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-