characteristic of native aspartate transcarbamoylase and alterations in the NMR spectrum when ligands bind to the active site . The NMR difference spectrum caused by the binding of the bisubstrate analog N-(phosphonacetyl)-L-aspartate to the hybrid containing 19F-labeled regulatory chains consisted of two troughs and a peak, suggesting that two tyrosines in the regulatory polypeptide chains were affected by the binding of ligand to the catalytic subunits . The increase in magnitude of the peak appeared to depend directly on the fractional saturation of the active sites . A peak with two distinct shoulders was observed in the 19F NMR spectrum of the hybrid containing fluorotyrosine in the catalytic chains when it was saturated with the ligand, whereas the spectrum for the unliganded enzyme consisted of a single peak . The NMR difference spectrum showed that the bisubstrate ligand perturbed at least two resonances, and these changes appeared to be tightly linked to the binding of the ligand.

J Biol Chem, 1985 Sep 25, 260(21), 11651 - 8
19F nuclear magnetic resonance studies of fluorotyrosine-labeled aspartate transcarbamoylase . Properties of the enzyme and its catalytic and regulatory subunits; Wacks DB et al.; Aspartate transcarbamoylase labeled with 3-fluorotyrosine was purified from an Escherichia coli strain which was auxotrophic for tyrosine and overproduced aspartate transcarbamoylase upon uracil starvation . The labeled enzyme in which about 85% of the tyrosines were replaced by fluorotyrosine exhibited high enzyme activity that varied in a sigmoidal manner with respect to the aspartate concentration . Also, the labeled enzyme was inhibited by CTP, activated by ATP, and exhibited a 2.6% decrease in sedimentation coefficient upon the addition of the active-site ligand, N-(phosphonacetyl)-L-aspartate . Thus, despite extensive replacement of tyrosines by fluorotyrosine, the modified enzyme was similar to native aspartate transcarbamoylase . The 19F nuclear magnetic resonance spectrum of isolated regulatory subunits labeled with fluorotyrosine consisted of a single peak . Addition of the activator, ATP, or the inhibitor, CTP, caused a loss of intensity at about 61.3 ppm upfield from a trifluoroacetic acid reference and an increase at about 61.5 ppm, but CTP also caused an increase at about 61.0 ppm . Five overlapping resonances were observed in the 19F NMR spectrum of unliganded catalytic subunits containing fluorotyrosine . Although the binding of the bisubstrate analog, N-(phosphonacetyl)-L-aspartate, or the combination of carbamoylphosphate and succinate caused similar disappearances of resonances, the addition of N-(phosphonacetyl)-L-aspartate caused the appearance of resonances not observed with carbamoylphosphate plus succinate . Carbamoylphosphate alone perturbed three or four resonances and the subsequent addition of succinate affected at least two.

J Biol Chem, 1985 Sep 25, 260(21), 11438 - 41
Escherichia coli DNA photolyase reverses cyclobutane pyrimidine dimers but not pyrimidine-pyrimidone (6-4) photoproducts; Brash DE et al.; The effect of purified Escherichia coli DNA photolyase on the UV light-induced pyrimidine-pyrimidone (6-4) photoproduct and cyclobutane pyrimidine dimer was investigated in vitro using enzyme purified from cells carrying the cloned phr gene (map position, 15.7 min) . Photoproducts were examined both as site-specific lesions in end-labeled DNA and as chromatographically identified products in uniformly labeled DNA . E . coli DNA photolyase removed cyclobutane dimers but had no activity on pyrimidine-pyrimidone (6-4) photoproducts . Photoreactivation can therefore be used to separate the biological effects of these two UV light-induced molecular lesions.

J Biol Chem, 1985 Sep 25, 260(21), 11845 - 51
The pairing activity of stable nucleoprotein filaments made from recA protein, single-stranded DNA, and adenosine 5'-(gamma-thio)triphosphate; Honigberg SM et al.; Under conditions that diminish secondary structure in single-stranded DNA, stable presynaptic filaments can be formed by recA protein in the presence of the nonhydrolyzable analog ATP gamma S, without the need for Escherichia coli single strand binding protein . Such stable presynaptic filaments resemble those formed in the presence of ATP and pair efficiently with homologous duplex DNA . Since this kind of stable filament does not displace a strand from the duplex molecule, it provides a model substrate to study synapsis independent of the earlier and later stages of the recA reaction . Even though detectable strand displacement did not occur in the presence of ATP gamma S, both single strand and double strand breaks in duplex DNA stimulated homologous pairing . These and related observations support the view that the presynaptic nucleoprotein filament and naked duplex DNA intertwine to form a nascent joint in which the duplex DNA is partially unwound, i.e . in which the pitch of the involved duplex segment is reduced.

Nucleic Acids Res, 1985 Sep 25, 13(18), 6439 - 45
Termination of a transcription unit comprising highly expressed genes in the archaebacterium Methanococcus voltae; Muller B et al.; The 3'termini of transcripts originating from genes organized in a highly expressed transcription unit were analyzed in the archaebacterium Methanococcus voltae . The putative termination signals were found in an AT-rich intergenic region following the 3'-terminal gene . The two detected signals both contain oligo(T) sequences . A possible stem/loop structure immediately precedes one of the oligo(T) tracts . This secondary structure is considered to have an additional function in stabilizing the transcripts.

J Biol Chem, 1985 Sep 25, 260(21), 11744 - 8
5'-Flanking DNA of the rat growth hormone gene mediates regulated expression by thyroid hormone; Casanova J et al.; Thyroid hormone has been shown to rapidly stimulate the rate of rat growth hormone gene transcription which parallels the kinetics of binding of 3,5,3'-triiodo-L-thyronine (L-T3) to its nuclear receptor (Yaffe, B . M., and Samuels, H . H . (1984) J . Biol . Chem . 259, 6284-6291) . We have constructed a chimeric gene to explore whether the 5'-flanking region of the rat growth hormone gene contains a DNA element which could mediate thyroid hormone control of growth hormone gene expression . The construct consists of 1.8 kilobase pairs of the 5'-flanking region extending 11 nucleotides downstream from the transcription initiation (cap) site ligated to Escherichia coli DNA containing the structural gene for the enzyme xanthine-guanine phosphoribosyltransferase . GC cells, a growth hormone producing rat pituitary cell line, were transfected with this chimeric gene and stable transformants in which the enzyme is regulated by L-T3 were isolated by positive selection using mycophenolic acid and xanthine . These stable transformants develop with relatively high frequency and show marked L-T3 stimulation of xanthine-guanine phosphoribosyltransferase mRNA which is initiated at the cap site of the growth hormone gene . This study provides the first evidence that the 5'-flanking region of the rat growth hormone gene contains a DNA regulatory element which can mediate control of gene expression by thyroid hormone.

Biochemistry, 1985 Sep 24, 24(20), 5343 - 50
Positional isotope exchange and kinetic experiments with Escherichia coli guanosine-5'-monophosphate synthetase; von der Saal W et al.; The kinetic mechanism of Escherichia coli guanosine-5'-monophosphate synthetase has been determined by utilizing initial velocity kinetic patterns and positional isotope exchange experiments . The initial velocity patterns of MgATP, XMP, and either NH3 or glutamine (as nitrogen source) were consistent with the ordered addition of MgATP followed by XMP and then NH3 . The enzyme catalyzes the exchange of 18O from the beta-nonbridge positions of {beta,beta,beta gamma,gamma,gamma,gamma-18O6}ATP into the alpha beta-bridge position only in the presence of XMP and Mg2+ . The exchange reaction did not require NH3 . The isotope exchange reaction increased as the XMP concentration increased and then decreased at saturating levels of XMP . These results also support the ordered addition of MgATP followed by XMP . GMP synthetase catalyzes the hydrolysis of ATP to AMP and PPi along with an ATP/PPi exchange reaction in the absence of NH3 . These data taken together support a mechanism in which the initial step in the enzymatic reaction involves formation of an adenyl-XMP intermediate . Psicofuranine, an irreversible inhibitor of the enzyme, acts by preventing the release or further reaction of adenyl-XMP with H2O or NH3 but does not suppress the isotope exchange or ATP/PPi exchange reactions . GMP synthetase has also been shown to require a free divalent cation for full activity . When Ca2+ replaces Mg2+ in the reaction, the positional isotope exchange reaction is enhanced but the reaction with NH3 to form GMP is greatly suppressed.

Biochemistry, 1985 Sep 24, 24(20), 5286 - 9
DNA methylation diminishes bleomycin-mediated strand scission; Hertzberg RP et al.; Three DNA duplexes differing substantially in sequence were derived from pBR322 plasmid DNA and supercoiled SV40 DNA by digestion with appropriate restriction endonucleases . Following treatment with the restriction methylase HhaI (recognition sequence: GCGC) or HhaI and HpaII (CCGG), the unmethylated and methylated DNAs were compared as substrates for the antitumor agent bleomycin . Bleomycin-mediated strand scission was shown to diminish substantially at a number of sites in proximity to the methylated cytidine moieties, especially where multiple sites had been methylated within a DNA segment of limited size . Detailed analysis of the DNA substrates revealed that both strands of DNA within a methylated region became more refractory to cleavage by bleomycin and that the protective effect could extend as many as 14 base pairs in proximity to the 5-methylcytidine moieties . Among the methylated DNA segments that became more resistant to bleomycin cleavage was a HpaII site of SV40 DNA, methylation of which has previously been shown to diminish the synthesis of the major late viral capsid protein following microinjection into Xenopus laevis oocytes . Study of the cleavage reaction at varying salt levels suggested that diminished bleomycin strand scission may be due, at least in part, to local conformational changes of the DNA to Z form (or other non-B-form structures) . The results are generally consistent with the hypothesis that one mechanism for the expression of selective therapeutic action by certain DNA damaging agents could involve the recognition of specific methylation patterns.

J Mol Biol, 1985 Sep 20, 185(2), 295 - 309
Intermediates in homologous pairing promoted by recA protein . Isolation and characterization of active presynaptic complexes; Tsang SS et al.; recA protein promotes homologous pairing and strand exchange by an ordered reaction in which the protein first polymerizes on single-stranded DNA . This presynaptic intermediate, which can be formed either in the presence or absence of Escherichia coli single-stranded binding protein (SSB), has been isolated by gel filtration and characterized . At saturation, purified complexes contained one molecule of recA protein per 3.6 nucleotide residues of single-stranded DNA . Complexes that had been formed in the presence of SSB contained up to one molecule of SSB per 15 nucleotide residues, but the content of SSB in different preparations of isolated complexes appeared to be inversely related to the content of recA protein . Even when they have lost as much as a third of their recA protein, presynaptic complexes can retain activity, because the formation of stable joint molecules depends principally on the binding of recA protein to the single-stranded DNA in the localized region that corresponds to the end of the duplex substrate.

J Mol Biol, 1985 Sep 20, 185(2), 261 - 72
Partition of unit-copy miniplasmids to daughter cells . III . The DNA sequence and functional organization of the P1 partition region; Abeles AL et al.; The boundaries of the P1 par (plasmid partition) region of the unit-copy plasmid P1 were defined to within 2.7 X 10(3) base-pairs of DNA . The DNA sequence of the region revealed two large open reading frames that could encode proteins of Mr 44,000 and Mr 38,000 . Both would be read in the same direction . The first open reading frame corresponds to the par A gene, the Mr 44,000 protein product of which was shown to be trans acting and essential for partition . The second open reading frame (parB) follows closely and may be cotranscribed with par A . The codon usage frequency for parB is consistent with its producing a protein product . The ParB protein was identified in cell extracts as a product with an apparent Mr of 45,000, suggesting that it behaves anomolously on gel electrophoresis . Following parB is the incB region, an incompatibility determinant thought to be the cis acting site that constitutes the putative attachment point on the DNA for the cellular partition apparatus . Subcloning of this site showed it to consist of a maximum of 174 base-pairs . The incB sequence is highly A + T-rich and contains a 20 base-pair inverted repeat . Another A + T-rich inverted repeat of similar size but different sequence is found between the putative parA promoter and the ribosome initiation sequence at the start of the parA open reading frame and may be involved in the autoregulation of ParA synthesis . The par region appears to contain a functional analog of the centromere of eukaryotic chromosomes . It is responsible for ensuring that newly replicated plasmids are properly distributed to daughter cells during cell division of its Escherichia coli host.

J Mol Biol, 1985 Sep 20, 185(2), 431 - 43
Substrate specificity of the DNA unwinding activity of the RecBC enzyme of Escherichia coli; Taylor AF et al.; The RecBC enzyme of Escherichia coli promotes genetic recombination of phage or bacterial chromosomes . The purified enzyme travels through duplex DNA, unwinding and rewinding the DNA with the transient production of potentially recombinogenic single-stranded DNA . The studies reported here are aimed at understanding which chromosomal forms allow the entry of RecBC enzyme and hence may undergo RecBC enzyme-mediated recombination . Circular duplex molecules, whether covalently closed, nicked or containing single-stranded gaps of 10 to 774 nucleotides, are not detectably unwound by RecBC enzyme . Linear duplex molecules are readily unwound if they have a nearly flush-ended terminus whose 5' and 3' ends are offset by no more than about 25 nucleotides; molecules with longer single-stranded tails are poorly bound by RecBC enzyme and are infrequently unwound . The single-strand endonuclease activity of RecBC enzyme can slowly cleave gapped circles to produce molecules presumably capable of being unwound . These results provide an enzymatic basis for the recombinogenicity of double-stranded DNA ends established from genetic studies of RecBC enzyme and Chi sites, recognition sites for RecBC enzyme-mediated DNA strand cleavage.

Nature, 1985 Sep 19-25, 317(6034), 267 - 70
Sequence of protein disulphide isomerase and implications of its relationship to thioredoxin; Edman JC et al.; The formation of disulphide bonds is essential to the structure and function of proteins . These bonds rapidly form either cotranslationally or immediately post-translationally in the lumen of the endoplasmic reticulum . Native disulphide pairing for such proteins has been achieved in vitro; however, the rates of reassembly are slow and the conditions non-physiological . To account for these observations, Anfinsen et al . proposed that a 'disulphide interchange protein' was the in vivo catalyst of disulphide bond rearrangement . Other groups discovered an activity with similar characteristics that catalysed the reductive cleavage of insulin and may be associated with insulin degradation, although this result has been disputed . The enzyme involved, protein disulphide isomerase (PDI; EC 5.3.4.1), may be the in vivo catalyst of disulphide bond formation . Here we describe the sequence of cloned rat liver PDI complementary DNA which predicts a protein with two distinct regions homologous with Escherichia coli thioredoxin, a known cofactor in oxidation-reduction reactions . Each of these regions contains the presumed active site sequence Trp-Cys-Gly-His-Cys-Lys, suggesting that PDI, similar in action to thioredoxin, catalyses disulphide bond interchange via an internal disulphide-sulphydryl interchange . The cDNA predicts a signal peptide consistent with the view that PDI is a luminal endoplasmic reticulum protein . PDI messenger RNA, although ubiquitous, is more highly concentrated in secretory cells.

Biochem Biophys Res Commun, 1985 Sep 16, 131(2), 675 - 80
m-Fluorotyrosine substitution in beta-galactosidase; evidence for the existence of a catalytically active tyrosine; Ring M et al.; The pH profiles of beta-galactosidase, having tyr replaced by m-fluorotyrosine, were compared to those of normal enzyme . The inflection point on the alkaline side was lowered about 1.5 pH units in the fluoro-enzyme, corresponding to the difference in the phenolic pKa values of m-fluorotyrosine and tyr . When glycosidic bond breakage was rate-limiting, the Vm at pH 7.0 was higher for the fluoro-enzyme . When hydrolysis was rate-limiting or when acceptors which made transgalactosylis rate-limiting were used, the Vm was lower for the fluoro-enzyme . This shows that a tyr in beta-galactosidase is a general-acid catalyst in the glycosidic bond breaking reaction and a tyr (probably the same one) is a general-base catalyst in the hydrolytic reaction.

Biochem Biophys Res Commun, 1985 Sep 16, 131(2), 994 - 1002
Control of isoleucine-valine biosynthesis in a valine-resistant mutant of Escherichia coli K-12 that simultaneously acquired azaleucine-resistance; Williams AL et al.; A mutant of Escherichia coli K-12 isolated as being growth resistant to L-valine (Valr) was shown also to exhibit growth resistance to 4-azaleucine (Azlr) . Transductional analysis indicated that Azlr is cotransduced with Valr at a frequency of 100% and both are linked to leu, ara, and carA . This mutation conferring valine and azaleucine growth resistance resulted in increased levels of isoleucine and valine biosynthetic enzymes as well as those of valyl- and isoleucyl-tRNA synthetases during growth in minimal and enriched media . Acquisition of Vals/Azls results in the restoration of normal regulation of both classes of ilv enzymes and normal patterns of the tRNA Ile species . The overall regulatory patterns observed for individual isoleucine and valine gene products suggest differential participation of isoleucine and valine and/or isoleucyl- and valyl-tRNA's in control of expression of the respective structural genes.

Eur J Biochem, 1985 Sep 16, 151(3), 521 - 4
Reversible dissociation of aspartokinase I/homoserine dehydrogenase I from Escherichia coli K 12 . The active species is the tetramer; Veron M et al.; Dimers of aspartokinase I/homoserine dehydrogenase I from Escherichia coli K 12 have been isolated under very mild conditions . The dimers which cannot be distinguished from the tetramers by their kinetic properties, reassociate in the presence of potassium ions or L-aspartate . The selective sensitivity of aspartokinase I/homoserine dehydrogenase I to mild proteolytic digestion of dimers has been used to probe the reassociation reaction under the conditions of aspartokinase assay . We demonstrate that rapid reassociation occurs and that the protein species present in the assay when dimers are used to test the activity is tetrameric . These results confirm the previously proposed model for the subunit association of aspartokinase I/homoserine dehydrogenase I.

Biochem Biophys Res Commun, 1985 Sep 16, 131(2), 780 - 5
Possible mechanism of the allosteric activation of cAMP receptor protein; Kypr J et al.; Secondary structure of cAMP receptor protein of E . coli was predicted and compared to its crystal structure in the complex with cAMP solved by McKay and Steitz . The two conformations coincide in the DNA binding domain but strikingly differ in the other domain which binds cAMP and causes protein dimerization . The comparison indicates that cAMP destabilizes a very long helix instead of which sheets are formed creating a hydrophobic pocket where cAMP binds . Consequently, the helix-sheets isomerization and a resulting change in the relative monomer disposition in the dimer appears to be the origin of cAMP-induced allosteric activation of the protein . Extremely long helices were also predicted in the regions of the regulatory subunit of cAMP-dependent protein kinase from bovine cardiac muscle where cAMP binds . It is thus likely that the proposed mechanism of the effect of cAMP on protein structure has wider implications.

Biochem Biophys Res Commun, 1985 Sep 16, 131(2), 756 - 62
Detection of a tetranuclear iron-sulfur center in fumarate reductase from Escherichia coli by electron paramagnetic resonance spectroscopy; Johnson MK et al.; Soluble fumarate reductase and fumarate reductase complex from Escherichia coli have been investigated by electron paramagnetic resonance spectroscopy . Both succinate- and dithionite-reduced samples show signals associated with a {2Fe-2S}1+ cluster that account maximally for slightly more than one spin/molecule . In addition, at temperatures below 20 K, dithionite-reduced samples exhibit broad and complex features, to high and low field of the {2Fe-2S}1+ signal, that are attributable to a spin coupled {4Fe-4S}1+ cluster . Preliminary attempts to quantify the signals indicate that the {4Fe-4S} cluster is present in an approximate 1:1 stoichiometry with the {2Fe-2S} cluster . The observed enhancement of the spin relaxation of the {2Fe-2S}1+ cluster on dithionite reduction is attributed to spin-spin interaction between the S = 1/2, reduced tetranuclear and binuclear clusters.

Biochem Biophys Res Commun, 1985 Sep 16, 131(2), 653 - 8
In vivo detection of a three iron cluster in fumarate reductase from Escherichia coli; Johnson MK et al.; Escherichia coli with plasmid amplified expression of fumarate reductase was grown anaerobically on a medium containing fumarate and glycerol and investigated by electron paramagnetic resonance spectroscopy . Anaerobically harvested cells exhibit an EPR signal characteristic of a reduced {2Fe-2S} cluster . Anaerobic addition of fumarate results in diminution of the reduced {2Fe-2S} signal and the appearance of the EPR signal associated with the oxidized 3Fe cluster . The results provide the first evidence for a trinuclear iron-sulfur cluster that exists in vivo, and suggest that the 3Fe cluster in purified fumarate reductase samples is not an artifact of the isolation procedure . The significance of this observation is discussed in relation to the physiological relevance of trinuclear iron-sulfur clusters.

Eur J Biochem, 1985 Sep 16, 151(3), 573 - 7
Comparison of the nucleotide sequences of the genes encoding the KS71A and F7(1) fimbrial antigens of uropathogenic Escherichia coli; Rhen M et al.; DNA fragments encompassing the genes for the KS71A and F7(1) fimbrial subunits of Escherichia coli strains KS71 (O4:K12) and AD110 (O6:K2), respectively, have been subjected to DNA sequencing . The nucleotide sequences of the two fimbrillin genes were identical and they encode a polypeptide of 187 amino acids of which 21 amino acids probably will constitute the signal sequence . The primary structure of these fimbrillins showed significant homology with the primary structure of other E . coli fimbrillins.

Eur J Biochem, 1985 Sep 16, 151(3), 505 - 14
The domain structure of tryptophan synthase . A neutron scattering study; Ibel K et al.; Tryptophan synthase from Escherichia coli is a complex of two alpha subunits and two beta subunits . Small-angle neutron scattering involving deuterium-labelled isomers revealed the quaternary structure of the enzyme at the level of the beta 2 subunit and the two structural domains P1 and P2 which constitute the alpha subunits . Within the alpha 2 beta 2 complex, the two alpha subunits are completely separated . They are situated on opposite sides of the beta 2 subunit . The most probable distance between the two alpha protomers is 10.5 +/- 1 nm; the nearest distance is 5.8 +/- 0.5 nm, and the largest distance is 13.5 +/- 0.5 nm . The two domains of the same alpha subunit are intimately juxtaposed . The distances between two like or unlike domains belonging to opposite alpha subunits are roughly equal . All domains exhibit about equal distances to the beta 2 subunit which is situated in the centre of the complex . Thus the cleft between P1 and P2, which probably contains the active site of the alpha subunit, makes intimate contact with the beta 2 subunit . Neutron scattering allows us to determine the shape of the beta 2 subunit within the complex . Comparison with the free dimer suggests a conformational change, upon assembly, from an elongated into a more compact form.

Biochem Biophys Res Commun, 1985 Sep 16, 131(2), 1033 - 40
The differential effects produced by daunomycin and adriamycin on RNA, polynucleotides, single stranded, supercoiled DNA, and nucleosomes; Pearlman LF et al.; Terbium, a sensitive probe whose fluorescence is strongly enhanced when bound to unpaired guanine and xanthine bases, has been employed to study the effects of adriamycin and daunomycin on a variety of nucleotide substrates . After treatment with either drug at concentrations of less than or equal to 1:500, the fluorescence of the probe was substantially abrogated . Daunomycin, however, produced a markedly greater effect than adriamycin with rRNA, linear calf thymus DNA, and polyriboguanylic acid . The difference between the drugs was experimentally significant, suggesting that changing the C9 side group from a methyl (daunomycin) to an alcohol (adriamycin) may result in a changed base sequence specificity . The distinction was also evident when changes in electrophoretic mobility of supercoiled and nucleosomal DNA was monitored, but only at much higher (1:25) drug:DNA ratios.

Experientia, 1985 Sep 15, 41(9), 1188 - 90
Alternariol, a dibenzopyrone mycotoxin of Alternaria spp., is a new photosensitizing and DNA cross-linking agent; DiCosmo F et al.; The mycotoxin alternariol (3,4',5-trihydroxy-6'-methyldibenzo {a} pyrone) but not alternariol monomethyl ether (3,4'-dihydroxy-5-methoxy-6'-methyldibenzo {a} pyrone) is phototoxic to Escherichia coli in the presence of near UV light (320-400 nm) . The phototoxicity bioassays with a DNA repair-deficient mutant of E . coli suggested that DNA may be the molecular target for photo-induced toxicity of alternariol . Interactions between alternariol and double-stranded, supercoiled DNA suggest that alternariol interacts with DNA by intercalation . No DNA breakage was detected in this system; however, alternariol forms a complex and cross-links double-stranded DNA in near UV light . These results suggest that alternariol is a new phototoxic, DNA-intercalating agent and is a DNA cross-linking mycotoxin in near UV light.

J Biol Chem, 1985 Sep 15, 260(20), 10986 - 90
Reconstitution of the Ubiquinone-dependent pyruvate oxidase system of Escherichia coli with the cytochrome o terminal oxidase complex; Carter K et al.; The aerobic respiratory chain of Escherichia coli is branched and contains two terminal oxidases . The chain predominant when the cells are grown with low aeration terminates with the cytochrome d terminal oxidase complex, and the branch present under high aeration ends with the cytochrome o terminal oxidase complex . Previous work has shown that cytochrome d complex functions as a ubiquinol-8 oxidase, and that a minimal respiratory chain can be reconstituted in proteoliposomes with a flavoprotein dehydrogenase (pyruvate oxidase), ubiquinone-8, and the cytochrome d complex . This paper demonstrates that the cytochrome o complex functions as an efficient ubiquinol-8 oxidase in reconstituted proteoliposomes, and that ubiquinone-8 serves as an electron carrier from the flavoprotein to the cytochrome complex . The maximal turnover (per cytochrome o) achieved in reconstituted proteoliposomes is at least as fast as observed in E . coli membrane preparations . Electron flow from the flavoprotein to oxygen in the reconstituted proteoliposomes generates a transmembrane potential of at least 120 mV, negative inside, which is sensitive to ionophore uncouplers and inhibitors of the terminal oxidase . These data demonstrate the minimal composition of this respiratory chain as a flavoprotein dehydrogenase, ubiquinone-8, and the cytochrome o complex . Previous models have suggested that cytochrome b556, also a component of the E . coli inner membrane, is required for electron flow to cytochrome o . This is apparently not the case . It now is clear that both of the E . coli terminal oxidases act as ubiquinol-8 oxidases and, thus, ubiquinone-8 is the branch point between the two respiratory chains.

J Biol Chem, 1985 Sep 15, 260(20), 11001 - 5
Effect of ATP on phosphofructokinase-2 from Escherichia coli . A mutant enzyme altered in the allosteric site for MgATP; Guixe V et al.; The activity of Escherichia coli phosphofructokinase-2 (Pfk-2) and of the mutant enzyme Pfk-2* was measured over a wide range of Mg2+ and ATP concentrations . MgATP2- inhibited only the Pfk-2 enzyme, with a degree of cooperativity of 1.5 . This inhibition was relieved upon increasing the fructose-6-P concentration or by lowering the pH of the reaction mixture . Other nucleotides used as phosphate donors instead of ATP did not inhibit . MgATP2- was the true substrate for both enzymes and their Km values for this compound were not affected by an increase of the free Mg2+ concentration . However, free Mg2+ partially relieved the MgATP2- inhibition of Pfk-2 under conditions where the ATP4- concentration was negligible, without changes in the degree of cooperativity . ATP4- acted as a strong competitive inhibitor of both Pfk-2 and Pfk-2* with respect to MgATP2- with Ki values of 10 and 8 microM, respectively . ADP, AMP, and cAMP did not prevent the MgATP2- inhibition of Pfk-2 . These results suggest the presence of an allosteric site for MgATP2- in Pfk-2 responsible for the MgATP2- inhibition, which is altered in Pfk-2* as a consequence of the structural mutation.

J Biol Chem, 1985 Sep 15, 260(20), 11207 - 15
The Fo subunits of the Escherichia coli F1Fo-ATP synthase are sufficient to form a functional proton pore; Aris JP et al.; The assembly of the Fo sector of the Escherichia coli ATP synthase has been studied using both structural and functional criteria for assembly . Cross-linking E . coli minicell membranes containing only the Fo subunits a, b, and c with dithiobis(succinimidyl propionate) (DSP) produces b2 and c2 dimers that are generated by cross-linking membranes containing the assembled holoenzyme . Five plasmids carrying the genes specifying the Fo polypeptides in a bacterial strain lacking all of the unc (ATP synthase) genes show a good correlation between Fo function and the amount of the membrane-bound Fo polypeptides . In this report we revise a conclusion reached previously (Klionsky, D.J., Brusilow, W.S.A., and Simoni, R.D . (1983) J . Biol . Chem . 258, 10136-10143) and present evidence that the Fo subunits alone are sufficient to assemble a functional proton pore.

J Biol Chem, 1985 Sep 15, 260(20), 11200 - 6
Assembly of a functional F1 of the proton-translocating ATPase of Escherichia coli; Klionsky DJ et al.; Assembly of the F1 portion of the proton-translocating ATPase of Escherichia coli was examined in vivo . Analysis of strains lacking genes which specify the Fo polypeptides a, b, and c showed that the F1 subunits were able to assemble into a complex in the absence of the Fo subunits . In addition we have investigated the effects of mutations in the individual genes which specify the F1 polypeptides on the assembly process . Mutations of the uncA(alpha), uncG(gamma), or uncD(beta) genes result in a defective assembly of the F1 complex . In contrast, mutations in the uncH(delta) or uncC(epsilon) genes did not prevent assembly of the core alpha beta gamma complex . In these cases, however, the partial F1 complexes were incapable of restoring energy-linked functions to F1-depleted membranes.

Nucleic Acids Res, 1985 Sep 11, 13(17), 6155 - 70
Poly(dAT) dependent trinucleotide synthesis catalysed by wheat germ RNA polymerase II . Effects of nucleotide substrates and cordycepin triphosphate; Dietrich J et al.; Kinetics of condensation of ribonucleotides to dinucleotides, leading to trinucleotide products formation, have been studied using wheat germ RNA polymerase II and poly(dAT) . Assay conditions can be selected under which both ApUpA and UpApU are formed in catalytic amounts . The kinetic parameters associated with these reactions indicate that the rate of trinucleotide formation might be affected by DNA sequence, as reported for E.coli RNA polymerase . Kinetics of disappearance of ApUpA and UpApU were studied under experimental conditions allowing poly(rAU) synthesis . The results can be interpreted as if after formation of a phosphodiester bond, a slow isomerisation step of the ternary transcription complex could occur . During this step, transcription complexes could dissociate with a finite probability, releasing trinucleotides in an abortive pathway . The above results are discussed in the view that, under these experimental conditions, wheat germ RNA polymerase II catalyses poly(rAU) synthesis, as if it is a non-processive enzyme . Cordycepin triphosphate can be condensed to a dinucleotide primer, yielding ApUpA . However the ATP analogue cannot be incorporated into longer products than a trinucleotide . On the other hand 3'-dATP behaves as a very potent inhibitor of translocation, with an inhibition constant of 0.15 microM, a value which is two orders of magnitude smaller than the Km value corresponding to ATP utilization in poly(rAU) synthesis . Simple models are proposed which allow a comparison with E.coli RNA polymerase, for which the results are well documented.

Nucleic Acids Res, 1985 Sep 11, 13(17), 6331 - 42
Elucidation of the mechanism of selective inhibition of mammalian DNA polymerase alpha by 2-butylanilinopurines: development and characterization of 2-(p-n-butylanilino)adenine and its deoxyribonucleotides; Khan NN et al.; 2-(p-n-Butylanilino)adenine (BuAA), an homolog of the DNA polymerase alpha (pol alpha)-specific inhibitor, N2-(p-n-butylphenyl)guanine (BuPG), was transformed to its 2'-deoxyribonucleoside, BuAdA, and the corresponding 2'-deoxyribonucleoside 5'-phosphates, BuAdAMP, BuAdADP, and BuAdATP . All five forms of BuAA are highly selective inhibitors of mammalian pol alpha, and the action of each is subject to specific competitive antagonism by dATP . BuAdADP, and BuAdATP, like the corresponding forms of BuPG, are very potent pol alpha inhibitors, displaying apparent Ki's of less than 3 nanomolar on natural activated templates . BuAdATP, like BuPdGTP, also inhibits pol alpha-catalysed reactions directed by non-complementary, thymine-deficient templates, and it does so via a mechanism subject to specific antagonism by its natural homolog, dATP . The results of the BuAdATP-homopolymer experiments complement those of analogous experiments with BuPdGTP and the dCTP-specific pol alpha inhibitor, aphidicolin, and strengthen the suggestion that mammalian pol alpha contains dNDP and dNTP binding sites which can recognize specific bases without direction by templates.

Nucleic Acids Res, 1985 Sep 11, 13(17), 6223 - 36
A novel transcription property of SP6 and T7 RNA polymerases: dependence on template structure; Schenborn ET et al.; The in vitro synthesis of extraneous RNA sequences by SP6 and T7 RNA polymerases from specific DNA templates is described . Transcription of templates prepared by digestion with restriction enzymes that leave 3' protruding ends resulted in the production of significant amounts of long, template-sized RNA transcripts which hybridized to vector DNA . Sequences copied from the noncoding template strand were among the extraneous transcripts . The presence of these sequences in probe preparations were detected in Southern and RNase protection hybridization assays . In contrast, transcription of DNA templates with blunt or 5' protruding ends yielded few RNA products as extraneous sequences.

Nucleic Acids Res, 1985 Sep 11, 13(17), 6171 - 83
Cloning and characterization of the gene for the yeast cytoplasmic threonyl-tRNA synthetase; Pape LK et al.; A fragment of DNA from the yeast nuclear gene MST1 that codes for the mitochondrial tRNAThr1 synthetase was used as a probe to screen for other yeast threonyl-tRNA synthetase genes . At low stringency, the MST1 probe hybridizes strongly to a 6.6 kb EcoRI fragment of yeast genomic DNA with the homologous gene and in addition hybridizes more weakly to a smaller 3.6 kb EcoRI fragment with a second threonyl-tRNA synthetase gene (THS1) . To clone THS1, a library was constructed by ligation to pUC18 of size selected (3-4.5 kb) EcoRI fragments of genomic DNA . Several clones containing the 3.6 kb EcoRI fragment were isolated . A 2,202 nucleotide long open reading frame corresponding to THS1 has been identified in the cloned fragment of DNA . The predicted protein encoded by THS1 is 38% identical to the E . coli threonyl-tRNA synthetase over the latter's length (642 amino acids) and is 42% identical to the predicted MST1 product over its 462 residues . In situ disruption of the chromosomal copy of THS1 is lethal to the cell, indicating that this gene codes for the cytoplasmic threonyl-tRNA synthetase.

Nucleic Acids Res, 1985 Sep 11, 13(17), 6137 - 54
Altered DNA conformations detected by mung bean nuclease occur in promoter and terminator regions of supercoiled pBR322 DNA; Sheflin LG et al.; Mung bean nuclease was used to probe for recognizable DNA unwinding and unpairing in the plasmid pBR322 . In negatively supercoiled DNA, but not relaxed DNA, cleavages occurred preferentially in non-coding regions of the genome . The types of nucleotide sequences cleaved and which non-coding regions were cleaved depended upon environmental conditions . At 37 degrees C, cleavages occurred in an 84 bp A+T-rich sequence in the terminator region of the ampicillin-resistance gene . Recognition is likely based on a novel DNA conformation which occurs in the longest, most dA+dT-rich region of pBR322 . In the presence of 1 mM Mg2+, cleavages occurred in inverted repeated sequences in the promoter regions of the RNA primer for DNA replication and ampicillin- and tetracycline-resistance genes as well as the terminator of RNA-1 . Potential loops of hairpin (cruciform) structures were cleaved . At 27 degrees C, cleavages occurred near a promoter activated by cAMP receptor protein in vitro and in the 3' non-coding region of the tetracycline-resistance gene . Thus, in supercoiled pBR322 DNA, recognizable DNA unwinding and unpairing occurs preferentially in regulatory regions for transcription and DNA replication.

Nucleic Acids Res, 1985 Sep 11, 13(17), 6105 - 24
The secondary structure of the 7SL RNA in the signal recognition particle: functional implications; Zwieb C; The secondary structure of the 7SL RNA in the signal recognition particle was determined by applying both a theoretical and an experimental approach . The compensatory base change approach was taken comparing the published sequences of human, Drosophila and Xenopus 7SL RNA's . The deduced secondary structure was confirmed by post-labeling of an RNase V1-nicked dog SRP with P32-pCp and RNA-ligase and analysis of the labeled RNA-fragments by non-denaturing/denaturing 2D polyacrylamide gel electrophoresis . Two interesting features in the secondary structure were revealed: Firstly, bases at positions 122 to 127 of the human 7SL RNA are not only able to pair with bases at positions 167 to 170, but also with a single-stranded region of the bases at positions 223 to 228, suggesting an alternative base pairing scheme for the 7SL RNA in all three organisms . In agreement with this finding, four different conformations were identified after transcription of the 7SL RNA from the genomic human clone . The involvement of the particular basepairing interaction postulated was confirmed by the analysis of a 7SL RNA deletion mutant (Sma1-409) . Secondly, a significant sequence homology of the paired bases at positions 236 to 255 and 104 to 109 in 7SL RNA with bases in 5S ribosomal RNA at the positions 84 to 110 was noticed, suggesting that 5S ribosomal and 7SL RNA interact with the same target during protein biosynthesis . These findings are summarized by proposing a mechanism for the translational arrest of protein synthesis by the signal recognition particle using specific sequences and an alternative configuration in the 7SL RNA.

Biochemistry, 1985 Sep 10, 24(19), 5062 - 70
Detection of high-affinity intercalator sites in a ribosomal RNA fragment by the affinity cleavage intercalator methidiumpropyl-EDTA-iron(II); Kean JM et al.; The affinity cleavage reagent methidiumpropyl-EDTA (MPE) {Hertzberg, R . P., & Dervan, P . B . (1982) J . Am . Chem . Soc . 104, 313-315} intercalates between base pairs in helical DNA and, when complexed with Fe(II), cleaves the DNA by oxidative degradation of the deoxyribose . We find that this reagent is useful for mapping structure in some RNA molecules . The reagent binds to poly(A)-poly(U) with the same or slightly lower affinity as the related ethidium intercalator, selectively binds double-helical in preference to single-stranded RNA, and when complexed with Fe(II) readily cleaves the RNA backbone . The reagent binds to three or four helical locations in tRNAPhe with an affinity of 10(5)-10(6) M-1 (0.1 M Na+, pH 7.6, 37 degrees C) . With a 345-base RNA fragment covering the S8/S15 protein binding region of Escherichia coli 16S ribosomal RNA, MPE-Fe(II) intercalates strongly at two helical sites: one is located at or near a single base bulge and the other at the end of a helix . Intense cutting is also seen in a region that is not part of a Watson-Crick helix . Ethidium bromide binds at these sites with high affinity (about 10(7) M-1 at 0.1 M Na+, pH 7.6, 37 degrees C) . The sites are all clustered in a region of the RNA thought to bind S15 . Tertiary folding of the RNA may distort helices in the molecule to create sites with particularly high affinities for intercalators; such sites may have functional significance in protein recognition or RNA-RNA interactions.

Biochemistry, 1985 Sep 10, 24(19), 5077 - 83
Preparation and characterization of various Escherichia coli RNA polymerases containing one or two intrinsic metal ions; Solaiman D et al.; The Escherichia coli DNA-dependent RNA polymerase (RPase) holoenzyme (alpha 2 beta beta' sigma) possesses 2 mol equiv of Zn: beta and beta' subunits each contain one Zn ion . An in vitro metal-substitution method developed earlier (method I) was used to remove the two intrinsic Zn ions and then to reconstitute other metal ions into the beta subunit of RPase . One Cd or Hg ion was successfully reconstituted into half-active enzymes (rec-Cd1- or rec-Hg1-RPase), while Mn or Ni ion was not incorporated . A new, simplified in vitro metal-substitution method (method II), which omitted the low-pH treatment and subsequent urea dialysis in method I, was devised in this study . Consequently, Zn or Cd could be incorporated into both the beta and beta' subunits, resulting in rec-Zn2- or rec-Cd2-RPase, respectively . However, only one Hg was incorporated, probably due to steric hindrance by the large size of the Hg ion, while Mn, Ni, or Cr was not bound by the reconstituted enzyme, which instead incorporated only one Zn . Analysis of the metal content of various reconstituted RPases indicated that without low-pH treatment Zn bound to both the beta and beta' subunits when Zn concentrations were higher than 2 X 10(-6)M, but it bound only to the beta' subunit at lower concentrations . Moreover, low-pH treatment destroys the metal binding site in the beta' subunit . The metal sites on the beta and beta' subunits did not have significant affinity for the transition metals such as Mn, Ni, and Cr.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1985 Sep 10, 24(19), 5090 - 8
Chemical synthesis and expression of a calmodulin gene designed for site-specific mutagenesis; Roberts DM et al.; A gene coding for a calmodulin was synthesized and expressed in Escherichia coli . The gene was produced by the enzymatic ligation of 61 chemically synthesized DNA fragments . The gene possesses 27 unique, regularly spaced, restriction endonuclease cleavage sites to facilitate gene mutagenesis by the replacement of specific gene segments with synthetic double-stranded DNA . An expression vector containing the calmodulin gene was used to transform E . coli . Purification and characterization of calmodulin (VU-1 calmodulin) expressed by these transformants showed that it lacks two posttranslational modifications: an amino-terminal blocking group and N epsilon, N epsilon, N epsilon-trimethyllysine at position 115 . The cyclic nucleotide phosphodiesterase activator properties of VU-1, higher plant, and vertebrate calmodulins were not statistically different . However, VU-1 calmodulin was found to activate nicotinamide adenine dinucleotide (NAD) kinase to a maximal level that was at least 3-fold higher than that found with higher plant and vertebrate calmodulins . This higher level of activation is also characteristic of calmodulins from Dictyostelium discoideum and Chlamydomonas reinhardtii {Roberts, D . M., Burgess, W . H., & Watterson, D . M . (1984) Plant Physiol . 75, 796-798; Marshak, D . R., Clarke, M., Roberts, D . M., & Watterson, D . M . (1984) Biochemistry 23, 2891-2899} . The only common feature among Dictyostelium, Chlamydomonas, and VU-1 calmodulins not found in higher plant and vertebrate calmodulins is an unmethylated lysine at position 115 . The results indicate that the lack of methylation of lysine-115 may contribute to the maximal level of NAD kinase activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1985 Sep 10, 24(19), 5147 - 52
Isotope-exchange enhancement studies of Escherichia coli glutamine synthetase; Clark DD et al.; Isotope-exchange enhancement studies, a variation on positional isotope-exchange enhancement as described by Raushel and Garrard {Raushel, F . M., & Garrard, L . J . (1984) Biochemistry 23, 1791-1795}, are used to establish the point in the biosynthetic reaction of Escherichia coli glutamine synthetase at which gamma-glutamyl phosphate is formed . In these experiments, the behavior of the reverse biosynthetic reaction, i.e., the reaction of ADP, L-glutamine, and phosphate to form NH4+, L-glutamate, and ATP, is examined as a function of the concentration of ammonium ion . By varying the concentration of NH4+, the ratio of the velocity of isotope exchange to the velocity of net reaction, as measured by the rate of 18O depletion from labeled phosphate and the rate of production of L-glutamate, respectively, can be modulated in a mechanism-dependent manner . Evidence is presented demonstrating the presence of a branch point in the mechanism . The enzyme-ATP-glutamate complex may partition in two ways, one involving binding of ammonium ion and the other involving the chemical transformation to form the enzyme-ADP-gamma-glutamyl phosphate complex . The alternate pathways then rejoin upon formation of the enzyme-ADP-NH4+-gamma-glutamyl phosphate complex . Because of the branch point, there is no absolute requirement that ammonium ion be absent or present in order for the formation of gamma-glutamyl phosphate to occur . At high concentrations of ammonia, one pathway through the branch can be eliminated, effectively making that portion of the pathway ordered, with ATP, L-glutamate, and NH4+ binding consistent with our previously reported steady-state kinetic mechanism {Meek, T . D., & Villafranca, J . J . (1980) Biochemistry 19, 5513-5519}.

FEBS Lett, 1985 Sep 9, 189(1), 81 - 4
New model systems to study DNA-protein recognition mechanisms; Jacob A et al.; dpG antibodies were fractionated on a cellulose-double-stranded DNA column . The flow-through fraction bound denatured DNA but not double-stranded DNA (dsDNA) . The dpG-eluted fraction bound dsDNA preferentially . The results show that proteins can recognise dpG in DNA by different mechanisms, some involving DNA unwinding.

J Mol Biol, 1985 Sep 5, 185(1), 177 - 88
RNA polymerase-DNA interactions in Streptomyces . In vitro studies of a S . lividans plasmid promoter with S . coelicolor RNA polymerase; Buttner MJ et al.; DNA fragments of the Streptomyces lividans plasmid pIJ101 have been tested for their ability to bind Streptomyces coelicolor RNA polymerase in vitro or to promote transcription in Streptomyces in vivo . One DNA fragment which does both was shown to encode a transcript which was expressed at low cell-density in cultures of pIJ101-containing cells . The transcript start was located on the DNA sequence of the fragment by nucleotide-primed RNA polymerase binding experiments and by S1 nuclease mapping . The pattern of DNase I protection, the sites of enhanced DNase I cleavage and the DNA sequence of the fragment suggest that the RNA polymerase holoenzyme form, which recognizes this promoter, is similar in its interaction with DNA to the major RNA polymerase of Escherichia coli . Regions showing 3/6 nucleotide homology with each of the -35 and -10 regions of the consensus sequence of E . coli promoters are present in the same positions relative to the transcript start . Symmetrical sequences which may be involved in the regulation of expression of the promoter and a potential polypeptide coding sequence can be identified.

J Mol Biol, 1985 Sep 5, 185(1), 93 - 104
Autogenous control of Escherichia coli threonyl-tRNA synthetase expression in vivo; Springer M et al.; The regulation of the expression of thrS, the structural gene for threonyl-tRNA synthetase, was studied using several thrS-lac fusions cloned in lambda and integrated as single copies at att lambda . It is first shown that the level of beta-galactosidase synthesized from a thrS-lac protein fusion is increased when the chromosomal copy of thrS is mutated . It is also shown that the level of beta-galactosidase synthesized from the same protein fusion is decreased if wild-type threonyl-tRNA synthetase is overproduced from a thrS-carrying plasmid . These results strongly indicate that threonyl-tRNA synthetase controls the expression of its own gene . Consistent with this hypothesis it is shown that some thrS mutants overproduce a modified form of threonyl-tRNA synthetase . When the thrS-lac protein fusion is replaced by several types of thrS-lac operon fusions no effect of the chromosomal thrS allele on beta-galactosidase synthesis is observed . It is also shown that beta-galactosidase synthesis from a promoter-proximal thrS-lac operon fusion is not repressed by threonyl-tRNA synthetase overproduction . The fact that regulation is seen with a thrS-lac protein fusion and not with operon fusions indicates that thrS expression is autoregulated at the translational level . This is confirmed by hybridization experiments which show that under conditions where beta-galactosidase synthesis from a thrS-lac protein fusion is derepressed three- to fivefold, lac messenger RNA is only slightly increased.

Nature, 1985 Sep 5-11, 317(6032), 71 - 2
The ras oncogene product p21 is not a regulatory component of adenylate cyclase; Beckner SK et al.; Harvey (Ha-MSV) and Kirsten (Ki-MSV) murine sarcoma viruses induce tumours in animals and transform various cells in culture because of the expression of the ras oncogene product, p21 (ref . 1) . Proto-oncogenes homologous with these genes are highly conserved evolutionarily and activated ras oncogenes have been detected in many human cancers . Whether c-ras oncogenes are directly responsible for human carcinogenesis is uncertain; however, it is clear that p21 mediates virus-induced transformation, although by an unknown mechanism . Epithelial and fibroblast cell lines transformed with Ha-MSV and Ki-MSV express p21 (ref . 8) and exhibit reduced adenylate cyclase activity . Like the guanine nucleotide regulatory proteins, Ns and Ni, which mediate stimulation and inhibition, respectively, of adenylate cyclase, p21 is a membrane-associated GTP binding protein, which exhibits GTPase activity . These similarities suggest that p21 and the adenylate cyclase regulatory proteins are related in cellular function, and that p21 depresses adenylate cyclase by inhibiting the activity of Ns or acting as Ni . We have therefore now examined the structural and functional similarities between p21 and Ns and Ni and find no evidence that p21 regulates adenylate cyclase activity by acting as one of these regulatory proteins.

J Biol Chem, 1985 Sep 5, 260(19), 10395 - 7
Spectroscopic studies of pyruvate oxidase flavoprotein from Escherichia coli trapped in the lipid-activated form by cross-linking; Mather MW et al.; Pyruvate oxidase is a flavoprotein dehydrogenase isolated from Escherichia coli which catalyzes the oxidative decarboxylation of pyruvate to acetate plus CO2 . The maximal turnover of the enzyme, measured using a ferricyanide reductase assay, is increased 20-to 30-fold by either of two methods . Proteolysis in the presence of the substrate (pyruvate) and cofactor (Mg2+-thiamin pyrophosphate) results in cleavage at a single locus near the carboxyl terminus and concomitant activation . Phospholipids and detergents can bind to the enzyme and result in a similar activation, which is presumed to be physiologically relevant, since the enzyme functions as a peripheral membrane enzyme . Previous studies showed that proteolytic activation of pyruvate oxidase results in substantial changes in the absorption spectrum of the oxidized form of the bound flavin . Up to this time, similar studies of the lipid-activated form of the enzyme have not been feasible, since it is necessary to reduce the flavoprotein in order to induce binding to the lipids . In this paper, glutaraldehyde cross-linking of the lipid-activated enzyme is used to trap the enzyme in this form . Spectroscopic studies show alterations of the flavin spectrum similar to those observed upon proteolytic activation . This alteration in the flavin binding site is consistent with kinetic studies which suggest that activation results from an acceleration in the rates of electron transfer both into and out of the bound flavin, which appears to be more "accessible" in the activated forms of the enzyme.

J Mol Biol, 1985 Sep 5, 185(1), 215 - 7
Induction of elongation factor Tu-GDP crystal polymorphism by polyethylene glycol contaminants; Jurnak F; Trypsin-modified elongation factor (EF-)Tu-GDP from Escherichia coli is known to crystallize in several different unit cells under apparently identical conditions . The crystal polymorphism was investigated and found to be correlated with the source of polyethylene glycol used in the crystallization procedure . The use of highly purified polyethylene glycol promoted the growth of a new crystal form belonging to space group P2(1)2(1)2(1) with cell dimensions of a = 71.9 A, b = 74.7 A, c = 170.9 A and two molecules per asymmetric unit . In extensive crystallization trials, substances that typically contaminate commercial preparations of polyethylene glycol were screened . The final results show that the presence of the divalent anions, HPO4(2-) or SO4(2-), at different concentrations induce the growth of two known crystal forms belonging to space groups C222(1) and P4(3)2(1)2 . The relevancy of the findings is discussed.

J Mol Biol, 1985 Sep 5, 185(1), 189 - 99
Quaternary structure changes in aspartate transcarbamylase studied by X-ray solution scattering . Signal transmission following effector binding; Herve G et al.; The result of binding the effectors ATP and CTP to aspartate transcarbamylase was studied by X-ray solution scattering . Binding of substrate analogues produces a substantial change in the solution scattering curve, allowing us to monitor the proportion of the different quaternary structure states present in solution . In the initial solution this ratio was made roughly unity by adding either carbamyl phosphate and succinate, or N-(phosphonacetyl)-L-aspartate (PALA) . ATP or CTP were then added, and their effect on the proportion of the different quaternary structure states was followed . When using carbamyl phosphate and succinate (weakly bound), ATP or CTP had a clear effect, as observed previously by monitoring the sedimentation rate (Changeux et al., 1968) . However, when PALA (strongly bound) was used, the effect of CTP was very much smaller, and that of ATP was undetectable . This result supports the explanation by Tauc et al . (1982), that nucleotides act mostly through changing the affinity of the active sites for substrate, and only to a small extent by directly modifying the quaternary structure equilibrium in the case of CTP.

J Biol Chem, 1985 Sep 5, 260(19), 10812 - 8
Purification and characterization of a periplasmic oligopeptide binding protein from Escherichia coli; Guyer CA et al.; We have purified and characterized an oligopeptide binding protein released from the periplasm of Escherichia coli W by mild osmotic shock . The purified protein was greater than 97% homogeneous as determined by either sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 60,000) or isoelectric focusing (pI = 5.95) . The binding protein has a Stokes radius of 30 A and a sedimentation coefficient (s(0)20,w) of 4.6 S . Based on these hydrodynamic studies, the native protein has a molecular weight of 56,000 . The tripeptide, Ala-Phe-{3H}Gly, which is transported via the shock-sensitive sensitive oligopeptide permease, binds to the purified protein in dilute solution with a Kd of 0.1 microM and a stoichiometry of approximately 1 to 1 . Results from this study support the hypothesis that this periplasmic oligopeptide binding protein functions in the initial recognition of peptide substrates for the oligopeptide permease system.

J Biol Chem, 1985 Sep 5, 260(19), 10680 - 8
In vitro expression of the intron-containing gene for T4 phage thymidylate synthase; Chu FK et al.; The mechanism of expression of the structural gene (td) of T4 phage thymidylate synthase, which contains a 1,017-base pair intron, was studied by employing a coupled transcription-translation system with a td containing recombinant plasmid (pKTd2) as template . The {3H}leucine-labeled protein products synthesized in this system were treated with antibody to the synthase and the resulting immunoprecipitate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Two labeled polypeptides were obtained, one with an Mr of 32,000 and the other with an Mr of 25,000 . The former corresponds in molecular weight to a subunit of T4-thymidylate synthase and the other to the 183-amino acid peptide encoded by exon I, the 5'-end of the interrupted td gene . When pKTd2 restricted in exon I was used as a template, labeled immunopeptides were not detected but, when restricted in the intron region or in exon II, only the 25,000 Mr exon I product was obtained . Both peptides (Mr = 25,000 and 32,000) were synthesized when the gene was restricted downstream to exon II . Active enzyme, as measured by the tritium release assay, was shown to form about 6 min after the td gene was added to the in vitro protein synthesizing system, and followed the appearance of mature mRNA, as evidenced by S1 nuclease protection studies . The enzyme increased linearly for another 14 min in conjunction with the appearance of the Mr = 32,000 immunopeptide . The exon I product, however, preceded the Mr = 32,000 peptide, indicating that a post-transcriptional processing event may be required for mature mRNA to be formed . Measurement of the RNA products from the td gene in a transcriptional system, with labeled probes from specific regions of the td gene, provided evidence in support of an RNA processing mechanism involving intron excision and exon splicing.

J Biol Chem, 1985 Sep 5, 260(19), 10563 - 8
Compensatory phosphorylation of isocitrate dehydrogenase . A mechanism for adaptation to the intracellular environment; LaPorte DC et al.; When Escherichia coli grows on acetate, the flow of isocitrate through the glyoxylate bypass is regulated, in part, through the phosphorylation of isocitrate dehydrogenase . In addition to its role in adaptation to alternative carbon sources, this phosphorylation system responds to variation in the intracellular level of isocitrate dehydrogenase . This system can compensate for changes in the cellular level of isocitrate dehydrogenase in excess of 10-fold, maintaining a nearly constant activity for isocitrate dehydrogenase during growth on acetate . The behavior of the phosphorylation system exhibited considerable strain-specific variation . This was most clearly demonstrated using mutants which lacked the ability to phosphorylate isocitrate dehydrogenase . In two strains, mutation of the gene for isocitrate dehydrogenase kinase/phosphatase rendered the cells unable to grow on acetate . In contrast, a third strain was relatively insensitive to a mutation in this gene . This lack of phenotypic expression appears to result from a lower cellular level of isocitrate dehydrogenase in this strain which renders the phosphorylation (and consequent inhibition) of isocitrate dehydrogenase less essential . The gene for isocitrate dehydrogenase kinase/phosphatase (aceK) was located in the glyoxylate bypass operon, downstream from the genes for isocitrate lyase and malate synthase.

J Biol Chem, 1985 Sep 5, 260(19), 10517 - 25
Interconversion of tight and loose couple 50 S ribosomes and translocation in protein synthesis; Burma DP et al.; On incubation of 50 S ribosomes, isolated from either tight couple (TC) or loose couple (LC) 70 S ribosomes, with elongation factor G (EG-G) and guanosine 5'-triphosphate, a mixture of TC and LC 50 S ribosomes is formed . There is almost complete conversion of LC 50 S ribosomes to TC 50 S ribosomes on treatment with EF-G, GTP, and fusidic acid . Similarly, TC 50 S ribosomes are converted to LC 50 S ribosomes, although partially, by treatment with EF-G and a GTP analogue like guanyl-5'-yl methylenediphosphate (GMP-P(CH2)P) or guanyl-5'-yl imidodiphosphate (GMP-P(NH)P) and including a polymer of 5'-uridylic acid (poly(U} in the incubation mixture . Furthermore, LC 23 S RNA isolated from LC 50 S ribosomes is converted to TC 23 S RNA on heat treatment, but similar treatment does not affect TC 23 S RNA . The interconversion was followed by several physical and biological characteristics of TC and LC 50 S ribosomes, like association capacities with 30 S ribosomes before and after kethoxal treatment, susceptibility to RNase I and polyphenylalanine-synthesizing capacity in association with 30 S ribosomes, as well as thermal denaturation profiles, circular dichroic spectra, and association capacity of isolated 23 S RNAs . These data strongly support the proposition that TC and LC 50 S ribosomes are the products of translocation during protein synthesis . The conformational change of 23 S RNA induced by EF-G and GTP is most probably responsible for the interconversion, and L7/L12 proteins play an important role in the process . A two-site model based on kethoxal data has also been proposed to explain the tightness and looseness of 70 S couples.

J Biol Chem, 1985 Sep 5, 260(19), 10487 - 94
Verification by mass spectrometry of the primary structure of human interleukin-2; Fukuhara K et al.; Proteolytic digests of interleukin-2 from a human leukemic T-cell line produced by Escherichia coli carrying a recombinant DNA were analyzed by fast atom bombardment mass spectrometry . The mass values of intense signals observed in the mass spectrum were consistent with peptides predicted from the nucleotide sequence of cDNA for human interleukin-2, an indication that the protein with the predicted amino acid sequence was produced by E . coli . BrCN and proteolytic digests of interleukin-2 obtained from cultured cells were also examined by fast atom bombardment mass spectrometry . The observed mass values were identical with those from interleukin-2 from E . coli except for that of the NH2-terminal sequence, in which the Thr residue at position 3 was bound to a sugar moiety . The mass spectra of the digests of the two interleukin-2 preparations and synthetic peptides with sequences from 117 to 128 and 121 to 128 predicted from the nucleotide sequence of cDNA for a human interleukin-2 indicated that Cys residues at positions 58 and 105 are linked by a disulfide bond and that the Cys residue at position 125 is free.

J Biol Chem, 1985 Sep 5, 260(19), 10478 - 81
Biochemical characterization of a paraquat-tolerant mutant of Escherichia coli; Kao SM et al.; The biochemical basis for paraquat tolerance was investigated using one of the paraquat-resistant Escherichia coli mutants previously isolated . When grown in the absence of paraquat (PQ2+), the specific activities of glucose-6-phosphate dehydrogenase and NADPH:PQ2+-diaphorase, both required for the expression of PQ2+ toxicity, were comparable in the wild type and the mutant . However, growth in the presence of 1 mM PQ2+ resulted in greater induction of these two enzymes in the wild type than in the mutant . Nevertheless, when the mutant was grown in 50 mM PQ2+, the activities of these two enzymes were comparable to those of the wild type grown in the presence of 1 mM PQ2+ . Measurement of cyanide-resistant respiration, an indication of intracellular superoxide generation, showed that the intracellular flux of superoxide mediated by subsaturating concentrations of paraquat was significantly lower in the mutant than in the wild type . Extracellular superoxide formation, as measured by superoxide dismutase-inhibitable cytochrome c reduction, was higher in the wild type than in the mutant whether grown in the absence or the presence of PQ2+ . The mutant did not show cross-resistance toward juglone or plumbagin, compounds known to exacerbate superoxide generation . The kinetics of {14C}PQ2+ uptake showed that the wild type accumulated PQ2+ against a concentration gradient, whereas the mutant seemed to do so only by facilitated diffusion . The results indicate that the impaired paraquat uptake system in the mutant results in the physiological and biochemical differences observed between the wild type and mutant.

J Biol Chem, 1985 Sep 5, 260(19), 10495 - 502
Expression of human terminal deoxynucleotidyl transferase in Escherichia coli; Peterson RC et al.; A cloned DNA fragment related to pT17 containing a partial cDNA sequence of human terminal deoxynucleotidyl transferase was used as a probe to screen for the full length cDNA sequence of the enzyme in a lambda gt11 library constructed from human lymphoblastoid KM-3 cDNA . A recombinant containing a 2068-base pair insert was isolated and recloned into the EcoRI site of the sequencing plasmic pUC-8 as two subclones, pT711 and pT106 . DNA sequencing and hybridization studies showed that pT711 contains the pT17 sequence and an additional 172 upstream nucleotides . pT711 represents the coding sequence for the carboxyl half of the terminal transferase protein . pT106, containing a 965-base pair insert, hybridizes to the same mRNA as pT711 on Northern blots and contains an open reading frame that is in phase with the reading frame of the insert in pT711 . Amino acid sequencing of the 58-kDa peptide of the calf thymus terminal transferase failed, indicating that the N terminus is blocked . N-Terminal sequencing of a 56-kDa form of the protein produced 24 amino acids corresponding to the translated human cDNA coding sequence starting at residue 398 of the insert in pT106 with 83% homology between bovine and human sequence . The initiation codon is assigned to an ATG sequence at nucleotide 329 of the insert in pT106 . Comparison of the translated human terminal transferase sequence with peptides from the calf thymus enzyme showed that the homology between the human and bovine enzyme is better than 90% among 263 amino acids determined . The coding sequences in pT106 and pT711 were recloned into an expression plasmid pUC-19 downstream from the lac promoter and in phase with the coding sequence of the lac Z gene . Lysates of bacteria carrying the reconstructed coding sequence of human terminal transferase contain a fused protein of 60 kDa that reacts with rabbit antibody to terminal transferase on immunoblots and exhibits enzyme activity . Isolation of this fused protein from bacterial lysates with mouse monoclonal antibody to human terminal transferase produces the expected protein of 60 kDa.

J Biol Chem, 1985 Sep 5, 260(19), 10392 - 4
Diphtheria toxin . Effect of substituting aspartic acid for glutamic acid 148 on ADP-ribosyltransferase activity; Tweten RK et al.; Photoaffinity labeling experiments with diphtheria toxin fragment A have implicated glutamic acid 148 as a constituent of the NAD binding site . To evaluate the role of this residue in ADP-ribosylation of elongation factor 2, we replaced it with aspartic acid by in vitro mutagenesis of a toxin gene fragment cloned in Escherichia coli . Fragment A containing aspartic acid at position 148 had less than 0.6% the ADP-ribosylation activity of wild-type fragment A . The mutation produced no change in sensitivity of fragment A to trypsin and little, if any, reduction in affinity of fragment A for NAD . These results indicate that glutamic acid 148 is essential for the ADP-ribosylation of elongation factor 2 and are consistent with other data suggesting that this residue may be at or near the catalytic center of the toxin.

J Biol Chem, 1985 Sep 5, 260(19), 10418 - 25
Monoclonal antibodies to Escherichia coli F1-ATPase . Correlation of binding site location with interspecies cross-reactivity and effects on enzyme activity; Dunn SD et al.; Twenty-one hybridoma cell lines which secret antibodies to the subunits of the Escherichia coli F1-ATPase were produced . Included within the set are four antibodies which are specific for alpha, six for beta, three for gamma, four for delta and four for epsilon . The antibodies were divided into binding competition subgroups . Two such competition subgroups are represented for the alpha, beta, and epsilon subunits, one for delta and three for gamma . The ability to bind intact F1-ATPase was demonstrated for some of the antibodies to alpha and beta, and for all of those to delta, while the antibodies to gamma and epsilon gave unclear results . All of the antibodies to alpha and beta which bound ATPase were found to have effects on the ATPase activity of purified E . coli F1-ATPase . One of those to alpha inhibited activity by about 30% . Another anti-alpha was mildly stimulatory . The four antibodies to beta which bound ATPase inhibited activity by 90% . In contrast, membrane-bound ATPase was hardly affected by the antibodies to alpha, but was inhibited by 40-60% by the antibodies to beta . The other antibodies to alpha and beta bound only free subunits, or partially dissociated ATPase, suggesting that their epitopes are buried between subunits in ATPase . These antibodies had no effects on activity . The ability of the antibodies to recognize ATPase subunits present in crude extracts from mitochondria, chloroplasts, and a variety of bacteria was tested using nitrocellulose blots of sodium dodecyl sulfate-polyacrylamide gels . One anti-beta specifically recognized proteins in the range of 50,000-60,000 daltons in each of the extracts, although the reaction with mitochondrial beta was weak . Some of the other antibodies had limited cross-reaction, but most were specific for the E . coli protein . In some species, those proteins which were recognized by the anti-beta ran with a higher apparent molecular weight than proteins which were recognized by an anti-alpha . All antibodies which exhibited cross-reactivity were found to recognize sites which were not exposed in intact ATPase, implying that the surfaces which lie between subunits are most highly conserved.

Eur J Biochem, 1985 Sep 2, 151(2), 311 - 7
Non-disruptive detection of DNA polymerases in nondenaturing polyacrylamide gels; Holler E et al.; A non-disruptive method is described with which DNA polymerases can be detected in homogeneous preparations and unfractionated cell extracts after electrophoresis in nondenaturing polyacrylamide gradient gels . The technique involves diffusion of DNA polymerase activity into an overlay assay agarose gel, the synthesis of radioactive DNA, removal of excess substrates and autoradiography . Cell extracts from a variety of organisms were studied using this method . The activity from Escherichia coli crude extracts migrated in a position corresponding to a higher molecular mass than did purified preparations of DNA polymerase I . DNA polymerases of higher organisms generally migrated in positions corresponding to 400--900 kDa, in some cases, close to 200 kDA.

Eur J Biochem, 1985 Sep 2, 151(2), 377 - 83
An improved procedure for purifying 5'-nucleotidase from various sources . Evidence for tissue and species differences in their molecular mass and affinity for F-actin; Dieckhoff J et al.; 5'-Nucleotidase from chicken gizzard smooth muscle has been extracted, using a sulfobetaine derivate of cholic acid, and purified to homogeneity by employing three chromatographic steps . It is shown that the purification scheme can be applied to 5'-nucleotidase from other sources, such as rat liver . On sodium dodecyl sulfate polyacrylamide gels, stained with silver nitrate, the purified enzyme from chicken gizzard shows a single polypeptide band with an apparent molecular mass of 79 kDa . The enzyme purified from rat liver exhibits a molecular mass of 73 kDa in agreement with published data {Bailyes, E.M., Soos, M., Jackson, P., Newby, A . C., Siddle, K . & Luzio, J.P . (1984) Biochem . J . 221, 369-377) . Gel filtration, using non-denaturating detergent solutions, indicates that the native enzyme may exist as a homodimer (152 kDa) or homotetramer (310 kDa) . Antibodies raised against the enzyme purified from chicken gizzard bind only 5'-nucleotidase, solubilized from chicken muscular sources, when immobilized, but not from chicken or rat liver . The existence of tissue specific variants of 5'-nucleotidase is therefore postulated and it appears that these particular isoforms can also be classified in membranous and secretory forms of 5'-nucleotidase . They also differ in their mode of interaction with actin . The AMPase activity of the membranous (= muscular) isoform is inhibited to a considerably higher percentage by F-actin than the enzyme isolated from rat liver.

Eur J Biochem, 1985 Sep 2, 151(2), 231 - 6
The mechanism of ion selectivity of OmpF-porin pores of Escherichia coli; Kobayashi Y et al.; The OmpF porin from the outer membrane of Escherichia coli acts as a lightly cation-selective pore, allowing the diffusion of small cations and cationic molecules, whose Mr are a little larger than the threshold exclusion limit . To ascertain the mechanism of this cation selectivity, we have examined a possible influence of cationic solutes on the fluorescence emission and the circular dichroic spectrum of tryptophan residues of the porin trimer, searching for conformational change(s) . The diffusion of cationic solutes was determined with the native and the amidated porins in the presence or the absence of the effector cations . The following results were obtained . (a) Cations, e.g . spermidine, caused fluorescence quenching in the native trimer, with a half-maximum fluorescence quenching at 11-18 microM . A change in the circular dichroic spectrum was also recorded at around 280 nm . (b) The dissociation constant of spermidine to the native trimer was calculated to be 16 microM as determined by the method of equilibrium dialysis . (c) The cation-caused fluorescence quenching was reversed when the carboxyl groups of the trimer were modified by the amidation reaction, though amidation of the trimer resulted in no significant change in the fluorescence intensity . (d) The diffusion rate of N-benzyloxycarbonyl-glycyl-L-prolyl-L-arginine p-nitroanilide through the native and the amidated porins was lowered in the presence and the absence, respectively, of cations . Both the extent of fluorescence quenching in the presence of cation and the rate of cation diffusion were inversely proportional to the number of amidated carboxyl residues . The relative fluorescence quenching of the porin trimer (the amidated versus the native) in the presence of cations was linearly related to the relative solute diffusion via the porin (the amidated versus the native) . These results suggested that cations caused a conformational change in the trimer, resulting in an easier diffusion of the solutes . The results suggested further that a limited number of carboxyl groups in the pore interior are involved in the cation selectivity of OmpF-porin pores.

Eur J Biochem, 1985 Sep 2, 151(2), 393 - 7
Biosynthesis of the O9 antigen of Escherichia coli . Synthetic glycosyldiphosphomoraprenols as probes for requirement of mannose acceptors; Jann K et al.; Synthetic monosaccharide derivatives (alpha-glucosyl, beta-glucosyl, alpha-mannosyl) and disaccharide derivatives (alpha-mannosyl-1,2-alpha-glucosyl, alpha-mannosyl-1,3-alpha-glucosyl, alpha-mannosyl-1,4-alpha-glucosyl, alpha-mannosyl-1,6-alpha-glucosyl) of diphosphomoraprenol were used as putative mannose acceptors in the biosynthesis of Escherichia coli O9 antigen . Membranes of E . coli O9 derived from the rfe mutant F 1357 were reconstituted with these compounds and then incubated with different concentrations of GDP-{14C}mannose . Of the monosaccharide derivatives tested, only alpha-glucodiphosphomoraprenol was a mannose acceptor and the only disaccharide derivative which accepted mannose was alpha-mannosyl-1,3-alpha-glucosyldiphosphomoraprenol . The alpha-glucosyl derivative accepted only one mannose unit at 4 microM GDP-{14C}mannose, and above 50 microM GDP-{14C}mannose about 25% of the product had a minimum size of about 30 mannose units . The alpha-mannosyl-1,3-alpha-glucosyl derivative was only a mannose acceptor at a GDP-{14C}mannose concentration of 50 microM and higher, and the product had a minimum size of about 30 mannose units . The results are discussed with respect to requirement of mannose acceptors.

Eur J Biochem, 1985 Sep 2, 151(2), 245 - 55
Studies of the GTPase domain of archaebacterial ribosomes; Beauclerk AA et al.; Ribosomes from the methanogens Methanococcus vannielii and Methanobacterium formicicum catalyse uncoupled hydrolysis of GTP in the presence of factor EF-2 from rat liver (but not factor EF-G from Escherichia coli) . In this assay, and in poly(U)-dependent protein synthesis, they were sensitive to thiostrepton . In contrast, ribosomes from Sulfolobus solfataricus did not respond to factor EF-2 (or factor EF-G) but possessed endogenous GTPase activity, which was also sensitive to thiostrepton . Ribosomes from the methanogens did not support (p)ppGpp production, but did appear to possess the equivalent of protein L11, which in E . coli is normally required for guanosine polyphosphate synthesis . Protein L11 from E . coli bound well to 23S rRNA from all three archaebacteria (as did thiostrepton) and oligonucleotides protected by the protein were sequenced and compared with rRNA sequences from other sources.

Eur J Obstet Gynecol Reprod Biol, 1985 Sep, 20(3), 181 - 9
Malakoplakia of the endometrium: a rare cause of postmenopausal bleeding; Chadha S et al.; A rare cause of postmenopausal bleeding in a 72-yr-old woman due to malakoplakia of endometrium is described . The light and electron microscopic features are described and it is postulated that malakoplakia is due to an abnormal macrophage response to Escherichia coli infection.

Am J Trop Med Hyg, 1985 Sep, 34(5), 921 - 4
The childhood health effects of an improved water supply system on a remote Panamanian island; Ryder RW et al.; The incidence of diarrhea, respiratory disease, and skin infections was prospectively determined after the introduction of a system which distributed unlimited quantities of high quality fresh water to each of the 150 housing units on Tupile, an island devoid of fresh water located off Panama's Caribbean coast and inhabited by 1,500 Cuna Indians . Tupile residents used 7.1 liters of water/person/day compared to the 2.3 usage rate of inhabitants on Achutupo, the control island . Despite ready availability of water in each household, Tupile residents continued to store water in contaminated vessels prior to use . Forty percent of stored water samples tested on Tupile and 45% on Achutupo were contaminated with E . coli organisms . There were 4.7 episodes/child year (E/Y) of acute diarrhea on Tupile compared with the 3.5 rate on Achutupo . The rotavirus infection rate on Tupile was 0.8 E/Y compared with 0.2 E/Y on Achutupo . Infection rates for Norwalk virus, respiratory syncytial virus and Coxsackie B 1-6 viruses were similar on both islands . Respiratory disease rates were high on both islands (2.2 E/Y on Tupile, 2.7 E/Y on Achutupo) . Achutupo had much higher rates of impetigo and scabies (0.6 E/Y and 2.5 E/Y, respectively) than Tupile (0.2 E/Y and 1.4 E/Y) . Provision of the water distribution system had a beneficial effect on the incidence of water-washed diseases (impetigo and scabies), but at best had no effect on diarrheal disease.

J Bacteriol, 1985 Sep, 163(3), 1021 - 37
Genetic structure of populations of Legionella pneumophila; Selander RK et al.; The genetic structure of populations of Legionella pneumophila was defined by an analysis of electrophoretically demonstrable allelic variation at structural genes encoding 22 enzymes in 292 isolates from clinical and environmental sources . Nineteen of the loci were polymorphic, and 62 distinctive electrophoretic types (ETs), representing multilocus genotypes, were identified . Principal coordinates and clustering analyses demonstrated that isolates received as L . pneumophila were a heterogeneous array of genotypes that included two previously undescribed species . For 50 ETs of L . pneumophila (strict sense), mean genetic diversity per locus was 0.312, and diversity was equivalent in ETs represented by isolates recovered from clinical sources and those collected from environmental sources . Cluster analysis revealed four major groups or lineages of ETs in L . pneumophila . Genetic diversity among ETs of the same serotype was, on average, 93% of that in the total sample of ETs . Isolates marked by particular patterns of reactivity to a panel of nine monoclonal antibodies were also genetically heterogeneous, mean diversity within patterns being about 75% of the total . Both Pontiac fever and the pneumonic form of legionellosis may be caused by isolates of the same ET . The genetic structure of L . pneumophila is clonal, and many clones apparently are worldwide in distribution . The fact that L . pneumophila is only 60% as variable as Escherichia coli raises the possibility that isolates recovered from clinical cases and man-made environments are a restricted subset of all clones in the species as a whole.

Carbohydr Res, 1985 Sep 1, 141(2), 239 - 53
Synthesis of alternate linear and branched repeating units of the Escherichia coli LP 1092 capsular polysaccharide containing 3-deoxy-alpha-D-manno-2-octulosonic acid (KDO) linked to secondary positions of D-ribose; Kosma P et al.; The oligosaccharides, methyl 3-O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-beta-D-ribofuranosid e, methyl 2-O-beta-D-ribofuranosyl-3-O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-beta-D-ribofuranosid e, and methyl O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2----2)-O-beta-D- ribofuranosyl-(1----2)-beta-D-ribofuranoside were prepared in high purity and good over-all yields . The constitutions of the trisaccharide derivatives correspond to the repeating units of the proposed linear and branched structures of the capsular polysaccharide(s) from Escherichia coli LP 1092 . The alpha-KDO-(2----3)-beta-D-Ribf and alpha-KDO-(2----2)-beta-D-Ribf units were synthesized by a modification of the Helferich procedure using methyl (4,5,7,8-tetra-O-acetyl-3-deoxy-alpha-D-manno-2-octulopyranosyl bromide)-onate and appropriate beta-D-ribofuranosyl derivatives . The constitutional and configurational assignments were based on the 250-MHz 1H-n.m.r.-spectra of protected derivatives of the oligosaccharides.

Proc Natl Acad Sci U S A, 1985 Sep, 82(18), 6100 - 3
Expression and assembly of active cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase in Escherichia coli containing stoichiometric amounts of large and small subunits; Tabita FR et al.; The genes for the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans were subcloned into plasmid pUC9 . After induction, both genes were expressed in Escherichia coli and the subunits were assembled into an active holoenzyme . The enzyme was purified from E . coli to high specific activity and was found to contain equimolar amounts of large and small subunits . The assembly of the hexadecameric ribulose bisphosphate carboxylase/oxygenase in E . coli should provide the basis for studies on the mechanism of assembly and the role of small subunits in catalysis.

Mutat Res, 1985 Sep, 146(2), 155 - 67
Regulation of expression of the cloned ada gene in Escherichia coli; Nakabeppu Y et al.; The ada gene of Escherichia coli K12, the regulatory gene for the adaptive response of bacteria to alkylating agents, was cloned in multicopy plasmids . O6-Methylguanine-DNA methyltransferase and 3-methyladenine-DNA glycosylase II, which are known to be inducible as part of the adaptive response, were produced in ada- cells bearing ada+ plasmids, even without treatment with alkylating agents . When such cells had been treated with methyl methanesulfonate, even higher levels of the enzyme activities were produced . Maxicell experiments revealed that the ada gene codes for a polypeptide with a molecular weight of 38 000 . We constructed a hybrid plasmid carrying an ada'-lacZ' fused gene, with the proper control region for ada expression . beta-Galactosidase synthesis from the fused gene was strongly induced only when cells were treated with low doses of methylating agents, but was weakly induced with relatively high doses of ethylating agents . The induction was autogenously regulated by the ada gene product, in a positive manner.

J Bacteriol, 1985 Sep, 163(3), 991 - 9
Role of translation and attenuation in the control of pyrBI operon expression in Escherichia coli K-12; Roland KL et al.; Expression of the pyrBI operon of Escherichia coli K-12, which encodes the subunits of the pyrimidine biosynthetic enzyme aspartate transcarbamylase, is negatively regulated by the intracellular levels of UTP . Previous experiments suggested a unique model for regulation of operon expression in which low UTP levels cause close coupling of transcription and translation of the pyrBI leader region . This close coupling suppresses transcriptional termination at an attenuator preceding the structural genes . In this study, we examined the regulatory role of translation and attenuation in operon expression . To determine whether the leader region is translated, we constructed a plasmid, designated pBHM17, in which the pyrBI promoter(s) and the first 11 codons for a putative 44-amino acid leader polypeptide are fused to codon 9 of lacZ . A transformant carrying this plasmid synthesized a beta-galactosidase fusion protein with the amino-terminal sequence of the leader polypeptide, demonstrating that the signals required for leader polypeptide synthesis function in vivo . Synthesis of the fusion protein was nearly insensitive to pyrimidine availability . In uracil-grown cells, the level of fusion protein synthesis encoded by plasmid pBHM17 was much greater than that encoded by a similar plasmid containing a pyrB::lacZ gene fusion, in which the pyrBI promoter-regulatory region is intact . These results indicate that the downstream leader sequence which includes the attenuator is required for regulation and functions as a transcriptional barrier . Oligonucleotide-directed mutagenesis was used to change the ATG leader polypeptide initiation codon of the intact pyrBI operon to ACG, which was shown to strongly inhibit translational initiation . This mutation greatly reduced operon expression and regulation as predicted by the attenuation control model.

Mol Gen Mikrobiol Virusol, 1985 Sep, (9), 44 - 7
{Purification of plasmid DNA by chromatographic methods}; Naumov GN; Chromatographic methods have been used to purify the DNA of plasmid RP1 . DNA was purified in two stages . DNA was precipitated by ethanol and separated from RNA and proteins in Sepharose 4B column after lysis of plasmid containing cells by alkaline solution of sodium dodecylsulphate . Separation of the total DNA preparation and isolation of plasmid DNA was achieved at the second stage by chromatography on the hydroxyapatite column . The resulting purified plasmid DNA was free of RNA, protein and linear fragments of chromosomal DNA . The plasmid DNA kept intact native structure and possessed the transforming activity . The DNA of RP1 yield after purification by the described technique presented 70-80 micrograms per g of wet biomass.

J Ultrastruct Res, 1985 Sep, 92(3), 180 - 9
Investigation of the 50 S ribosomal subunit by electron microscopy and image analysis; Verschoor A et al.; In electron micrographs of 50 S (large) subunits from Escherichia coli ribosomes, the highly preferred crown view is inferred to represent the roughly hemispherical particle lying with its flat or concave face against the carbon film . Single particle averaging allows the reproducible details of the crown view particle to be recognized . Multivariate image analysis shows the most variable morphological features of this view to be the two side protrusions, the L7/L12 stalk and the L1 ridge, both of which show apparent positional variations . The invariance of the features of the particle body implies that the movements of the side protrusions are not merely a result of perspective changes produced by major rotations of the particle body out of its quasistable, flat-lying position . A bending point localized on the L7/L12 stalk is conjectured to represent a functional "hinge" that may be related to the secondary/tertiary structure of the L7/L12 dimeric protein.

Mikrobiologiia, 1985 Sep-Oct, 54(5), 730 - 4
{Protective action of antioxidants on Escherichia coli cells immobilized in polyacrylamide gel}; Starostina NG et al.; The object of this work was to find out whether antioxidants could be used for weakening the effect of free radicals on Escherichia coli cells immobilized in polyacrylamide gel . Some of the antioxidants soluble in lipids and water (ionol, Epigid, glutathione) protected the cells against the action of free radicals produced in the process of acrylamide polymerization, and increased the viability of the immobilized bacteria.

J Biochem (Tokyo), 1985 Sep, 98(3), 793 - 7
Assignment of catalytically essential cysteine residues in aspartase by selective chemical modification with N-(7-dimethylamino-4-methylcoumarynyl)maleimide; Ida N et al.; N-(7-Dimethylamino-4-methylcoumarynyl)maleimide (DACM), a fluorescent reagent for sulfhydryl groups, was employed to determine the functionally essential cysteine residues in aspartase from Escherichia coli . Analysis of the tryptic peptides containing DACM-labeled residues by reverse phase HPLC revealed that Cys-140 and Cys-430 were selectively modified, among 11 residues whose loci were recently determined by a DNA sequencing study (Takagi, J.S., et al . (1985) Nucl . Acids Res . 13, 2063-2074) . When the modification was carried out in the presence of Mg2+ and L-aspartate, the enzyme activity remained unchanged and no cysteine residue was modified . This suggests that two cysteine residues are located at the L-aspartate binding site and that at least one of them is involved in the catalytic reaction.

J Biochem (Tokyo), 1985 Sep, 98(3), 681 - 5
Purification and subunit structure of recBC DNase from Escherichia coli harboring a recB and recC genes-inserted plasmid; Umeno M et al.; recBC DNase of Escherichia coli has been purified from the transformant, HB101/pFS11-04 (recB+ recC+), by successive ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-150 gel filtration, hydroxyapatite chromatography, DNA cellulose affinity chromatography, and second DEAE-cellulose chromatography . The purified enzyme was obtained in an overall yield of 3% . The enzyme protein appeared as a single pure component on native polyacrylamide gel electrophoresis . The purified enzyme was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis . The results show that recBC DNase consists of two nonidentical subunits with molecular weights of 125,000 and 135,000, and isoelectric points of 5.6 and 5.7, respectively.

Mol Biol (Mosk), 1985 Sep-Oct, 19(5), 1378 - 85
{Deacylated tRNA binds with 50S subunits of Escherichia coli ribosomes at a special site not corresponding to the P'-site}; Parfenov DV et al.; In the presence of methanol 50S ribosomal subunits reveal two independents sites for binding of deacylated tRNA and/or AcPhe-tRNA . The site with lower affinity was identified with the donor (P') site as the dissociation constant (Ka) for AcPhe-tRNA was equal to the Michaelis constant for its reaction with puromycin both at 0 degrees C and 25 degrees C . Log Ka increases linearly with methanol concentration . This suggests that there are no conformational transitions of the interacting components, the affinity increases only quantatively due to lowering of the dielectric constant of water, and the site can exist even in the absence of methanol, but its Ka may be too low to be measured . It follows from these data that the higher-affinity site, which is observed both in the absence and presence of methanol, cannot be the P' site as it was generally believed . By all its properties it is more like the additional E site, which has been recently found on 70S ribosomes . Specifically, its affinity for deacylated tRNA is about 1000-fold higher than for AcPhe-tRNA (in the P'-site they are almost the same).

Mol Biol (Mosk), 1985 Sep-Oct, 19(5), 1269 - 72
{Ribosomal proteins directly interacting with fMet-tRNAfMet in the 30S initiation complex}; Broude NE et al.; By means of ultraviolet-induced (254nm) RNA-protein cross-links it is shown, that tRNAfMet inside the preinitiation complex, formed by binding of fMet-tRNAfMet with 30S subunit of E . coli ribosome and RNA of the phage MS2 in the presence of initiation factors, directly interacts with proteins S4, S5, S9, S11, S14 and S15-S17.

EMBO J, 1985 Sep, 4(9), 2377 - 83
An inner membrane protein N-terminal signal sequence is able to promote efficient localisation of an outer membrane protein in Escherichia coli; Jackson ME et al.; To test the importance of N-terminal pre-sequences in translocation of different classes of membrane proteins, we exchanged the normal signal sequence of an Escherichia coli outer membrane protein, OmpF, for the pre-sequence of the inner membrane protein, DacA . The DacA-OmpF hybrid was efficiently assembled into the outer membrane in a functionally active form . Thus the pre-sequence of DacA, despite its relatively low hydrophobicity compared with that of OmpF, contains all the essential information necessary to initiate the translocation of OmpF to the outer membrane . Since processing of DacA was also shown to be dependent upon SecA we conclude that the initiation of translocation of this inner membrane polypeptide across the envelope occurs by the same mechanism as outer membrane and periplasmic proteins . The N-terminal 11 amino acids of mature OmpF, which in the hybrid are replaced by the N-terminal nine amino acids of DacA, carry no essential assembly signals since the hybrid protein is apparently assembled with equal efficiency to OmpF.

Biol Chem Hoppe Seyler, 1985 Sep, 366(9), 901 - 6
Two-dimensional polyacrylamide gel electrophoresis of ribosomal proteins in the nanogram range; Brockmoller J et al.; A two-dimensional gel electrophoresis system to identify and check the purity of ribosomal proteins from different organisms with nanogram quantities is described . This procedure combines the method of Geyl et al . for the separation of ribosomal proteins of Escherichia coli, and the microscale electrophoresis system for proteins described by Neuhoff and Poehling, with several modifications . The first gel dimension is carried out in capillaries and the second in the form of slab gels, both are run in newly designed chambers suitable for 10-20 samples . This electrophoresis system enables a reduction of the running time from 2 days to 2 hours and an increase in sensitivity, with Coomassie blue staining, from 3-5 micrograms for the normal 100 X 100 mm gels to 50-100 ng . The resolution of all ribosomal proteins on the micro-gel (30 X 38 X 0.5 mm) is similar to the separation on the mini-gel of 100 X 100 X 3 mm as described by Geyl et al.

Z Geburtshilfe Perinatol, 1985 Sep-Oct, 189(5), 235 - 8
{Changes in prostaglandins in endotoxin-induced shock in pregnant minipigs}; Holzgreve W et al.; Six pregnant mini-pigs received a continuous endotoxin infusion of 1 mg/kg body weight . E.-coli-lipopolysaccharide, two control animals were treated in the same way without endotoxin application . Blood was drawn from the fetal umbilical cord after exposure through a uterine window . In contrast to the controls, concentrations of prostaglandin F2 alpha increased in animals which received endotoxin infusions . The prostaglandin-E2-concentrations in the treated pregnant animals decreased by half during the first hour of the experiment, whereas they staid fairly constant in the untreated group of animals . The thromboxane-B2-concentration increased from 70 to 110 pg/ml three hours after endotoxin application in contrast to the control group . The fetuses in both groups did not show any significant changes during the experiments . The results indicate that prostaglandin F2 alpha is released as a special endotoxin effect in adult mini-pigs . Our measurements in the fetus make a direct endotoxin effect across the placenta unlikely . The fetuses of the mini-pigs, however, died as a result of the maternal cardio-vascular collaps.

Vopr Virusol, 1985 Sep-Oct, 30(5), 558 - 61
{Expression of the HBsAg gene of the hepatitis B virus under the control of the promoter region of gene PH05 in yeast cells}; Granovskii NN et al.; Recombinant plasmids have been constructed containing the complete HBsAg gene combined with the promotor region of the yeast gene of acid phosphatase . The plasmids are capable of replication in E . coli and Saccharomyces cerevisiae cells . In cultivation of yeast cells containing these plasmids in a synthetic medium with a low phosphorus content a protein is synthesized possessing the properties of hepatitis B virus surface antigen.

J Gen Microbiol, 1985 Sep, 131 ( Pt 9), 2327 - 33
Properties of wild-type strains of enterotoxigenic Escherichia coli which produce colonization factor antigen II, and belong to serogroups other than O6; Scotland SM et al.; Enterotoxigenic strains of Escherichia coli, which belonged to serogroups other than O6 and produced colonization factor antigen II, usually produced only coli surface antigen 3 (CS3) and gave weak mannose-resistant haemagglutination of bovine erythrocytes . A non-autotransferring plasmid, NTP165, from a strain of E . coli O168 . H16 coded for heat-stable enterotoxin, heat-labile enterotoxin and CS antigens . The CS antigens expressed after acquisition of plasmid NTP165 depended on the recipient strain: a biotype A strain of serotype O6 . H16 expressed CS1 and CS3; a biotype C strain of serotype O6 . H16 expressed CS2 and CS3; strain K12 and strain E19446 of serotype O139 . H28 expressed only CS3 . An exceptional wild-type strain, E24377, of serotype O139 . H28 produced CS1 and CS3 when isolated; a variant of E24377 which had lost the plasmid coding for CS antigens produced both CS1 and CS3 after the introduction of NTP165.

J Gen Microbiol, 1985 Sep, 131 ( Pt 9), 2285 - 92
Mutation affecting resistance of Escherichia coli K12 to nalidixic acid; Hrebenda J et al.; A new mutation, nalD, determining resistance of Escherichia coli to nalidixic acid (NAL) is reported . The nalD mutant described is resistant to NAL at 37 degrees C but sensitive at 30 degrees C . It is defective in penetration of NAL and glycerol through the outer membrane at 37 degrees C . The nalD mutation is located half-way between 89 and 89.5 min on the E . coli genetic map.

J Gen Microbiol, 1985 Sep, 131 ( Pt 9), 2263 - 7
Exclusion by the IncI plasmid R144 determined by measuring DNA transfer in Escherichia coli conjugations; Hartskeerl RA et al.; Exclusion specified by the IncI plasmid R144 was determined by measuring the amount of donor DNA transferred to appropriate recipient cells . When recipient cells harboured an R144-derived Exc+ recombinant plasmid, the exclusion value determined in that way was comparable with the exclusion value determined by measuring the efficiency of transconjugant colony formation . When recipient cells harboured the plasmid R144drd-3, the exclusion value determined by measuring the amount of donor DNA transferred to recipient cells appeared more valid than the value determined by measuring transconjugant colony formation.

Biochem Int, 1985 Sep, 11(3), 301 - 9
Single amino acid substitutions at conserved residues of human interferon-alpha can effect antiviral specific activity; Nisbet IT et al.; Site-specific in vitro mutagenesis was used to direct various amino acid substitutions at conserved positions within the sequence of human interferon-alpha 1 (IFN-alpha 1) . The antiviral specific activity of IFN-alpha 1, expressed in M13 as a fusion protein {IFN-alpha 1 (phi WT)}, could be altered by single amino acid substitutions . The substitution of glycine for tyrosine at position 123 results in a loss of more than 99% of the antiviral specific activity on human cells, but causes no significant change in the antiviral specific activity on primary bovine cells . The tyrosine at position 123 is thus implicated in determining human cell specificity . Based on analysis of IFN-alpha 2, IFN-alpha 1 contains two dulsulphide bridges between cysteine residues 29 and 139 and cysteine residues 1 and 99 . IFN-alpha 1 also contains a fifth cysteine residue at position 86 . IFN-alpha 1 (phi WT) carrying three serine for cysteine substitutions at positions 1, 86 and 99 retains 23% of the antiviral specific activity of IFN-alpha 1 (phi WT) on human cells . However, the antiviral activity on bovine cells is not significantly affected by this modification . The presence of the disulphide bridge between residues 1 and 99 thus appears to be required for full antiviral activity on human but not bovine cells . A single serine for cysteine substitution at position 29 reduces the antiviral specific activity on both human and bovine cells by some 95% . This data shows that the disulphide bridge between residues 29 and 139 is critical for the antiviral activity of IFN-alpha's.

Pflugers Arch, 1985 Sep, 405(1), 1 - 4
Comparison of the action of prostaglandin with endotoxin on thermoregulatory response thresholds; Hashimoto M et al.; Prostaglandin E2 (PGE2) and lipopolysaccharide (LPS) derived from E . coli were injected into the lateral cerebral ventricle of rabbits at 30 degrees C ambient temperature . The threshold core temperatures for ear cutaneous vasoconstriction (Thv) and shivering (Thsh) were determined by whole-body cooling with an intestinal thermode . Each threshold, as determined at the plateau phase of LPS fever and PGE2 hyperthermia respectively, were compared with the control values before LPS and PGE2 injection . Thsh was not changed by the injection of LPS, while Thv was increased . After PGE2 injection both Thsh and Thv were increased in comparison to their control levels . These changes paralleled the elevation of core temperature . The present study does not exclude prostaglandins as humoral mediators involved in some of the central processes generating fever, but suggest at the same time that there are additional properties of LPS fever for which prostaglandins do not account.

J Infect, 1985 Sep, 11(2), 167 - 71
An outbreak of food-borne enterotoxigenic Escherichia coli diarrhoea in England; Riordan T et al.; An outbreak of diarrhoea with abdominal pain occurred among members of the staff of a school and their guests after a social function at which a cold buffet was served . Sixty people attended the function and 43 subsequently completed questionnaires . Of these, 27 had diarrhoea . The median incubation period was 36 h and the range 12-66 h . Food history analysis showed an association between diarrhoea and eating curried turkey mayonnaise . Stool specimens from 13 of those who developed diarrhoea were examined: Escherichia coli 06.H16 (producing heat-stable and heat-labile enterotoxins) was found in nine specimens and E . coli 027.H20 (producing heat-stable enterotoxin) in 11 specimens . Eight patients had both strains and only one was negative for enterotoxigenic E . coli . Food samples were not available for examination . Enterotoxigenic E . coli should be considered as a possible cause in well-defined outbreaks of food-borne diarrhoea with abdominal pain.

Am J Vet Res, 1985 Sep, 46(9), 1971 - 4
Bovine neutrophils treated with chemotactic agents: morphologic changes; Forsell JH et al.; Neutrophils were isolated from the peripheral blood of cattle . After the neutrophils were incubated with zymosan-activated serum, the neutrophils changed from spherical to a bipolar shape . Ninety percent of the neutrophils became bipolar in 5 to 10 minutes . Bacterial cell filtrate and casein also induced bipolar shape changes in neutrophils, but N-formyl-L-methionyl-L-leucinyl-L-phenylalanine did not . The neutrophil-shape-change response was a rapid in vitro assay to evaluate early chemotactic events.

Age Ageing, 1985 Sep, 14(5), 282 - 4
Late infection of joint prostheses; Dhala AA et al.; Late infection of an orthopaedic prosthesis can cause serious illness in elderly patients . Four cases are described to show the importance of recognizing this condition.

J Pathol, 1985 Sep, 147(1), 41 - 8
Effects of endotoxin-treatment on inflammatory cell infiltrates in murine Meth A sarcoma; Kuper CF et al.; The effect of intravenously injected endotoxin on inflammatory cells within solid Meth A tumours was studied and central hyperaemia, necrosis and early collapse were observed macroscopically at 4, 24 and 48 h, respectively . The effects were studied in semithin sections and cytocentrifuge preparations of the tumours . The inflammatory cell reaction evoked by the tumours in untreated animals was relatively slight . It was located predominantly around the lateral margins of the tumours and only a few inflammatory cells were found inside the tumour . Prominent effects of endotoxin included a transient increase of mononuclear inflammatory cells in the centre of the tumour by 4 h and a reduction of the influx of lymphocytes, observed in and around the margin of control tumours, by 48 h . Mast cells formed an important part of the inflammatory cell infiltrate, but no distinct changes in number and appearance were observed with time or following treatment . Total host cell numbers within tumours did not increase significantly upon endotoxin-treatment . Results suggest that a direct cytotoxic action of host cells cannot account for the extensive tumour damage observed . Rather, endotoxin-induced regression seems to be related to decreased lymphocyte numbers.

Br J Ind Med, 1985 Sep, 42(9), 591 - 5
Influence of organic solvent mixtures on biological membranes; Gustafson C et al.; A simple experimental model was used to study the influence of organic solvents and solvent mixtures on the integrity of biological membranes . Radiolabelled membranes were prepared biosynthetically by growing Escherichia coli in the presence of 14C-oleic acid; the bulk of the radioactivity was incorporated into 14C-phosphatidylethanolamine, the predominant phospholipid species in E coli membranes . The radiolabelled bacteria were incubated at 37 degrees C in the presence of solvent, and the mixture filtrated through a Millipore 0.45 micron filter . This filtration retained radiolabel associated with the bacteria, and only radiolabel released as a result of solvent action was allowed through the filter . The radioactivity in the filtrate was then counted and expressed as a percentage of the total radioactivity . Results showed that aliphatic alcohols released membrane constituents in relation to their hydrocarbon chain length (1-propanol greater than 2-propanol greater than ethanol greater than methanol); the effects of aliphatic alcohols were potentiated by acetone, ethyl methyl ketone, ethylene glycol, and N,N'-dimethylformamide, and the effects of ethanol were potentiated by 1-butanol, benzyl alcohol, and ethylacetate . These findings point to the possibility that certain mixtures of organic solvents are more damaging to membranes than the components of the mixture would indicate, and suggest that the experimental model used might help in showing mixtures that are particularly harmful.

Am Rev Respir Dis, 1985 Sep, 132(3), 569 - 75
A fibrinolytic inhibitor of human alveolar macrophages . Induction with endotoxin; Chapman HA Jr et al.; Alveolar macrophages are intimately involved with fibrin during the course of acute and chronic inflammatory processes in the lung . In this study, the capability of macrophages to impede fibrinolysis was investigated . Human alveolar macrophages obtained by lavage from normal volunteers released a fibrinolytic inhibitor during the first 24 h of in vitro culture but only inconsistently and in some cases (7 of 15) not at all . Lysates of freshly lavaged cells had no activity . Endotoxin, 5 to 100 ng/ml, consistently induced intracellular accumulation and extracellular release of a fibrinolytic inhibitor by cultured macrophages . Induction required protein synthesis . The intracellular and secreted forms of the inhibitor were true plasminogen activator (PA) inhibitors, as judged by their ability to block urokinase-mediated conversion of 125I-plasminogen . On average, 10(7) endotoxin-stimulated macrophages secreted sufficient PA inhibitor during a 24-h culture in vitro to neutralize 2 picomoles urokinase (16 international units) . Analysis of the interaction of the human macrophage PA inhibitor with 125I-urokinase (apparent size, 33 kilodaltons) by SDS-gel electrophoresis showed that the enzyme and inhibitor mostly dissociated in SDS, but a stable complex occurred at 60 to 65 kilodaltons and a broad band of enzyme or enzyme-inhibitor complexes between 33 and 40 kilodaltons . Either heat treatment of the inhibitor or active site inhibition of urokinase with p-nitrophenylguanidinobenzoate blocked both types of interaction . The pattern of interaction was virtually indistinguishable from that of a partially purified human placental urokinase inhibitor but different from that of serum protease inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Am Rev Respir Dis, 1985 Sep, 132(3), 494 - 9
Effects of OKY-046, a selective thromboxane synthetase inhibitor, on endotoxin-induced lung injury in unanesthetized sheep; Kubo K et al.; We tested the effects of OKY-046, a selective thromboxane synthetase inhibitor, on endotoxin-induced lung injury in unanesthetized sheep in order to evaluate the role of thromboxane (Tx) in this injury . Escherichia coli endotoxin (1 microgram/kg) infusion produced a biphasic response . The early period (Phase 1) was a transient pulmonary hypertension . The late period (Phase 2) was a more prolonged period characterized by a marked high flow of lung lymph with a high concentration of protein, suggesting increased pulmonary vascular permeability . During Phase 1, there were remarkable increases in TxB2 and 6-keto-PGF1 alpha concentrations in lung lymph and in plasma samples obtained from the pulmonary artery (PA) and the left atrium (LA) . The increase in plasma TxB2 level of the LA was greater than that of the PA . During Phase 2, TxB2 levels returned to the baseline values, whereas 6-keto-PGF1 alpha levels remained elevated . Pretreatment with OKY-046 prevented the pulmonary hypertension and increases in TxB2 levels during Phase 1 . However, OKY-046 had little effect on lung lymph balance during Phase 2 . We conclude that the early pulmonary hypertension induced by endotoxin is mediated mainly by release of TxA2 from the lungs, and TxA2 is not attributed to the increased pulmonary permeability during the late period.

Am J Surg, 1985 Sep, 150(3), 301 - 5
Characterization of neutrophil iodination for the assessment of phagocytic and opsonic function in septic patients; Kellerman JS et al.; To increase our understanding of the nature of surgical infection, further studies on the host defense abilities of infected patients are required . Therefore, a more thorough investigation of the iodination method for the measurement of polymorphonuclear leukocyte function and serum opsonic activity was undertaken to characterize its application in surgical infection . A significant relationship was found between the phagocytic indices derived from different standard neutrophils or sera measured on the same day . When expressed as a value of normal phagocytic indices minus abnormal phagocytic indices, this relationship was constant from day to day despite wide variations in the absolute phagocytic index values . This finding enables direct comparisons to be made between the values obtained both from the same patient and from different patients during the course of their illness by reference to daily control values . We also found that the system was sufficiently sensitive to detect, in a dose-responsive manner, the changes induced in normal neutrophil phagocytosis and serum opsonic activity by a specific bacterial challenge with either K . pneumoniae or E . coli . In addition, zymosan, which is utilized in the iodination reaction but also has immunoadjuvant properties, was found to enhance neutrophil function but depress serum opsonic activity in the face of such bacterial challenges . We conclude that the iodination technique is a credible method for the indirect measurement of polymorphonuclear phagocytosis and serum opsonic function in the face of a bacterial challenge and can be reliably employed in studies of septic patients provided these findings are taken into account.

Proc Natl Acad Sci U S A, 1985 Sep, 82(18), 6090 - 4
Protein RepC is involved in copy number control of the broad host range plasmid RSF1010; Haring V et al.; Essential replication (rep) genes of the broad host range plasmid RSF1010 have been cloned onto controlled expression vectors and their protein products have been visualized, after induction, by NaDodSO4/polyacrylamide gel electrophoresis of whole cell lysates . During this induction the replication of a coresident RSF1010 replicon, pKT210, was analyzed by quantitative DNA X DNA hybridization . The initiation of pKT210 replication was stimulated 6-fold by a simultaneous overproduction of the RepA and RepC proteins compared to cells in which only the RepA protein was overproduced . An enhanced synthesis of the RepB protein resulted in a 1.6-fold stimulation of pKT210 replication, whereas an overproduction of the RepA protein alone had no effect . Purified RepC protein has been shown to bind preferentially to DNA carrying the replication origin of RSF1010 . Within this segment it was bound specifically to those DNA fragments that contained the 20-base-pair direct repeats of the origin region . These results suggest that RepC protein acts as a positive replication regulator, that its concentration is rate-limiting, and that the replication rate of RSF1010 is controlled, at least in part, at the level of RepC synthesis.

Proc Natl Acad Sci U S A, 1985 Sep, 82(18), 6060 - 4
Cloning and expression in Escherichia coli of the cDNA for murine tumor necrosis factor; Pennica D et al.; A murine tumor necrosis factor (MuTNF) cDNA was isolated from a cDNA library prepared by using mRNA from the murine macrophage-like cell line PU5-1.8 induced with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate . The cDNA encodes a polypeptide consisting of a 79 amino acid pre sequence followed by a mature MuTNF sequence of 156 amino acids . The 235 amino acid murine pre-TNF polypeptide is 79% homologous to the human pre-TNF protein . There is one potential N-linked glycosylation site on MuTNF, in contrast to human TNF, which lacks any such site . The MuTNF cDNA, when engineered for expression in Escherichia coli, was found to direct the synthesis of biologically active MuTNF as determined by its cytotoxicity against several transformed cell lines.

Proc Natl Acad Sci U S A, 1985 Sep, 82(17), 5681 - 4
Cloning, expression in Escherichia coli, and reconstitution of human myoglobin; Varadarajan R et al.; A full-length cDNA clone for human myoglobin has been isolated from a human skeletal muscle cDNA library . The clone as isolated has a cDNA insert approximately one kilobase long and has 5' and 3' untranslated regions of approximately 80 and 530 base pairs, respectively . The sequence of the translated region corresponds exactly to that predicted for human myoglobin . The cDNA was expressed in high yield in Escherichia coli as a fusion protein consisting of the first 31 amino acids of the phage lambda cII gene, the tetrapeptide Ile-Glu-Gly-Arg, and the myoglobin sequence by following the approach of Nagai and Thogersen {Nagai, K . & Thogersen, M . C . (1984) Nature (London) 309, 810-812} . The fusion product was isolated, reconstituted with heme, cleaved with trypsin, and purified to generate a protein whose properties are indistinguishable from those for authentic human myoglobin . Myoglobin can be readily prepared on a gram scale by using these methods.

Proc Natl Acad Sci U S A, 1985 Sep, 82(17), 5656 - 60
Translational efficiency of the Escherichia coli adenylate cyclase gene: mutating the UUG initiation codon to GUG or AUG results in increased gene expression; Reddy P et al.; Roy et al . {Roy, A., Haziza, C . & Danchin, A . (1983) EMBO J . 2, 791-797} established that translation of Escherichia coli adenylate cyclase initiates at a UUG codon, and they suggested this might decrease the efficiency of translation . We investigated the effect of varying the initiation codon on the expression of the adenylate cyclase (cya) gene . Using oligonucleotide-directed mutagenesis, we changed the UUG initiation codon to GUG and the more common initiator AUG and assayed for cya gene expression in a number of ways . First, the GUG initiation codon, in place of UUG, doubled cya expression when cya was expressed from the dual cya P1/P2 promoters . The corresponding AUG codon construct was nonviable . Second, when the cya gene was placed under the transcriptional control of the thermoinducible phage lambda PL promoter, the relative amounts of cya gene product were 1:2:6 for the UUG, GUG, and AUG initiation codons, respectively . Finally, the cya P2 promoter, Shine-Dalgarno sequence, and the DNA corresponding to the first 86 codons of cya were fused to DNA encoding the E . coli galactokinase gene beginning at the second codon . The relative amounts of the fusion polypeptides, which had galactokinase activity, were 1:2:3 for the UUG, GUG, and AUG initiation codons, respectively . These results demonstrate that the cya UUG initiation codon limits cya expression at the level of translation.

Mutat Res, 1985 Sep, 146(2), 185 - 9
On mutation fixation resulting from nitrosoguanidine-induced DNA damage in Escherichia coli K12 ada-; Orrego C et al.; Brief treatment with nitrosoguanidine of E . coli defective in the removal of the pre-mutagenic lesion, gives rise to cells that segregate mutant clones . No depletion in the number of such cells occurs for at least 4 generations of growth in liquid medium . It is concluded that pre-mutagenic lesions persist and result in copying errors by an otherwise normal replication machinery.

J Bacteriol, 1985 Sep, 163(3), 938 - 42
Control of utilization of L-arginine, L-ornithine, agmatine, and putrescine as nitrogen sources in Escherichia coli K-12; Shaibe E et al.; The regulation of the synthesis of the enzymes involved in the utilization of L-arginine, L-ornithine, agmatine, and putrescine as a sole nitrogen source in Escherichia coli K-12 was examined . The synthesis of agmatine ureohydrolase, putrescine aminotransferase, and pyrroline dehydrogenase is dually controlled by catabolite repression and nitrogen availability . Catabolite repression of agmatine ureohydrolase, but not that of putrescine aminotransferase or pyrroline dehydrogenase, is relieved by the addition of cAMP . Agmatine ureohydrolase synthesis in addition is subject to induction by L-arginine and agmatine . Arginine decarboxylase and ornithine decarboxylase synthesis is not sensitive to catabolite repression or to stimulation by nitrogen limitation or subject to substrate induction.

J Bacteriol, 1985 Sep, 163(3), 933 - 7
Metabolic pathway for the utilization of L-arginine, L-ornithine, agmatine, and putrescine as nitrogen sources in Escherichia coli K-12; Shaibe E et al.; The pathway for the utilization of L-arginine, agmatine, L-ornithine, and putrescine as the sole nitrogen source by Escherichia coli K-12 has been elucidated . Mutants impaired in the utilization of one or more of the above compounds were isolated, and their growth on the different compounds as a sole source of nitrogen and the activities of enzymes of the putative pathway were examined . Our results show that L-arginine is first decarboxylated to agmatine, which is hydrolyzed to urea and putrescine . L-Ornithine is decarboxylated to putrescine . Putrescine is transaminated to gamma-aminobutyraldehyde, which is oxidized to gamma-aminobutyric acid . gamma-Aminobutyric acid is degraded to succinate . The gene for putrescine aminotransferase was located at 89 min on the E . coli K-12 chromosome, and the gene for gamma-aminobutyraldehyde (pyrroline) dehydrogenase was mapped at approximately 30 min.

J Bacteriol, 1985 Sep, 163(3), 870 - 6
Evidence for RecA protein association with the cell membrane and for changes in the levels of major outer membrane proteins in SOS-induced Escherichia coli cells; Garvey N et al.; Membrane fractions from Escherichia coli cells expressing DNA damage-inducible (SOS) functions contain elevated quantities of RecA protein (L . J . Gudas and A . B . Pardee, J . Mol . Biol . 101:459-477, 1976) . We used two-dimensional polyacrylamide gel electrophoresis to separate membrane proteins from several strains to determine whether this effect is an artifact due to contamination of membranes during preparation by the large amount of cytoplasmic RecA present in SOS-induced cells . We found that amplification of RecA+ protein without a DNA-damaging treatment does not result in increased RecA-membrane association, whether recA is depressed specifically by an operator-constitutive recA allele or coordinately with other SOS genes by a lexA mutation that inactivates their common repressor . In contrast, large amounts of RecA appear in membrane fractions from undamaged cells of an SOS-constitutive strain carrying recA730, which encodes a spontaneously SOS-activated RecA . We conclude that the increased association of RecA with the membrane fraction requires the presence of the activated form of RecA, and that this association may contribute significantly to the SOS response . We describe also striking effects of SOS expression on the levels of the outer membrane proteins OmpA, OmpC, and OmpF.

J Bacteriol, 1985 Sep, 163(3), 817 - 23
Repair of cis-platinum-DNA adducts by ABC excinuclease in vivo and in vitro; Husain I et al.; cis-Platinum compounds, which are used in cancer chemotherapy, are thought to exert their effect by damaging DNA . It is known that this damage is partially repaired in Escherichia coli . Using cis-Pt-treated pBR322 DNA as a probe, we investigated the role of nucleotide excision repair in the removal of Pt-DNA adducts . We found that the nucleotide excision pathway was the major mechanism for repairing Pt adducts in transforming plasmid DNA but that a recA-dependent pathway also contributed to plasmid survival . When cis-Pt-damaged pBR322 was treated with the purified nucleotide excision enzyme ABC excinuclease in vitro, a fraction of the adducts was removed by the enzyme; this removal resulted in a corresponding increase in transformation efficiency.

J Bacteriol, 1985 Sep, 163(3), 1288 - 9
supN ochre suppressor gene in Escherichia coli codes for tRNALys; Uemura H et al.; We describe the cloning and nucleotide sequence of a new tRNALys gene, lysV, in Escherichia coli . An ochre suppressor allele of this gene, supN, codes for a tRNALys with anticodon UUA, presumably derived by a single base change from a wild-type UUU anticodon . The sequence of the supN tRNALys is identical to the sequence of ochre suppressor tRNAs encoded by mutant alleles at the lysT locus . This locus, which contains the two previously known tRNALys genes of E . coli, is located far from the lysV locus on the chromosome.

J Bacteriol, 1985 Sep, 163(3), 1267 - 9
Single transporter for sulfate, selenate, and selenite in Escherichia coli K-12; Lindblow-Kull C et al.; A Michaelis-Menten kinetic analysis of the transport of sulfate, selenate, and selenite into Escherichia coli K-12 showed that the three dianions were transported by the same carrier . Km values, used as a measure of the affinity of each ligand for the carrier, showed that sulfate was bound 5 times more tightly than selenate and 37 times more tightly than selenite . The specificity ratio, Vmax/Km, also indicated that sulfate was the preferred ligand . There was little difference in the ratios for selenate and selenite.

Infect Immun, 1985 Sep, 49(3), 528 - 32
Correlation between adherence to HeLa cells and serogroups, serotypes, and bioserotypes of Escherichia coli; Scaletsky IC et al.; Four hundred fifty Escherichia coli strains of 45 O serogroups and subgroups and 112 serotypes were studied to determine their patterns of adherence to HeLa cells . Adherence was exhibited by strains of 17 O serogroups and subgroups, but within these groups more than one adherence pattern was frequently observed . However, within each serotype, the adherence pattern was highly consistent . Localized adherence (LA) was observed much more frequently in serotypes that we considered to be enteropathogenic E . coli serotypes (93%) than in other serotypes (14%), whereas diffuse adherence (DA) occurred predominantly among nonenteropathogenic E . coli strains . Determination of biochemical characteristics showed that within O serogroups, nonmotile strains tended to have the same behavior as motile strains with the LA adherence pattern, suggesting that they were derived from these motile strains . LA and non-LA strains of the same serotype differed biochemically . LA appears to be a property of most E . coli commonly considered to be enteropathogenic and should assist attempts to determine which E . coli are enteropathogenic and to elucidate their pathogenic mechanisms.

J Virol, 1985 Sep, 55(3), 533 - 46
Monoclonal antibodies specific for adenovirus early region 1A proteins: extensive heterogeneity in early region 1A products; Harlow E et al.; Hybridomas secreting monoclonal antibodies specific for the adenovirus early region 1A (E1A) proteins were prepared from BALB/c mice immunized with a bacterial trpE-E1A fusion protein . This protein is encoded by a hybrid gene that joins a portion of the Escherichia coli trpE gene and a cDNA copy of the E1A 13S mRNA (Spindler et al., J . Virol . 49:132-141, 1984) . Eighty-three hybridomas that secrete antibodies which recognize the immunogen were isolated and single cell cloned . Twenty-nine of these antibodies are specific for the E1A portion of the fusion protein . Only 12 of the monoclonal antibodies can efficiently immunoprecipitate E1A polypeptides from detergent lysates of infected cells . E1A polypeptides were analyzed on one-dimensional, sodium dodecyl sulfate-polyacrylamide gels and two-dimensional, isoelectric focusing polyacrylamide gels . The E1A proteins that are specifically immunoprecipitated by the monoclonal antibodies are heterogeneous in size and charge and can be resolved into approximately 60 polypeptide species . This heterogeneity is due not only to synthesis from multiple E1A mRNAs, but also at least in part to post-translational modification . Several of the monoclonal antibodies divide the E1A polypeptides into immunological subclasses based on the ability of the antibodies to bind to the antigen . In particular, two of the monoclonal antibodies bind to the polypeptides synthesized from the 13S E1A mRNA, but not to other E1A proteins.

Int J Radiat Biol Relat Stud Phys Chem Med, 1985 Sep, 48(3), 381 - 8
Sites of termination of in vitro DNA synthesis on psoralen phototreated single-stranded templates; Piette J et al.; Single-stranded DNA has been photochemically induced to react with 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and used as substrate for DNA replication with E . coli DNA polymerase I large fragment . By using the dideoxy sequencing procedure, it is possible to map the termination sites on the template photoreacted with HMT . These sites occur at the nucleotides preceding each thymine residue (and a few cytosine residues), emphasizing the fact that in a single-stranded stretch of DNA, HMT reacts with each thymine residue without any specificity regarding the flanking base sequence of the thymine residues . In addition, termination of DNA synthesis due to psoralen-adducted thymine is not influenced by the efficiency of the 3'-5'exonuclease proof-reading activity of the DNA polymerase.

Mutat Res, 1985 Sep, 146(2), 143 - 7
Induction of 3-methyladenine DNA glycosylase II is recA+-independent; Evensen G; The recA1 mutation was transduced into the tag-2 mutant of E . coli, thus making a strain deficient in the induction of SOS repair as well as in the constitutive repair of 3-alkylated adenines in DNA . The double mutant recA tag is more sensitive to methyl methanesulfonate exposure than either single mutant, indicating that recA and tag mutations block different pathways in repair of alkylation damage . The double mutant is more deficient in host cell reactivation of alkylated phages than the tag single mutant . However, alkylation induction of the double mutant with N-methyl-N'-nitro-N-nitrosoguanidine resulted in killing adaptation of the cells to methyl methanesulfonate and restored the host cell reactivation capacity for alkylated lambda phage to wild-type levels . These adaptive responses can be ascribed to the induction of 3-methyladenine DNA glycosylase II which is shown by enzyme analysis to proceed normally in the recA mutant background . The results imply that the induction of the alkA gene encoding 3-methyladenine DNA glycosylase II is independent of SOS induction.

Virology, 1985 Sep, 145(2), 304 - 12
Reinitiation during lambda DNA replication resulting from either cis-Pt treatment or infection of a P2 lysogenic strain; Schnos M et al.; Nested areas of replication are observed in phage lambda replicative intermediates and arise from reinitiation from the lambda origin . Reinitiation occurs when the first round of lambda replication takes place in the presence of the drug cis-Pt or when lambda infects a host which has been preincubated with the drug . In the latter case it is shown that the infection proceeds during the expression of SOS functions induced in the host as a result of the drug treatment . When lambda infects a host lysogenic for phage P2, an interference process occurs which prevents formation of lambda phage . The lambda DNA does, however, undergo at least one round of replication but is abnormal in that lambda origins reinitiate to form nested areas of replication similar to those resulting from exposure to the drug cis-Pt.

Mol Cell Biol, 1985 Sep, 5(9), 2307 - 15
Novel method for identifying sequence-specific DNA-binding proteins; Levens D et al.; We developed a general method for the enrichment and identification of sequence-specific DNA-binding proteins . A well-characterized protein-DNA interaction is used to isolate from crude cellular extracts or fractions thereof proteins which bind to specific DNA sequences; the method is based solely on this binding property of the proteins . The DNA sequence of interest, cloned adjacent to the lac operator DNA segment is incubated with a lac repressor-beta-galactosidase fusion protein which retains full operator and inducer binding properties . The DNA fragment bound to the lac repressor-beta-galactosidase fusion protein is precipitated by the addition of affinity-purified anti-beta-galactosidase immobilized on beads . This forms an affinity matrix for any proteins which might interact specifically with the DNA sequence cloned adjacent to the lac operator . When incubated with cellular extracts in the presence of excess competitor DNA, any protein(s) which specifically binds to the cloned DNA sequence of interest can be cleanly precipitated . When isopropyl-beta-D-thiogalactopyranoside is added, the lac repressor releases the bound DNA, and thus the protein-DNA complex consisting of the specific restriction fragment and any specific binding protein(s) is released, permitting the identification of the protein by standard biochemical techniques . We demonstrate the utility of this method with the lambda repressor, another well-characterized DNA-binding protein, as a model . In addition, with crude preparations of the yeast mitochondrial RNA polymerase, we identified a 70,000-molecular-weight peptide which binds specifically to the promoter region of the yeast mitochondrial 14S rRNA gene.

J Biochem (Tokyo), 1985 Sep, 98(3), 859 - 62
Nucleotide sequence of the 5' flanking region responsible for the enhancement of the expression of yeast enolase 1 gene; Uemura H et al.; Several plasmids carrying different length of the 5' flanking region of yeast (Saccharomyces cerevisiae) enolase 1 gene (ENO1) which is fused in frame to the Escherichia coli lacZ gene were constructed by recombination in vitro . Promoter activity of ENO1 was assayed by measuring beta-galactosidase activity of the fused gene product . Comparison of the promoter activity of these plasmids suggests that the sequences required for a strong promoter activity lie within the DNA segment -724 to -353 base pairs (bp) upstream from the start of ENO1 coding sequence . The nucleotide sequence of this region was determined.

Biochimie, 1985 Sep, 67(9), 987 - 97
In vivo and in vitro effect of mutations in tetA promoter from pSC101: insertion of poly(dA.dT) stretch in the spacer region does not inactivate the promoter; Jacquet MA et al.; Two mutants, mapping at the HindIII site (between the consensus sequences) of the pSC101 tetA promoter, were studied: MA2 corresponds to a 4 bp deletion between positions -12 and -15; B30 bears a 44 bp insertion C(TA)21 G at the HindIII site . Both mutants were assayed in vivo (ability of the plasmid to confer resistance to tetracycline, plasmid-directed protein synthesis, S1-mapping of mRNA) and in vitro (abortive initiation assay) . Compared to w.t., MA2 is a poor promoter in vivo; RNA polymerase binding, complex activation and rate of initial oligonucleotide synthesis are strongly reduced in vitro; this is in keeping with the known effects of altering the consensus elements in E . coli promoters . In contrast, B30 shows in vivo a promoter activity only slightly reduced in comparison to that of the w.t . tetA promoter; both in vivo and in vitro, the transcription start site is outside and downstream the (TA)21 stretch, 5-7 bp upstream that found in the w.t . To adjust the behaviour of B30 and the claimed consensus distance between the E . coli promoter consensus sequences, some structural modification in the (TA)21 stretch -either spontaneous or induced by RNA polymerase- can be hypothesized . Unless the (TA)21 stretch itself plays the role of a relatively good promoter, the results suggest that promoter-specific elements may be distributed along the DNA sequences over distances longer, but seldom less, than the 17 +/- 2 bp consensus distance.

Biochimie, 1985 Sep, 67(9), 959 - 71
Construction and identification of recombinant plasmids carrying cDNAs coding for ovine alpha S1-, alpha S2-, beta-, kappa-casein and beta-lactoglobulin . Nucleotide sequence of alpha S1-casein cDNA; Mercier JC et al.; An ovine mammary cDNA library has been constructed from total poly(A)+ RNA isolated from the mammary gland of a lactating ewe, using a classical procedure . Blunt-ended double-stranded cDNAs prepared with reverse transcriptase and nuclease S1 were tailed with dCTP, inserted into the dGMP-tailed PstI site of plasmid pBR322 and cloned in E . coli . Five series of homologous clones representing abundant messenger RNAs (strong hybridization with a single-stranded cDNA probe generated from total poly(A)+ RNA) were selected using each time a different predominant cloned ds-cDNA as probe, then identified by positive hybridization-translation of the cognate mRNA and subsequent immunoprecipitation and electrophoresis of the protein . The lengths of alpha s1-, alpha s2-, beta-, kappa-casein and beta-lactoglobulin mRNAs are in the range of 1.2, 1.1, 1.25, 1.0 and 0.85 kb, respectively, as determined by Northern blotting analysis . Five homologous mRNAs of similar sizes were identified in the porcine species by dot blot hybridization and Northern analyses . The nucleotide sequence of alpha s1-casein mRNA was determined by sequencing, according to Maxam and Gilbert, both a 1080 bp long cloned ds-cDNA and a ss-cDNA (268 nucleotides) generated by 5' extension of a 5' terminal truncated radiolabeled fragment (83 bp) of the relevant ds-cDNA, used as primer for reverse transcription . The 3' non coding region (431 nucleotides, excluding the poly(A) tail) represents 70% of the length of the coding region (618 nucleotides) flanked by a 61 nucleotide 5' region . Comparison of sequences of ovine and bovine, rat and guinea-pig alpha s1-casein mRNAs has revealed a greater homology in the 3' and especially 5' non coding regions . In the reading frame, the conserved regions are essentially those corresponding to the signal peptide and phosphopeptide domains . The derived 206 amino acid sequence of ovine pre-alpha s1-casein differs from that of its bovine counterpart (genetic variant B) by 24 amino acid substitutions and a deletion of 8 amino acid residues occurring in the polypeptide chain of the mature protein . Such a variation (84% homology only) in two phylogenetically closely related species indicates a high rate of evolution of alpha s1-casein.

Biochimie, 1985 Sep, 67(9), 1053 - 7
Sequence analysis of the glyW region in Escherichia coli; Tucker SD et al.; We have determined the DNA sequence of a 1-kilobase segment of the Escherichia coli chromosome . The segment contains glyW, a duplicate gene for tRNA3Gly, and its flanking regions . An insertion sequence, previously known to have occurred spontaneously within the sequenced fragment, was identified as IS1 . Possible sites for initiation and termination of transcription were identified by comparing them with the sequences of model promoter regions and termination structures . The results suggest that the expression of glyW may depend upon the expression of the preceding gene, pgsA, by transcriptional or translational overlap, by cotranscription of these two genes, or both.

Mol Biol (Mosk), 1985 Sep-Oct, 19(5), 1242 - 50
{Immunity to repeated transposition of the insertion sequence IS21}; Danilevich VN et al.; The ability of pBR325 derivatives carrying a copy of IS21-element to accept the second copy of this element from plasmid pRP19.6, a temperature-sensitive for replication mutant of RPI containing the duplicated IS21 was studied . It was shown that the frequency of IS21 transposition into plasmids pBR32S::IS21 differing by localization IS21 was lower by two orders of magnitude as compared to that of pBR325 . The restriction endonuclease analysis revealed that the insertion of the second copy of IS21 resulted in the formation of pBR325 derivatives carrying the tandem repeated copies of IS21 . It was also shown that the plasmids pBR325::IS21 were capable of increasing the frequency of pRP19.6 insertion into the bacterial chromosome from 3-9 to 200-300 times depending on IS21 localization . On the basis of the results obtained and literature data the possible mechanism of the transposition immunity is discussed.

Mol Biol (Mosk), 1985 Sep-Oct, 19(5), 1194 - 205
{Effectiveness of expression of the chloramphenicol acetyltransferase gene controlled by foreign regulatory regions in Escherichia coli cells . I . Construction of vectors for the cloning of transcription regulatory elements}; Mashko SV et al.; New plasmids pML2.1 and pML4 were constructed for cloning the transcription regulatory regions . In the pML2.1 the structural part of chloramphenicol acetyltransferase gene of the pBR325 is under control of the lacUV5-promotor . Because the unique BamH1 cleavage site is in the joint region, one may use it for cloning transcription termination regions and selecting recombinant clones with the AprCms phenotype . As for the pML4, the foreign fragment integration is carried directly before the structural part of cat-gene and it is expressed only if the promotor regions are present . The plasmids were sequenced and their restriction maps were established . Small molecular weight (about 2,0 MDa, AprCmr) or only Apr intact genes and convenient disposition of many unique cleavage sites by restriction endonucleases make these plasmids useful for different genetic engineering experiments.

EMBO J, 1985 Sep, 4(9), 2301 - 7
A single amino acid alteration in the initiation protein is responsible for the DNA overproduction phenotype of copy number mutants of plasmid R6K; Inuzuka M et al.; A novel type of high copy-number (cop) mutants of a mini-R6K plasmid were isolated . The mutations were mapped in the pir gene which encodes the pi initiation protein for plasmid R6K DNA replication . They resulted in an alteration by substitution of a single amino acid: threonine to isoleucine at the 108th position for the cop41, and proline to serine at the 113th position for the cop50, of the 305 amino acid pi protein . The cop41 mutation in the pi protein was found to be trans-dominant over the wild-type allele in the copy control of plasmid R6K . Moreover, it was shown that the altered pi protein was not overproduced in maxicells carrying this mutant plasmid and had a higher affinity to the repeated sequence which is present in the pir promoter region . Most likely the mutated pi protein also interacts more efficiently with the same repeated sequences, a target of pi, in the replication origin region and increases the frequency of the initiation event per cell division.

Can J Biochem Cell Biol, 1985 Sep, 63(9), 969 - 76
Site-directed cleavage of immunoglobulin gene segments by lymphoid cell extracts; D'Agostaro G et al.; To study the enzyme(s) involved in the site-specific recombination of immunoglobulin (Ig) gene segments, we designed an assay to detect V-J joining in vitro . The DNA from a hybrid phage (lambda VJCK) containing the VK41 gene segment separated by a 6-kilobase spacer region from the entire J-CK sequence was incubated with lymphoid cell extracts and packaged in vitro . Phages carrying genomic deletions were selected by screening for ethylenediaminetetraacetic acid resistance . Although no site-specific V-J fusion events were detected, the packaging efficiency of lambda VJCK DNA was 10(2)- to 10(3)-fold lower than that of lambda DNA . This suggested the presence in the cell extracts of an endonucleolytic activity with a specificity for the mouse DNA sequences . To detect the endonuclease cleavage products, plasmids containing VK or JK gene segments were used as a DNA substrate and the products of the in vitro reaction were visualized by autoradiography in Southern blots . Double-stranded cleavages were observed to occur near the 5' end of each one of the five JK gene segments and near the 3' end of a VK gene segment . A plasmid containing the mouse I-A beta gene was found to be resistant to cleavage, thus confirming the specificity of the endonucleolytic activity for sequences associated with the mouse Ig gene segments.

Vopr Virusol, 1985 Sep-Oct, 30(5), 544 - 9
{Discovery of the sequences of herpes simplex virus type 1 in the genomes of normal and transformed mouse and hamster cell lines}; Tsar'kov SG et al.; Sequences capable of hybridization with cloned fragments of herpes simplex virus type I (HSVI) DNA were found in genome DNA of Syrian hamster fibroblast lines transformed either by intact HSVI genome (cell line 14.012.81) or this virus DNA fragments (cell line EH/A44) . The method of pinpoint hybridization demonstrated the presence in DNA of the cell lines under study of sequences capable of hybridization both with unique areas and with HSVI genome replicas . A correlation was established between the tumorigenic properties of the transformed lines and the number of HSVI sequences present in genomes of these lines . The genome of mouse cell line BaLB/3T3 was found to contain sequences homologous to HSVI replica areas, their number exceeding that of virus sequences present in the genome of nontransformed hamster cells (BHK/21 line).

Plasmid, 1985 Sep, 14(2), 167 - 70
One-kilobase direct repeats of plasmid pSa; Valentine CR; One-kilobase, direct repeats were found on either side of the chloramphenicol resistance gene of plasmid pSa . The right repeat corresponded to the region coding for sulfanilamide resistance . The repeats were not identical as judged by distances between restriction enzyme sites, hybridization, and by the ability to confer resistance to sulfanilamide.

Plasmid, 1985 Sep, 14(2), 162 - 6
Transfer of transposable drug-resistance elements Tn5, Tn7, and Tn76 to Azotobacter beijerinckii: use of plasmid RP4::Tn76 as a suicide vector; Owen DJ et al.; Transposable elements Tn5, Tn7, and Tn76 were transferred to Azotobacter beijerinckii . Evidence was obtained for the transposition of Tn5 but cells of the majority of presumptive transposition isolates had abnormal morphologies and rapidly lost viability when subcultured . Data are presented that indicate that plasmid RP4::Tn76 behaves as a suicide vector upon transfer to this host, allowing the isolation of A . beijerinckii::Tn76 isolates at a high frequency . Nitrogen-fixing mutants and leucine and adenine auxotrophs were isolated from cultures in which the transposition of Tn76 occurred.

Plasmid, 1985 Sep, 14(2), 134 - 42
A versatile multiple- and single-copy vector system for the in vitro construction of transcriptional fusions to lacZ; Linn T et al.; A multiple-copy (plasmid) vector and a single-copy (lambda) vector were constructed for the in vitro formation of transcriptional fusions to lacZ . In both vectors the transcription of lacZ is dependent upon the attachment of a promoter upstream, but beta-galactosidase is independently translated from the hybrid mRNA . These vectors are based on the W205 trp-lac deletion, but most of the trpBA DNA has been removed . Promoters are fused to the 3' end of trpA rather than the HindIII site at the 5' end of trpB used in other vectors containing the W205 deletion . This modification avoids the polar effects encountered when either an untranslated sequence, or a sequence translated in a different reading frame is fused to trpB . Hence the level of beta-galactosidase synthesized by fusions in these new vectors accurately reflects the frequency of transcription from the attached promoter . A polyrestriction site linker precedes the lacZ gene in both vectors and allows the direct ligation of promoter containing DNA fragments produced by a large collection of restriction endonucleases.

J Gen Microbiol, 1985 Sep, 131 ( Pt 9), 2443 - 7
Tn2440, a composite tetracycline resistance transposon with direct repeated copies of IS160 at its flanks; Nies BA et al.; The tetracycline resistance region of the multi-resistance plasmid pBP16 is flanked by direct repeats of the insertion sequence IS160 . The tetracycline resistance region plus the flanking IS elements can transpose as a discrete unit . The composite transposon, designated Tn2440, has a size of 4.0 kb.

Virus Res, 1985 Sep, 3(2), 181 - 90
The complete sequence of bluetongue virus serotype 10 segment 3 and its predicted VP3 polypeptide compared with those of BTV serotype 17; Ghiasi H et al.; The complete sequence of the large RNA segment 3 (L3) of bluetongue virus serotype 10 (BTV-10) has been determined from DNA copies of the viral RNA cloned in the E . coli plasmid pBR322 . The L3 viral RNA is 2772 nucleotides long with a single open reading frame of 2706 nucleotides . The L3 predicted primary gene product (VP3) is 103 342 daltons and has a net charge at neutral pH of -5 . The sequence of the L3 RNA species differs by 126 point mutations from that of BTV-17 (i.e., 95.5% homology; see M . Purdy, J . Petre and P . Roy, J . Virol . 51, 754-759, 1984) . The predicted L3 primary gene products of the two viruses differ by 9 amino acids . These differences correspond to 9 point mutations and represent 0.15% of the sites where nucleotide substitution could cause an amino acid change . By contrast, another 114 point mutations in the genome correspond to 6.5% of the available sites where nucleotide substitutions could be silent (i.e., where a nucleotide substitution may not cause an amino acid change) . Three point mutations are in the 3' non-coding region of the RNA species . The quantitative differences between the coding and silent mutations are interpreted as representing the result of gene product conservation.

Somat Cell Mol Genet, 1985 Sep, 11(5), 499 - 504
High spontaneous mutation frequency of BPV shuttle vector; Ashman CR et al.; A recombinant shuttle vector containing the entire bovine papillomavirus (BPV) genome, sequences from pBR322, and the Escherichia coli gpt gene was used to transform mouse C127 cells . Plasmid extracted from the transformed mouse cells was used to transform a Gpt- derivative of E . coli HB101, and the relative frequency of plasmids carrying a mutation in the gpt gene was determined . Approximate mutant frequencies of 3-16 X 10(-3) were observed for plasmid molecules which had been passaged through the mammalian cells . Restriction digest analysis indicated that most of the mutant plasmid molecules had gross rearrangements in their DNA structures.

Proc Natl Acad Sci U S A, 1985 Sep, 82(18), 6104 - 8
Isolation and characterization of the product of the methionine-regulatory gene metJ of Escherichia coli K-12; Smith AA et al.; We have modified a previously isolated metJ plasmid by removing a segment of DNA including the rop gene . Bacterial strains carrying this plasmid produce elevated levels of the metJ gene product, presumably because of the high number of gene copies in the cell . We have isolated the metJ gene product in nearly homogeneous form from such a strain . The subunit size and the amino acid composition are the same as those predicted from the DNA sequence of the metJ gene . Sedimentation equilibrium measurements show that the native metJ gene product is a dimer . The purified dimer protects a short segment of DNA in the regulatory region of the metB and metJ genes from hydrolysis by DNase I.

Proc Natl Acad Sci U S A, 1985 Sep, 82(18), 6045 - 9
Role of the SulB (FtsZ) protein in division inhibition during the SOS response in Escherichia coli: FtsZ stabilizes the inhibitor SulA in maxicells; Jones C et al.; Induction of the SOS response in Escherichia coli by DNA-damaging treatments results in the synthesis of the SulA polypeptide, and this is sufficient to cause the resulting inhibition of cell division . Mutations at either sulA (sfiA) or sulB (sfiB) suppress this division inhibition . The SulB protein is identical to FtsZ, a protein required for normal division in E . coli . In the presence of FtsZ, the half-life of SulA synthesized in maxicells is approximately 12 min . In contrast, in the absence of FtsZ or in the presence of a mutant form of FtsZ (SulB114) that prevents division inhibition in vivo, SulA is extremely unstable with a half-life of only 3 min . Both FtsZ and SulA are isolated with the inner membrane of E . coli maxicells in the presence of MgCl2 . We propose that the SulA inhibitor interacts directly with FtsZ in vivo to block the essential division function of this protein.

Mutat Res, 1985 Sep, 146(2), 149 - 54
Ability of various alkylating agents to induce adaptive and SOS responses: a study with lacZ fusion; Otsuka M et al.; We used alkA'-lacZ' and umuC'-lacZ' fused genes and determined the ability of various alkylating agents to induce adaptive and SOS responses . The degree of induction of expression of these genes was quantitatively measured by a simple colorimetric assay of beta-galactosidase activity . SN1 type methylating agents, such as N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea, were more effective inducers for the alkA than for the umuC system, while SN1 type ethylating agents, such as N-ethyl-N'-nitro-N-nitrosoguanidine and N-ethyl-N-nitrosourea, were more potent inducers for the umuC than for the alkA system . Similar but less striking effects on the two systems were obtained with SN2 type alkylating agents.

J Bacteriol, 1985 Sep, 163(3), 890 - 4
DNA sequence and complementation analysis of a mutation in the rplX gene from Escherichia coli leading to loss of ribosomal protein L24; Nishi K et al.; A mutation in Escherichia coli leads to the loss of ribosomal protein L24, severely impaired growth, and a temperature-sensitive phenotype . The mutation was shown to be in rplX, the gene for protein L24, and was due to the alteration of an AAA codon to a TAA stop codon at position 61 in rplX that resulted in a 20-amino acid peptide instead of the 104 amino acids of wild-type L24 protein . rplX genes from three temperature-resistant and fast growing pseudorevertants of the mutant were cloned and sequenced . They were found to have different base substitutions in the TAA codon, resulting in the reappearance of a full-sized protein L24 moiety . Complementation of the slow growth in trans could be achieved with several plasmids containing at least the spc promoter and intact L14 and L24 genes . Plasmids containing genes distal to rplX could further stimulate growth, and the wild type arose when the entire spc operon and the alpha operon were present . In all cases, protein L24 was expressed by the plasmids . Therefore, slow growth could be explained by polarity extending to the alpha operon . However, temperature sensitivity could not be complemented by any of the plasmids in trans, although we found that this phenotype was caused by the mutation in the rplX gene.

J Bacteriol, 1985 Sep, 163(3), 1196 - 202
Identification of livG, a membrane-associated component of the branched-chain amino acid transport in Escherichia coli; Nazos PM et al.; Branched-chain amino acids are transported into Escherichia coli by two osmotic shock-sensitive systems (leucine-isoleucine-valine and leucine-specific transport systems) . These high-affinity systems consist of separate periplasmic binding protein components and at least three common membrane-bound components . In this study, one of the membrane-bound components, livG, was identified . A toxic analog of leucine, azaleucine, was used to isolate a large number of azaleucine-resistant mutants which were defective in branched-chain amino acid transport . Genetic complementation studies established that two classes of transport mutants with similar phenotypes, livH and livG, were obtained which were defective in one of the membrane-associated transport components . Since the previously cloned plasmid, pOX1, genetically complemented both livH and livG mutants, we were able to verify the physical location of the livG gene on this plasmid . Recombinant plasmids which carried different portions of the pOX1 plasmid were constructed and subjected to complementation analysis . These results established that livG was located downstream from livH with about 1 kilobase of DNA in between . The expression of these plasmids was studied in minicells; these studies indicate that livG appears to be membrane bound and to have a molecular weight of 22,000 . These results establish that livG is a membrane-associated component of the branched-chain amino acid transport system in E . coli.

J Bacteriol, 1985 Sep, 163(3), 1172 - 9
Regulation of single and multicopy his operons in Escherichia coli; Riccio A et al.; We fused segments of the Escherichia coli his regulatory region to galK in single-copy and multicopy vectors . These fusions demonstrated that (i) derepression of his by histidine starvation is due exclusively to attenuation; (ii) the his promoter is metabolically regulated; and (iii) both regulatory systems operate when the his regulatory region is present on a multicopy plasmid . Thus, there is no evidence for titration of his regulatory elements . Deletions of the his anti-attenuator region, carried on multicopy plasmids, cause low-level galK expression . This expression is not stimulated by histidine starvation, but is growth rate dependent . We replaced the his attenuator with the efficient lambda terminator, to . In the context of the his regulatory region, however, lambda to only partially terminates transcription.

J Bacteriol, 1985 Sep, 163(3), 1153 - 7
Identification of the merR gene of R100 by using mer-lac gene and operon fusions; Foster TJ et al.; Transcriptional (operon) and translational (gene) fusions between the R100 merR gene and lacZ were constructed in vitro in a pBR322 plasmid carrying the mer genes derived from plasmid R100 . The translational fusions were oriented in the opposite direction to and divergently from the merTCAD genes . This shows that the reading frame previously thought to be merR was incorrect . Expression of the gene fusion was repressed in trans by a compatible plasmid carrying the R100 merR+ gene, as was a similarly oriented transcriptional fusion . In contrast, expression of beta-galactosidase by the lac fragment located at the same site but in the opposite orientation was at a lower level and was not repressed by merR+.

J Bacteriol, 1985 Sep, 163(3), 1095 - 100
Identification of the Escherichia coli deoR and cytR gene products; Singer JT et al.; The protein products encoded by the Escherichia coli deoR and cytR structural genes have been identified based on results obtained from E . coli maxicells harboring (i) recombinant plasmids carrying wild-type deoR and cytR genes, (ii) deletion derivatives of the deoR+ and cytR+ plasmids, (iii) plasmids containing site-specific mutations in the deoR and cytR structural genes, and (iv) plasmids which have transposon Tn1000 inserted into the deoR and cytR structural genes . Analysis of the protein profiles obtained from all the maxicell experiments demonstrated that the deoR gene encodes a protein with a subunit molecular weight of 30,500 and that the product of the cytR gene is a protein with a subunit molecular weight of 37,000.

J Bacteriol, 1985 Sep, 163(3), 1074 - 9
Isolation and expression of the Escherichia coli gene encoding malate dehydrogenase; Sutherland P et al.; An oligodeoxynucleotide specific for a pentapeptide sequence corresponding to amino acid residues 32 through 36 of Escherichia coli malate dehydrogenase was chemically synthesized and used to identify the mdh gene on plasmid pLC32-38 from the Clarke-Carbon recombinant library . Cells transformed with this plasmid exhibited a 10-fold increase in malate dehydrogenase activity . A 1.2-kilobase PvuII fragment which hybridized with the oligodeoxynucleotide probe was subcloned, and the identity of the mdh structural gene was confirmed by partial nucleotide sequence analysis . The expression of the mdh gene, as measured by both Northern blotting and enzyme assays, was found to vary over a 20-fold range with different culture conditions.

J Bacteriol, 1985 Sep, 163(3), 1067 - 73
F factor inhibition of conjugal transfer of broad-host-range plasmid RP4: requirement for the protein product of pif operon regulatory gene pifC; Miller JF et al.; By the use of deletions, point mutations, and gene fusions, we show that the protein product of the F factor pifC gene is responsible for F factor inhibition of plasmid RP4 conjugal transfer . Deletion analysis of pif sequences carried by pSC101-F chimeric plasmids demonstrated that removal of all or part of the pifC coding sequence greatly decreased or abolished the ability of these plasmids to inhibit RP4 transfer . Amber mutations in the pifC gene eliminated inhibition in an Su- host strain but not in and Su+ (supF) host . Plasmids carrying nonpolar pifC mutations did not decrease the efficiency of RP4 transfer when present in trans . Whereas pifC+ plasmids inhibited RP4 transfer, the presence of RP4 in the same cell as F' lac increased F'lac Pif activity approximately 1,000-fold . This effect most likely resulted from the binding of the pifC product to RP4 DNA and concomitant derepression of the F factor pif operon . PifC inhibited trans mobilization of pMS204, a nonconjugative plasmid carrying the RP4 oriT locus, by the RP1 derivative pUB307 . pMS204 had no trans effect on pif operon expression, whereas pUB307 increased F'lac Pif expression, as did RP4 . Our results suggest that the pifC product inhibits expression of one or more RP4 genes, the products of which are required for conjugal transfer of RP4 and are required in trans for mobilization of nonconjugal RP4 oriT containing plasmids.

J Bacteriol, 1985 Sep, 163(3), 1055 - 9
Physical characterization of the cloned protease III gene from Escherichia coli K-12; Dykstra CC et al.; Analysis of the cloned protease III gene (ptr) from Escherichia coli K-12 has demonstrated that in addition to the previously characterized 110,000-Mr protease III protein, a second 50,000-Mr polypeptide (p50) is derived from the amino-terminal end of the coding sequence . The p50 polypeptide is found predominantly in the periplasmic space along with protease III, but does not proteolytically degrade insulin, a substrate for protease III . p50 does not appear to originate from autolysis of the larger protein . Protease III is not essential for normal cell growth since deletion of the structural gene causes no observed alterations in the phenotypic properties of the bacteria . A 30-fold overproduction of protease III does not affect cell viability . A simple new purification method for protease III is described.

Cell, 1985 Sep, 42(2), 629 - 38
tnpM: a novel regulatory gene that enhances Tn21 transposition and suppresses cointegrate resolution; Hyde DR et al.; We have identified a new gene, tnpM, in Tn21 that encodes the 12.6 kilodalton modulator protein . The Tn21 modulator enhances Tn21 transposition and suppresses resolution of cointegrate replicons in vivo . A putative binding site may be located in the N-terminal portion of the TnpR (resolvase) structural gene sequences . Tn501 transposition and cointegrate resolution can be regulated by the subcloned tnpM gene of Tn21 in trans-complementation experiments . Examination of the Tn501 DNA sequence also reveals a potential tnpM coding sequence upstream of the Tn501 resolvase gene . We conclude that Tn21 and Tn501 are different from Tn3 and Tn1000 both in genome organization and in regulation of transposition functions.

Cell, 1985 Sep, 42(2), 611 - 21
Structure of the eukaryotic transcription apparatus: features of the gene for the largest subunit of Drosophila RNA polymerase II; Biggs J et al.; The Drosophila melanogaster RpII215 locus encodes the largest subunit of RNA polymerase II . We have now mapped the 7 kb transcript of the locus and have determined that it contains four exons and three introns . By sequencing 2582 nucleotides from the promoter-proximal end of the RpII215 locus, we have precisely mapped the start site of transcription and the splice sites of the first intron . Segments of the amino acid sequence predicted by the only long open reading frame of the RpII215 gene transcript display striking homology with corresponding segments of the beta subunit of E . coli RNA polymerase.

Virology, 1985 Sep, 145(2), 356 - 61
Transposon-mediated mutagenesis of a baculovirus; Fraser MJ et al.; Spontaneous mutants of insect nuclear polyhedrosis viruses of Autographa californica (AcMNPV) and Galleria mellonella (GmMNPV) were analyzed . These mutants produce few polyhedra in infected cells and have insertions of host cell DNA . All the insertions mapped to two adjacent sites in the genome . The junctions between two host insertions and the viral DNA were sequenced . One of the insertions contained a perfect 7-bp inverted terminal repeat, and had caused a direct duplication of 4 bp of viral DNA at the point of insertion . Therefore, this insertion sequence has properties of a transposon of the host cell Trichoplusia ni.

Virology, 1985 Sep, 145(2), 330 - 4
In vitro growth of two related leporipoxviruses in lymphoid cells; Strayer DS et al.; We examined the in vitro growth patterns of two leporipoxviruses, malignant rabbit fibroma virus (MV) and Shope fibroma virus (SFV), in lymphoid cells . MV replicates well in normal spleen cells in vitro . At low m.o.i . (0.001), dramatic virus growth occurs in unstimulated cell cultures . This growth is enhanced by addition of the T lymphocyte mitogen, concanavilin A, or the B lymphocyte mitogen, Escherichia coli lipopolysaccharide . Shope fibroma virus does not grow in lymphocytes in culture, with or without mitogen stimulation . MV itself profoundly inhibits lymphocyte mitogenesis, while SFV does not . MV and SFV added to normal lymphocytes do not appear to alter their viability in culture . Thus, MV appears to be novel in its ability to replicate to high titer in resting lymphocytes . This growth pattern may be useful in understanding MV-induced immunologic dysfunction.

J Biochem (Tokyo), 1985 Sep, 98(3), 629 - 36
Alcohol induced B-A transition of DNAs with different base compositions studied by circular dichroism; Nara-Inui H et al.; The circular dichroic (CD) spectra of natural DNAs (from Cl . perfringens, T2 phage, calf thymus, E . coli, and M . lysodeikticus) as well as duplexes of synthetic DNAs (poly(dA) X poly(dT), poly(dA-dT), and poly(dG-dC} were measured in water-ethanol mixtures with 0.3 mM NaCl . A conformational change from the B to the A form was observed for the natural DNAs on adding ethanol . The ethanol concentration that induces the transition and the extent of the change in the CD spectrum are different for the five natural DNAs depending on their GC contents . The higher the GC content is, the more easily the transition to the A form takes place . The results indicate that the GC content of a DNA is an important factor for induction of the B-A transition . The results for the synthetic DNAs show that their properties cannot be inferred by simple extrapolation of those of natural DNAs . Coexisting ions and the molecular weight of a DNA were also found to affect the induction of the B-A transition.

EMBO J, 1985 Sep, 4(9), 2357 - 63
Proton conduction by subunit a of the membrane-bound ATP synthase of Escherichia coli revealed after induced overproduction; von Meyenburg K et al.; Transcriptional fusions between the phage lambda promotor pR and ATP synthase genes, atp, on plasmid pBR322 were constructed in order to study the effects upon growth and physiology of Escherichia coli of induced overproduction of H+-ATPase subunits . Constitutive overproduction of the complete enzyme had earlier been found to result in decreased growth rate and cytological defects . When a 15-fold overproduction of subunit a alone, or together with subunit c, or with all other ATP synthase subunits was suddenly induced, the following effects were observed . Inhibition of growth and protein synthesis within 10 min of induction, which effect was suppressed by N,N'-dicyclohexylcarbodiimide, also when the chromosomal atp genes coding for the Fo subunits a, b and c were deleted . Partial collapse of the membrane potential delta psi at 4-6 min after induction paralleled by inhibition of thiomethylgalactoside and guanosine transport . Respiration and alpha-methylglucoside transport was not affected . The partial collapse of delta psi, and the specific inhibition of proton-driven transport systems is taken to show that the subunit a has--when suddenly overproduced and inserted into the membrane--a protonophoric activity . It is suggested that this protonophoric activity of subunit a is related to the function of this subunit in the Fo sector in H+-ATPases.

J Gen Microbiol, 1985 Sep, 131 ( Pt 9), 2319 - 26
The possession of three novel coli surface antigens by enterotoxigenic Escherichia coli strains positive for the putative colonization factor PCF8775; Thomas LV et al.; A possible colonization factor, E8775, has previously been described for enterotoxigenic Escherichia coli strains of serogroups O25, O115 and O167 . Re-examination of these strains by immunodiffusion has revealed that the antigenic nature of this factor, renamed putative colonization factor (PCF) 8775, is more complex than was first thought . All the strains of serogroup O25 tested possessed two antigenic components, termed CS4 and CS6, and gave mannose-resistant haemagglutination (MRHA) of human and bovine erythrocytes . Spontaneous variants possessing CS6 only did not give MRHA . Strains of serogroups O115 and O167 had the antigenic components CS5 and CS6, and gave MRHA of human, bovine and guinea-pig erythrocytes . Using immune electron microscopy, the components CS4 and CS5 were identified as fimbriae . No fimbriae were associated with CS6.

J Clin Microbiol, 1985 Sep, 22(3), 457 - 8
Concurrent infection of pigs with enterotoxigenic Escherichia coli of different serogroups; Francis DH et al.; Naturally occurring dual infections with Escherichia coli of different serogroups occurred in 12 pigs 2 to 14 days of age . In each case, one isolate was hemolytic and produced K88 pili and the other was nonhemolytic and produced either K99 or 987P pili.

Arch Biochem Biophys, 1985 Sep, 241(2), 364 - 70
Deletion of seven amino acid residues from the gamma subunit of Escherichia coli H+-ATPase causes total loss of F1 assembly on membranes; Kanazawa H et al.; A mutant gene for the gamma subunit of H+-translocating ATPase was cloned from Escherichia coli mutant NR70 isolated by B . P . Rosen {J . Bacteriol . 116, 1124-1129 (1973)} . Determination of its nucleotide sequence revealed a deletion of 21 base pairs between nucleotide residues 64 and 84, resulting in a deletion of seven amino acid residues (LysAlaMetGluMetValAla) from the amino-terminal portion . This deletion resulted in the loss of a hydrophobic domain of the subunit estimated by an analysis of its hydropathic character . Since F1 subunits are reported not to be assembled on the normal F0 portion of NR70, it is concluded that the hydrophobic domain deleted in the mutant subunit is important for assembly of the F1 portion . Introduction of a plasmid pNR70 carrying the mutant allele of NR70 into a wild-type strain gave no recombinants resistant to neomycin . This result suggested that the neomycin-resistant phenotype is not directly related to the defect in the gamma subunit of NR70.

Proc Natl Acad Sci U S A, 1985 Sep, 82(17), 5724 - 7
An invertible element of DNA controls phase variation of type 1 fimbriae of Escherichia coli; Abraham JM et al.; The expression of type 1 fimbriae (pili) of Escherichia coli is turned on and off at the transcriptional level at a high frequency (10(-3) per cell per generation) in a process termed phase variation . Using Southern blot and DNA sequence analysis, we have detected a genomic rearrangement in the switch region immediately upstream of the fimbrial structural gene . This rearrangement involves an invertible 314-base-pair segment of DNA whose alternating orientation apparently results in the on-and-off activation of a promoter that determines the state of fimbrial expression.

J Infect Dis, 1985 Sep, 152(3), 560 - 5
Detection of an adherence factor of enteropathogenic Escherichia coli with a DNA probe; Nataro JP et al.; A DNA probe to detect genes conferring localized adherence of enteropathogenic Escherichia coli (EPEC) to Hep-2 cells was evaluated by using E . coli isolates from the stools of Peruvian infants with and without diarrhea . The probe was both sensitive and specific and revealed that Hep-2 adherence (because of the EPEC adherence factor {EAF} was more frequent in some O serogroups of EPEC than in others . Those serogroups in which EAF is almost always found have been designated class I EPEC; serogroups in which EAF is rarely found have been designated class II . Both class I (EAF-positive) and class II EPEC are associated with diarrheal disease.

J Infect Dis, 1985 Sep, 152(3), 550 - 9
The diarrheal response of humans to some classic serotypes of enteropathogenic Escherichia coli is dependent on a plasmid encoding an enteroadhesiveness factor; Levine MM et al.; Isolates of the most common O serogroups of enteropathogenic Escherichia coli (EPEC) associated with infant diarrhea (designated class I) adhere to Hep-2 cells; the genes for this adhesin, termed EPEC adherence factor (EAF), are located on plasmids 50-70 MDa in size . Volunteers ingested 10(10) organisms of an O127:H6 Hep-2-adhesive class I strain (E2348/69) or its plasmid-minus, nonadhesive derivative . Diarrhea occurred in nine of 10 volunteers who ingested the parent strain (mean, 1,178 ml) but in only two of nine who took the plasmid-minus variant (mean, 433 ml; P less than .006) . All volunteers ill from strain E2348/69 mounted serum IgA and IgG responses to a 94-kDa plasmid-associated outer membrane protein of E2348/69; this protein was found in other class I EPEC but not in enterotoxigenic or meningitic strains . The 50-70-MDa EAF plasmid seems necessary for full expression of pathogenicity in EPEC that exhibit Hep-2 adhesiveness . EPEC isolates of certain other, less common, O serogroups (O44, O86, and O114) are rarely Hep-2 adhesive . These EPEC, designated class II, possess distinct 50-70 MDa plasmids lacking EAF genes . Diarrhea was caused by 10(8) or 10(10) organisms of an O114:H2 class II EPEC strain (mean, 1,156 ml) in six of 11 volunteers . This result confirmed that class II EPEC are pathogenic by a mechanism not involving Hep-2 adhesiveness.

J Bacteriol, 1985 Sep, 163(3), 1191 - 5
Multiple controls exerted on in vivo expression of the pepN gene in Escherichia coli: studies with pepN-lacZ operon and protein fusion strains; Gharbi S et al.; Three physiological conditions were shown to promote transcriptional regulation of pepN expression: phosphate limitation, the nature of the source of carbon and energy, and anaerobiosis . The transcriptional level of regulation can be deduced from the observation of these effects in strains carrying operon fusion pepN-lacZ . Mutations in the various genes phoB, phoM, phoR, crp, and fnr (oxrA) did not affect pepN expression.

J Bacteriol, 1985 Sep, 163(3), 1109 - 13
Effect of some D-amino acids on the steady-state level of glutamine synthetase in Escherichia coli; Berberich MA; D-Glutamate can elicit an increase in the specific activity of glutamine synthetase (GS) when added to cells growing in the presence of high ammonia nitrogen . This effect is independent of glutamate dehydrogenase or glutamate synthase activities and could not be provoked by the addition of the various metabolites which participate in the regulation of GS in the covalent modification system . Neither could an increase in GS level be elicited by addition of any of the D-amino acids which function as allosteric effectors or inhibitors of GS activity . The increase in GS level could also be provoked by addition of D-lysine, D-threonine, or glycine to cells growing in an ammonia-rich medium . The increase in GS level generated by a mixture of D-glutamate, D-lysine, D-threonine, and glycine approximates the increase in GS level observed during step-down of a wild-type Escherichia coli culture from ammonia-sufficient to ammonia-limited growth conditions . Studies with mutants exhibiting alterations in GS regulation indicated that the increase elicited by the addition of D-amino acids depends on the presence of the wild-type glnD allele, although no direct correlation between a positive response and the state of adenylylation of GS can be made.

Infect Immun, 1985 Sep, 49(3), 581 - 6
Immunochemical characterization of P pili from invasive Escherichia coli; Hanley J et al.; P pili (or fimbriae) are present on most pyelonephritogenic Escherichia coli strains, and they mediate binding to erythrocytes and epithelial cells . To determine the antigenic diversity of P pili, we purified the pili from 14 bacteremic E . coli strains which caused mannose-resistant hemagglutination . Pilus preparations consisted of one to three bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ranged in molecular weight from 14,000 to 19,500 . There was no single band common to all the strains . An enzyme-linked immunosorbent inhibition assay detected 20 ng of pilus antigen . When four different rabbit antisera were used, only two or fewer heterologous strains could inhibit the enzyme-linked immunosorbent assay . Immunoblots yielded the same results . Protein sequences of four P pili had identical N termini . These results show that despite having identical amino-terminal sequences, P pili are antigenically heterogeneous . The receptor-binding domains which are likely to be identical in all strains must be immunorecessive.

Mol Biol Evol, 1985 Sep, 2(5), 359 - 69
Evolution of transposable elements: an IS10 insertion increases fitness in Escherichia coli; Chao L et al.; Strains of Escherichia coli carrying Tn10, a transposon consisting of two IS10 insertion sequences flanking a segment encoding for a tetracycline-resistance determinant, gain a competitive advantage in chemostat cultures . All Tn10-bearing strains that increase in frequency during competition have a new IS10 insertion that is found in the same location in the genome of those strains . We mapped, by a gradient of transmission, the position of the new IS10 insertion . We examined 11 isolates whose IS10 insertion was deleted by recombinational crossing-over, and in all cases the competitive fitness of the isolates was decreased . These results show that the IS10-generated insertion increases fitness in chemostat cultures . We named the insertion fit::IS10 and suggest that transposable elements may speed the rate of evolution by promoting nonhomologous recombination between preexisting variations within a genome and thereby generating adaptive variation.

Mol Cell Biol, 1985 Sep, 5(9), 2414 - 22
Macronuclear structure of the G surface antigen gene of Paramecium primaurelia and direct expression of its repeated epitopes in Escherichia coli; Meyer E et al.; The gene encoding the G surface antigen of Paramecium primaurelia was cloned from a macronuclear DNA library by a screening procedure involving differential hybridization with cDNA probes synthesized from polyadenylated RNAs of cells expressing one of two alternate antigens . S1 mapping experiments and sequencing of the cloned DNA and the mRNA showed that the cloned gene corresponded to the high-molecular-weight mRNA that had been indirectly identified as that of the G surface antigen . Because the genetic code of Paramecium spp . is different from the "universal" code, this mRNA cannot be correctly translated in vitro; direct proof that it encoded the antigenic determinants of this protein was therefore obtained through expression of fragments of the coding sequence in Escherichia coli by using the expression vector lambda gt11 . Studies on the structure of this gene revealed that the central part of the coding sequence contained at least five tandem repeats of 222 base pairs, encoding immunogenic domains of the protein . We also showed that, like other surface antigen genes of trypanosomes and paramecia, this gene lay next to a chromosome end and that no rearrangement of its immediate genomic environment was associated with its expression.

Vaccine, 1985 Sep, 3(3 Suppl), 172 - 4
Expression of haemagglutinin gene; Nobusawa E et al.; Antigenic drift of the haemagglutinin (HA) molecule of type A influenza viruses has been thought to occur by the accumulation of a series of point mutations in the antigenically important regions of the molecule . In order to study the antigenic sites on the HA molecule, an attempt was made to use the site-specific mutagenesis method.

Mol Cell Biochem, 1985 Sep, 68(1), 23 - 30
Captan alters transcription in Escherichia coli permeabilized by toluene; Lewis RA et al.; RNA synthesis was measured in toluenized E . coli by the incorporation of radiolabeled precursor into either acid precipitable or phenol extracted RNA . Exposure to captan (100 microM) caused a 2.6 fold increase in the apparent rate of RNA synthesis . When captan was tested for its effect on the initiation of RNA synthesis, using either rifampicin-treated cells or by measuring the incorporation of gamma {32P}ATP or gamma {32P}GTP, no change was observed in the number of RNA chains being initiated . Thus, captan does not exert its influence at the level of initiation of nascent chains . However, captan did have an effect on chain growth . From calculations of the incorporation of precursors molecules, RNAs isolated from treated cells were measured to be an average of 2.7 times longer than those from untreated cells . RNA chain lengths were also analyzed by polyacrylamide gel electrophoresis . By this latter technique it was also shown that cells exposed to captan synthesized RNAs that were longer than those of untreated cells . Alterations in the degradation of RNA molecules do not account for the captan mediated response in RNA synthesis.

J Clin Microbiol, 1985 Sep, 22(3), 425 - 7
Detection of Escherichia coli adhesins with DNA probes; Lanser JA et al.; DNA hybridization assays for genes encoding the Escherichia coli adhesins K88 and K99 were developed . These assays were used to screen a variety of animal E . coli strains, and the results were compared with results obtained by serological methods . All methods compared well for determining the presence of adhesin K99 . However, the DNA hybridization detected more K88-positive strains than did either enzyme immunoassay or slide agglutination . Agreement was improved when strains were grown on blood or nutrient agar instead of Minca medium.

Eur J Immunol, 1985 Sep, 15(9), 966 - 9
Inhibition of mast cell sensitization in vitro by a human immunoglobulin epsilon-chain fragment synthesized in Escherichia coli; Coleman JW et al.; An immunoglobulin epsilon-chain fragment was synthesized in E . coli by cloning and expression of the gene coding for the second, third and fourth constant domains of the human IgE heavy chain . The bacterial CH2-4 polypeptide product was assembled by oxidation into a covalently linked dimeric epsilon-chain molecule presumably analogous to the Fc region of native IgE . This bacterial Fc epsilon preparation, within the concentration range 0.01-10 micrograms/ml, inhibited sensitization of human lung mast cells, determined as histamine released upon challenge with specific antigen . Monomer CH2-4 epsilon-chain polypeptide, prepared by reduction and alkylation of the active bacterial Fc epsilon fragment, was inactive as an inhibitor of sensitization . The molar potency of the active bacterial Fc epsilon product was approximately one fourth of that of native IgE . Since the bacterial Fc epsilon is nonglycosylated, carbohydrate does not make an essential contribution to the Fc receptor binding activity of IgE . These results show that a functionally active immunoglobulin molecule can be synthesized by gene cloning and expression in E . coli.

Surgery, 1985 Sep, 98(3), 371 - 7
Mechanism of the adjuvant effect of hemoglobin in experimental peritonitis . IX: The infection-potentiating effect of hemoglobin in Escherichia coli peritonitis is strain specific; Pruett TL et al.; Hemoglobin solutions have been said to consistently increase the lethality of otherwise nonlethal bacterial inocula in experimental models of Escherichia coli peritonitis . We tested the capacity of stroma-free hemoglobin to potentiate the lethality of each of 26 separate clinical isolates of E . coli . The LD50 of each strain with and without stroma-free hemoglobin was then correlated with the ability of that strain to express putative "virulence characteristics": the expression of 0 (lipopolysaccharide) and K (capsular) antigens, the ability to produce colicin V, the capacity to hemagglutinate mammalian red cells in the presence of 1% mannose, and the ability to secrete alpha-hemolysin . No perfect correlations were found . The LD50 of only four of the 26 strains of E . coli was affected by hemoglobin . Each of these four strains could hemagglutinate red cells and secreted alpha-hemolysin . Many other strains whose lethality was not increased by hemoglobin also had these virulence properties . We must conclude that the infection-potentiating effect of hemoglobin cannot be shown for most clinical isolates of E . coli and that the mechanism cannot be correlated with the usual "virulence characteristics" of E . coli.

Proc Natl Acad Sci U S A, 1985 Sep, 82(18), 6300 - 4
Carboxyl-terminal domain of the Epstein-Barr virus nuclear antigen is highly immunogenic in man; Milman G et al.; The carboxyl-terminal one-third of the Epstein-Barr virus nuclear antigen (EBNA-1) encoded by the BamHI restriction fragment K was synthesized in Escherichia coli by use of a high-expression plasmid . The resultant 28-kDa EBNA fusion polypeptide, comprising 5-10% of the total soluble bacterial protein, was purified to apparent homogeneity by phosphocellulose and hydroxylapatite column chromatography . Both rabbit monospecific antibodies and mouse monoclonal antibodies against 28-kDa EBNA gave nuclear immunofluorescence staining on Epstein-Barr virus (EBV)-infected lymphoblastoid cell lines and recognized the appropriate intact EBNA polypeptide bands on immunoblots . An ELISA with the purified 28-kDa EBNA as antigen was used to quantitate anti-EBNA antibody in human serum samples . The ELISA method was approximately 100-fold more sensitive than the classical anticomplement immunofluorescence assay . Anti-EBNA antibody was detected in sera from 100% of normal individuals who were seropositive for the viral capsid antigen, and low anti-EBNA titers were detected in serum from most patients with acute infectious mononucleosis . The assay gave the expected pattern of titers in sera from patients with rheumatoid arthritis, Burkitt lymphoma, or nasopharyngeal carcinoma, thus confirming the validity of this purified reagent for assessing EBNA antibody status . Approximately 10% of normal individuals and rheumatoid arthritis patients had anti-EBNA titers as high as those seen in nasopharyngeal carcinoma patients . In these high-titer individuals, greater than 1% of the total IgG are antibodies that recognize 28-kDa EBNA, which indicates that the carboxyl-terminal domain of EBNA is highly immunogenic.

Proc Natl Acad Sci U S A, 1985 Sep, 82(17), 5608 - 11
Is the 5S RNA a primitive ribosomal sequence?
Nazar RN, Wong WW.
A tandemly arranged cluster of 55 RNA-like sequences in the middle of ribosomal 26S to 28S rRNAs from divergent eukaryotic organisms raises the possibility that the larger ribosomal RNAs were built up, at least in part, by gene amplification events and suggests an intriguing evolutionary relationship between the 55 rRNA and the larger rRNA molecules.

J Bacteriol, 1985 Sep, 163(3), 973 - 82
Incompatibility mutants of IncFII plasmid NR1 and their effect on replication control; Wu RP et al.; DNA from the replication control region of plasmid NR1 or of the Inc- copy mutant pRR12 was cloned into a pBR322 vector plasmid . These pBR322 derivatives were mutagenized in vitro with hydroxylamine and transformed into Escherichia coli cells that harbored either NR1 or pRR12 . After selection for the newly introduced pBR322 derivatives only, those cells which retained the unselected resident NR1 or pRR12 plasmids were examined further . By this process, 134 plasmids with Inc- mutations in the cloned NR1 or pRR12 DNA were obtained . These mutants fell into 11 classes . Two of the classes had plasmids with deletions or insertions in the NR1 DNA and were not examined further . Plasmids with apparent point mutations were classified by examining (i) their ability to reconstitute a functional NR1-derived replicon (Rep+ or Rep-), (ii) the copy numbers of the Rep+ reconstituted replicons, (iii) the cross-reactivity of incompatability among the various mutant classes and parental plasmids, and (iv) the trans effects of the mutants on the copy number and stable inheritance of a coresident plasmid.

J Bacteriol, 1985 Sep, 163(3), 1142 - 6
Identification and mapping of regions of the plasmid pKM101 which influence the growth rate and resistance to phleomycin E of Escherichia coli WP2; Hall RM; The effects of deletion of various regions of the pKM101 genome on several phenotypes conferred by pKM101 in Escherichia coli WP2 cells were investigated . Differences in the response of cells carrying pKM101 or various pKM101 deletion derivatives to the mutagenic effects of phleomycin E can be attributed to differences in sensitivity to the lethal effects of phleomycin E . Resistance to phleomycin E is conferred by the pKM101 mucAB genes (or an adjacent gene) but observed only with pKM101 derivatives which have lost a 2.2-kilobase (BalI-KpnI-2) segment which completely includes the pKM101 endonuclease gene nuc . A pKM101 slow-growth determinant, distinct from the slo gene, has also been identified and localized in the 2.4-kilobase (BalI-KpnI-3) segment which is adjacent to the nuc gene . Loss of this region does not appear to substantially influence the toxic or mutagenic effects of phleomycin E.

Infect Immun, 1985 Sep, 49(3), 719 - 23
Kinetics and characterization of interferon production by murine spleen cells stimulated with Legionella pneumophila antigens; Blanchard DK et al.; Formalin-killed Legionella pneumophila bacterial cells, as well as a purified cell wall preparation (designated F-1 antigen) containing lipopolysaccharide (LPS), stimulated production of interferons (IFNs) in mouse spleen cell cultures . L . pneumophila whole-cell vaccine induced an IFN that was pH 2 labile and neutralized by anti-IFN-gamma indicating that IFN-gamma was the dominant form present . F-1 antigen induced a mixture of IFNs, depending upon the age of the culture and cell types present . In freshly prepared whole-spleen cultures and in 2-h adherent cultures, F-1 induced predominantly IFN-alpha/beta . In whole-spleen cultures that were allowed to age for 24 to 48 h before stimulation, F-1 was seen to induce mostly IFN-gamma, with low levels of IFN-alpha/beta present . Since only IFN-alpha/beta was produced in T-cell-depleted populations (at 2 h or at 48 h), it is suggested that T cells are responsible for IFN-gamma production in aged cultures . Additionally, heat-treated F-1, Escherichia coli LPS, and heat-treated E . coli LPS all induced similar levels of IFN-gamma in whole-splenocyte or nonadherent cell cultures which were incubated 48 h before stimulation . This suggests that LPS present in F-1 is responsible for IFN-gamma production and that an activated cell population is required . These results show that L . pneumophila antigens can induce the production of various types of IFN in mouse spleen cell cultures through several mechanisms.

Hepatology, 1985 Sep-Oct, 5(5), 763 - 9
Mitochondrial antibodies in primary biliary cirrhosis: species and nonspecies specific determinants of M2 antigen; Lindenborn-Fotinos J et al.; Sera from patients with primary biliary cirrhosis reacted with four major bands in beef heart mitochondria and ATPase extract when analyzed by immunoblot after sodium dodecyl sulfate-polyacrylamide gel electrophoresis . These four immunologically reactive bands corresponded to protein bands with molecular weights of about (a) 80,000; (b) 63,000; (c) 56,000; and (d) 43,000 to 46,000 . An additional immunoreactive band was found with some high-titered primary biliary cirrhosis sera at 36,000 . No association with any ATPase subunits was found, except for band c which migrated between the alpha- and beta-subunit of ATPase . Most ATPase fractions did not contain this band c, indicating that M2 determinants, as defined by immunoblot, are not identical with any ATPase subunit . Species and nonspecies-specific determinants of M2 were identified using mitochondria from rat liver and human heart and liver . Antigenic bands a, c and d were nonspecies-specific . Band b and e occurred only in beef heart . An additional determinant at about 38,000 was detected using human heart and liver mitochondria . Primary biliary cirrhosis sera showed a typical reaction with two protein bands of Escherichia coli, one at about 85,000 to 90,000 and the other at 60,000 . Antibodies against both determinants could be absorbed with submitochondrial particles of beef heart showing that E . coli shares cross-reacting determinants with mitochondria . Sera from 56 primary biliary cirrhosis patients were tested using beef heart mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

J Immunol, 1985 Sep, 135(3), 1739 - 45
Reactivity of anti-mitochondrial autoantibodies in primary biliary cirrhosis: definition of two novel mitochondrial polypeptide autoantigens; Frazer IH et al.; Sera that contained autoantibodies to mitochondria (AMA) by immunofluorescence were examined by immunoblotting for reactivity with mitochondrial polypeptides from various mammalian species, yeast, and E . coli . Mitochondrial polypeptides were separated by polyacrylamide gel electrophoresis, were immobilized on nitrocellulose, and were exposed to sera . The sera tested included 18 AMA-positive sera from patients with primary biliary cirrhosis (PBC), two AMA-positive sera from patients without PBC, and 53 AMA-negative sera . All AMA-positive sera reacted with either one or the other, or usually both of two human mitochondrial polypeptides of 70 kilodalton (kD) and 45 kD . The 53 AMA-negative sera were not reactive with the 70 kD polypeptide, but six reacted with the 45 kD polypeptide . The reactivity of the 70 kD and the 45 kD polypeptide was destroyed by brief exposure to trypsin . The counterpart of the 70 kD reactive polypeptide in human mitochondria was a 65 to 70 kD polypeptide in rat and mouse mitochondria, and a 55 kD polypeptide in yeast and in E . coli . The apparent 45 kD polypeptide was similar in all mitochondrial preparations tested, but no counterpart could be identified in E . coli . Beef heart mitochondria were used to show that the reactive polypeptides were present in a semipurified preparation of the F1 portion of mitochondrial H+ ATPase; however, sera did not react with the beta subunit of ATPase, proposed as a candidate mitochondrial autoantigen . The present molecular characterization of two particular antigens should lead to the more precise identification of these antigens, and also to a clearer insight into the pathogenesis of PBC.

Biochim Biophys Acta, 1985 Aug 30, 846(2), 305 - 12
Glucocorticoid effects on membrane lipid mobility during differentiation of murine B lymphocytes; Keating KM et al.; The lateral motion of membrane lipids on lipopolysaccharide-stimulated murine B lymphocytes was measured using photobleaching recovery techniques . The mobility of the phospholipid analog 3,3'-dioctadecylindocarbocyanine iodide (DiI) was measured at 37 degrees C on B lymphocytes 48 h after stimulation by various concentrations of lipopolysaccharide . DiI mobility on lymphoblasts from cultures stimulated with 10 micrograms/ml lipopolysaccharide was reduced 50% compared with unstimulated, small B cells . However, both lower and higher lipopolysaccharide concentrations caused some decrease in lipid mobility . Lipid mobility was measured on B cells stimulated with 10 micrograms/ml lipopolysaccharide at zero time, on lymphoblasts at 18, 24, 48 and 72 h, and on immunoglobulin (Ig) -secreting lymphocytes at 96 h . The diffusion coefficient of DiI on both control and lipopolysaccharide-treated cells at zero time is 6.3 X 10(-9) cm2 X s-1 . This value remains unchanged for unstimulated cells over 72 h . Lipid mobility of lipopolysaccharide-activated lymphoblasts decreased during incubation with lipopolysaccharide to 5.0, 3.4, 2.8 and 2.4 X 10(-9) cm2 X s-1 after 18, 24, 48 and 72 h, respectively . DiI mobility on immunoglobulin (Ig) -secreting lymphocytes identified at the foci of Protein A-coated sheep red blood cells plaques is 8.6 X 10(-9) cm2 X s-1, a value similar to that of unstimulated B cells . The effect of introducing various concentrations of a synthetic glucocorticoid, triamcinolone acetonide (TA), to 48 h lipopolysaccharide-stimulated cells for 6 h was examined . Maximal TA effect was observed at a concentration of 10(-7) M, which caused an increase in lipid mobility to 7.5 X 10(-9) cm2 X s-1 . Exposing resting B cells (t = 0) or lymphoblasts (t = 24, 48 or 72 h) to TA for 3 h had no effect on lipid mobility . Treatment for 6 h with 10(-7) MTA increased DiI diffusion to 12.6, 9.9, 7.5 and 6.8 X 10(-9) cm2 X s-1 on control cells and on 24, 48 and 72 h lipopolysaccharide-activated lymphoblasts, respectively . A longer incubation of 12 h with 10(-7) MTA caused no further change in lipid lateral diffusion . The response was glucocorticoid-specific . In lymphoblasts (48 h) incubated an additional 6 h with 10(-7) MTA and a 100-fold excess of cortexolone or progesterone, the increase in lipid mobility was substantively blocked; estradiol and testosterone had no effect on lipid lateral diffusion.(ABSTRACT TRUNCATED AT 400 WORDS)

Science, 1985 Aug 30, 229(4716), 869 - 71
Passive immunization against cachectin/tumor necrosis factor protects mice from lethal effect of endotoxin; Beutler B et al.; A highly specific polyclonal rabbit antiserum directed against murine cachectin/tumor necrosis factor (TNF) was prepared . When BALB/c mice were passively immunized with the antiserum or with purified immune globulin, they were protected against the lethal effect of the endotoxin lipopolysaccharide produced by Escherichia coli . The prophylactic effect was dose-dependent and was most effective when the antiserum was administered prior to the injection of the endotoxin . Antiserum to cachectin/TNF did not mitigate the febrile response of endotoxin-treated animals, and very high doses of endotoxin could overcome the protective effect . The median lethal dose of endotoxin in mice pretreated with 50 microliters of the specific antiserum was approximately 2.5 times greater the median lethal dose for controls given nonimmune serum . The data suggest that cachectin/TNF is one of the principal mediators of the lethal effect of endotoxin.

Biochemistry, 1985 Aug 27, 24(18), 4931 - 8
Identification of the site of cross-linking in 16S rRNA of an aromatic azide photoaffinity probe attached to the 5'-anticodon base of A site bound tRNA; Ciesiolka J et al.; The site of Escherichia coli 16S ribosomal RNA cross-linked to the 5'-anticodon base of A site bound E . coli valyl-tRNA was identified . Cross-linking was via the affinity probe 6-{(2-nitro-4-azidophenyl)amino}caproate (NAK) or 3-{{2-{(2-nitro-4-azidophenyl)amino}ethyl}dithio}propionate (SNAP) attached to the carboxyl group of the 5'-anticodon base 5-(carboxyethoxy)uridine via an ethylenediamine spacer {Gornicki, P., Ciesiolka, J., & Ofengand, J . (1985) Biochemistry (preceding paper in this issue)} . With both probes, RNase T1 digestion of the isolated 16S RNA-tRNA covalent complex, 5'-32P postlabeling, and gel electrophoresis yielded two oligonucleotides larger than any fragments from non-cross-linked tRNA or rRNA . Appearance of the oligomers was dependent on the presence of the probe on the tRNA . Unmodified tRNA in the A and/or P sites did not yield any product . The presence of elongation factor Tu in the incubation mixture was also required . Dithiothreitol (DDT) treatment of the SNAP-induced covalent complex prior to electrophoresis also abolished the oligomers . Only the larger of the two oligomers (present in a 3:1 ratio) was sequenced . The SNAP dimer was cleaved with DTT, and the rRNA and tRNA oligomers were separated and sequenced as monomers . The NAK dimer was sequenced without cleavage by taking advantage of the differences in electrophoretic mobility among sequence and/or composition isomers of the same length . In both cases, the rRNA oligomer was identified as UACACACCG1401, and the nucleotide cross-linked was shown to be the C1400 residue . The expected tRNA modification site was also identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1985 Aug 27, 24(18), 4924 - 30
Cross-linking of the anticodon of P and A site bound tRNAs to the ribosome via aromatic azides of variable length: involvement of 16S rRNA at the A site; Gornicki P et al.; The topography of the ribosomal decoding site was explored by affinity labeling from the 5'-anticodon base, 5-(carboxymethoxy)uridine-34, of P or A site bound tRNA1Val . A nitrophenyl azide was attached to the carboxyl group of this nucleotide via side chains varying in length from 18 to 24 A . Binding of acetylvalyl-tRNA to the P site was codon dependent and that of valyl-tRNA to the A site was both codon and elongation factor Tu (EFTu) dependent . Cross-linking to both A and P sites was irradiation, probe, codon, and, in the case of the A site, EFTu dependent . Putative P-site cross-linked aminoacyl-tRNA was reactive with puromycin . The yield of cross-linking was little affected by placement of the tRNA at the A or P site but varied considerably with the length and structure of the probe side chain . When the distance from the pyrimidine C-5 atom to the azide group was 23 A, 42-45% cross-linking was obtained at each site, but when the distance was decreased to 18 A, only 7-12% was found . Placing an S-S bond in the center of the 23-A leash decreased the A-site yield to about half, while insertion of a CONH group decreased A-site cross-linking about 8-fold . P-site cross-linking was more sensitive to mercaptan quenching (50% at 0.5 mM) than was that at the A site (50% at greater than 2.0 mM) but both were partially shielded from solvent.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1985 Aug 27, 24(18), 4802 - 6
Base tilt of DNA in various conformations from flow linear dichroism; Edmondson SP et al.; We have measured the isotropic absorption (Aiso) and linear dichroism (LD) of Escherichia coli DNA in 0.01 M Na+ (10.4 base pairs per turn of B form), 5.5 M NH4F (10.2 base pairs per turn of B form), and 80% trifluoroethanol (A form) into the vacuum UV spectral region . The reduced dichroism spectrum (LD divided by Aiso) of DNA in the A conformation differed from those of the B conformations, demonstrating that LD is a sensitive method for distinguishing DNA conformation . The reduced dichroism spectra of the B conformations were similar, indicating little change in the orientation of the bases for DNA in high salt . The wavelength dependence of the reduced dichroism indicates that the angle between the base planes and the helix axis is less than 76 degrees for all three conformations of DNA.

Biochemistry, 1985 Aug 27, 24(18), 4777 - 84
Photochemical cross-linking of tRNA1Arg to the 30S ribosomal subunit using aryl azide reagents attached to the anticodon loop; Chen JK et al.; The 2-thiocytidine residue at position 32 of tRNA1Arg from Escherichia coli was modified specifically with three photoaffinity reagents of different lengths, and the corresponding N-acetylarginyl-tRNA1Arg derivatives were cross-linked to the P site of E . coli 70S ribosomes by irradiation . Covalent attachment was dependent upon the presence of a polynucleotide template and exposure to light of the appropriate wavelength . From 4% to 6% of the noncovalently bound tRNA became cross-linked to the ribosome as a result of photolysis, and attachment to the P site was confirmed by the reactivity of arginine in the covalent complexes toward puromycin . Analysis of the irradiated ribosomes by sucrose-gradient sedimentation at low Mg2+ concentration revealed that the tRNA was associated exclusively with the 30S subunit in all cases . Two of the N-acetylarginyl-tRNA1Arg derivatives were attached primarily to ribosomal proteins whereas the third was cross-linked mainly to 16S RNA . Partial RNase digestion of the latter complex demonstrated that the tRNA had become attached to the 3' third of the rRNA molecule . In addition, the tRNA-rRNA bond was shown to be susceptible to cleavage by hydroxylamine and mercaptoethanol.

Biochemistry, 1985 Aug 27, 24(18), 4754 - 61
Substrate specificity and protonation state of ornithine transcarbamoylase as determined by pH studies; Kuo LC et al.; The ornithine transcarbamoylase catalyzed reaction and its inhibition by L-norvaline have been investigated between pH 5.5 and 10.5 . The steady-state turnover rate (kcat) of the enzyme from Escherichia coli increases with pH and plateaus above pH 9 . Its change with pH conforms to a single protonation process with an apparent pKa of 7.3 . The effect of pH on the apparent Michaelis constant (KMapp) of L-ornithine suggests that this diamino acid in its cationic form is not the substrate . Treating only the zwitterions of ornithine as substrate, the pH profile of the pseudo-first-order rate constant (kcat/KMz) of the reaction is a bell-shaped curve characterized by pKa's of 6.2 and 9.1 and asymptotic slopes of +/- 1 . Similar pKa's (6.3 and 9.3) are obtained for the pKi profile of zwitterionic L-norvaline, a competitive inhibitor . The pKi profile further indicates that the alpha-amino group of the inhibitor must be charged for binding . Together, these pH profiles provide sufficient information to suggest that only the minor zwitterionic species of ornithine, H2N(CH2)3CH(NH3+)COO-, binds the enzyme productively . The selection of this substrate form by the enzyme leads to a Michaelis complex in which ornithine is poised for nucleophilic attack . Following such binding, the need for deprotonation of the delta-NH3+ group is avoided, and transcarbamoylation becomes energetically more feasible . Reaction schemes accounting for the effects of pH are proposed for the enzymic reaction.

Biochemistry, 1985 Aug 27, 24(18), 4721 - 6
Regulation of the kinetics of the interaction of Escherichia coli RNA polymerase with the lambda PR promoter by salt concentration; Roe JH et al.; The rate of formation of transcriptionally competent open complexes between Escherichia coli RNA polymerase (RNAP) and the lambda PR promoter is extraordinarily sensitive to the nature and concentration of the electrolyte ions in the solution . The pseudo-first-order time constant of open complex formation tau obsd, determined in excess RNAP at 25 degrees C as a function of NaCl concentration, is proportional to the concentration product {Na+}12 {RNAP}-1 . Consequently, tau obsd is far more sensitive to changes in the salt concentration than to changes in the concentration of RNAP . The origin of this effect is the release of the thermodynamic equivalent of 12 monovalent ions in the process of closed complex formation at the lambda PR promoter . In more complex ionic mixtures, ion-specific stoichiometric effects on tau obsd are observed . These are not ionic strength effects but are instead both valence and species specific . Both the association and dissociation rate constants of RNAP at the lambda PR promoter are strongly salt dependent, varying (in NaCl) as {Na+}-12 and {Na+}8, respectively . Consequently, the equilibrium constant characterizing open complex formation at this promoter varies with {Na+}-20 . Electrostatic interactions and counterion release are the major contributors to the binding free energy driving open complex formation in a dilute salt solution . Since the in vivo ionic environment of E . coli (and other cells) is highly variable, these large salt effects are almost certainly of physiological significance . Variations in the intracellular concentrations of inorganic and organic ions, including polyamines, must exert both global and also promoter-specific regulatory effects on the initiation of transcription, as well as on numerous other protein-nucleic acid interactions.

Nucleic Acids Res, 1985 Aug 26, 13(16), 5919 - 26
A convenient technique to compare the efficiency of promoters in Escherichia coli; Vidal-Ingigliardi D et al.; We describe a technique which allows one to insert any promoter in front of the chromosomal malPQ operon . This can be done easily by using only one plasmid, one strain, and two simple selections . Properties of the final chromosomal fusion are such that the level of amylomaltase, the product of the malQ gene, measures quantitatively the efficiency of the inserted promoter . This method was utilized to compare the efficiency of four well-known promoters: lacZp, trp, tac, lambdaPR and three malT activated promoters: malPp, malkP and malEp.

Nucleic Acids Res, 1985 Aug 26, 13(16), 5927 - 36
The primary structure of the DeoR repressor from Escherichia coli K-12; Valentin-Hansen P et al.; The nucleotide sequence of the deoR gene of E . coli, which codes for the DeoR repressor, has been determined . This gene codes for a polypeptide that is 252 amino acids residues in length . Computer-assisted analysis of the nucleotide sequence strongly suggests that the DNA binding domain of the DeoR repressor is located in the N-terminal part of the protein . After the coding region there is a dyad symmetry similar to a palindromic unit present outside many structural genes on the E . coli chromosome.

Nucleic Acids Res, 1985 Aug 26, 13(16), 5869 - 82
Cloning and characterization of the cDNAs for human and rabbit interleukin-1 precursor; Furutani Y et al.; DNA sequence complementary to the mRNA for rabbit interleukin-1 precursor (preIL-1) has been cloned from the cDNA library constructed using partially purified poly(A)+RNA from induced rabbit alveolar macrophages by mRNA hybridization-translation assay . By using this cDNA as a probe, human IL-1 cDNA was isolated from the cDNA library prepared using poly(A)+RNA from induced HL-60 cells, a human monocyte-like cell line . The amino acid sequences of the human and rabbit preIL-1 deduced from the cDNA sequences reveal their primary structures which consists of 271 and 267 amino acid residues, respectively . The amino acid sequence is 64% conserved between human and rabbit . The difference in number of amino acid residues results from the carboxy-terminal extention of 4 amino acid residues in human preIL-1 . Expression of the cloned human cDNA in E . coli yielded biologically active IL-1.

J Biol Chem, 1985 Aug 25, 260(18), 10069 - 74
Response of recA-dependent operons to different DNA damage signals; Smith CL; The responses of three recA-dependent operons (recA, lambda, and phi 80), with three different repressors, to five DNA damage treatments were compared . Each operon shows a unique induction onset time constant over a wide dose range . However, the extent of response is variable within the same dose range . Individual Escherichia coli cells show a graded SOS response to different levels of DNA damage . Apparently phage repressors have evolved to discriminate between lethal and sublethal DNA damage to a host by a wide variety of agents . Multiple induction signals could account for the complex behavior of this system . For example, the absence of the recBC enzyme from a cell leads to complex changes in SOS induction onset times and extents.

J Biol Chem, 1985 Aug 25, 260(18), 10378 - 87
Regulation of Escherichia coli purF . Analysis of the control region of a pur regulon gene; Makaroff CA et al.; Escherichia coli purF has been determined to be the distal gene of a polycistronic operon . The first gene of the purF operon encodes a hydrophobic 17.9-kDa protein of unknown function . Deletion analyses indicate that the 17.9-kDa protein plays no role in the regulation of purF in cis . mRNA hybridization studies establish that purF is regulated at the transcriptional level . Enzyme and mRNA levels are repressed 11-17-fold by excess adenine . A single mRNA start site at nucleotide +1 was identified for transcripts synthesized in vivo . Two sites, at +1 and approximately +30, were used for transcription initiation in vitro . The purF promoter is localized between nucleotides -96 and -7 with sequences upstream of -71 necessary for high level expression . Initial evidence suggests that transcription is subject to stringent control . Deletion analyses localize the purF control element to a region between nucleotides -71 and +35 . A putative control site between nucleotides -35 to +3 strongly resembles a 5' flanking sequence in the co-regulated gene purM . This site contains an imperfect inverted repeat sequence that is characteristic of sites recognized by regulatory proteins and is a candidate for the purF operator . This is the first detailed analysis of a gene involved in de novo purine nucleotide biosynthesis.

J Biol Chem, 1985 Aug 25, 260(18), 10063 - 8
Primary structure of histidine-tRNA synthetase and characterization of hisS transcripts; Freedman R et al.; Histidine-tRNA synthetase is one of the smallest bacterial aminoacyl-tRNA synthetases . It is less than one-half the size of the largest aminoacyl-tRNA synthetases . The entire nucleotide sequence of the Escherichia coli hisS locus was determined . The coding region is comprised of 424 codons, and the sequence was determined for 200 nucleotides on the 5'- and 3'-sides of the coding region . The translated nucleotide sequence was confirmed extensively by independent amino acid sequence information obtained by Edman degradations of purified peptides and by measurements of peptide masses by fast atom bombardment mass spectrometry . A significant sequence alignment of four bacterial aminoacyl-tRNA synthetases was reported recently (Webster, T., Tsai, H., Kula, M., Mackie, G., and Schimmel, P . (1984) Science 226, 1315-1317) . Although the four enzymes vary considerably in length, this match occurs within the first 100 amino acids of each of the four enzymes and is in the segment believed to be part of the catalytic core . But no strong alignment could be found of the histidine sequence with these four tRNA synthetase sequences . This enzyme may be derived, therefore, from a different progenitor . Previous work suggested that three places in the hisS 5'-noncoding sequence could be promoter sites for RNA polymerase (Eisenbeis, S . J., and Parker, J . (1982) Gene 18, 107-114) . We detected a 1400-nucleotide RNA species by RNA blot analysis with a hisS-specific probe . S1 nuclease mapping demonstrated a 5'-end to the RNA species occurs at -67 +/- 1, relative to the first nucleotide of the coding region . This position coincides with the predicted start site for transcription from one of the previously proposed promoter sites.

J Biol Chem, 1985 Aug 25, 260(18), 10263 - 7
Purification of recA-based fusion proteins by immunoadsorbent chromatography . Characterization of a major antigenic determinant of Escherichia coli recA protein; Krivi GG et al.; Monoclonal antibodies to Escherichia coli recA protein were prepared, characterized, and used as affinity reagents for the purification of recA and recA:somatostatin fusion proteins . The monoclonal antibodies recognize an antigenic determinant or determinants located between amino acids 260 and 330 of recA . Addition of a fragment of the recA gene coding for these amino acids to an unrelated gene (beta-galactosidase) allowed the resulting beta-galactosidase fusion protein to be recognized by the recA monoclonal antibodies.

J Mol Biol, 1985 Aug 20, 184(4), 725 - 34
Repair of psoralen and acetylaminofluorene DNA adducts by ABC excinuclease; Sancar A et al.; Escherichia coli UvrA, UvrB and UvrC proteins acting in concert remove the major ultraviolet light-induced photoproduct, the pyrimidine dimer, from DNA in the form of a 12 to 13-nucleotide long single-stranded fragment . In vivo data indicate that the UvrABC enzyme is also capable of removing other nucleotide diadducts as well as certain nucleotide monoadducts from DNA and initiating the repair process that leads to removal of interstrand crosslinks caused by some bifunctional chemical agents . We have determined the action mechanism of the enzyme on nucleotide monoadducts produced by 4'-hydroxymethyl-4,5',8-trimethylpsoralen and N-acetoxy-N-2-acetylaminofluorene . In both cases we find that the enzyme hydrolyzes the eighth phosphodiester bond 5' and the fifth phosphodiester bond 3' to the modified base . This cutting pattern is similar to that observed with diadduct substrate, the only difference being that while the enzyme incises the fourth or fifth phosphodiester bond 3' to the pyrimidine dimer it always hydrolyzes the fifth bond relative to monoadducts . Our results also suggest that ABC excinuclease cuts the same two phosphodiester bonds on both sides of a T whether that T has a psoralen monoadduct or is involved in psoralen-mediated interstrand crosslink.

J Mol Biol, 1985 Aug 20, 184(4), 587 - 98
Supercoiling response of the lac ps promoter in vitro; Borowiec JA et al.; The rate of open promoter complex formation was measured on lac ps promoter DNA templates differing in negative superhelicity . The templates ranged from fully relaxed to those with numbers of superhelical turns exceeding that of form I plasmid DNA . The observed transcription response had two clearly distinguished phases: an initial rapid rise in rate followed eventually by a precipitous inhibition . The stimulation phase involved a nearly 40-fold increase in rate, which peaks at superhelical densities near that of isolated form I plasmid DNA . The introduction of more negative superhelical turns leads to inhibition . The magnitude of the response and the observation of both increases and decreases suggest that minor differences in superhelicity in vivo could lead to significant increases or decreases in transcription rate . The increase in rate was found to be directly proportional to the free energy of supercoiling; that is, to the square of the superhelical density . We suggest that the energy may be used both for enhanced DNA melting and for changes in DNA structure that alter the helical "face" with which RNA polymerase must interact . A quantitative method is presented that allows simple estimation of differences in the supercoiling response among promoters, both in the presence and in the absence of added factors.

J Mol Biol, 1985 Aug 20, 184(4), 599 - 610
Promoter mutations affecting divergent transcription in the Tn10 tetracycline resistance determinant; Daniels DW et al.; The tetracycline resistance determinant in transposon Tn10 consists of two genes, the tetA resistance gene and the tetR repressor gene, that are transcribed from divergent overlapping promoters . We determined the levels of pulse-labeled tet messenger RNA in Escherichia coli strains with the Tn10 tet genes on a multicopy plasmid . Addition of the inducer 5a,6-anhydrotetracycline results in a 270- to 430-fold increase in tetA mRNA and a 35- to 65-fold increase in tetR mRNA . As judged by the relative molar amounts of tetA and tetR mRNA synthesized under maximally inducing conditions, the tetA promoter (tetPA) is 7 to 11 times more active than the two tetR promoters (tetPR1 and tetPR2) combined . We characterized ten mutations in tetPA, including nine single-base-pair substitutions and a 30-base-pair deletion . All of the single-base-pair changes reduce the agreement with the consensus sequence for promoters recognized by E . coli RNA polymerase . Mutations in highly conserved nucleotides result in a 200- to 600-fold reduction in tetPA activity in vivo . Unexpectedly, tetPA mutations reduce by two- to fourfold the combined activity in vivo of tetPR1 and tetPR2, in spite of their locations outside the -35 and -10 regions of tetPR1 and tetPR2 . For two tetPA mutations, the negative effect on tetPR activity was also demonstrated in tetR- tetPR-lacZ operon fusion strains, thus eliminating the possibility that it is due to variations in either plasmid copy-number or induction efficiency . The pleiotropic effects of tetPA mutations are discussed in terms of the expectation that the overlapping tet promoters compete for RNA polymerase.

J Mol Biol, 1985 Aug 20, 184(4), 577 - 85
Pyrimidine dimers are not the principal pre-mutagenic lesions induced in lambda phage DNA by ultraviolet light; Wood RD; Experiments were performed to examine the role of cyclobutyl pyrimidine dimers in the process of mutagenesis by ultraviolet (u.v.) light . Lambda phage DNA was irradiated with u.v . and then incubated with an Escherichia coli photoreactivating enzyme, which monomerizes cyclobutyl pyrimidine dimers upon exposure to visible light . The photoreactivated DNA was packaged into lambda phage particles, which were used to infect E . coli uvr- host cells that had been induced for SOS functions by ultraviolet irradiation . Photoreactivation removed most toxic lesions from irradiated phage, but did not change the frequency of induction of mutations to the clear-plaque phenotype . This implies that cyclobutyl pyrimidine dimers can be lethal, but usually do not serve as sites of mutations in the phage . The DNA sequences of mutants derived from photoreactivated DNA showed that almost two-thirds (16/28) were transitions, the same fraction found for u.v . mutagenesis without photoreactivation . These results show that in this system, the lesion inducing transitions (the major type of u.v.-induced mutation) is not the cyclobutyl pyrimidine dimer; a strong candidate for a mutagenic lesion is the Pyr(6-4)Pyo photoproduct . On the other hand, photoreactivation of SOS-induced host cells before infection with u.v.-irradiated phage reduced mutagenesis substantially . In this case, photoreversal of cyclobutyl dimers serves to reduce expression of the SOS functions that are required in the process of targeted u.v . mutagenesis.

J Mol Biol, 1985 Aug 20, 184(4), 677 - 701
Primary structure and subunit stoichiometry of F1-ATPase from bovine mitochondria; Walker JE et al.; The enzyme complex F1-ATPase has been isolated from bovine heart mitochondria by gel filtration of the enzyme released by chloroform from sub-mitochondrial particles . The five individual subunits alpha, beta, gamma, delta and epsilon that comprise the complex have been purified from it, and their amino acid sequences determined almost entirely by direct protein sequence analysis . A single overlap in the gamma-subunit was obtained by DNA sequence analysis of a complementary DNA clone isolated from a bovine cDNA library using a mixture of 32 oligonucleotides as the hybridization probe . The alpha, beta, gamma, delta and epsilon subunits contain 509, 480, 272, 146 and 50 amino acids, respectively . Two half cystine residues are present in the alpha-subunit and one in each of the gamma- and epsilon-chains; they are absent from the beta- and delta-subunits . The stoichiometry of subunits in the complex is estimated to be alpha 3 beta 3 gamma 1 delta 1 epsilon 1 and the molecular weight of the complex is 371,135 . Mild trypsinolysis of the F1-ATPase complex, which has little effect on the hydrolytic activity of the enzyme, releases peptides from the N-terminal regions of the alpha- and beta-chains only; the C-terminal regions are unaffected . Sequence analysis of the released peptides demonstrates that the N terminals of the alpha- and beta-chains are ragged . In 65% of alpha-chains, the terminus is pyrrolidone carboxylic acid; in the remainder this residue is absent and the chains commence at residue 2, i.e . lysine . In the beta-subunit a minority of chains (16%) have N-terminal glutamine, or its deamidation product, glutamic acid (6%), or the cyclized derivative, pyrrolidone carboxylic acid (5%) . A further 28% commence at residue 2, alanine, and 45% at residue 3, serine . The delta-chains also are heterogeneous; in 50% of chains the N-terminal alanine residue is absent . The sequences of the alpha- and beta-chains show that they are weakly homologous, as they are in bacterial F1-ATPases . The sequence of the bovine delta-subunit of F1-ATPase shows that it is the counterpart of the bacterial epsilon-subunit . The bovine epsilon-subunit is not related to any known bacterial or chloroplast H+-ATPase subunit, nor to any other known sequence . The counterpart of the bacterial delta-subunit is bovine oligomycin sensitivity conferral protein, which helps to bind F1 to the inner mitochondrial membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

FEBS Lett, 1985 Aug 19, 188(1), 73 - 6
Final steps of the maturation of Omp F, a major protein from the outer membrane of Escherichia coli; Barbas JA et al.; Pulse-labelling experiments with E . coli cells allowed us to follow the incorporation of de novo proteins into the outer membrane of the cell envelope . Labelled membrane samples containing increasingly different levels of newly synthesized Omp F protein were subjected to chemical cross-linking with a bifunctional cleavable reagent in order to investigate the process of trimer formation of the protein . From the results obtained, we conclude that the formation of functional Omp F trimers is substantially delayed to, and can be distinguished from, the incorporation of Omp F monomers to the outer membrane.

Arch Biochem Biophys, 1985 Aug 15, 241(1), 118 - 31
The relationship between translational initiation and messenger RNA inactivation in down-shifted Escherichia coli; Jacobson LA et al.; The parameters of protein synthesis and functional inactivation of global messenger RNA (mRNA) were examined in a Tic+ strain of Escherichia coli during the 30-min period following a shift-down from glucose-minimal to succinate-minimal medium . The rate of mRNA inactivation and the relative translational initiation frequency were both most severely depressed immediately after the shift-down and increased slowly thereafter . If glucose was restored to the medium at any time after shift-down, mRNA inactivation immediately resumed its normal (preshift) rate and the protein-forming capacity was increased . These changes in mRNA inactivation rate do not reflect an altered mRNA composition in the down-shifted cells . The relative rate of mRNA inactivation was linearly proportional to the relative translational initiation frequency over a 10-fold range of initiation frequencies . Low initiation frequencies represent increased "dwell" of the ribosomes at the initiation site before the commencement of polypeptide chain initiation . We propose that initiating ribosomes protect mRNA from an inactivating endonucleolytic cleavage at or near the ribosome binding site.

J Biol Chem, 1985 Aug 15, 260(17), 9805 - 17
The structure, biochemical properties, and immunogenicity of neurofilament peripheral regions are determined by phosphorylation state; Carden MJ et al.; Treatment of freshly isolated, bovine neurofilaments with Escherichia coli alkaline phosphatase removes over 90% of the phosphate groups from serine residues of the Mr 200,000 and 150,000 polypeptide components (NF200 and NF150) . Dephosphorylated NF200 and NF150 remain associated with filaments, but migrate in sodium dodecyl sulfate gels with reduced apparent molecular weights . Unusual migration appears to be due to modification at regions of these polypeptides that are peripheral to the neurofilament backbone as defined by limited chymotryptic digestion . Over 90 monoclonal antibodies recognizing epitopes located within the peripheral domain of native NF200 all show reduced affinity for dephosphorylated NF200 . A single monoclonal antibody binds within the filament-associated domain of NF200 and its recognition of NF200 is unaffected upon treatment of neurofilaments with phosphatase . Around 50% of our monoclonal antibodies that bind NF150 monospecifically and at epitopes within its peripheral domain have reduced affinities for NF150 from phosphatase-treated filaments, while the remaining 50% bind native and dephosphorylated NF150 equally well . The smallest neurofilament component (NF70) contains few phosphate groups, most of which remain after treatment of neurofilaments with phosphatase . The resulting form of NF70 migrates normally in gels and its recognition by antibodies is unchanged . We conclude that phosphorylation modifies the structure of the two larger neurofilament polypeptides along domains that are peripheral to the filamentous backbone and that these effects are more pronounced for NF200 than for NF150.

Arch Biochem Biophys, 1985 Aug 15, 241(1), 106 - 17
The size and heterogeneity of the messenger RNA associated with 70 S monosomes from down-shifted Escherichia coli; Wnek AP et al.; After an energy source shift-down, Escherichia coli accumulates 70 S ribosome-mRNA complexes ("70 S monosomes") . The monosome mRNA strands are predominantly primary transcription products with purine nucleoside 5'-triphosphate and 5'-diphosphate termini present at a 1:2 ratio . The number-average chain length is 564 +/- 30 nucleotides, indicating that the population represents primarily monocistronic mRNAs . Digestions with endonucleases and exonucleases indicate that the ribosomes lie near the 5' ends of the mRNA strands and that the majority of the mRNA strands contain 5'-proximal "leader" sequences (average 10 nucleotides) outside the protective boundary of the ribosome . These data are consistent with the hypothesis that the increased functional stability of mRNA in down-shifted cells may result from protection by bound ribosomes of endonuclease-susceptible site(s) near the 5' ends of the mRNA strands.

J Biol Chem, 1985 Aug 15, 260(17), 9929 - 35
Hybridization selection of covalent nucleic acid-protein complexes . 2 . Cross-linking of proteins to specific Escherichia coli mRNAs and DNA sequences by formaldehyde treatment of intact cells; Schouten JP; Proteins cross-linked to pBR322 mRNAs and DNA by formaldehyde treatment of intact Escherichia coli cells have been detected with the use of a novel detection method . Among the proteins cross-linked to pBR322 mRNAs were S1, S21, and at least six other proteins of the small ribosomal subunit, initiation factor 1, elongation factor (EF) Tu, and very small amounts of EF-G and EF-Ts . The single strand binding protein, the HU-proteins, and RNA polymerase subunits alpha and beta were among the proteins cross-linked to pBR322 DNA . The results obtained suggest that the procedures described, can also be used to study interactions between different nucleic acid-bound polypeptides . The results are discussed in relation to the working mechanism of formaldehyde, and are compared to the results obtained with cross-linking induced by ultraviolet light . The methods presented should also be of use for the study of nucleic acid-protein interactions in other organisms.

J Biol Chem, 1985 Aug 15, 260(17), 9916 - 28
Hybridization selection of nucleic acid-protein complexes . 1 . Detection of proteins cross-linked to specific mRNAs and DNA sequences by irradiation of intact Escherichia coli cells with ultraviolet light; Schouten JP; A method is presented for the detection in crude lysates of subnanogram amounts of proteins covalently bound to a specific nucleic acid sequence . The sensitivity of this method enabled us to study proteins cross-linked to specific DNA and mRNA sequences by irradiation of intact Escherichia coli cells with ultraviolet light . Among the proteins cross-linked to pBR322 DNA, the single strand binding protein, the HU-proteins, and the RNA polymerase beta and sigma subunits were present . Some, but not all proteins were cross-linked to 5-bromodeoxyuridine-substituted DNA more efficiently than to normal DNA . Ribosomal protein S1 is by far the most prominent protein cross-linked to mRNAs . Among the proteins cross-linked in smaller amounts to mRNAs are translation initiation factor IF 1, and at least six proteins of the 30 S ribosomal subunit, among which is S21 . No 50 S proteins, nor IF-2, IF-3 or any of the elongation factors could be detected . Some UV-induced nucleic acid-protein cross-links were found to be heat-labile . It is concluded that the method employed may be used to compare the proteins interacting with different mRNAs, as well as single-copy DNA sequences from bacteria and eucaryotes with low complexity genomes.

J Biol Chem, 1985 Aug 15, 260(17), 9727 - 33
Dependence of maltose transport and chemotaxis on the amount of maltose-binding protein; Manson MD et al.; Maltose-binding protein (MBP) is essential for maltose transport and chemotaxis in Escherichia coli . To perform these functions it must interact with two sets of cytoplasmic membrane proteins, the MalFGK transport complex and the chemotactic signal transducer Tar . MBP is present at high concentrations, on the order of 1 mM, in the periplasm of maltose-induced or malTc constitutive cells . To determine how the amount of MBP affects transport and taxis, we utilized a series of malE signal-sequence mutations that interfere with export of MBP . The MBP content in shock fluid from cells carrying the various mutations ranged from 4 to 23% of the malE+ level . The apparent Km for maltose transport varied by less than a factor of 2 among malE+ and mutant strains . At a saturating maltose concentration 9% (approximately 90 microM) of the malE+ amount of MBP was required for half-maximal uptake rates . Transport exhibited a sigmoidal dependence on the amount of periplasmic MBP, indicating that MBP may be involved in a cooperative interaction at some stage of the transport process . The chemotactic response to a saturating maltose stimulus exhibited a first-order dependence on the amount of periplasmic MBP . Thus, interaction of a single substrate-bound MBP with Tar appears sufficient to initiate a chemotactic signal from the transducer . A half-maximal chemotactic response occurred at 25% of the malE+ MBP level, suggesting that in vivo the KD for binding of maltose-loaded MBP to Tar is quite high (approximately 250 microM).

J Biol Chem, 1985 Aug 15, 260(17), 9624 - 9
Substrate specificity of aspartate transcarbamylase . Interaction of the enzyme with analogs of aspartate and succinate; Foote J et al.; The ability of aspartate transcarbamylase from Escherichia coli to catalyze carbamylation of amino acids other than the natural substrate, L-aspartate, was examined . Cysteine, cysteate, cysteinesulfinate, and 3-nitroalanine showed kcat values at pH 7 of 0.16, 0.58, 5.2, and 62 s-1, respectively, while kcat with aspartate was 320 s-1 . In a parallel study, competitive inhibition constants of 3-nitropropionate, 3-mercaptopropionate, 3-sulfopropionate, and 3-sulfinopropionate were found to be high, about 0.1 M, compared with that of succinate, 0.56 mM . Although cysteinesulfinate had low activity as a substrate, the pH dependences of kcat and kcat/Km in H2O and D2O observed with the compound closely paralleled those of aspartate . The results of these studies suggest that substrate specificity and reactivity are achieved in part by a strong, highly specific interaction of one or more active site residues with the beta-carboxylate of L-aspartate . Unlike the sigmoidal kinetics found with aspartate, saturation of native aspartate transcarbamylase by cysteine sulfinate showed a lack of cooperativity, even under conditions of activation of the reaction by ATP and inhibition by CTP . The cysteinesulfinate reaction was increased 9-fold by the bisubstrate analog N-phosphonacetyl-L-aspartate . These results were interpreted in terms of an inability of cysteinesulfinate to cause the allosteric conformational change promoted by aspartate.

Eur J Biochem, 1985 Aug 15, 151(1), 59 - 65
Nucleotide sequence of the pyrD gene of Escherichia coli and characterization of the flavoprotein dihydroorotate dehydrogenase; Larsen JN et al.; Dihydroorotate dehydrogenase (EC 1.3.3.1) was purified to near electrophoretic homogeneity from the membranes of a strain of Escherichia coli carrying the pyrD gene on a multicopy plasmid . The preparation had a specific activity of 120 mumol min-1 mg-1 and contained flavin mononucleotide (FMN) in amounts stoichiometric to the dihydroorotate dehydrogenase subunit (Mr = 37000) . The flavin group was reduced when dihydroorotate was added in the absence of electron acceptors . The complete sequence of 1357 base pairs of an EcoRI-EcoRI DNA fragment containing the pyrD gene was established . Dihydroorotate dehydrogenase is encoded by a 336-triplets open reading frame . The molecular mass (Mr = 36732), the amino acid composition and the N-terminal sequence of the predicted polypeptide agree well with the data obtained by analysis of the purified protein . A region of the amino acid sequence (residues 292-303, i.e . Ile-Ile-Gly-Val-Gly-Gly-Ile-Asp-Ser-Val-Ile-Ala) shows distinct homology to the cofactor binding site of other flavoproteins . No hydrophobic regions large enough to span the cytoplasmic membrane were observed . By the S1-nuclease technique an mRNA start was mapped 34 +/- 2 nucleotide residues upstream of the beginning of the coding frame of pyrD . The leader region contains no similarity to the attenuators of the pyrB and pyrE genes of E . coli.

J Biol Chem, 1985 Aug 15, 260(17), 9875 - 83
Amplification and purification of UvrA, UvrB, and UvrC proteins of Escherichia coli; Thomas DC et al.; The UvrA, UvrB, and UvrC proteins of Escherichia coli are subunits of a DNA repair enzyme, ABC exci nuclease . In order to amplify these proteins, we have joined the artificial canonical promoter tac (Amann E., Brosius, J., and Ptashne, M . (1983) Gene (Amst.) 25, 167-178) to the uvr genes to obtain plasmids that express these genes under the control of the lac repressor . When cells carrying the tac-uvr plasmids are induced by the gratuitous lac inducer isopropyl-beta-D-galactoside the Uvr proteins are overproduced reaching a level of 10-20% of total cellular proteins after 6-8 h of induction . We have developed methods to purify all three Uvr proteins, UvrA, UvrB, and UvrC, in milligram quantities and to near homogeneity from these overproducing cells . The purified UvrA protein is an ATPase but UvrB and UvrC proteins are not . However, UvrB protein stimulates the ATPase activity of UvrA protein by a factor of 1.5 in the presence of double-stranded DNA and by a factor of about 2.6 in the presence of UV-irradiated DNA but not in the absence of DNA.

J Biol Chem, 1985 Aug 15, 260(17), 9775 - 83
Accessibility of lysyl residues of Escherichia coli B/r porin (OmpF) to covalent labeling reagents of different sizes . An approach for a three-dimensional structure of a channel-forming protein; Schlaeppi JM et al.; The three-dimensional structure of Escherichia coli B/r porin (OmpF) was studied by chemical modification using activated sugars of different size . Galactose and galactosides of different penetration properties through the porin channel were oxidized by galactose oxidase, and the 6-aldehydes formed were linked to amino groups in porin by reduction with NaBH3CN . Tryptic fragments of modified and unmodified porin were separated by reversed-phase high pressure liquid chromatography and identified by amino acid and amino-terminal analysis from the known primary structure of OmpF . Modification of purified native porin trimers in beta-octylglucoside revealed three classes of amino groups: (i) those not modified by any sugars; (ii) those modified only by small sugars that diffuse rapidly through the pore, such as galactose or melibiose; and (iii) those modified by either small or large sugars, the latter including pore-impermeant sugars such as stachyose . The results suggest that the three classes of amino groups correspond, respectively, to groups buried in the trimeric molecule, those in the interior of the pore and those exposed on the surface of porin . In addition modification experiments performed on whole cells suggested that all the reactive groups modified by the pore-impermeant sugars (class iii) are located on the surface of porin exposed on the outside of the outer membrane.

Biochemistry, 1985 Aug 13, 24(17), 4694 - 703
Photoinactivation and photoaffinity labeling of tryptophan synthase alpha 2 beta 2 complex by the product analogue 6-azido-L-tryptophan; Miles EW et al.; The photoaffinity reagent 6-azido-L-tryptophan was synthesized by chemical methods . It binds reversibly in the dark to the alpha 2 beta 2 complex of tryptophan synthase of Escherichia coli and forms a quinonoid intermediate with enzyme-bound pyridoxal phosphate (lambda max = 476 nm) . The absorbance of this chromophore has been used for spectrophotometric titrations to determine the binding of 6-azido-L-tryptophan (the half-saturation value {S}0.5 = 6.3 microM) . Photolysis of the quinonoid form of the alpha 2 beta 2 complex results in time-dependent inactivation of the beta 2 subunit but not of the alpha subunit . The extent of photoinactivation is directly proportional to the absorbance at 476 nm of the quinonoid intermediate prior to photolysis . The substrate L-serine is a competitive inhibitor of 6-azido-L-tryptophan binding and photoinactivation . The competitive inhibitors L-tryptophan, D-tryptophan, and oxindolyl-L-alanine also protect against photoinactivation . The results demonstrate that 6-azido-L-tryptophan is a quasi-substrate for the alpha 2 beta 2 complex of tryptophan synthase and that photolysis of the enzyme-quasi-substrate quinonoid intermediate results in photoinactivation . The modified alpha 2 beta 2 complex retains its ability to bind pyridoxal phosphate and to cleave indole-3-glycerol phosphate, a reaction catalyzed by the alpha subunit . 6-Azido-L-tryptophan (side-chain 1,2,3-14C3 labeled) was synthesized enzymatically from 6-azidoindole and uniformly labeled L-{14C}serine by the alpha 2 beta 2 complex of tryptophan synthase on a preparative scale and has been isolated . Incorporation of 14C label from 6-azido-L-{14C}tryptophan is stoichiometric with inactivation . Our finding that most of the incorporated 14C label is bound in an unstable linkage suggests that an active site carboxyl residue is the major site of photoaffinity labeling by 6-azido-L-tryptophan.

Biochemistry, 1985 Aug 13, 24(17), 4527 - 33
Quantitative determination of the 5-(hydroxymethyl)uracil moiety in the DNA of gamma-irradiated cells; Frenkel K et al.; 5-(Hydroxymethyl)uracil (HMUra) is a chemically stable derivative of thymine formed through the action of ionizing radiation which we previously identified in the DNA of gamma-irradiated HeLa cells {Teebor, G . W., Frenkel, K., & Goldstein, M . S . (1984) Proc . Natl . Acad . Sci . U.S.A . 81, 318-321} . In this report, we determine whether HMUra can be used as a marker of exposure of DNA to ionizing radiation . Dose-response curves for its formation in {3H}thymidine-labeled DNA were constructed by exposing the DNA to increasing amounts of gamma-radiation and measuring the HMUra content . DNA was irradiated both in solution and in intact cells . HMUra was identified as the 2'-deoxyribonucleoside 5-(hydroxymethyl)-2'-deoxyuridine (HMdU) by subjecting the irradiated DNA to enzymatic digestion and analyzing the mixture of 2'-deoxyribonucleosides by high-pressure liquid chromatography . The identity of the radiogenically formed HMdU was confirmed by acetylation and the structure of the acetyl derivative obtained by mass and nuclear magnetic resonance spectroscopies . At two different DNA concentrations in solution, the same number of thymidine moieties were converted to HMdU, indicating that within this range of concentration the formation of HMdU was mediated through the indirect action of ionizing radiation . Equal amounts of HMdU were formed in single- and double-stranded DNA at each radiation dose, indicating that DNA conformation did not affect HMdU formation . Surprisingly, the G value (number of HMdU molecules formed/100 eV) was higher in irradiated cellular DNA than in DNA irradiated in solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1985 Aug 13, 24(17), 4549 - 52
1H NMR studies of lambda cro repressor . 1 . Selective optimization of two-dimensional relayed coherence transfer spectroscopy; Weber PL et al.; Two-dimensional relayed coherence transfer NMR spectroscopy (RELAY) has been used to corroborate side chain spin system identities in crowded regions of the 1H NMR spectrum of the lambda cro repressor protein . The mixing time in the RELAY experiments was optimized for specific preselected spin systems by using recently developed methods {Bax, A., & Drobny, G . (1985) J . Magn . Reson, 61, 306-320}, which utilize the transverse relaxation time (T2) of the molecule and relevant J couplings for the defined spin system . We demonstrate that a mixing time of 26 ms gives rise to strong C alpha H-C gamma H3 RELAY cross peaks for all valine, threonine, and isoleucine residues, while RELAY cross peaks for other spin systems are weak or are not observed . This allows for rapid and unambiguous identification of the side chain resonances for valine, isoleucine, threonine, and alanine (by elimination) . The use of optimized RELAY for analyzing and identifying spin systems in complex spectra is discussed.

Nucleic Acids Res, 1985 Aug 12, 13(15), 5545 - 61
Fate of exogenous recombinant plasmids introduced into mouse and human cells; Biamonti G et al.; We have constructed a number of plasmids selectable in both E . coli and mouse or human cells . Human DNA sequences were inserted and the recombinant plasmids were used to transfect either mouse or human cells by the Ca-phosphate precipitation technique . We have observed that: (i) competent cells uptake large amounts of plasmid DNA; (ii) input plasmids persist in transformed mammalian cells as free unreplicating circular molecules for up to 20 generations; such persistence does not depend on the presence of selective markers; (iii) plasmids incorporated into mouse L-cells undergo widespread rearrangements (in the absence of replication) entailing mostly deletions of both human and bacterial sequences which yield smaller products; the latter appear to be more stable in a subsequent transformation cycle . Surprisingly such rearrangements are almost totally absent in transformed human KB-cells . This property of human KB-cells may prove useful for the development of a vector apt at cloning and expressing human DNA sequences . Unlike what has been observed in yeast, no "autonomously replicating sequence" can be detected in mammalian cells by randomly cloning human DNA sequences into a selectable plasmid and screening for an increased transformation efficiency.

Nucleic Acids Res, 1985 Aug 12, 13(15), 5471 - 83
Mammalian DNA helicase; Hubscher U et al.; A forked DNA was constructed to serve as a substrate for DNA helicases . It contains features closely resembling a natural replication fork . The DNA was prepared in large amounts and was used to assay displacement activity during isolation from calf thymus DNA polymerases alpha holoenzyme . One form of DNA polymerase alpha holoenzyme is possibly involved leading strand replication at the replication fork and possesses DNA dependent ATPase activity (Ottiger, H.-P . and Hubscher, U . (1984) Proc . Natl . Acad . Sci . USA 81, 3993-3997) . The enzyme can be separated from DNA polymerase alpha by velocity sedimentation in conditions of very low ionic strength and then be purified by chromatography on Sephacryl S-200 and ATP-agarose . At all stages of purification, DNA dependent ATPase and displacement activity profiles were virtually superimposable . The DNA dependent ATPase can displace a hybridized DNA fragment with a short single-stranded tail at its 3'hydroxyl end only in the presence of ATP, and this displacement relies on ATP hydrolysis . Furthermore, homogeneous single-stranded binding proteins from calf thymus as well as from other tissues cannot perform this displacement reaction . By all this token the DNA dependent ATPase appears to be a DNA helicase . It is suggested that this DNA helicase might act in concert with DNA polymerase alpha at the leading strand, possibly pushing the replication fork ahead of the polymerase.

Nucleic Acids Res, 1985 Aug 12, 13(15), 5457 - 69
Separation of complementary strands of plasmid DNA using the biotin-avidin system and its application to heteroduplex formation and RNA/DNA hybridizations in electron microscopy; Delius H et al.; A method for the separation of complementary strands with the help of the biotin-avidin system is described . Restriction fragments were terminally labeled at both ends with biotinylated nucleotides . The DNA was cut by a second restriction enzyme, and the fragments were bound to an avidin agarose column . The non-biotinylated strands were eluted with 0.1 M NaOH, and the biotin-labeled strands were subsequently released from the column by elution with 50% guanidine isothiocyanate/formamide . Contamination of the separated strands by complementary single strands was less than 4%.-Separated linear single strands of the vector pEMBL were prepared . On annealing with recombinant circular DNA a substitution loop is formed which provides position and orientation markers for the unambiguous electron microscopic analysis of heteroduplexes or hybrids formed with the inserted sequences . -The terminal biotin label was visualized by complex formation with a streptavidin-ferritin conjugate.

J Mol Biol, 1985 Aug 5, 184(3), 441 - 53
Temperature dependence of the rate constants of the Escherichia coli RNA polymerase-lambda PR promoter interaction . Assignment of the kinetic steps corresponding to protein conformational change and DNA opening; Roe JH et al.; The kinetics of formation and of dissociation of open complexes (RPo) between Escherichia coli RNA polymerase (R) and the lambda PR promoter (P) have been studied as a function of temperature in the physiological range using the nitrocellulose filter binding assay . The kinetic data provide further evidence for the mechanism R + P in equilibrium I1 in equilibrium I2 in equilibrium RPo, where I1 and I2 are kinetically distinguishable intermediate complexes at this promoter which do not accumulate under the reaction conditions investigated . The overall second-order association rate constant (ka) increases dramatically with increasing temperature, yielding a temperature-dependent activation energy in the range 20 kcal (near 37 degrees C) to 40 kcal (near 13 degrees C) (1 kcal = 4.184 kJ) . Both isomerization steps (I1----I2 and I2----RPo) appear to be highly temperature dependent . Except at low temperatures (less than 13 degrees C) the step I1----I2, which we attribute to a conformational change in the polymerase with a large negative delta Cp degrees value, is rate-limiting at the reactant concentrations investigated and hence makes the dominant contribution to the apparent activation energy of the pseudo first-order association reaction . The subsequent step I2----RPo, which we attribute to DNA melting, has a higher activation energy (in excess of 100 kcal) but only becomes rate-limiting at low temperature (less than 13 degrees C) . The initial binding step R + P in equilibrium I1 appears to be in equilibrium on the time-scale of the isomerization reactions under all conditions investigated; the equilibrium constant for this step is not a strong function of temperature and is approximately 10(7) M-1 under the standard ionic conditions of the assay (40 mM-Tris . HCl (pH 8.0), 10 mM-MgCl2, 0.12 M-KC1) . The activation energy of the dissociation reaction becomes increasingly negative at low temperatures, ranging from approximately -9 kcal near 37 degrees C to -30 kcal near 13 degrees C . Thermodynamic (van't Hoff) enthalpies delta H degrees of open complex formation consequently are large and temperature-dependent, increasing from approximately 29 to 70 kcal as the temperature is reduced from 37 to 13 degrees C . The corresponding delta Cp degrees value is approximately -2.4 kcal/deg . We propose that this large negative delta Cp degrees value arises primarily from the burial of hydrophobic surface in the conformational change (I1 in equilibrium I2) in RNA polymerase in the key second step of the mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

J Mol Biol, 1985 Aug 5, 184(3), 375 - 87
Transformation of yeast with linearized plasmid DNA . Formation of inverted dimers and recombinant plasmid products; Kunes S et al.; The molecular products of DNA double strand break repair were investigated after transformation of yeast (Saccharomyces cerevisiae) with linearized plasmid DNA . DNA of an autonomous yeast plasmid cleaved to generate free ends lacking homology with the yeast genome, when used in transformation along with sonicated non-homologous carrier DNA, gave rise to transformants with high frequency . Most of these transformants were found to harbor a head-to-head (inverted) dimer of the linearized plasmid . This outcome of transformation contrasts with that observed when the carrier DNA is not present . Transformants occur at a much reduced frequency and harbor either the parent plasmid or a plasmid with deletion at the site of the cleavage . When the linearized plasmid is introduced along with sonicated carrier DNA and a homologous DNA restriction fragment that spans the site of plasmid cleavage, homologous recombination restores the plasmid to its original circular form . Inverted dimer plasmids are not detected . This relationship between homologous recombination and a novel DNA transaction that yields rearrangement could be important to the cell, as the latter could lead to a loss of gene function and lethality.

J Biol Chem, 1985 Aug 5, 260(16), 9443 - 51
Isolation and characterization of the orotidine 5'-monophosphate decarboxylase domain of the multifunctional protein uridine 5'-monophosphate synthase; Floyd EE et al.; The multifunctional protein uridine 5'-monophosphate (UMP) synthase catalyzes the final two reactions of the de novo biosynthesis of UMP in mammalian cells by the sequential action of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate (OMP) decarboxylase (EC 4.1.1.23) . This protein is composed of one or two identical subunits; the monomer weighs of 51,500 daltons . UMP synthase from mouse Ehrlich ascites cells can exist as three distinct species as determined by sucrose density gradient centrifugation: a 3.6 S monomer, a 5.1 S dimer, and a 5.6 S conformationally altered dimer . Limited digestion of each of these three species with trypsin produced a 28,500-dalton peptide that was relatively resistant to further proteolysis . The peptide appears to be one of the two enzyme domains of UMP synthase for it retained only OMP decarboxylase activity . Similar results were obtained when UMP synthase was digested with elastase . OMP decarboxylase activity was less stable for the domain than for UMP synthase; the domain can rapidly lose activity upon storage or upon dilution . The size of the mammalian OMP decarboxylase domain is similar to that of yeast OMP decarboxylase . If the polypeptides which are cleaved from UMP synthase by trypsin are derived exclusively from either the amino or the carboxyl end of UMP synthase, then the size of a fragment possessing the orotate phosphoribosyltransferase domain could be as large as 23,000 daltons which is similar in size to the orotate phosphoribosyltransferase of yeast and of Escherichia coli.

J Mol Biol, 1985 Aug 5, 184(3), 529 - 33
Sites of dnaA protein-binding in the replication origin of the Escherichia coli K-12 chromosome; Matsui M et al.; On the basis of the observation that dnaA protein binds preferentially to DNA fragments carrying the Escherichia coli chromosomal replication origin (oriC), the binding sites were investigated by DNase I footprinting . As a result, three strong binding sites were identified in the minimal oriC sequence . The respective binding sites were 16 to 17 base-pairs long, and contained a common sequence (5') T-G-T-G-(G/T)-A-T-A-A-C (3') in the middle, although their polarities were not the same . Since mutants defective in function for autonomous replication have been isolated in the corresponding positions of the common sequence at each binding site, dnaA protein-binding at these sites seems to be significant for replication initiation.

J Mol Biol, 1985 Aug 5, 184(3), 399 - 412
Structure and expression of the cell division genes ftsQ, ftsA and ftsZ; Yi QM et al.; The essential cell division genes ftsQ, ftsA and ftsZ map in a cluster of cell envelope genes located at two minutes on the Escherichia coli genetic map and appear to constitute an atypical operon . These closely clustered genes are all transcribed in the same direction and yet each of these genes was independently cloned on a multicopy plasmid and found to be expressed . Tn5 insertion mutagenesis of this region confirmed that these genes were independently expressed, indicating that each of these genes has its own promoter . However, maximum expression of the distal ftsZ gene required the promoter for the proximal ftsQ gene . The proximal ftsZ promoter was located within the ftsA structural gene and the proximal ftsA promoter was located within the ftsQ structural gene . No transcription terminators were evident from the DNA sequence analysis of this region . The sequence analysis also revealed that the termination codon for ftsQ overlapped the initiation codon of the ftsA gene and that the ftsA and ftsZ genes were separated by 60 base-pairs . The ftsQ gene product has a calculated Mr of 31,432 and the ftsA gene product has a calculated Mr of 45,327 . The structure and expression of these genes are discussed.

J Biol Chem, 1985 Aug 5, 260(16), 9427 - 34
The succinate dehydrogenase of Escherichia coli . Immunochemical resolution and biophysical characterization of a 4-subunit enzyme complex; Condon C et al.; Using EPR spectroscopy to monitor the integrity of the enzyme, conditions have been established which allow specific immunoprecipitation of the succinate dehydrogenase complex of Escherichia coli . The enzyme complex precipitated from Lubrol PX-solubilized membranes by monospecific antiserum in the presence of a cocktail of protease inhibitors contains four polypeptides of apparent MrS 71,000, 26,000, 17,000, and 15,000 . The 71-kDa flavopeptide is readily susceptible to proteolysis, and the enzyme complex shows unusual facile dissociation . Spectroscopic measurements indicate the presence of a {2Fe-2S} cluster (Center 1), a {3Fe-xS} cluster (Center 3), and a b-type cytochrome . In addition, a change in relaxation of Center 1 at low potentials is indicative of Center 2 . Midpoint redox potentials of Centers 1-3 for both the membrane-bound and detergent-solubilized enzyme were estimated to be +10 mV, -175 mV, and +65 mV, respectively.

J Biol Chem, 1985 Aug 5, 260(16), 9346 - 56
Characterization and derivation of the gene coding for mitochondrial carbamyl phosphate synthetase I of rat; Nyunoya H et al.; The nucleotide sequence of rat carbamyl phosphate synthetase I mRNA has been determined from the complementary DNA . The mRNA comprises minimally 5,645 nucleotides and codes for a polypeptide of 164,564 Da corresponding to the precursor form of the rat liver enzyme . The primary sequence of mature rat carbamyl phosphate synthetase I indicates that the precursor is cleaved at one of two leucines at residues 38 or 39 . The derived amino acid sequence of carbamyl phosphate synthetase I is homologous to the sequences of carbamyl phosphate synthetase of Escherichia coli and yeast . The sequence homology extends along the entire length of the rat polypeptide and encompasses the entire sequences of both the small and large subunits of the E . coli and yeast enzymes . The protein sequence data provide strong evidence that the carbamyl phosphate synthetase I gene of rat, the carAB gene of E . coli, and the CPA1 and CPA2 genes of yeast were derived from common ancestral genes . Part of the rat carbamyl phosphate synthetase I gene has been characterized with two nonoverlapping phage clones spanning 28.7 kilobases of rat chromosomal DNA . This region contains 13 exons ranging in size from 68 to 195 base pairs and encodes the 453 carboxyl-terminal amino acids of the rat protein . Southern hybridization analysis of rat genomic DNA indicates the carbamyl phosphate synthetase I gene to be present in single copy.

J Biol Chem, 1985 Aug 5, 260(16), 9316 - 25
Replication of pBR322 DNA in vitro with purified proteins . Requirement for topoisomerase I in the maintenance of template specificity; Minden JS et al.; The replication of plasmid pBR322 DNA has been reconstituted with purified proteins from Escherichia coli . Initiation of the leading-strand requires RNA polymerase holoenzyme, DNA polymerase I, RNase H, and DNA gyrase . Initiation of the lagging-strand requires the primosomal proteins (the dnaB, dnaC, and dnaG proteins, replication factor Y (protein n') and proteins i, n, and n") and the single-stranded DNA binding protein . DNA polymerase III holoenzyme is required for extensive elongation of the nascent DNA chains . The products of this replication reaction are primarily nonsegregated daughter molecules . However, the addition of small amounts of soluble extract from E . coli results in the completion and segregation of these molecules to give mature form I DNA, suggesting that additional factors are required for this process . Topoisomerase I is necessary to make the replication system specific for pBR322 DNA as a template, indicating that the linking number of the DNA, determined by an equilibrium between the opposing activities of topoisomerase I and DNA gyrase, plays a crucial role in determining the reactivity of the DNA molecule toward initiating DNA replication . The function of the proteins involved in the replication of this closed-circular, double-stranded, superhelical DNA is discussed.

J Biol Chem, 1985 Aug 5, 260(16), 9326 - 35
Purification and characterization of murine retroviral reverse transcriptase expressed in Escherichia coli; Roth MJ et al.; Expression of a region of the Moloney murine leukemia virus (M-MuLV) pol gene in Escherichia coli resulted in the synthesis of reverse transcriptase activity which could be detected in crude extracts . Construction of deletions at the 3' terminus of this gene resulted in a 4-fold increase in the level of the reverse transcriptase activity in the soluble fraction of crude lysates and yielded the high level production of a stable protein species of Mr = 71,000 . Purification of this protein by column chromatography on DEAE-cellulose, phosphocellulose, polyribocytidylic acid-agarose, and hydroxylapatite indicated that it was a multifunctional enzyme containing RNase H and reverse transcriptase activity . The Mr = 71,000 species had a sedimentation coefficient of 4.65 S by glycerol gradient centrifugation, indicating that the enzyme was a monomer . Using poly(A)+ mRNAs primed with oligo(dT), the enzyme synthesized double-stranded DNA copies between 1.3 and 9.9 kilobases in length . Synthesis of long cDNA required 8 mM Mg2+, 4 mM Mn2+, 2 mM dNTPs, and saturating levels of enzyme . Actinomycin D efficiently limited the enzyme to the first strand synthesis . Additional characteristics of the fusion protein are described.

J Biol Chem, 1985 Aug 5, 260(16), 9085 - 7
Thermal regulation of beta-galactosidase synthesis using anti-sense RNA directed against the coding portion of the mRNA; Ellison MJ et al.; The in vivo production of RNA that is complementary to the mRNA of a particular target gene (anti-sense RNA) appears to be an effective tool for the regulation of genes in Escherichia coli (Coleman, J., Green, P.J., and Inouye, M . (1984) Cell 37, 429-436) . These investigators demonstrated that short anti-sense transcripts which are complementary to the ribosome binding site of the target mRNA are overwhelmingly the most effective in blocking protein synthesis . We have constructed plasmids which produce thermally regulated anti-sense transcripts of three regions of the E . coli lac Z gene coding sequence, and have examined the relative effects of these constructs on the synthesis of the lac Z gene product, beta-galactosidase . We conclude that there is a strong correlation between the length of RNA complementarity and the suppression of beta-galactosidase synthesis . Furthermore, a significant inhibition of translation can be obtained when the anti-sense transcript lacks complementarity to the 5' noncoding region of the mRNA, provided that the extent of complementarity with the coding sequence is considerable.

J Biochem (Tokyo), 1985 Aug, 98(2), 395 - 406
Structural requirements of lipid A responsible for the functions: a study with chemically synthesized lipid A and its analogues; Homma JY et al.; To confirm the revised lipid A structure of Escherichia coli and to establish the structure responsible for its functions, biological activities of the synthetic compounds based on the presented structure of E . coli lipid A were investigated . Compound 506, 2-deoxy-6-O-(2-deoxy-2-{(R)-3-dodecanoyloxytetradecanoylamino}-3-O {(R)3-tetradecanoyloxytetradecanoyl}-beta-D-glucopyranosyl}-3-O-{(R) -3-hydroxytetradecanoyl}-2-{(R)-3-hydroxytetradecanoylamino}-alpha -D-glucopyranose 1,4'-bis(phosphate), exhibited activities identical to those of natural E . coli lipid A in eliciting Shwartzman reaction and tests on lethality, pyrogenicity, interferon- and tumor necrosis factor-inducing activities as well as in B-cell activating activity and Limulus amebocyte lysate gelating activity . With the exception of the Shwartzman reaction the monophosphorylated synthetic compounds at either the 1 or 4' position showed slightly lower activities than the compound with the bisphosphorylated compound (Compound 506) . The compound without the phosphate group showed no or only very weak activities . The structural requirements for each activity (i.e . binding position and composition of fatty acids and presence of phosphate groups) are discussed taking into account the results of previous investigations.

Mutat Res, 1985 Aug, 151(1), 129 - 36
Influence of treatment temperature on the genotoxic effects of cisplatin in CHO cells: cytotoxicity, mutagenicity and induction of lesions in DNA; Plooy AC et al.; In cells exposed in vitro to the cytotoxic and mutagenic antitumor drug cisplatin (cis-Pt(NH3)2Cl2), various adducts with nuclear DNA are formed . A comparative study was made of the influence of temperature variation during treatment of cultured Chinese hamster ovary (CHO) cells with cisplatin on cytotoxicity, mutation induction and Pt-DNA adduct formation . Before and after treatment (1 h at 32, 37 or 40 degrees C) cells were kept at 37 degrees C . Cytotoxicity increased with temperature; D0 values were 29.6 +/- 1.6, 21.1 +/- 1.2 and 11.4 +/- 0.6 microM at 32, 37 and 40 degrees C, respectively . Pt-DNA binding to DNA at 40 degrees C was 2.0 (+/- 0.3) times as high as at 32 degrees C . This factor remained practically constant over a 24-h post-treatment incubation of the cells, during which about 60% of DNA-bound Pt were removed . As the increase in cytotoxicity between 32 and 40 degrees C was roughly in proportion to that in Pt binding, no substantial changes in the spectrum of adducts appeared to occur . The induction of DNA interstrand cross-links, studied at 32 and 40 degrees C, varied linearly with dose . Influence of temperature on cross-link formation was comparable to that on total Pt binding . Amounts of cross-links highly increased during 24 h after treatment . Plots of cross-links against survival after treatments at 32 and 40 degrees C almost coincided . Induction of 6-thioguanine-resistant (HGPRT) mutants at various cisplatin concentrations did not show a clear temperature dependency . Consequently, equitoxic treatments were significantly more mutagenic at 32 degrees C than at 40 degrees C, the opposite of what has been reported for E . coli.

Mayo Clin Proc, 1985 Aug, 60(8), 523 - 30
Entamoeba polecki infection in Southeast Asian refugees: multiple cases of a rarely reported parasite; Gay JD et al.; Recently, Entamoeba polecki was identified for the first time in our parasitology laboratory in stool specimens from eight Southeast Asian refugees . This ameba has been reported infrequently in the Western world; most reported cases have been from the New Guinea region . In most previously described patients and in our patients, no definite gastrointestinal symptoms could be directly attributed to E . polecki infection . Morphologically, E . polecki may mimic the pathogen E . histolytica and also nonpathogens such as E . coli . These species are most readily distinguished by studying encysted forms . In contrast to E . histolytica and E . coli, E . polecki characteristically has uninucleate cysts . Both pigs and monkeys naturally harbor E . polecki, but four of the patients in this series had no apparent contact with these animals . Other modes of infection may be human-to-human transmission or acquisition from other domestic animals . Six of our eight patients were treated successfully with one course of metronidazole in a regimen similar to that used for E . histolytica infection . In the two other patients, repeated courses of therapy eradicated the infection . Because of the recent increase in number of Southeast Asian immigrants to the United States, E . polecki may be identified more frequently than in the past . Physicians and laboratory personnel should be familiar with this organism, because it may be confused with E . histolytica or may act as a pathogen.

J Bacteriol, 1985 Aug, 163(2), 595 - 9
Bdellovibrio bacteriovorus synthesizes an OmpF-like outer membrane protein during both axenic and intraperiplasmic growth; Rayner JR et al.; Outer membrane preparations of Bdellovibrio bacteriovorus grown intraperiplasmically on Escherichia coli containing OmpF were prepared by the Triton X-100 procedure of Schnaitman (J . Bacteriol . 108:545-552, 1971) . They contained a protein that migrated to almost the same position as E . coli OmpF in sodium dodecyl sulfate-acrylamide gradient gel electrophoresis and to the same position as E . coli OmpF when urea was incorporated into the gel . The mobility of this protein increased relative to that of OmpC in urea-containing gels as does E . coli OmpF . However, the same protein was also produced during axenic growth and during intraperiplasmic growth on prey lacking OmpF . The peptide profile generated by partial proteolysis of this protein showed no homology to that produced from E . coli OmpF . We conclude that B . bacteriovorus synthesizes an OmpF-like protein . Previous claims that the bdellovibrio incorporates an intact E . coli OmpF are not consistent with these observations.

Proc Natl Acad Sci U S A, 1985 Aug, 82(16), 5305 - 9
Inducible transcription of the unrearranged kappa constant region locus is a common feature of pre-B cells and does not require DNA or protein synthesis; Nelson KJ et al.; Transcription of unrearranged kappa constant region (kappa 0) loci is dramatically induced in pre-B cells transformed by the Abelson murine leukemia virus when the cells are exposed to bacterial lipopolysaccharide (LPS) . Transcriptional activity, detected both by accumulation of the 8-kilobase kappa 0 RNA product and by nuclear run-on measurements, is evident within a few hours after exposure to LPS and continues to increase over a 24-hr period . During this time, transcription of rearranged mu heavy-chain loci remains at the basal constitutive level . In accord with previous studies of the B-cell lymphoma 70Z/3, this transcriptional activation is accompanied by the appearance of a DNase I-hypersensitive site in the kappa enhancer region but not by any detectable hypomethylation of the locus . Moreover, the present studies demonstrate that induction of kappa transcription can occur in the absence of DNA or protein synthesis . These results have led us to propose a model in which an external signal such as LPS or a functionally equivalent lymphokine may initiate kappa transcription in pre-B cells by modifying or overriding the activity of an enhancer-specific factor.

Dev Biol, 1985 Aug, 110(2), 321 - 30
Ecdysterone selectively stimulates the expression of a 23000-Da heat-shock protein-beta-galactosidase hybrid gene in cultured Drosophila cells; Lawson R et al.; To study the regulated expression of cloned heat-shock genes in homologous cells, hybrid Drosophila heat-shock-Escherichia coli beta-galactosidase genes were constructed . Segments of the ecdysterone-inducible 23,000-Da heat-shock protein (hsp23) gene and of two other hsp genes (hsp84 and 70), which are not hormone regulated, were functionally linked to the bacterial coding sequence, and the resulting hybrid genes were introduced into cultured, hormone-responsive Drosophila cells by transfection . All hybrid genes directed the synthesis of E . coli-specific beta-galactosidase in heat-treated cells . hsp23 hybrid gene expression was stimulated strongly by ecdysterone, while the activities of the other hybrid genes were not affected at all by the hormone . A hybrid gene with only 147 bp of hsp23 promoter sequence could not be activated by either heat or ecdysterone treatment . Thus, far upstream sequences contain signals required for the regulated expression of the hsp23 gene in Drosophila cells.

Eur J Biochem, 1985 Aug 1, 150(3), 485 - 90
A mutant from Escherichia coli which lacks ribosomal proteins S17 and L29 used to localize these two proteins on the ribosomal surface; Stoffler-Meilicke M et al.; A mutant of Escherichia coli has been isolated which lacked ribosomal proteins S17 and L29, as judged by two-dimensional gel electrophoresis . A battery of immunological tests was used to confirm this result . Ribosomes of this mutant were used as a control for the localization of proteins S17 and L29 on the surface of the ribosomal subunits of E . coli . Protein S17 has been localized on the 30S subunit body, 3-5 nm away from the lower pole, while protein L29 is located at the back of the 50S particle on the opposite side to the interface.

Biokhimiia, 1985 Aug, 50(8), 1374 - 6
{Effect of serotonin on the yield of UV- and x ray-induced DNA damage}; Ivanova EV et al.; Using two-dimensional thin-layer chromatography, the effect of serotonin on the yield of thymine dimers and on cleavage of the N-glycosidic bond in the DNA irradiated with ultraviolet (UV) light and X-ray was studied . Bound serotonin was shown to reduce the synthesis of UV-induced thymine dimers but had no effect on the number of X-ray-induced breaks in the N-glycoside bonds in thymidine residues . The data obtained are discussed in terms of the mechanisms of serotonin involvement in the photoprotection of yeast cells from the lethal action of UV and X-ray irradiations.

J Toxicol Sci, 1985 Aug, 10 Suppl 1, 123 - 8
{Mutagenicity test of halopredone acetate}; Ohuchida A et al.; The mutagenicity of halopredone acetate (THS-201) was investigated by means of reverse mutation test in seven bacterial strains (S . typhimurium TA100, TA1535, TA98, TA1538, TA1537 and E . coli WP2, WP2 uvrA) and chromosomal aberration test in cultured Chinese hamster cells (CHL) . In the reverse mutation test, no significant increase of revertant was observed at dose levels from 50 to 5000 micrograms/plate in the absence and presence of mammalian metabolic activation system . THS-201 caused no increase of chromosomal aberrants at dose levels of 1.6, 8.0, 40.0 and 200 micrograms/ml irrespective of metabolic activation . These results indicated that THS-201 has no mutagenic activity.

J Biochem (Tokyo), 1985 Aug, 98(2), 379 - 84
A more sensitive enzyme immunoassay of anti-insulin IgG in guinea pig serum with less non-specific binding of normal guinea pig IgG; Kohno T et al.; An enzyme immunoassay of anti-insulin IgG in guinea pig serum was improved in sensitivity by reducing the non-specific binding of normal guinea pig IgG and enhancing the specific binding of anti-insulin IgG . Silicone rubber pieces or polystyrene balls were coated with normal rabbit IgG, followed by coupling of insulin using glutaraldehyde . The insulin-normal rabbit IgG-coated silicone rubber pieces or polystyrene balls were incubated with normal rabbit IgG and then with diluted guinea pig anti-insulin serum in the presence of normal rabbit IgG at a lower temperature (20 degrees C) . Finally, the solid phases were incubated with rabbit (anti-guinea pig IgG) Fab'-horseradish peroxidase conjugate to measure the amount of guinea pig IgG bound . The detection limit of anti-insulin IgG in guinea pig serum was improved 10 to 100-fold compared to that of enzyme immunoassay performed by incubating insulin-bovine serum albumin-coated solid phases with diluted guinea pig anti-insulin serum at 37 degrees C and then with rabbit (anti-guinea pig IgG) Fab' conjugated to beta-D-galactosidase from Escherichia coli, according to a previous report (Kato, K., et al . (1978) J . Biochem . 84, 93-102).

EMBO J, 1985 Aug, 4(8), 2109 - 12
Localization of ribosomal protein S2 on the surface of the 30S subunit from Escherichia coli, using monoclonal antibodies; Schwedler-Breitenreuter G et al.; Protein S2 has been localized on the surface of the 30S subunit of Escherichia coli by immuno-electron microscopy . The antibody was obtained from a fusion of myeloma cells with spleen cells of mice, which had been immunized with intact 30S ribosomal subunits of E . coli . The binding site of the antibody was on the head of the small subunit, just above the small lobe, in the region where protein S3 has also been localized . S2 is the first ribosomal protein to have been mapped exclusively with monoclonal antibody.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1985 Aug, 260(1), 1 - 7
Comparison between enterotoxic activity and methanol solubility in heat-stable enterotoxins (STa and STb) from Escherichia coli of human, porcine and bovine origins; Gonzalez EA et al.; We have investigated the enterotoxic activity of culture filtrates and their methanol extracted fractions from 10 ST (STa or STb) producing Escherichia coli strains from human, porcine and bovine origin, in the infant mouse test (IMT) as well as in the rabbit intestinal loop test (RILT) . Unconcentrated culture filtrates and methanol-soluble fractions from the eight STa-producing strains were positive in the IMT while methanol-insoluble fractions obtained from these STa-positive strains, like methanol-soluble and -insoluble fractions from the two strains producing only STb, lacked activity in the IMT . Unconcentrated culture filtrates from all ST-producing strains were unable to cause fluid accumulation in the rabbit ligated intestinal loops after 6 h incubation . When this material, concentrated 5-fold, was tested again, the culture filtrates and methanol-soluble fractions from all STa-producing strains yielded strongly positive fluid accumulation in the RILT, whereas culture filtrates and their methanol extracted fractions from the strains producing only STb, like methanol-insoluble fractions from four STa-producing strains, caused slight fluid secretion in the rabbit intestinal loops.

J Gen Microbiol, 1985 Aug, 131 ( Pt 8), 2079 - 85
Identification of a new locus in the Escherichia coli cotransduction gap that represents a new genetic component of the L-asparagine utilization system; Chesney RH et al.; A temperature-sensitive Escherichia coli mutant defective for the ability to utilize L-asparagine as a sole nitrogen source was isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis . The mutation (asu) produces two distinct phenotypic effects . Mutant strains grow poorly at high temperature on minimal plates containing asparagine as the sole nitrogen source; this effect is greatly exacerbated by the presence of methionine . Mutant strains utilize L-asparagine as a nitrogen source three to four times more efficiently at permissive temperatures than the wild-type strains . The mutation maps at 32.4 min on the E . coli chromosome, within the E . coli cotransduction gap . Mutant strains produce normal amounts of thermo-stable L-asparaginase I activity . The mutation therefore affects a component of the asparagine utilization system other than the catabolism of asparagine within the cell; it probably affects asparagine uptake.

Genetika, 1985 Aug, 21(8), 1253 - 60
{Molecular mechanisms of the initiation of complete and mosaic mutations}; Dianov GL et al.; To study the molecular basis of the origin of complete and mosaic mutants, pBR322 plasmid with one- or two-stranded DNA damage was constructed by limited chemical modification of the plasmid DNA . Damage of one strand of DNA resulted in induction of mosaic mutants . Data were obtained indicating that complete mutations arise as a result of damage of two strands in the region of the mutagenized gene.

Appl Environ Microbiol, 1985 Aug, 50(2), 298 - 303
Destruction of the outer membrane permeability barrier of Escherichia coli by heat treatment; Tsuchido T et al.; Heat treatment of a wild-type Escherichia coli strain at 55 degrees C in 50 mM Tris-hydrochloride buffer with or without 10 mM magnesium sulfate or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at pH 8.0 caused an increase in cell surface hydrophobicity . By determining the location of n-hexadecane droplets attached to cells by phase-contrast microscopy, the septal and polar regions of heated cells appeared to become the most frequently hydrophobic . Some of the lipopolysaccharide molecules in the outer membrane were released from heated cells, and the cells became susceptible to the hydrolytic action of added phospholipase C . Heat-treated cells also became permeable to the hydrophobic dye crystal violet, which was added externally . The release of part of the outer membrane by heat treatment appeared to bring about the disorganization of the outer membrane structure and, as a consequence, to result in the partial disruption of the permeability barrier function of the outer membrane . Tris was found to enhance damage to the outer membrane by heat.

DNA, 1985 Aug, 4(4), 273 - 81
High-level expression in Escherichia coli of biologically active bovine growth hormone; George HJ et al.; High-level synthesis of bovine growth hormone (bGH) in Escherichia coli was achieved by maximizing gene transcription and optimizing the translational efficiency of bGH mRNA . Nearly all of the recombinant hormone was found in the pellet fraction after bacterial cell lysis . This property allowed the purification of bGH nearly to homogeneity . Protein sequence analysis indicated that greater than 93% of the purified hormone had the amino-terminal methionine residue removed by E . coli, yielding mature bGH . In a hypophysectomized rat assay system, purified bacterial-produced bGH demonstrated growth-promoting activity equivalent to that of pituitary-derived bovine growth hormone.

Am J Vet Res, 1985 Aug, 46(8), 1770 - 4
Endotoxic shock in the awake young pig: absence of beneficial effect of prednisolone sodium succinate treatment; Schrauwen EM et al.; In nonanesthetized young pigs, the influence of prednisolone sodium succinate therapy on a 65% lethal dose of Escherichia coli endotoxin was studied by evaluating clinical signs, several hemodynamic variables, survival rate, and changes seen at necropsy . Endotoxin infusion induced reproducible clinical signs characterized by nausea, vomiting, dyspnea, cyanosis, and moderate excitement followed by severe CNS depression . Among the hemodynamic variables, there were decreases in arterial blood pressure and cardiac output and increases in pulmonary arterial pressure, heart rate, and total peripheral and pulmonary vascular resistances . Core temperature and arterial pH did not change significantly . Survival rate at 30 hours after the start of the endotoxin infusion was 35% . According to the necropsy, marked edema and hemorrhages were in several organs . Treating the experimental animals with prednisolone sodium succinate (3 injections of 10 mg/kg of body weight after the start of the endotoxin infusion) did not influence any of the monitored hemodynamic variables, except for arterial blood pressure, which was higher at the end of the hemodynamic recording period (270 minutes after the start of the endotoxin infusion) . Clinical signs, survival rate, and changes at necropsy were similar in both treated and nontreated pigs . This lack of effect can be due to an inappropriate dosage of the steroid or failure of steroid treatment to alleviate endotoxin-mediated effects.

Am J Vet Res, 1985 Aug, 46(8), 1768 - 9
A porcine-murine hybridoma that secretes porcine monoclonal antibody of defined specificity; Raybould TJ et al.; Spleen cells from a pig hyperimmunized with Escherichia coli were fused with nonproducer mouse plasmacytoma cells . Stable hybridoma lines secreting porcine immunoglobulins were obtained . One line secreted monoclonal porcine immunoglobulin G, which reacted specifically with E coli in an enzyme-linked immunosorbent assay and precipitated a polypeptide of molecular weight of 50,000 . This is the first report of a stable porcine-murine hybridoma producing porcine antibody of defined specificity.

J Pharm Sci, 1985 Aug, 74(8), 892 - 4
Dynamics of disinfection of selected preservatives against Escherichia coli; Hurwitz SJ et al.; Mathematical models were determined relating preservative concentration and D values (decimal reduction times at 25 degrees C; pH 6.9-7.1) against Escherichia coli in aqueous medium . Preservatives investigated were 2-bromo-2-nitro-1,3-propanediol (Bronopol), N-(hydroxymethyl)-N-(1,3-dihydroxymethyl-2, 5-dioxo-4-imidazolidinyl)-N'-(hydroxymethyl)urea (Germall II), phenethyl alcohol, and benzyl alcohol . Linear regression was used to determine D values {i.e., the time required for a particular concentration of preservative at a specified pH, temperature, and medium to cause a 90% reduction of viable organisms (E . coli)} from a number of concentrations of each preservative . Linear regression of the log D values versus the log of the concentration (a minimum of 4 concentrations per preservative) were used to derive power curves . Concentration exponents, eta values (the logarithmic values relating changes in rates of kill for specified changes in concentrations) and A values (extrapolated D values at 1% concentration), were determined . Correlation coefficients for these power fits ranged from -0.987 to -0.999 . Plots depicting the closeness of fit of the models to the actual data are shown.

J Biochem Biophys Methods, 1985 Aug, 11(2-3), 145 - 52
Estimation of protein molecular weights by high performance molecular-sieve chromatography on agarose columns in sodium dodecyl sulphate solution; Eriksson KO; Calibration curves showing the linear relationship between -log KD and (molecular weight)2/3 are presented for proteins subjected to molecular-sieve chromatography in a 0.1% sodium dodecyl sulphate solution on crosslinked gels of different agarose concentrations (5, 9, 12 and 20%) . These gels permit separation and estimation of molecular weights of proteins in the range 5000 to about 400 000, 130 000, 130 000, and 100 000, respectively . Separations of model proteins, heavy and light chains of gamma-globulin, and cyanogen bromide fragments of cellulase are shown.

Eur J Radiol, 1985 Aug, 5(3), 224 - 5
Renal abscess: report of a case with sonographic, urographic and CT evaluation; Weigert F et al.; Publication Types:

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